US20040089616A1

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Publication number: US20040089616A1
Application number: US10/684,707
Authority: US
Inventors: Gregory Kellogg; Stephen Kieffer-Higgins; Mona Jensen; Shari Ommert; Mikayla Kob; Andrea Pierce; Keith Morneau; Hsin Lin
Original assignee: Tecan Trading AG
Current assignee: Tecan Trading AG
Priority date: 1997-05-23
Filing date: 2003-10-14
Publication date: 2004-05-13
Also published as: US6632399B1

Abstract

This invention provides methods and apparatus for performing microanalytic and microsynthetic analyses and procedures. Specifically, the invention provides a microsystem platform for use with a micromanipulation device to manipulate the platform by rotation, thereby utilizing the centripetal force resulting from rotation of the platform to motivate fluid movement through microchannels embedded in the microplatform. The microsystem platforms of the invention are also provided having microfluidics components, resistive heating elements, temperature sensing elements, mixing structures, capillary and sacrificial valves, and methods for using these microsystems platforms for performing biological, enzymatic, immunological and chemical assays.

Description

This application claim priority to U.S. Ser. No. 09/083,678, filed May 22, 1998. This application is also related to U.S. Ser. No. 08/995,056, filed Dec. 19, 1997, U.S. Ser. No. 08/910,726, filed Aug. 12, 1997, U.S. Ser. No. 08/768,990, filed Dec. 18, 1996 and U.S. Ser. No. 08/761,063, filed Dec. 5, 1996, the disclosures of every one of which are explicitly incorporated by reference herein.[0001]

BACKGROUND OF THE INVENTION

1. Field of the Invention[0002]

This invention relates to methods and apparatus for performing microanalytic analyses and procedures on fluid samples. In particular, the invention relates to microminiaturization of genetic, biochemical and chemical processes related to analysis, synthesis and purification. Specifically, the invention provides a microsystem platform that is rotationally manipulated by a micromanipulation device, thereby utilizing the centripetal forces resulting from rotation of the platform to motivate fluid movement through microchannels embedded in the microplatform. The microsystem platforms of the invention are provided having microfluidics components, chambers and reservoirs, resistive heating elements, temperature sensing elements, mixing structures, capillary and sacrificial valves, and methods for using these microsystems platforms for performing biological, enzymatic, immunological and chemical assays.[0003]

2. Summary of the Related Art[0004]

Assays for detecting analytes in fluid samples, particularly complex fluids such as biological fluid samples, are used for a variety of diagnostic, environmental, synthetic and analytical purposes in the medical, biological, chemical, biochemical and environmental arts.[0005]

Certain analytes are important for diagnosis and monitoring of acute and chronic disease in humans. For example, diabetes is the fourth major cause of morbidity and mortality in the U.S., even though the biochemical basis for the disease has been known for at least a century and drugs (primarily insulin) and methods for managing the disease are robust and widely used. It has been recognized that sensitive monitoring of blood sugar levels, either directly or by detecting levels of metabolites and disease-associated modifications (such as the relative fraction of glycated hemoglobin in the bloodstream) is important in managing the disease. However, fast and inexpensive ways of performing the assays necessary for efficient management of multiple indicators of the disease state are not currently available.[0006]

In the field of medical, biological and chemical assays, mechanical and automated fluid handling systems and instruments are known in the prior art.[0007]

U.S. Pat. No. 4,279,862, issued Jul. 21, 1981 to Bertaudiere et al. disclose a centrifugal photometric analyzer.[0008]

U.S. Pat. No. 4,381,291, issued Apr. 26, 1983 to Ekins teach analytic measurement of free ligands.[0009]

U.S. Pat. No. 4,515,889, issued May 7, 1985 to Klose et al. teach automated mixing and incubating reagents to perform analytical determinations.[0010]

U.S. Pat. No. 4,676,952, issued Jun. 30, 1987 to Edelmann et al. teach a photometric analysis apparatus.[0011]

U.S. Pat. No. 4,745,072, issued May 17, 1998 to Ekins discloses immunoassay in biological fluids.[0012]

U.S. Pat. No. 5,061,381, issued Oct. 29, 1991 to Burd discloses a centrifugal rotor for performing blood analyses.[0013]

U.S. Pat. No. 5,122,284, issued Jun. 16, 1992 to Braynin et al. discloses a centrifugal rotor comprising a plurality of peripheral cuvettes.[0014]

U.S. Pat. No. 5,160,702, issued Nov. 3, 1993 to Kopf-Sill and Zuk discloses rotational frequency-dependent[0015]
valves
using capillary forces and siphons, dependent on
wettablility” of liquids used to prime said siphon.
U.S. Pat. No. 5,171,695, issued Dec. 15, 1992 to Ekins discloses determination of analyte concentration using two labeling markers.[0016]
U.S. Pat. No. 5,173,193, issued Dec. 22, 1992 to Schembri discloses a centrifugal rotor for delivering a metered amount of a fluid to a receiving chamber on the rotor.[0017]
U.S. Pat. No. 5,242,803, issued Sep. 7,1993 to Burtis et al. disclose a rotor assembly for carrying out an assay.[0018]
U.S. Pat. No. 5,409,665, issued Apr. 25, 1995 to Burd discloses a cuvette filling in a centrifuge rotor.[0019]
U.S. Pat. No. 5,413,009, issued Jul. 11, 1995 to Ekins discloses a method for analyzing analytes in a liquid.[0020]
U.S. Pat. No. 5,472,603, issued Dec. 5, 1995 to Schembri discloses an analytical rotor comprising a capillary passage having an exit duct wherein capillary forces prevent fluid flow at a given rotational speed and permit flow at a higher rotational speed.[0021]
Anderson, 1968,[0022]Anal. Biochem.28: 545-562 teach a multiple cuvette rotor for cell fractionation.
Renoe et al., 1974[0023]Clin. Chem.20: 955-960 teach a
minidisc
module for a centrifugal analyzer.
Burtis et al., 1975,[0024]Clin. Chem.20: 932-941 teach a method for a dynamic introduction of liquids into a centrifugal analyzer.
Fritsche et al., 1975,[0025]Clin. Biochem.8: 240-246 teach enzymatic analysis of blood sugar levels using a centrifugal analyzer.
Burtis et al., 1975,[0026]Clin Chem.21: 1225-1233 teach a multipurpose optical system for use with a centrifugal analyzer.
Hadjiioannou et al., 1976,[0027]Clin. Chem.22: 802-805 teach automated enzymatic ethanol determination in biological fluids using a miniature centrifugal analyzer.
Lee et al., 1978,[0028]Clin. Chem.24: 1361-1365 teach a automated blood fractionation system.
Cho et al., 1982,[0029]Clin. Chem.28: 1956-1961 teach a multichannel electrochemical centrifugal analyzer.
Bertrand et al., 1982,[0030]Clinica Chimica Acta119: 275-284 teach automated determination of serum 5′-nucleotidase using a centrifugal analyzer.
Schembri et al., 1992,[0031]Clin Chem.38: 1665-1670 teach a portable whole blood analyzer.
Walters et al., 1995,[0032]Basic Medical Laboratory Technologies,3rd ed., Delmar Publishers: Boston teach a variety of automated medical laboratory analytic techniques.
Recently, microanalytical devices for performing select reaction pathways have been developed.[0033]
U.S. Pat. No. 5,006,749, issued Apr. 9, 1991 to White disclose methods apparatus for using ultrasonic energy to move microminiature elements.[0034]
U.S. Pat. No. 5,252,294, issued Oct. 12, 1993 to Kroy et al. teach a micromechanical structure for performing certain chemical microanalyses.[0035]
U.S. Pat. No. 5,304,487, issued Apr. 19, 1994 to Wilding et al. teach fluid handling on microscale analytical devices.[0036]
U.S. Pat. No. 5,368,704, issued Nov. 29, 1994 to Madou et al. teach microelectrochemical valves.[0037]
International Application, Publication No. WO93/22053, published 11 Nov. 1993 to University of Pennsylvania disclose microfabricated detection structures.[0038]
International Application, Publication No. WO93/22058, published 11 Nov. 1993 to University of Pennsylvania disclose microfabricated structures for performing polynucleotide amplification.[0039]
Columbus et al., 1987,[0040]Clin. Chem.33: 1531-1537 teach fluid management of biological fluids.
Ekins et al., 1994[0041]Ann. Biol. Clin.50: 337-353 teach a multianalytic microspot immunoassay.
Wilding et al., 1994,[0042]Clin. Chem.40: 43-47 disclose manipulation of fluids on straight channels micromachined into silicon.
One drawback in the prior art microanalytical methods and apparati has been the difficulty in designing systems for moving fluids on microchips through channels and reservoirs having diameters in the 10-500 μm range. Microfluidic systems require precise and accurate control of fluid flow and valving to control chemical reactions and analyte detection. Conventional pumping and valving mechanisms have been difficult to incorporate into microscale structures due to inherent conflicts-of-scale. These conflicts of scale arise in part due to the fact that molecular interactions arising out of mechanical components of such components, which are negligible in large (macroscopic) scale devices, become very significant for devices built on a microscopic scale.[0043]
While devices and pumping and valving mechanisms have been developed which overcome some of these conflict-of-scale difficulties, there are other inherent problems with these systems. A number of microanalytical platforms have been developed which use electrokinetic forces for fluid pumping: electroosmotic flow devices; electrohydrodynamic devices; and electrophoretic devices. An inherent drawback in these systems is that they rely on precise control of pH and free charges in the fluid being pumped. This makes them incapable of pumping most raw biological samples, such as blood and urine, and creates difficulties in pumping organic solvents. In cases where these systems may be used, pre-conditioning of the fluid to enhance electrokinetic effects is usually required.[0044]
Systems that use centripetal force to effect fluid movement in microstructures address the need for a pumping mechanism to effect fluid flow, but cannot alone solve these scale-related drawbacks of conventional fluidics reduced to microfluidics scale. There remains a need for a simple, flexible, reliable, rapid and economical microanalytic and microsynthetic reaction platform for performing biological, biochemical and chemical analyses and syntheses that can move fluids within the structural components of a microsystems platform. Such a platform should be able to move nanoliter-to microliter amounts of fluid, including reagents and reactants, at rapid rates to effect the proper mixing of reaction components, removal of reaction side products, and isolation of desired reaction products and intermediates. There remains a need in the art for centripetally-motivated microfluidics platforms capable of precise and accurate control of flow and metering of fluids in both microchip-based and centrifugal microplatform-based technologies.[0045]

SUMMARY OF THE INVENTION

This invention provides microsystems platforms as disclosed in co-owned and co-pending U.S. Ser. No. 08/761,063, filed Dec. 5, 1996 and U.S. Ser. No. 09/083,678, filed May 22, 1998, each of which is incorporated by reference herein. Specifically, this invention provides microfluidics platforms for performing biological, enzymatic, immunological and chemical assays.[0046]
It is an advantage of the centrifugal rotors and Microsystems platforms of the invention that an imprecise amount of a fluid comprising a biological sample can be applied to the rotor or platform and a precise volumetric amount of the biological sample is delivered to a fluid reservoir comprising a reaction vessel or other component of the rotor or platform for performing chemical, biochemical, immunological or other analyses. It is an advantage of the centrifugal rotors and Microsystems platforms of the invention that metering of said precise amount of a biological fluid sample, for example, a drop of blood, is provided as an intrinsic property of the metering capillary channel of the rotor or platform, thereby avoiding variability introduced by centripetal metering of the sample into a reaction reservoir. It is a further advantage of the centrifugal rotors and Microsystems platforms of the invention that an operator can avoid having to precisely measure an amount of a fluid comprising a biological sample for application to the rotor or microsystem platform, thereby permitting end-users, including consumers, having a lower level of sophistication to use a medically diagnostic or other embodiment of the rotor or microsystem platform of the invention.[0047]
It is an advantage of the centrifugal rotors and Microsystems platforms of the invention that fluid movement into and out of fluid reservoirs on the rotor or platform is precisely determined by displacement of a first fluid, such as biological sample, from a fluid reservoir by a second fluid contained in a second reservoir on the rotor or platform. It is also an advantage of the centrifugal rotors and Microsystems platforms of the invention that approximately complete replacement of the volumetric capacity of a first reservoir can be achieved by using fluid displacement as disclosed herein, thereby providing for maximum recovery of a first fluid sample upon displacement by a second fluid, or maximum delivery and replacement of the first fluid by the second fluid. This aspect of the invention is advantageous for providing sequential chemical or biochemical reaction steps wherein mixing of the reagents is not desired.[0048]
It is also an advantage of the centrifugal rotors and Microsystems platforms of the invention that these platforms provide an integrated microfluidics system containing components and structures for performing microanalytic assays whereby fluid flow on the platform is motivated by centripetal force and controlled by capillary and/or sacrificial valves. The invention provides such integrated platforms whereby an operator is required simply to apply a sample, most preferably an imprecise volume of a fluid sample, to an entry port on the disk surface, and a complex series of analytical steps are performed without further operator manipulation on the platform. Movement of fluids on the disk, and the sequence of analytical reaction steps performed thereupon, is a consequence of changes in rotor speed and/or the opening of sacrificial valves as directed by an instruction set contained in a program contained on the disk itself or in the memory of the micromanipulation apparatus that controls disk rotation and performance.[0049]
The Microsystems platforms also provide disks that can perform a multiplicity of analytical reactions on either several samples or a particular sample, whereby the reactions are performed sequentially or individually. In addition, a wide variety of analytic reactions can be performed on the Microsystems platforms of the invention, as further described below.[0050]
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.[0051]

DESCRIPTION OF THE DRAWINGS

FIGS. 1 through 8 are schematic representations of microfluidics arrays and components for performing direct analyte detection assays using the Microsystems platforms of the invention.[0052]
FIGS. 9 through 11 are schematic representations of microfluidics arrays and components for performing separations of analyte from a fluid sample using the Microsystems platforms of the invention.[0053]
FIGS. 12 and 13 are schematic representations of microfluidics arrays and components for performing both direct analyte detection assays and analyte separations using the Microsystems platforms of the invention.[0054]
FIGS. 14A and 14B illustrate the results of the assays disclosed in Example 4 FIG. 15 is a photograph showing DNA recovery using the assays described in Example 4.[0055]
FIG. 16 is a schematic diagram of a snap-in glucose assay component of a Microsystems platform of the invention.[0056]

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides centrifugal rotors and Microsystems platforms for providing centripetally-motivated fluid micromanipulation.[0057]
For the purposes of this invention, the term “sample” will be understood to encompass any fluid, solution or mixture, either isolated or detected as a constituent of a more complex mixture, or synthesized from precursor species.[0058]
For the purposes of this invention, the term “in fluid communication” or “fluidly connected” is intended to define components that are operably interconnected to allow fluid flow between components. In preferred embodiments, the platform comprises a rotatable platform, more preferably a disk, whereby fluid movement on the disk is motivated by centripetal force upon rotation of the disk.[0059]
For the purposes of this invention, the term “a centripetally motivated fluid micromanipulation apparatus” is intended to include analytical centrifuges and rotors, microscale centrifugal separation apparati, and most particularly the microsystems platforms and disk handling apparati of International Application No. WO97/21090, incorporated by reference.[0060]
For the purposes of this invention, the term “Microsystems platform” is intended to include centripetally-motivated microfluidics arrays as disclosed in International Application No. WO97/21090.[0061]
For the purposes of this invention, the terms “capillary”, “microcapillary and “microchannel” will be understood to be interchangeable and to be constructed of either wetting or non-wetting materials where appropriate.[0062]
For the purposes of this invention, the term “fluid chamber” will be understood to mean a defined volume on a rotor or Microsystems platform of the invention comprising a fluid.[0063]
For the purposes of this invention, the term “entry port” will be understood to mean a undefined volume on a rotor or microsystems platform of the invention comprising a means for applying a fluid to the rotor or platform.[0064]
For the purposes of this invention, the term “capillary junction” will be understood to mean a junction of two components wherein one or both of the lateral dimensions of the junction are larger than the corresponding dimensions of the capillary. In wetting or wettable systems, such junctions are where capillary valving occurs, because fluid flow through the capillaries is stopped at such junctions. In non-wetting or non-wettable junctions, the exit from the chamber or reservoir is where the capillary junction occurs. In general, it will be understood that capillary junctions are formed when the dimensions of the components change from a small diameter (such as a capillary) to a larger diameter (such as a chamber) in wetting systems, in contrast to non-wettable systems, where capillary junctions form when the dimensions of the components change from a larger diameter (such as a chamber) to a small diameter (such as a capillary).[0065]
For the purposes of this invention, the term “biological sample” or “biological fluid sample” will be understood to mean any biologically-derived analytical sample, including but not limited to blood, plasma, serum, lymph, saliva, tears, cerebrospinal fluid, urine, sweat, plant and vegetable extracts, semen, cell culture fluids, cellular lysate aqueous or non-aqueous fractions of the above and ascites fluid.[0066]
For the purposes of this invention, the term “air displacement channels” will be understood to include ports in the surface of the platform that are contiguous with the components (such as chambers and reservoirs) on the platform, and that comprise vents and microchannels that permit displacement of air from components of the platforms and rotors by fluid movement.[0067]
For the purposes of this invention, the term “capillary action” will be understood to mean fluid flow in the absence of rotational motion or centripetal force applied to a fluid on a rotor or platform of the invention.[0068]
For the purposes of this invention, the term “capillary microvalve” will be understood to mean a capillary comprising a capillary junction whereby fluid flow is impeded and can be motivated by the application of pressure on a fluid, typically by centripetal force created by rotation of the rotor or platform of the invention.[0069]
The microplatforms of the invention (preferably and hereinafter collectively referred to as discs or disks for the purposes of this invention), the terms “microplatform”, “Microsystems platform” and “disc” or “disk” are considered to be interchangeable, and are provided to comprise one or a multiplicity of microsynthetic or microanalytic systems. Such microsynthetic or microanalytic systems in turn comprise combinations of related components as described in further detail herein that are operably interconnected to allow fluid flow between components upon rotation of the disk. These components can be fabricated as described below either integral to the disk or as modules attached to, placed upon, in contact with or embedded in the disk. The invention also comprises a micromanipulation device for manipulating the disks of the invention, wherein the disk is rotated within the device to provide centripetal force to effect fluid flow on the disk. Accordingly, the device provides means for rotating the disk at a controlled rotational velocity, for stopping and starting disk rotation, and advantageously for changing the direction of rotation of the disk. Both electromechanical means and control means, as further described herein, are provided as components of the devices of the invention. User interface means (such as a keypad and a display) are also provided, as further described in International Application WO97/21090.[0070]
Fluid (including reagents, samples and other liquid components) movement is controlled by centripetal acceleration due to rotation of the platform. The magnitude of centripetal acceleration required for fluid to flow at a rate and under a pressure appropriate for a particular microsystem is determined by factors including but not limited to the effective radius of the platform, the position angle of the structures on the platform with respect to the direction of rotation and the speed of rotation of the platform.[0071]
The capillary junctions and microvalves of the invention are based on the use of rotationally-induced fluid pressure to overcome capillary forces. Fluids which completely or partially wet the material of the microchannels (or reservoirs, reaction chambers, detection chambers, etc.) which contain them experience a resistance to flow when moving from a microchannel of narrow cross-section to one of larger cross-section, while those fluids which do not wet these materials resist flowing from microchannels (or reservoirs, reaction chambers, detection chambers, etc.) of large cross-section to those with smaller cross-section. This capillary pressure varies inversely with the sizes of the two microchannels (or reservoirs, reaction chambers, detection chambers, etc., or combinations thereof), the surface tension of the fluid, and the contact angle of the fluid on the material of the microchannels (or reservoirs, reaction chambers, detection chambers, etc.) Generally, the details of the cross-sectional shape are not important, but the dependence on cross-sectional dimension results in microchannels of dimension less than 500 μm exhibit significant capillary pressure. By varying the intersection shapes, materials and cross-sectional areas of the components of the Microsystems platform of the invention, valves are fashioned that require the application of a particular pressure on the fluid to induce fluid flow. This pressure is applied in the disks of the invention by rotation of the disk (which has been shown above to vary with the square of the rotational frequency, with the radial position and with the extent of the fluid in the radial direction). By varying capillary valve cross-sectional dimensions as well as the position and extent along the radial direction of the fluid handling components of the microsystem platforms of the invention, capillary valves are formed to release fluid flow in a rotation-dependent manner, using rotation rates of from 100 rpm to several thousand rpm. This arrangement allows complex, multistep fluid processes to be carried out using a pre-determined, monotonic increase in rotational rate. The theoretical principles underlying the use of capillary junctions and microvalves are disclosed in International Patent Application, Publication No. WO98/07019, incorporated by reference.[0072]
The instant invention provides Microsystems platforms comprising microfluidics components, heating elements, temperature sensing elements, capillary valves, sacrificial valves and a rotor design for transmitting electrical signals to and from the Microsystems platforms of the invention. The invention provides fluidics components for capillary metering of precise amounts of a volume of a fluid sample from the application of a less precise volume of a fluid sample at an entry port on the microsystem platform. These embodiments of the invention provide for delivery of precise amounts of a sample such as a biological fluid sample without requiring a high degree of precision or accuracy by the operator or end-user in applying the fluid to the platform, and is advantageous in embodiments of the Microsystems platforms of the invention that are used by consumers and other relatively unsophisticated users. The invention also provides laminar flow-dependent replacement of a fluid in a first chamber by a second displacement fluid in a second chamber on the platform. These embodiments of the invention provide approximately complete replacement of a fluid in one chamber on the platform with fluid from another, and thereby provide means for practicing sequential chemical reactions and other sequential processes on the platform under conditions wherein mixing of the two fluids is disadvantageous. The invention also provides turbulent flow mixing components, which permit thorough mixing of different fluid components on the platform, and in particular, the invention provides mixing chambers fluidly connected with fluid reservoirs containing equal amounts of two or more different fluids or unequal amounts of two or more different fluids. In addition, the invention provides fluid reservoirs fluidly connected with mixing chamber of the invention and shaped to determine the relative rate of flow of each of the different fluids into the mixing chamber. In these embodiments, gradients of two fluids differing in viscosity, solute concentration or concentration of suspended particulates can be produced using the mixing chambers of the invention, as disclosed; U.S. Ser. No. 09/083,678 incorporated by reference. Such gradients can be transferred to reservoirs on the platform for further analytical manipulations, and can form the basis for controlled testing of concentration-dependent effects of various catalysts, drugs, toxins or other biological or chemical agents.[0073]
Platforms of the invention such as disks and the components comprising such platforms are advantageously provided having a variety of composition and surface coatings appropriate for a particular application. Platform composition will be a function of structural requirements, manufacturing processes, and reagent compatibility/chemical resistance properties. Specifically, platforms are provided that are made from inorganic crystalline or amorphous materials, e.g. Silicon, silica, quartz, inert metals, or from organic materials such as plastics, for example, poly(methyl methacrylate) (PMMA), acetonitrile-butadiene-styrene (ABS), polycarbonate, polyethylene, polystyrene, polyolefins, polypropylene and metallocene. These may be used with unmodified or modified surfaces. Surface properties of these materials may be modified for specific applications. Surface modification can be achieved by silanization, ion implantation and chemical treatment with inert-gas plasmas (i.e., gases through which electrical currents are passed to create ionization). Also provided by the invention are platforms made of composites or combinations of these materials, for example, platforms manufactures of a plastic material having embedded therein an optically transparent glass surface comprising for example the detection chamber of the platform. Microplatform disks of the invention are preferably fabricated from thermoplastics such as teflon, polyethylene, polypropylene, methylmethacrylates and polycarbonates, among others, due to their ease of molding, stamping and milling. Alternatively, the disks can be made of silica, glass, quartz or inert metal. A fluid handling system is built by sequential application of one or more of these materials laid down in stepwise fashion onto the thermoplastic substrate. Alternatively, the entire disc can be injection molded, embossed or stamped. Disks of the invention are fabricated with an injection molded, optically-clear base layer having optical pits in the manner of a conventional compact disk (CD). The optical pits provide means for encoding instrument control programming, user interface information, graphics, data analysis, and sound specific to the application and driver configuration. The driver configuration depends on whether the micromanipulation device is a handheld, benchtop or floor model, and also on the details of external communication and other specifics of the hardware configuration. This layer is then overlaid with a reflective surface, with appropriate windows for external detectors, specifically optical detectors, being left clear on the disk. Other layers of polycarbonate of varying thickness are laid down on the disk in the form of channels, reservoirs, reaction chambers and other structures, including provisions on the disk for valves and other control elements. These layers can be pre-fabricated and cut with the appropriate geometries for a given application and assembled on the disk. Layers comprising materials other than polycarbonate can also be incorporated into the disk. The composition of the layers on the disk depend in large part on the specific application and the requirements of chemical compatibility with the reagents to be used with the disk. Electrical layers can be incorporated in disks requiring electric circuits, such as electrophoresis applications and electrically-controlled valves. Control devices, such as integrated circuits, laser diodes, photodiodes and resistive networks that can form selective heating areas or flexible logic structures can be incorporated into appropriately wired recesses, either by direct fabrication of modular installation onto the disk. Reagents that can be stored dry can be introduced into appropriate open chambers by spraying into reservoirs using means similar to inkjet printing heads, and then dried on the disk. A top layer comprising access ports and air vents, ports or shafts is then applied. Liquid reagents are then injected into the appropriate reservoirs, followed by application of a protective cover layer comprising a thin plastic film.[0074]
The platforms of the invention are preferably provided with a multiplicity of components, either fabricated directly onto the platform, or placed on the platform as prefabricated modules. In addition to the integral components, certain devices and elements can be located external to the platform, optimally positioned on a device of the invention in relation to the platform, or placed in contact with the platform either while rotating or when at rest. Components optimally comprising the platforms of the invention or a controlling device in combination therewith include detection chambers, reservoirs, valving mechanisms, detectors, sensors, temperature control elements, filters, mixing elements, and control systems.[0075]
This invention provides microsystems platforms comprising the following components.[0076]

1. Fluidics Components

The platforms of the invention are provided comprising microfluidics handling structures in fluidic contract with one another. In preferred embodiments, fluidic contact is provided by capillary or microchannels comprising the surface of the platforms of the invention. Microchannel sizes are optimally determined by specific applications and by the amount of delivery rates required for each particular embodiment of the platforms and methods of the invention. Microchannel sizes can range from 0.02 mm to a value close to the thickness of the platform. Microchannel shapes can be trapezoid, circular or other geometric shapes as required. Microchannels preferably are embedded in a platform having a thickness of about 0.1 to 100 mm, wherein the cross-sectional dimension of the microchannels across the thickness dimension of the platform is less than 500 μm-800 μm and from 1 to 90 percent of said cross-sectional dimension of the platform. In these embodiments, which is based on the use of rotationally-induced fluid pressure to overcome capillary forces, it is recognized that fluid flow is dependent on the orientation of the surfaces of the components. Fluids which completely or partially wet the material of the microchannels, reservoirs, detection chambers, etc. (i.e., the components) of the platforms of the invention which contain them experience a resistance to flow when moving from a component of narrow cross-section to one of larger cross-section, while those fluids which do not wet these materials resist flowing from components of the platforms of the invention of large cross-section to those with smaller cross-section. This capillary pressure varies inversely with the sizes of the two components, or combinations thereof, the surface tension of the fluid, and the contact angle of the fluid on the material of the components. Generally, the details of the cross-sectional shape are not important, but the dependence on cross-sectional dimension results in microchannels of dimension less than 500 μm exhibit significant capillary pressure. By varying the intersection shapes, materials and cross-sectional areas of the components of the platform of the invention, “valves” are fashioned that require the application of a particular pressure on the fluid to induce fluid flow. This pressure is applied in the disks of the invention by rotation of the disk (which has been shown above to vary with the square of the rotational frequency, with the radial position and with the extent of the fluid in the radial direction). By varying capillary valve cross-sectional dimensions as well as the position and extent along the radial direction of the fluid handling components of the platforms of the invention, capillary valves are formed to release fluid flow in a rotation-dependent manner, using rotation rates of from 100 rpm to several thousand rpm. This arrangement allows complex, multistep fluid processes to be carried out using a predetermined, monotonic increase in rotational rate.[0077]
A first example of the microfluidics arrays provided by this invention is shown in FIGS. 1A through 1D. A Microsystems platform is provided by the invention that is specifically designed for performing an assay for detecting a chemical species in a complex mixture, preferably an aqueous mixture. These Figures illustrate an array advantageously used for any such assay; detection of blood glucose concentration is illustrated herein. It will be understood that in this description, the use of the words “cell” and “particulate” will be interchangeable, and that cells are a particular example of a specific particulate species comprising the fluid samples, and most preferably the biological fluid samples, analyzed using the present invention.[0078]
A Microsystems platform provided by the invention and specifically designed for performing blood glucose assay is illustrated in FIG. 1A. Disk embodiments of the platforms of the invention were fashioned, for example, from machined acrylic or injection-molded polycarbonate, polystyrene, polypropylene, acetonitrile-butadiene-styrene, or high-density polyethylene (HDPE). The overall disc dimensions include an outer radius of from about 1 cm to about 15 cm and an inner radius of from about 0.1 cm to about 1 cm, wherein the disk was mounted on the spindle of a rotary device. The thickness of the disc ranged from about 0.2 mm to about 1.5 cm. All surfaces coming into contact with blood on the platform may be advantageously treated with heparin, EDTA or other anticoagulants to facilitate fluid flow thereupon[0079]
The components of the blood glucose assay were prepared as follows. Fluid[0080]sample entry port101 having a depth in the platform surface from about 0.15 cm to about 3 mm and lateral dimensions of from about 0.1 cm to about 2.5 cm were constructed on the platform, and designed to accommodate a volume of from about 5 to about 200 μL. This entry port was fluidly connected with ametering capillary102 having a square cross-sectional diameter of from about 0.02 mm to about 2 cm and proximal ends rounded with respect toentry port101; the length of this metering chamber array was sufficient to contain a total volume of from about 15 to about 150 μL. The entry port was also constructed to be fluidly connected with anoverflow capillary121 having a cross-sectional diameter of from about 0.02 mm to about 2 mm and proximal ends rounded with respect toentry port101. The overflow capillary was fluidly connected with anoverflow chamber122 having a depth in the platform surface of from about 0.02 mm to about 9 mm, greater than the depth of theoverflow capillary121. Each of the chambers on the platform were also connected with air ports or air channels, such as114, that have dimensions of from about 0.02 mm to about 2 mm deep and permitted venting of air displaced by fluid movement on the platform. Acapillary junction115 that is from about 0.03 mm to about 2.2 mm deep is present in the air channel to prevent fluid flow into the air channel.
[0081]Entry port101 was positioned on the platform from about 1 cm to about 12 cm from the center of rotation.Metering chamber102 extended from about 0.25 cm to about 2 cm fromentry port101. The extent of the length ofoverflow capillary121 was greater than the extent of the length ofmetering capillary102. The position ofoverflow chamber122 was, for example, from about 1.2 cm to about 14 cm from the axis of rotation.
In an alternative embodiment of the fluid metering structures of the invention, shown in FIG. 1B, fluid[0082]sample entry port101 is comprised of a funnel having a depth in the platform surface from about 0.75 mm to 5 mm, lateral dimensions of from about 2 mm to 2 cm in the long dimension and from about 2 mm to 2 cm in the short dimension and positioned from about 0.75 cm to about 5 cm from the center of rotation, and designed to accommodate a volume of about 5 to about 200 μL. The bottom surface of the entry port consists of aslot120 fluidly connected to microfluidics structures formed in the other (under) side of the microfluidics surface of the platform.Entry port101 is connected toentry passageway102 having a rectangular cross-sectional diameter of from about 0.1 mm to 5 mm wide and from about 0.1 mm to 5 mm deep and extending from about 0.1 cm to about 3 cm fromentry port101. Theentry passageway102 is fluidly connected toblood entry chamber103.Blood entry chamber103 has a depth of from about 0.1 mm to 5 mm, lateral dimensions of from about 1 mm to about 4 cm, and is positioned from about 0.75 cm to about 2.5 cm from the center of rotation.Blood entry chamber103 further comprisesblood metering volume104 designed to accommodate a volume of from about 1 μL to about 50 μL and anoverflow passageway105. The overflow passageway was fluidly connected with anoverflow capillary106 having a depth in the platform surface of from about 0.1 mm to about 1 mm.Overflow capillary121 is further fluidly connected withoverflow chamber122 which is comprised of two parts, a shallowouter portion108 having a depth from about 0.05 mm to about 0.5 mm and a deeperinner portion109 having a depth of from about 0.1 mm to 5 mm.Overflow chamber122 is positioned from about 4 cm to about 5.8 cm from the axis of rotation. The distal end of theblood overflow capillary121 is chosen to be farther from the center of rotation than the distal end ofblood chamber104. Each of the chambers on the platform were also connected with air ports orair channels114, that are from about 0.1 mm to about 5 mm deep and permit venting of air displaced by fluid movement on the platform. Acapillary junction115 that is from about 0.03 mm to about 2.2 mm deep is present in the air channel to prevent fluid flow into the air channel. In alternative embodiments, these vents may be multiply connected to one another through a manifold such that fluid flow merely displaces air within the structure, rather than forcing it through vents in the platform surface.
Alternatively, an unmetered amount of a blood sample is placed directly in[0083]blood fluid chamber104, which in this embodiment is open to the surface of the disk to accept blood application. In these embodiments, the amount of blood fluid to be assayed is controlled by the capacity ofassay chamber107 or thematrix106 contained therein as described below.
As described herein for performing blood glucose assays (and as understood in the art that essentially the same microfluidics structures can be used for a multiplicity of blood analyte assays or, more generally, for analyte assays in any fluid sample, most preferably a biological fluid sample), a capillary barrier prevents movement of the fluid sample directly into the[0084]assay chamber107. In the metering structure shown in FIG. 1A, fluid (or more properly for this illustrative example, blood)metering capillary102 acted as a capillary barrier that prevented blood fluid flow frommetering capillary102 at a first, non-zero rotational speed f₁, ranging from about 200 rpm to about 450 rpm and sufficient to permit fluid flow comprising overflow from theentry port101 throughoverflow capillary121 and intooverflow chamber122. This capillary boundary was constructed to be overcome at a second rotational speed f₂, ranging from about 250 rpm to about 900 rpm (where f₂>f₁). In the alternative embodiments shown in FIGS. 1B and 1C,blood fluid chamber104 acted as a capillary barrier that was maintained during rotation at a rotational speed sufficient to motivated excess fluid sample from theentry port101 to theoverflow chamber122, and was overcome at a second rotational speed greater than the first rotational speed to permit fluid sample flow intoassay chamber107.
[0085]Blood metering capillary102 andblood fluid chamber104 were in different alternatives of the microsystems platforms of the invention fluidly connected to capillary110 that was from about 0.02 mm to about 2 mm deep and had a cross-sectional diameter of from about 0.02 mm to about 2 mm and was connected to capillary orsacrificial valve111. Sacrificial orcapillary valve111 was further fluidly connected withcapillary112 that was from about 0.02 mm to about 2 mm deep and had a cross-sectional diameter of from about 0.02 mm to about 2 mm, and further toassay chamber107. In capillary valve embodiments, the junction betweencapillary110 andcapillary112 createscapillary valve111, wherein saidcapillary valve111 was from about 0.03 mm to about 2.2 mm deep and had a cross-sectional diameter of from about 0.03 mm to about 2.2 mm. In order to function as a capillary valve, the junction betweencapillary110 and capillary112 must have a depth and/or cross sectional area greater than that ofcapillary110 in a disc fabricated from hydrophilic materials such as acrylic. In sacrificial valve embodiments, intermediate melting temperature materials (including, for example, waxes as described above) are placed in the lumen ofcapillary110 forming a fluid-tight seal. In these embodiments,chamber111 is fluidly connected to the sacrificial valve so that melted wax from the release sacrificial valve is sequestered in the chamber.
[0086]Rectangular assay chamber107 was constructed in the surface of the platform to have a depth of from about 0.2 mm to about 3 mm, most preferably comprising a circular orrectangular depression113 connected tocapillary112.Depression113 was constructed to have a volumetric capacity of from half to twice the assay volume.Assay chamber107 also comprised a pad ormatrix106 of a hydrophilic substance. Materials used to prepare said matrices include but are not limited to derivatized nylons, nitrocellulose, fiberglass and polyesters, most preferably having a pore size of 0.2-2.0 μm, most preferably comprising a positively-charged nylon matrix having a pore size of about 0.8 μm. The upper limit on pore size ofmatrix106 is chosen to inhibit or prevent blood cell entry into the matrix. The matrix is positioned inassay chamber107 to be in fluidic contact withdepression113, more preferably coveringdepression113, and most preferably having a surface area greater than the surface area ofdepression113. The matrix was further impregnated with immobilized reagents which produce a detectable product proportional to the amount or concentration of glucose in a blood sample. Most preferably, the detectable product is a colored product, i.e., a product absorbing light at a detectable, most preferably a visible, wavelength.
In preparing matrices according to the invention, reagents advantageously used to detect and more preferably quantitate an amount of a component of a biological fluid sample are impreganted into the matrix. As a non-limiting example, glucose is detected according to the invention using a glucose oxidase assays system, as described in additional detail below. Matrices for performing such assays using the microfluidics platforms of the invention are prepared by saturating the matrix membrane with an 8 mL solution of distilled water containing 0.12[0087]g 2,5-ferandione polymer with methoxylene (CAS: 9011-16-9), 10 mg EDTA, 200 mg Polypep® Low Viscosity (Sigma Chemical Company, St. Louis, Mo.), 668 mg sodium citrate, 28.75 mg glucose oxidase and 27.3 mg peroxidase. The saturated membrane is then dried and then saturated with 5 mL acetonitrile and 5 mL distilled water containing 40 mg 3-methyl-2-benzothiazolinone hydrazone and 80 mg 3-dimethylaminobenzoic acid. The saturated membrane is dried and applied to the microfluidics disk of the invention for use.
In an alternative embodiment shown in FIG. 15,[0088]assay chamber107 is comprised of a rectangular cavity in the surface of the platform having a depth of from about 0.2 mm to about 3 mm which is fluidly connected at its end proximal to the axis of rotation to capillary112 and toair displacement channels114 andports115. The second member of the assay chamber is a rectangular piece made from the same material as the platform or other material and designed to snap into the cavity forming liquid-tight seals around all edges. The snap-in piece has two faces, an A face and a B face. The A face consists of afluid entry channel112 connected todepression113;depression113 is further connected toair displacement channels114.Depression113 is from about 0.05 mm to about 5 mm deep and has a dimensions from about 0.5 mm by 4 mm, having a volumetric capacity of from about half to about twice the assay volume applied to the disc. A pad ormatrix106 is attached to the A face of the snap-in piece, comprising a hydrophilic substance possessing a pore size of 0.2-2.0 μm, most preferably comprising a positively-charged nylon matrix having a pore size of about 0.8 μm. The upper limit on pore size ofmatrix106 is chosen to inhibit or prevent blood cell entry into the matrix.. The matrix is positioned inassay chamber107 to be in fluidic contact withdepression113, more preferably coveringdepression113, and most preferably having a surface area greater than the surface area ofdepression113. The matrix is further impregnated with immobilized reagents which produce a detectable product proportional to the amount or concentration of glucose in a blood sample. Most preferably, the detectable product is a colored product, i.e., a product absorbing light at a detectable, most preferably a visible, wavelength.
As illustrated in FIGS. 2A through 2F, in the use of this platform an imprecise volume (ranging from 20-150 μL) of blood was applied to the[0089]entry port101. In embodiments of the platform comprising air displacement channels, the fluid wicked intoair channel114 and was stopped bycapillary junction115. Fluid also wicked intometering capillary102 andoverflow capillary121. Fluid flowed through themetering capillary102 andoverflow capillary121 at no rotational speed until the fluid reached capillary junctions at the junction betweenmetering capillary102 andcapillary110 andoverflow capillary121 andoverflow chamber122.Metering capillary102 was constructed to define a precise volume from about 15 to about 60 μL of fluid betweenentry port101 andcapillary junction111, which was designed to be at least the amount of the fluid placed by the user inentry port101.
After sample loading by a user and filling of[0090]metering capillary102 andoverflow capillary121 at no rotational speed, the platform was spun at a first rotational speed f₁, ranging from about 50 rpm to about 600 rpm, which was sufficient to motivate fluid flow through theoverflow capillary121 in this microfluidics array having anentry port101 with a depth of from about 0.2 mm to about 3 mm,metering capillary102 with dimensions of about 0.5 mm×0.5 mm in cross-section and a length of about 2.2-3.8 cm from the center of rotation and anoverflow capillary121 with dimensions of about 0.5 mm×0.5 mm in cross-section and a length of about 5.4 cm from the center of rotation.
Due to the greater distance of the end of[0091]overflow capillary121 from the center of rotation than the end ofmetering capillary102, at rotational speed f₁fluid flowed throughoverflow capillary121 intooverflow chamber122. The platform was spun until all excess fluid is evacuated fromentry port101 and intooverflow chamber122, except the fluid contained inmetering chamber102.
At a second rotational speed f[0092]₂of from about 100 rpm to about 1000 rpm, the precise amount of fluid contained inmetering capillary102 was delivered intoassay chamber107. In embodiments comprising asacrificial valve111 in-line withcapillary110 at a position betweencapillary110 and112 shown in FIG. 2B, release of the sacrificial valve resulted in fluid flow throughcapillary112 and intoassay chamber107. In said embodiments, fluid flow is achieved at rotational speed f₂with removal of the sacrificial valve. In embodiments of the platforms of the invention comprisingcapillary valve111 at a position betweencapillary110 and112 shown in FIG. 2B, capillary110 preferably filled along with filling ofmetering capillary102 until blood reachedcapillary junction111 at the junction betweencapillary110 andcapillary112; in such embodiments, the capillary junction had a depth of from about 0.03 mm to about 2.2 mm, or at least greater than the depth ofcapillary110.
Blood flowing into[0093]assay chamber107 is preferentially directed todepression113 in the assay chamber; the dimensions ofdepression113 are conveniently chosen to be able to contain substantially all of the blood fluid of the sample metered throughmetering capillary102 into assay chamber107 (FIG. 2C). Displaced air flows throughair channel114, and may be vented to the surface of the disc or in communication withblood fluid chamber104.
As blood flows into[0094]depression113, the fluid component of the blood is driven by pressure and hydrophilic forces intomatrix106; the pore size of the matrix is chosen to prevent the cellular components of the blood from entering the matrix (FIG. 1.4). In preferred embodiment, the cellular component of the blood is retained indepression113 and the fluid component is efficiently distributed by wicking and by centripetal force intomatrix106. In an alternative embodiment,alternative matrix118 comprises at least two distinct elements that are compressed or adhered to one another in the assays chamber. The first element is similar to the reagent-containingmatrix106 described above; however, this embodiment of the matrix has a pore size that is not limited by the size of cellular components of the blood, and can be any pore size deemed optimal on experimental, economic, manufacturing or availability grounds. The second element comprises a filtering layer having a pore size that prevents cells and cellular debris from entering this portion of the matrix. In a preferred embodiment, the two elements and rearranged inassay chamber107 so that second matrix element is in contact withdepression113 wherein the blood aliquot is first contacted with the second matrix component. Blood fluid flows into and through the second matrix element and into the first matrix element, whereby cellular components of blood are prevented from enteringassay chamber107 by the pore size of second matrix element. Preferably, the dimensions of the matrix element is about 1 cm by about 0.75 cm and has a thickness of about 0.05 cm.
As the fluid component of the blood wicks into[0095]matrix106, dried reagents are solubilized and the reaction of the blood component catalyzed by said reagents proceeds. The timescale over which these chemical reactions take place is chosen to be long compared with the time it takes for the fluid component of blood to saturate thematrix106. The time it takes for the blood fluid to saturate the matrix is dependent on the capacity of thematrix106 to absorb the fluid and the delivery speed of the blood fluid to theassay chamber107, which in turn is dependent on the rotational velocity and the cross-sectional dimensions ofcapillary110. In preferred embodiments, the reaction(s) goes to completion within about 1 min. Reaction of the blood component(s) with the reagents produce colored product which is then detected (FIG. 2E). In preferred embodiments, detection is performed spectrophotometrically, including absorbance, transmittance, reflection, fluorescence, and chemiluminescence, although visual inspection is also contemplated in alternative embodiments of the invention.
In an alternative embodiment,[0096]assay chamber107 further comprises adetection cell117 that is laterally adjacent to the portion ofassay chamber107 comprising depression113 (shown in FIGS. 3A through 3E). This embodiment uses a variation ofalternative matrix118 wherein the matrix comprises a blood separation element and a blood fluid wicking element. These elements are arranged inassay chamber107 so that the blood separation element is in contact withdepression113 and the blood fluid wicking element is compressed or attached to the blood separation element along its entire extent. A blood fluid wicking element having dimensions as described above and sufficient to encompass the entire surface extent of the blood separation element and to further extend throughout the complete extent ofassay chamber107, includingdetection cell117. Reagents are only present in the portion of the blood fluid wicking element in that portion of the element comprisingdetection cell117; this arrangement is shown in detail in FIG. 3A.
In the practice of this embodiment of the invention, blood delivered to[0097]depression113 wicks through the blood separation element and into the blood fluid wicking element (FIGS. 3B and 3C). As the region of blood fluid wicking element abovedepression113 saturates with blood plasma, the blood fluid wicks laterally into the portion of thematrix118 comprising detection cell117 (FIG. 3D). Wetting of this portion of the matrix, which comprises the immobilized reagents and initiates the glucose detection reaction(s) as described in Example 1, with the production of a colored product (FIG. 3E). The amount of colored product produced is detected and the amount of glucose in the blood sample determined thereby.
Another alternative embodiment of a blood glucose assay Microsystems platform is shown in FIGS. 4A through 4E. Construction of the disk embodiments of the platforms of the invention were as described above.[0098]
The blood application and metering components and their dimensions and relationships to one another are identical to those described above, comprising sample entry port chamber[0099]201,metering capillary202, overflow capillary203, and overflow chamber205. As in Example 1, each of the overflow and fluid chambers is also connected with air ports or air channels, such as214, and capillary junction(s)215, that permit venting of air displaced by fluid movement on the platform.
[0100]Metering capillary202 is fluidly connected to capillary210 that was from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is connected to capillary orsacrificial valve211. Sacrificial orcapillary valve211 is further fluidly connected withcapillary212 that is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm; capillary212 is further fluidly connected toassay chamber207. Where capillary210 is connected withcapillary valve211, saidcapillary valve211 is from about 0.03 mm to about 2.2 mm deep and has a cross-sectional diameter of from about 0.03 mm to about 2.2 mm.
[0101]Assay chamber207 comprises a depression in the surface of the platform having a depth of from about 0.2 mm to about 3 mm preferably comprising a circular orrectangular depression213 connected tocapillary212 and most preferably wherein a metered or otherwise controlled amount of blood can be contained in the assay chamber.Assay chamber207 also comprises a pad ormatrix206 of a hydrophilic substance possessing a pore size of from about 0.2 to about 2 μm, most preferably about 0.8 μm. The upper limit on pore size ofmatrix206 is chosen to inhibit or prevent blood cell entry into the matrix, and to promote entry or wicking of the fluid component of blood (plasma or serum) to enter the body of the matrix. The matrix is positioned inassay chamber207 to be in fluidic contact withdepression213, more preferably coveringdepression213, and most preferably having a surface area greater than the surface area ofdepression213; in embodiments not havingdepression213 as a component ofassay chamber207, the matrix substantially fills the volumetric extent of the assay chamber. The matrix is further impregnated with immobilized reagents which produce a detectable product proportional to the amount or concentration of glucose in a blood sample. Most preferably, the detectable product is a colored product, i.e., a product absorbing light at a detectable, most preferably a visible, wavelength.
In an alternative embodiment,[0102]assay chamber207 comprises a surface wherein reagents219 are directly dried or otherwise immobilized on the surface of the chamber, and in such embodiments the assay chamber does not comprisematrix206.
[0103]Assay chamber206 is further fluidly connected withcapillary220 at a position preferably most distal to the axis of rotation.Capillary220 is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm, and is further fluidly connected withwaste reservoir221.Waste reservoir221 is positioned from about 1.2 cm to about 14 cm from the axis of rotation, and has a depth in the platform of from about 0.2 mm to about 3 mm.Assay chamber206 is also fluidly connected withcapillary222 at a position preferably most proximal to the axis of rotation.Capillary222 is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm, and is further fluidly connected withwash buffer reservoir223. Washbuffer reservoir223 has a depth in the platform of from about 0.2 mm to about 3 mm and is positioned from about 1.2 cm to about 14 cm from the axis of rotation, and in any event more proximal to the axis thanassay chamber206. Fluid flow fromwash buffer reservoir223 throughcapillary222 is controlled by eithercapillary valve224 orsacrificial valve225. Where capillary222 was connected withcapillary valve224, saidcapillary valve224 is from about 0.03 mm to about 2.2 mm deep and has a cross-sectional diameter of from about 0.03 mm to about 2.2 mm.
As illustrated in FIGS. 4A through 4D, in the use of this platform an imprecise volume (ranging from 20-150 μL of fluid) of blood is applied to the entry port[0104]201. Application of blood to the platform and delivering a metered amount of blood tometering capillary202 was achieved as described above. Alternatively, an unmetered amount of blood is introduced onto the platform directly intoassay chamber207. Preferably, from about 15 μL to about 150 μL of blood is delivered toassay chamber207.
At a rotational speed f[0105]₂of about 100-1000 rpm, the precise amount of fluid contained inmetering capillary202 is delivered intoassay chamber207. In embodiments comprising asacrificial valve211 in-line withcapillary210 at a position betweencapillary210 andassay chamber207 as shown in FIG. 3.1, release of the sacrificial valve results in fluid flow throughcapillary212 and intoassay chamber207. In said embodiments, fluid flow is achieved at rotational speed f₂with removal of the sacrificial valve. In embodiments of the platforms of the invention comprisingcapillary valve211 at a position betweencapillary210 and212 as shown in FIG. 3B, capillary210 preferably fills along with filling ofblood metering capillary202 until blood reachescapillary junction211 at the junction betweencapillary210 andcapillary212; in such embodiments, the capillary junction had a depth of from about 0.03 mm to about 2.2 mm. At a rotational speed f₂of about 100-1000 rpm, the fluid contained inblood metering capillary202 is delivered into assay chamber207 (FIG. 3C).
Blood flowing into[0106]assay chamber207 is directed tomatrix206 in the assay chamber; the dimensions ofmatrix206 are conveniently chosen to be able to contain substantially all of the blood fluid of the sample metered throughmetering capillary202 intoassay chamber207. Displaced air flows throughair channel214, and may be vented to the surface of the disc or in communication withblood metering capillary202 or entry port201. Alternatively, in embodiments wherein the blood sample is unmetered, the capacity of thematrix206 is sufficient to absorb a controlled amount of blood. In further alternative embodiments, wherein assay chamber does not comprisematrix206, the assay chamber is provided having a capacity for a controlled volume of blood.
As blood flows into[0107]assay chamber207, the fluid component of the blood wicks intomatrix206; the pore size of the matrix is chosen to prevent the cellular components of the blood from entering the matrix. In preferred embodiment, the cellular component of the blood is retained inassay chamber207 and the fluid component is efficiently wicked uniformly throughoutmatrix206.
As the fluid component of the blood wicks into[0108]matrix206, dried reagents are solubilized and the reaction of the blood component catalyzed by said reagents proceeds. The timescale over which these reactions take place is chosen to be long compared with the time it takes for the fluid component of blood to saturate thematrix206; however, in preferred embodiments, the reaction(s) goes to completion within about 0.5 to about 5 min. Reaction of the blood component(s) with reagents produce colored product which is then detected. In preferred embodiments, detection is performed spectrophotometrically, although visual inspection is also contemplated in alternative embodiments of the invention.
After a time sufficient to produce a detectable amount of a colored product, the wash buffer is released from[0109]wash buffer reservoir223 throughcapillary222 and intoassay chamber207. In embodiments comprising asacrificial valve225 in-line withcapillary222 at a position betweencapillary222 andassay chamber207 shown in FIG. 3.3, release of the sacrificial valve results in fluid flow throughcapillary222 and intoassay chamber207. In said embodiments, fluid flow is achieved at rotational speed f₃of about 250 rpm to about 1500 rpm with removal of the sacrificial valve. In embodiments of the platforms of the invention comprisingcapillary valve224 at a position betweencapillary222 andassay chamber207 shown in FIG. 3D, capillary222 preferably fills along with filling of blood fluid chamber204 until blood reachedcapillary junction224; in such embodiments, the capillary junction had a depth of from about 0.03 mm to about 2.2 mm. At a higher rotational speed f₄of about 400-2000 rpm, the fluid contained inwash reservoir223 is delivered into assay chamber207 (FIG. 3D). Because the fluid flow of wash buffer into the assay chamber and fluid from the assay chamber to the waste chamber is laminar, there is very little mixing of the washing fluid with the fluid initially in the assay chamber. The wash fluid displaces the fluid sample in the assay by pushing it into the waste chamber. The exit ofcapillary220 intochamber221 is at a radial position such thatassay chamber207 must remain filled with fluid during this washing process. The quality of fluid removal is such that no more than 1 part in 1000 of the fluid in the chamber207 (which has not been imbibed into matrix206) remains. The fluid which has wicked intomatrix206 is not removed during this wash because of the small pore size of the matrix which resists fluid flow; furthermore, color reagents do not diffuse out ofmatrix206 if the wash time is relatively short (less than a few hundred seconds). As a result, interfering blood fluid components such as hemoglobin are removed fromchamber207 while substantially leaving behind color reagents inmatrix206. These are then interrogated spectrophotometrically in assay chamber207 (FIG. 4E).
Another alternative embodiment of a blood glucose assay Microsystems platform is shown in FIGS. 5A through 5E. Construction of the disc embodiments of the platforms of the invention were as described above.[0110]
The blood application and metering components and their dimensions and relationships to one another are identical to those described above, comprising sample entry port chamber[0111]301,metering capillary302, overflow capillary303, and overflow chamber305. As in Example 1, each of the overflow and fluid chambers is also connected with air ports or air channels, such as314, and capillary junction(s)315, that permit venting of air displaced by fluid movement on the platform.
[0112]Metering capillary302 is fluidly connected to capillary310 that is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is connected to capillary orsacrificial valve311. Sacrificial orcapillary valve311 is further fluidly connected withcapillary312 that is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm, and is further fluidly connected toassay chamber307. Where capillary310 is connected withcapillary valve311, saidcapillary valve311 is from about 0.03 mm to about 2.2 mm deep and has a cross-sectional diameter of from about 0.03 mm to about 2.2 mm.
[0113]Assay chamber307 comprises a pad ormatrix306 of a hydrophilic substance possessing a pore size of from about 0.2 μm to about 2 μm, most preferably about 0.8 μm. The upper limit on pore size ofmatrix306 is chosen to inhibit or prevent blood cell entry into the matrix. The matrix is further impregnated with immobilized reagents which produce a detectable product proportional to the amount or concentration of glucose in a blood sample. Most preferably, the detectable product is a colored product, i.e., a product absorbing light at a detectable, most preferably a visible, wavelength.
As illustrated in FIGS. 5A through 5E, in the use of this platform an imprecise volume (ranging from about 15 μL to about 150 μL of fluid) of blood is applied to the entry port[0114]301. Application of blood to the platform and delivering a metered amount of blood toblood metering capillary302 was achieved as described above. Alternatively, an unmetered amount of blood is introduced onto the platform directly intoassay chamber307, preferably, from about 15 μL to about 150 μL.
At a rotational speed f[0115]₁of 100-1000 rpm the precise amount of fluid contained inmetering chamber302 is delivered intoassay chamber307. In embodiments comprising asacrificial valve311 in-line withcapillary310 at a position betweencapillary310 and312 shown in FIG. 5A, release of the sacrificial valve results in fluid flow throughcapillary312 and intoassay chamber307. In said embodiments, fluid flow is achieved at rotational speed f₁with removal of the sacrificial valve. In embodiments of the platforms of the invention comprisingcapillary valve311 at a position betweencapillary310 and312 shown in FIG. 5B, capillary310 preferably fills along with filling ofblood metering capillary302 until blood reachescapillary junction311 at the junction betweencapillary310 andcapillary312; in such embodiments, the capillary junction has a depth of from about 0.03 mm to about 2.2 mm. At a higher rotational speed f₂of about 250 rpm to about 1500 rpm, the fluid contained inblood metering capillary302 is delivered into assay chamber307 (FIG. 5C).
Blood flows into[0116]assay chamber307 with displaced air flowing throughair channel314, and may be vented to the surface of the disc or in communication withblood metering capillary302 or entry port301 (FIG. 5C). As blood flows intoassay chamber307, the fluid component of the blood wicks intomatrix306; the pore size of the matrix is chosen to prevent the cellular components of the blood from entering the matrix. In an alternative embodiment, alternative matrix318 comprises at least two distinct elements that are compressed or adhered to one another in the assays chamber. The first element is similar to the reagent-containingmatrix306 described above; however, this embodiment of the matrix has a pore size that is not limited by the size of cellular components of the blood, and can be any pore size deemed optimal on experimental, economic, manufacturing or availability grounds. The general dimensions of the matrix are equivalent to the dimensions disclosed above, so that the matrices are advantageously standardized and interchangeable on the microsystems platforms of the invention. The second element comprises a filtering layer having a pore size that prevents cellular components from entering this portion of the matrix. In a preferred embodiment, these elements are arranged inassay chamber307 so that the second matrix element is in contact with the surface ofassay chamber307 wherein the blood aliquot is first contacted with the second matrix component. Blood fluid wicks into and through second matrix element and into the first matrix element, whereby cellular components of blood are prevented from enteringassay chamber307 by the pore size of second matrix element.
As the fluid component of the blood wicks into[0117]matrix306, dried reagents are solubilized and the reaction of the blood component catalyzed by said reagents proceeds as described below. The timescale over which these reactions take place is chosen to be long compared with the time it takes for the fluid component of blood to saturate thematrix306; however, in preferred embodiments, the reaction(s) goes to completion within about 0.5 to about 5 min. Reaction of the blood component(s) with reagents produce colored product (FIG. 5D). After sufficient time for the reaction to proceed, the platform is centrifuged at a speed of from about 800 rpm to about 200 rpm, wherein said speed pellets the cellular component of the blood, particularly the red blood cell component thereof, to the “bottom” or most axially distal portion ofassay chamber307. Detection of the colored product of the glucose detecting reaction inmatrix306 is performed at a position inassay chamber307 radially more proximal to the axis of rotation than the position to which the cellular fraction has been pelleted (FIG. 5E). In preferred embodiments, detection is performed spectrophotometrically, although visual inspection is also contemplated in alternative embodiments of the invention. The amount of colored product produced is detected and the amount of glucose in the blood sample determined thereby.
Another alternative embodiment of a blood glucose assay Microsystems platform is shown in FIGS. 6A through 6E. Construction of the disk embodiments of the platforms of the invention were as described above.[0118]
The blood application and metering components and their dimensions and relationships to one another are identical to those described above, comprising sample entry port chamber[0119]401,metering capillary402, overflow capillary403, and overflow chamber405. As in Example 1, each of the overflow and fluid chambers is also connected with air ports or air channels, such as414, and capillary junction(s)415, that permit venting of air displaced by fluid movement on the platform.
[0120]Metering capillary402 is fluidly connected to capillary410 that is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is connected to capillary orsacrificial valve411. Sacrificial orcapillary valve411 is further fluidly connected withcapillary412 that is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm.Capillary412 is further fluidly connected tocell separation chamber407. Where capillary410 is connected withcapillary valve411, saidcapillary valve411 is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm.
[0121]Cell separation chamber407 comprised a depression in the surface of the platform having a depth of from about 0.02 mm to about 3 cm, most preferably comprising a circular orrectangular depression413 connected tocapillary412. Cell separation chamber also comprises afilter406 consisting of a porous material whose pores are sized (from about 0.2 μm to about 2 μm) to filter red blood cells.Filter406 is in contact with and more preferably adhered to the surface ofcell separation chamber407 and most extends overdepression413. Blood flowing intocell separation chamber407 is directed todepression413 bycapillary412. Asdepression413 fills, serum is both wicked and driven by rotation-induced pressure through thefilter406 to the upper surface ofcell separation chamber407, leaving red blood cells and other cellular components trapped beneathfilter406 indepression413. The volume of blood fluid at the upper surface ofcell separation chamber407 ranges from about 15 μL to about 150 μL.
[0122]Cell separation chamber407 is fluidly connected at a radial position distal from the axis of rotation to capillary420 that is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm.Capillary420 is further fluidly connected withassay chamber421 that is from about 0.02 mm to about 3 cm deep and has a cross-sectional diameter of from about 0.02 mm to about 10 cm.Assay chamber421 comprises a pad ormatrix422 of a hydrophilic substance possessing any convenient pore size, for example, a pore size of 0.2-2.0 μm, such as a pore size of about 0.8 μm. The matrix is further impregnated with immobilized reagents which produce a detectable product proportional to the amount or concentration of glucose in a blood sample. Most preferably, the detectable product is a colored product, i.e., a product absorbing light at a detectable, most preferably a visible, wavelength.
As illustrated in FIGS. 6A through 6E, in the use of this platform an imprecise volume (ranging from 15 to about 150 μL of fluid) of blood is applied to the entry port[0123]401. Application of blood to the platform and delivering a metered amount of blood tometering capillary402 was achieved as described in Example 1 above. At a rotational speed f₁of 100-1000 rpm, the precise amount of fluid contained inmetering capillary402 is delivered intoassay chamber407. Alternatively, an unmetered amount of blood is introduced onto the platform directly intoassay chamber407, preferably, from about 15 μL to about 150 μL.
Fluid movement into[0124]metering capillary402 is accompanied by filling ofcapillary410. In embodiments comprising asacrificial valve411 in-line withcapillary410 at a position betweencapillary410 and412 shown in FIG. 6A, release of the sacrificial valve results in blood fluid flow throughcapillary412 and intocell separation chamber407. In said embodiments, fluid flow is achieved at rotational speed f₁with removal of the sacrificial valve. In embodiments of the platforms of the invention comprisingcapillary valve411 at a position betweencapillary410 and412 shown in FIG. 6A, capillary410 preferably fills along with filling ofmetering capillary402 until blood reachescapillary junction411 at the junction betweencapillary410 andcapillary412. In such embodiments, the capillary junction has a depth of from about 0.03 mm to about 2.2 mm. In these embodiments, the fluid contained inmetering capillary402 is delivered intocell separation chamber407 by rotation of the disc at a higher rotational speed f₂of from about 250 rpm to about 1500 rpm (FIG. 6B).
Blood flows into[0125]cell separation chamber407 with displaced air flowing throughair channel414, and may be vented to the surface of the disc or in communication withmetering capillary402 or entry port401 (FIG. 6C). As blood flows intocell separation chamber407, the fluid component of the blood wicks intomatrix406; the pore size of the matrix is chosen to prevent the cellular components of the blood from entering the matrix. In a preferred embodiment,matrix406 is arranged incell separation chamber407 so that the matrix is in contact with or more preferably adhered to the lower surface ofcell separation chamber407. Blood fluid, such as plasma or serum, traversesmatrix406 by wicking and under rotation-induced pressure, saturating the matrix and filling a space formed between the top surface of the matrix and the top surface of cell separation chamber407 (FIG. 6C).
Blood fluid exits[0126]cell separation chamber407 throughcapillary420 and flows into assay chamber421 (FIG. 6D), with displaced air flowing throughair channel414, and may be vented to the surface of the disc or in communication with cell separation chamber407 (FIG. 5.4). Blood fluid flows intoassay chamber421 and wicks intomatrix422. As the blood fluid wicks intomatrix422, dried reagents are solubilized and the reaction of the blood component catalyzed by said reagents proceeds as described below. The timescale over which these reactions take place is chosen to be long compared with the time it takes for the fluid component of blood to saturate thematrix422; however, in preferred embodiments, the reaction(s) goes to completion within about 0.5 min to about 5 min. Reaction of the blood component(s) with reagents produce a colored product (FIG. 6E). In preferred embodiments, detection of colored product is performed spectrophotometrically, although visual inspection is also contemplated in alternative embodiments of the invention. The amount of colored product produced is detected and the amount of glucose in the blood sample determined thereby.
Another alternative embodiment of a blood glucose assay microsystems platform is shown in FIGS.[0127]7A-7D. Construction of the disk embodiments of the platforms of the invention were as described above.
The blood application and metering components and their dimensions and relationships to one another are identical to those described above, comprising sample entry port chamber[0128]501,metering capillary502, overflow capillary503, and overflow chamber505. As in Example 1, each of the overflow and fluid chambers is also connected with air ports or air channels, such as514, and capillary junction(s)515, that permit venting of air displaced by fluid movement on the platform.
[0129]Metering capillary502 is fluidly connected withcapillary510, having a cross-sectional diameter of from about 0.02 mm to about 2 mm and extending from about 1 mm to about 5 cm from the blood fluid chamber.Capillary510 is also fluidly connected with mixingchamber515 that is from about 0.02 mm to about 3 cm deep and having a cross-sectional diameter of from about 0.02 mm to about 10 cm, and is positioned from about 1.2 cm to about 14 cm from the center of rotation. Mixingchamber515 is also fluidly connected withcapillary520 having a cross-sectional diameter of from about 0.02 mm to about 2 mm.Capillary520 is further fluidly connected withreagent chamber521, whereinreagents508 for detecting blood glucose and determining the concentration thereof are stored.Reagent chamber521 is from about 0.02 mm to about 3 cm deep and having a cross-sectional diameter of from about 0.02 mm to about 10 cm, and is positioned from about 1.2 cm to about 14 cm from the center of rotation. In certain embodiments,reagents508 are stored on the disc in solution; in these embodiments, fluid flow throughcapillary520 is preferably controlled usingsacrificial valve525. In alternative embodiments,reagents508 are stored inreagent chamber521 in dry form. In these embodiments, capillary520 can preferably comprisesacrificial valve525 orcapillary valve525 positioned between mixingchamber515 andreagent chamber521. In these embodiments,reagent chamber521 further comprises means for a user to add an appropriate amount of a reagent diluent530, or the platform further comprises diluent chamber531 fluidly connected toreagent chamber521 by way of capillary532. In these embodiments, reagent diluent chamber531 that is from about 0.02 mm to about 3 cm deep and having a cross-sectional diameter of from about 0.02 mm to about 10 cm, and is positioned from about 1.2 cm to about 14 cm from the center of rotation. In these embodiments,reagents508 are solubilized inreagent chamber521 immediately prior to or during use.
Mixing[0130]chamber515 is fluidly connected bycapillary channel536 having a cross-sectional diameter of from about 0.02 mm to about 2 mm and extending from about 1 mm to about 5 cm from the mixing chamber.Capillary channel536 is further fluidly connected with mixedfluid receiving chamber537. Mixedfluid chamber537 is from about 0.02 mm to about 3 cm deep and having a cross-sectional diameter of from about 0.02 mm to about 10 cm, and is positioned from about 1.2 cm to about 14 cm from the center of rotation. Alternatively,capillary536 is further fluidly connected tosecond mixing chamber540.Second mixing chamber540 is from about 0.02 mm to about 3 cm deep and having a cross-sectional diameter of from about 0.02 mm to about 10 cm, and is positioned from about 1.2 cm to about 14 cm from the center of rotation. Second mixing chamber is fluidly connected by acapillary channel546 which is further connected with mixedfluid receiving chamber537. In these embodiments,capillary channel546 has a cross-sectional diameter of from about 0.02 mm to about 2 mm and extends from about 1 mm to about 5 cm from the second mixing chamber.
[0131]Capillary channels510 and520, andcapillary channels536 and546, may be offset in their connection with the mixing chamber(s). As a consequence, fluid flowing throughcapillary channels536 and546 is forced to encounter the opposite wall of mixingchambers515 and540 before fluid flow can proceed through further capillary channels. The fluid streams entering a small channel flow in a laminar fashion and therefore mix only by diffusion; the mixing chamber allows the fluids to move in a turbulent fashion and thus mix more effectively. This results in the creation of turbulence in the mixed laminar fluid stream incapillary channels536 and546 caused by the conjoint flow of fluid from the input capillaries without appreciable mixing. The turbulence created by the structure of mixingchambers515 and540 is sufficient to disrupt laminar flow and cause fluid mixing in the chamber prior to continued fluid flow throughcapillary channel546 and into mixedfluid receiving chamber537.
As illustrated in FIGS. 7A through 7D, in the use of this platform a volume of blood is applied to[0132]metering capillary502, either directly or using the metering components of the platform described above. Fluid enters the each of thecapillaries510 and520 and stops at capillary junction(s) or sacrificial valve(s)525.
At a rotational speed f[0133]₁of 100 to 1000 rpm, the fluids from each capillary flow pastcapillary junction525 and through mixing chamber515 (FIG. 7B). Alternatively, fluid flow is activated by release ofsacrificial valves525. Fluid flow within mixingchamber515 is turbulent, in contrast to fluid flow throughcapillaries510 and520, which is primarily laminar, so that mixing occurs predominantly in mixingchamber515. Fluid flow proceeds throughchannel536 and then either throughsecond mixing chamber540 or directly throughcapillary546 into mixed fluid receiving chamber537 (FIG. 7C).
Glucose detection reagents mixed with blood reacts in mixed fluid receiving chamber[0134]537 (FIG. 7C). The timescale over which these reactions take place preferably goes to completion within about 0.5 min to about 5 min. Reaction of the blood component(s) withreagents508 produce a colored product (FIG. 7D). After sufficient time for the reaction to proceed, the platform is centrifuged at a speed of about 500 rpm to about 3000 rpm, wherein said speed pellets the cellular component of the blood, particularly the red blood cell component thereof, to the “bottom” or most axially distal portion of mixedfluid receiving chamber537. Detection of the colored product of the glucose detecting reaction is performed at a position in mixedfluid receiving chamber537 radially more proximal to the axis of rotation than the position to which the cellular fraction has been pelleted (FIG. 7D). In preferred embodiments, detection is performed spectrophotometrically, although visual inspection is also contemplated in alternative embodiments of the invention. The amount of colored product produced is detected and the amount of glucose in the blood sample determined thereby.
Another alternative embodiment of a blood glucose assay Microsystems platform is shown in FIGS. 8A through 8D. Construction of the disk embodiments of the platforms of the invention were as described above.[0135]
The blood application and metering components and their dimensions and relationships to one another are identical to those described above, comprising sample entry port chamber[0136]601,metering capillary602, overflow capillary603, and overflow chamber605. As in Example 1, each of the overflow and fluid chambers is also connected with air ports or air channels, such as634, and capillary junction(s)635, that permit venting of air displaced by fluid movement on the platform.
[0137]Metering capillary602 is fluidly connected to capillary610 that is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is connected to capillary orsacrificial valve611. Sacrificial orcapillary valve611 is further fluidly connected withcapillary612 that is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm.Capillary612 is further fluidly connected tocell separation chamber607.Cell separation chamber607 is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 002 mm to about 3 cm, and is positioned from about 1.2 cm to about 14 cm from the center of rotation. Where capillary610 is connected withcapillary valve611, saidcapillary valve611 is from about 0.03 mm to about 2.2 mm deep and has a cross-sectional diameter of from about 0.03 mm to about 2.2 mm.
[0138]Cell separation chamber607 comprises a depression in the surface of the platform having a depth of from about 0.02 mm to about 3 cm, most preferably comprising a circular orconcave depression613 connected tocapillary612.Depression613 has a depth of from about 0.02 mm to about 1 cm and a volume of from 15 μL to about 150 μL.Cell separation chamber607 also comprises afilter606 consisting of a porous material whose pores are sized to filter red blood cells, ranging from about 0.2 μm to about 2 μm.Filter606 is in contact with and more preferably adhered to the surface ofcell separation chamber607 and most preferably extends overdepression613. Blood flowing intocell separation chamber607 is directed todepression613 bycapillary612. Asdepression613 fills, serum is both wicked and driven by rotation-induced pressure through thefilter606 to the upper surface ofcell separation chamber607, leaving red blood cells and other cellular components trapped beneathfilter606 indepression613. The volume of blood fluid at the upper surface ofcell separation chamber607 is from about 15 μL to about 150 μL.
[0139]Cell separation chamber607 is fluidly connected at a radial position distal from the axis of rotation to capillary620 that is from about 0.02 mm to about 2 mm deep, has a cross-sectional diameter of from about 0.02 mm to about 2 mm and extends from about 1 mm to about 5 cm fromcell separation chamber607.Capillary620 is further fluidly connected with mixingchamber615 that is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm, and is positioned from about 1.2 cm to about 14 cm from the center of rotation.Capillary620 is further fluidly connected withreagent chamber621, whereinreagents608 for detecting blood glucose and determining the concentration thereof is stored.Reagent chamber621 is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm, and is positioned from about 1.2 cm to about 14 cm from the center of rotation. In certain embodiments,reagents608 are stored on the disc in solution; in these embodiments, fluid flow throughcapillary620 is preferably controlled usingsacrificial valve625. In alternative embodiments,reagents608 are stored inreagent chamber621 in dry form. In these embodiments, the platform further comprises diluent chamber631 fluidly connected toreagent chamber621 by way of capillary632. Diluent chamber631 is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm, and is positioned from about 1.2 cm to about 14 cm from the center of rotation. Capillary632 has a cross-sectional diameter of from about 0.02 mm to about 2 mm, and is positioned from about 1.2 cm to about 14 cm from the center of rotation. In these embodiments,reagents608 are solubilized inreagent chamber621 immediately prior to or during use.
Mixing[0140]chamber615 is fluidly connected bycapillary channel636 having a cross-sectional diameter of from about 0.02 mm to about 2 mm and extending from about 1 mm to about 5 cm from the mixing chamber.Capillary channel636 is further fluidly connected with mixedfluid receiving chamber637. Mixedfluid chamber637 is from about 0.02 mm to about 2 mm deep, has a cross-sectional diameter of from about 0.02 mm to about 2 mm, and is positioned from about 1.2 cm to about 14 cm from the center of rotation.
Alternatively,[0141]capillary636 is further fluidly connected tosecond mixing chamber640.Second mixing chamber640 is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm, and is positioned from about 1.2 cm to about 10 cm from the center of rotation. Second mixing chamber is fluidly connected by acapillary channel646 which is further connected with mixedfluid receiving chamber637.Capillary channel646 has a cross-sectional diameter of from about 0.02 mm to about2 mm and extends from about 1 mm to about 5 cm from the second mixing chamber.
[0142]Capillary channels610 and620, andcapillary channels636 and646, may be offset in their connection with the mixing chamber(s). As a consequence, fluid flowing throughcapillary channels636 and646 are forced to encounter the opposite wall of mixingchambers615 and640 before fluid flow can proceed through further capillary channels. This results in the creation of turbulence in the mixed laminar fluid stream incapillary channels636 and646 caused by the conjoint flow of fluid from the input capillaries without appreciable mixing. The turbulence created by the structure of mixingchambers615 and640 is sufficient to disrupt laminar flow and cause fluid mixing in the chamber prior to continued fluid flow throughcapillary channel646 and into mixedfluid receiving chamber637. Mixedfluid receiving chamber637 is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm, and is positioned from about 1.2 cm to about 14 cm from the center of rotation.
As illustrated in FIGS. 8A through 8D, in the use of this platform a volume of blood is applied to[0143]metering capillary602, either directly or using the metering components of the platform described above. Blood flows intocell separation chamber607 with displaced air flowing through air channel654, and may be vented to the surface of the disc or in communication with blood fluid chamber604 (FIG. 8B). As blood flows intocell separation chamber607, the fluid component of the blood wicks intomatrix606; the pore size of the matrix (from about 0.2 mm to about 2 μm) is chosen to prevent the cellular components of the blood from entering the matrix. In a preferred embodiment,matrix606 is arranged incell separation chamber607 so that the matrix is in contact with or more preferably adhered to the lower surface ofcell separation chamber607. Blood fluid, such as plasma or serum, traversesmatrix606 by wicking and under rotation-induced pressure, saturating the matrix and filling a space formed between the top surface of the matrix and the top surface of cell separation chamber607 (FIG. 8C).
Blood fluid exits[0144]cell separation chamber607 throughcapillary610 and flows into mixingchamber615. Similarly, at a rotational speed of from about 200 rpm to about 200 rpm sufficient to overcomecapillary valve625, or upon release ofsacrificial valve625, solubilizedreagents608 flow throughcapillary620 and into mixing chamber615 (FIG. 8C). Fluid flow within mixingchamber615 is turbulent, in contrast to fluid flow throughcapillaries610 and620, which is primarily laminar, so that mixing occurs predominantly in mixingchamber615. Fluid flow proceeds throughchannel636 and then either throughsecond mixing chamber640 or directly throughcapillary646 into mixed fluid receiving chamber637 (FIG. 8D).
Glucose detection reagents mixed with blood in mixed fluid receiving chamber[0145]637 (FIG. 8D). The timescale over which these reactions take place preferably goes to completion within about 0.5 min. to about 5 min. Reaction of the blood component(s) withreagents608 produce a colored product (FIG. 8D). Detection of the colored product of the glucose detecting reaction is performed in mixedfluid receiving chamber637. In preferred embodiments, detection is performed spectrophotometrically, although visual inspection is also contemplated in alternative embodiments of the invention. The amount of colored product produced is detected and the amount of glucose in the blood sample determined thereby.
The microfluidics structures of the invention are used to detect the amount or concentration of a chemical species in a solution or complex mixture, most preferably an aqueous solution or mixture. In certain preferred embodiments, the chemical species to be detected in glucose in blood. In one embodiment, blood glucose in blood is detected using a hexokinase assay.[0146]
In this assay, hexokinase converts glucose to glucose-6-phosphate (G-6-P) in the presence of magnesium cation. G-6-P is then converted to 6-phosphogluconate (6-PG) by glucose-6-phosphate dehydrogenase in the presence of NADP producing a stoichiometric amount of NADPH. NADPH is oxidized by reaction with phenazine methosulfate (PMS) as an intermediate, which is then oxidized by reaction with indotetrotazolium chloride, which forms a colored product that absorbs visible light at 520 nm. Optimum reagent component concentrations vary according to the specifics of the application of this chemical assay, as well understood and practiced by one versed in the art. This reaction scheme is illustrated as follows:[0147]
The amount of glucose in the blood fluid is directly related to the amount of reduced indotetrotazolium chloride, and the concentration of reduced indotetrotazolium chloride was related to glucose concentration using light spectroscopy as described below, as understood by those with skill in the art. All of these reagents are preferably provided as dried[0148]reagents108 applied to the disc, for example, comprisingmatrix106 shown in FIGS. 1 through 8. These reagents can be lyophilized or air dried directly onto the surface of the disk, for example by “ink-jet” methods, or can be applied as dried beads or other particulate components.
In a variation of this reaction scheme, reduction of NADP is assayed directly without the use of PMS or indotetrazolium chloride. In this embodiment, the concentration of oxidized NADP is detected spectrophotometrically by illuminating the sample with light at 340 nm, and detecting absorbance. The amount of glucose in the sample is inversely proportional to the amount of oxidized NADP present in the sample after reaction. In these embodiments, the amount of NADP must be precisely controlled to be certain that the inverse proportionality between oxidized NADP and glucose is maintained.[0149]
Detection is performed by transmission, reflection or reflectance spectroscopy. In transmission spectroscopy, light at a wavelength of 520 nm produced by a narrow band light source, most preferably using a light emitting diode (LED) with a filter, enters one face of an assay chamber, more preferably at a position in the chamber that comprises a detection cell, and the light transmitted through the detection cell is detected using a photomultiplier tube, photodiode, photodiode array, or avalanche photodiode. The photomultiplier tube is calibrated so that the amount of transmitted light detected is interpreted according to Beer's law to determine optical density and hence concentration of glucose in the sample by the well-known linear relation between the logarithm of the incident intensity/transmitted intensity and the concentration of colored (absorbing) product.[0150]
Alternatively, production of colored reaction products is detected by reflection spectroscopy. Light at a wavelength of 520 nm is produced by a narrow band light source, most preferably a further combination with a monochromator, grating, or filter. Monochromatic light sources such as lasers or rare gas lamps as well as quasi-monochromatic light sources such as LEDs may also be used and enters one face of an assay chamber, more preferably at a position in the chamber that comprises a detection cell. In preferred embodiments, the face of the assay chamber or detection cell opposite to the face illuminated by the light source comprises a reflective surface, preferably formed using a reflective material, including but not limited to an aluminum layer, a metallized glass, a mirror, and high gloss paint, or a diffusely reflecting surface such as TiO[0151]₂, underneath the colored fluid, which advantageously decreases the contribution of scratches or rotor wobble. Illuminated light is reflected back through the detection cell at a direct or through an oblique angle and is detected using a photomultiplier tube photo diode, photodiode array, or avalanche photodiode.
For embodiments of the platforms of the invention wherein the assay chamber comprises a solid or porous matrix, production of colored reaction products is most preferably detected by a variation on reflection spectroscopy described above. Light at a wavelength of 520 nm is produced by a narrow band light source, most preferably in further combination with a monochromator is produced that enters one face of an assay chamber, more preferably at a position in the chamber that comprises a detection cell. Light is absorbed from the colored reaction products comprising the matrix and is scattered from the matrix which comprises a diffusely-scattering material. The diffusely scattered, reflected light is detected.[0152]
In an alternative reaction protocol, glucose oxidase is used to produce hydrogen peroxide by oxidation of glucose in the blood sample. In this reaction scheme, glucose oxidase converts glucose to gluconic acid and hydrogen peroxide; a twice-stoichiometric amount of hydrogen peroxide is produced relative to the amount of glucose present in the blood sample. The hydrogen peroxide then oxidizes a dye precursor present in the assay chamber or preferably within a detection cell, yielding a colored product. A variety of dye precursors are useful in the practice of this aspect of the invention, including but not limited to O-dianisidine, O-toluidine, O-tolidine, benzidine, 2,2′-azinodi-(3-ethylbenzthiazoline sulfonic acid), 3-methyl-2-benzthiazolinone hydrazone plus N,N-dimethylaniline, phenyl plus 4-aminophenzanone, sulfonated 2,4-dichlorophenol plus 4-aminophenzanone, 3-methyl-2-benzothiazolinone hydrazone plus 3-(dimethylamino) benzoic acid, 2-methoxy-4-allyl phenol and 4-aminoantipyrene-dimethylaniline. Optimum reagent component concentration vary according to the specifics of the application of this chemical assay, as well understood and practiced by one versed in the art. This reaction scheme is illustrated as follows:[0153]
The amount of glucose in the blood fluid is directly related to the amount of oxidized dye produced. The concentration of oxidized dye produced is determined by Beers law and light spectroscopy measurements. The total amount of oxidized dye produced is then used to calculate the sample glucose concentration. Alternatively and preferably, the rate of oxidized dye production is measured and that rate related to the sample glucose concentration. Calibration, to relate glucose concentration to optical measurements calculated by either method above, are well understood by one versed in this art. All of these reagents are preferably provided as dried[0154]reagents108 applied to the disc, for example, comprisingmatrix106 shown in FIGS. 1 through 8. These reagents can be applied, by for example methods, including but not limited to, filling, spraying, dipping, rolling and stamping a solution containing reagent components followed by lyophilization or air drying. Several applications may be made sequentially, with different components.
The invention also provides microsystem platforms for performing separations of particular components of a solution or complex mixture. In particular, the invention provides disc embodiments of the platforms of the invention comprising separation chambers containing components or matrices that specifically bind and retain particular chemical species comprising a chemical solution or complex mixture. This aspect of the invention is illustrated by a microfluidics array for separating glycated hemoglobin from a blood sample, as shown in FIG. 9.[0155]
Construction of the disk embodiments of the platforms of the invention were as described above. The blood application and metering components and their dimensions and relationships to one another are identical to those described above, comprising sample entry port chamber[0156]701,metering capillary702, overflow capillary703, and overflow chamber705. As in Example 1, each of the overflow and fluid chambers is also connected with air ports or air channels, such as754, and capillary junction(s)755, that permit venting of air displaced by fluid movement on the platform.
[0157]Metering capillary702 is fluidly connected to capillary710 that is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm.Capillary710 is further fluidly connected to mixingchamber715 that is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm, and is positioned from about 1.2 cm to about 14 cm from the center of rotation.Lysis buffer chamber716 containing blood lysis buffer is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm, and is positioned from about 1.2 cm to about 14 cm from the center of rotation.Lysis buffer chamber716 is positioned more proximally to the axis of rotation that mixingchamber715, and has a volumetric capacity of from about 15 μL to about 150 μL of lysis buffer, composed of 0.1% Triton X100 in 50 mM Tris pH 9.5.Lysis buffer chamber715 is fluidly connected throughcapillary718 to mixingchamber715.
Mixing[0158]chamber715 is fluidly connected to capillary717 that is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is connected tosecondary metering structure719.Secondary metering structure719 is from about 0.02 mm to about 3 cm deep, and is positioned from about 1.2 cm to about 14 cm from the center of rotation.Secondary metering structure719 is constructed to comprise three sections. A first metering section is arranged proximal to the entry position ofcapillary717 and is separated from a second metering section by a septum that extends from the distal wall of the structure to a position just short of the proximal wall of the structure. This arrangement produces a fluid connection between the first metering section having a volumetric capacity of from about 5 μL to about 15 μL and second metering section having a volumetric capacity of from about 5 μL to about 15 μL. An overflow chamber is positioned adjacent to the second metering section and separated by a septum that extends from the distal wall of the structure to a position just short of the proximal wall of the structure. This arrangement produces a fluid connection between the second metering section and the overflow sections of thesecondary metering structure719.
[0159]Capillary721 is in fluid connection withsecondary metering structure719 at the distal wall of the first metering section.Capillary721 is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is fluidly connected to boronateaffinity matrix chamber722. Boronateaffinity matrix chamber722 is from about 0.02 mm to about 0.3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm and is positioned from about 1.2 cm to about 14 cm from the axis of rotation. Boronateaffinity matrix chamber722 further comprises 20-50 μL boronate-functionalized agarose beads having a mean diameter of about 60 μm; the beads are maintained in thechamber722 using aporous frit727. Fluid flow throughcapillary721 is connected to capillary orsacrificial valve723.Capillary724 is in fluid connection withsecondary metering structure719 at the distal wall of the second metering section.Capillary724 is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is connected to readwindow726. Readwindow726 is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm and is positioned from about 1.2 cm to about 14 cm from the axis of rotation. Read window is comprised of a material transparent to light at a wavelength of about 430 nm. Fluid flow throughcapillary724 is connected to capillary orsacrificial valve725.
Boronate[0160]affinity matrix chamber722 is further fluidly connected tocapillary728.Capillary728 is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is connected to columnpreparation buffer reservoir729. Columnpreparation buffer reservoir729 is from about 0.02 mm to about 3 cm deep and has a cross-sectional diameter of from about 0.02 mm to about 10 cm and is positioned from about 1.2 cm to about 12 cm from the axis of rotation, more proximal than boronateaffinity matrix chamber722. Columnpreparation buffer reservoir729 comprises from about 100 μL to about 500 μL of column preparation buffer comprising magnesium chloride, taurine, D,L-methionine, sodium hydroxide, antibiotics and stabilizers (obtained from IsoLab as described in the Examples below). Fluid flow throughcapillary728 is connected to capillary orsacrificial valve735.
Boronate[0161]affinity matrix chamber722 is further fluidly connected tocapillary730.Capillary730 is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02mm to about 2 mm and is connected to column washbuffer reservoir731. Columnwash buffer reservoir731 is from about 0.02 mm to about 3 cm deep and has a cross-sectional diameter of from about 0.02 mm to about 10 cm and is positioned from about 1.2 cm to about 14 cm from the axis of rotation, more proximal than boronateaffinity matrix chamber722. Columnwash buffer reservoir731 comprises from about 100 μL to about 500 μL of column preparation buffer as described above. Fluid flow throughcapillary730 is connected to capillary orsacrificial valve736.
In alternative embodiments, column[0162]preparation buffer reservoir729 and column washbuffer reservoir731 can be the same reservoir, or can be fluidly connected as shown in FIG. 9A.
Boronate[0163]affinity matrix chamber722 is further fluidly connected tocapillary732.Capillary732 is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is connected to readwindow726.
[0164]Read window726 is further fluidly connected tocapillary733.Capillary733 is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is connected to wastereservoir734.Waste reservoir734 is from about 0.02 mm to about 3 cm deep and has a cross-sectional diameter of from about 0.02 mm to about 10 cm and is positioned from about 1.2 cm to about 14 cm from the axis of rotation.
As illustrated in FIG. 9A, in the use of this platform a volume of blood from about 15 μL to about 150 μL is applied to[0165]metering capillary702, either directly or using the metering components of the platform described above. Blood flowing throughcapillary710 and lysis buffer flowing throughcapillary718 are mixed in mixingchamber715 by overcoming capillary valve711 or release of sacrificial valve711. A volume of lysis buffer from about 25 μL to about 90 μL was mixed with the blood sample. Fluid-flow within mixingchamber715 is turbulent, in contrast to fluid flow throughcapillaries710 and718, which is primarily laminar, so that mixing occurs predominantly in mixingchamber715. Fluid flow proceeds throughchannel717 and intosecondary metering structure719.
The mixture of lysis buffer and blood, comprising a lysed blood sample, flows at a rotational speed f[0166]₂from about 200 rpm to about 2000 rpm intosecondary metering structure719. The lysed blood sample enters and fills the first section ofsecondary metering structure719. Continued lysed blood sample flow intosecondary metering structure719 then fills the second section ofsecondary metering structure719. Any additional lysed blood sample then empties into the overflow chamber ofsecondary metering structure719. Most preferably, a sufficient volume of lysis buffer and blood sample is applied to the disc to fill at least the first and second metered sections ofsecondary metering structure719.
After the lysed blood sample is completely transferred to[0167]secondary metering structure719, capillary orsacrificial valve735 is released, allowing from 100 μL to about 500 μL of column preparation buffer to flow at rotational speed f₂throughcapillary730 and intoboronate affinity matrix722. Continued or discontinuous rotation motivates column preparation buffer throughboronate affinity matrix722, capillary732, readwindow726, capillary733 and intowaste reservoir734.
After the column preparation buffer is applied to boronate[0168]affinity matrix chamber722, capillary orsacrificial valve723 is released, allowing the metered lysed blood sample from the first metered section ofsecondary metering structure719 throughcapillary721 and intoboronate affinity matrix722 and allowed to incubate in the affinity matrix chamber for from about 0.5 to about 5 min. Capillary orsacrificial valve736 is then released, allowing from 100 μL to about 500 μL of column wash buffer to flow at rotational speed f_? throughcapillary730 and intoboronate affinity matrix722. Continued or discontinuous rotation motivates column preparation buffer throughboronate affinity matrix722, capillary732 and into readwindow726. During fluid flow of wash buffer through the boronate affinity matrix chamber, read window is preferably illuminated by light at a wavelength of 430 nm and the concentration of hemoglobin in the sample after glycated hemoglobin has been removed by the boronate affinity matrix is determined thereby.
Capillary or[0169]sacrificial valve725 is released at rotational speed f₃of from about 100-1000 rpm and the metered lysed blood sample from the second metered section ofsecondary metering structure719 flows throughcapillary724 and into readwindow726. Read window is then illuminated by light at a wavelength of 430 nm and the concentration of hemoglobin in the sample determined transmission, reflection, or reflectance spectroscopy. The amount of glycated hemoglobin in the sample is determined by subtracting the amount of hemoglobin obtained in the first reading from the amount of hemoglobin obtained in the second reading.
An alternative embodiment of the glycated hemoglobin assay microsystem platform of the invention is shown in FIG. 10. Construction of the disk embodiments of the platforms of the invention were as described above. The blood application and metering components and their dimensions and relationships to one another are identical to those described above, comprising sample entry port chamber[0170]801,metering chamber802, overflow capillary803, and overflow chamber805. As in Example 1, each of the overflow and fluid chambers is also connected with air ports or air channels, such as854, and capillary junction(s)855, that permit venting of air displaced by fluid movement on the platform.
[0171]Metering capillary802 is fluidly connected to capillary810 that is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm.Capillary810 is further fluidly connected to mixingchamber815 that is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm, and is positioned from about 1.2 cm to about 14 cm from the center of rotation.Lysis buffer chamber816 containing blood lysis buffer is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm, and is positioned from about 1.2 cm to about 14 cm from the center of rotation.Lysis buffer chamber816 is positioned more proximally to the axis of rotation that mixingchamber815, and has a volumetric capacity of from about 15 μL to about 150 μL of lysis buffer, composed of 0.1% Triton X100 in 50 mM Tris pH 9.5.Lysis buffer chamber815 is fluidly connected throughcapillary818 to mixingchamber815.
Mixing[0172]chamber815 is fluidly connected to capillary817 that is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is connected tosecondary metering structure819.Secondary metering structure819 is from about 0.02 mm to about 3 cm deep, and is positioned from about 1.2 cm to about 14 cm from the center of rotation.Secondary metering structure819 is constructed to comprise two sections. A metering section is arranged proximal to the entry position ofcapillary817 and is separated from an overflow section by a septum that extends from the distal wall of the structure to a position just short of the proximal wall of the structure. This arrangement produces a fluid connection between the metering section having a volumetric capacity of from about 5 μL to about 15 μL and the overflow chamber.
Capillary[0173]821 is in fluid connection withsecondary metering structure819 at the distal wall of the first metering section. Capillary821 is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is fluidly connected to boronateaffinity matrix chamber822. Boronateaffinity matrix chamber822 is from about 0.02 mm to about 0.3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm and is positioned from about 1.2 cm to about 14 cm from the axis of rotation. Boronateaffinity matrix chamber822 further comprises 20-50 μL boronate-functionalized agarose beads having a mean diameter of about 60 μm; the beads are maintained in thechamber822 using aporous frit827. Boronateaffinity matrix chamber822 further comprises at least one surface that is translucent to light of at least wavelength of about 430 nm, permitting direct illumination and interrogation of the amount of glycated hemoglobin that is bound thereto. Fluid flow through capillary821 is connected to capillary orsacrificial valve23.
Boronate[0174]affinity matrix chamber822 is further fluidly connected tocapillary828.Capillary828 is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is connected to columnpreparation buffer reservoir829. Columnpreparation buffer reservoir829 is from about 0.02 mm to about 3 cm deep and has a cross-sectional diameter of from about 0.02 mm to about 10 cm and is positioned from about 1.2 cm to about 12 cm from the axis of rotation, more proximal than boronateaffinity matrix chamber822. Columnpreparation buffer reservoir829 comprises from about 100 μL to about 500 μL of column preparation buffer comprising magnesium chloride, taurine, D,L-methionine, sodium hydroxide, antibiotics and stabilizers (obtained from IsoLab as described in the Examples below). Fluid flow throughcapillary828 is connected to capillary orsacrificial valve835.
Boronate[0175]affinity matrix chamber822 is further fluidly connected tocapillary830.Capillary830 is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is connected to column washbuffer reservoir831. Columnwash buffer reservoir831 is from about 0.02 mm to about 3 cm deep and has a cross-sectional diameter of from about 0.02 mm to about 10 cm and is positioned from about 1.2 cm to about 14 cm from the axis of rotation, more proximal than boronateaffinity matrix chamber822. Columnwash buffer reservoir831 comprises from about 100 μL to about 500 μL of column preparation buffer as described above. Fluid flow throughcapillary830 is connected to capillary orsacrificial valve836.
In alternative embodiments, column[0176]preparation buffer reservoir829 and column washbuffer reservoir831 can be the same reservoir, or can be fluidly connected as shown in FIG. 10A.
Boronate[0177]affinity matrix chamber822 is further fluidly connected tocapillary832.Capillary732 is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm.Capillary832 is further fluidly connected withwaste reservoir834.Waste reservoir834 is from about 0.02 mm to about 3 cm deep and has a cross-sectional diameter of from about 0.02 mm to about 10 cm and is positioned from about 1.2 cm to about 14 cm from the axis of rotation.
As illustrated in FIG. 10A, in the use of this platform a volume of blood from about 15 μL to about 150 μL is applied to[0178]metering capillary802, either directly or using the metering components of the platform described above. Blood flowing throughcapillary810 and lysis buffer flowing throughcapillary818 are mixed in mixingchamber815 by overcoming capillary valve811 or release of sacrificial valve811. A volume of lysis buffer from about 25 μL to about 90 μL was mixed with the blood sample. Fluid flow within mixingchamber815 is turbulent, in contrast to fluid flow throughcapillaries810 and818, which is primarily laminar, so that mixing occurs predominantly in mixingchamber815. Fluid flow proceeds throughchannel817 and intosecondary metering structure819.
The mixture of lysis buffer and blood, comprising a lysed blood sample, flows at a rotational speed f[0179]₂from about 200 rpm to about 2000 rpm intosecondary metering structure819. The lysed blood sample enters and fills the metering section ofsecondary metering structure819. Any additional lysed blood sample then empties into the overflow chamber ofsecondary metering structure819. Most preferably, a sufficient volume of lysis buffer and blood sample is applied to the disc to fill at least the metered sections ofsecondary metering structure819.
After the lysed blood sample is completely transferred to[0180]secondary metering structure819, capillary orsacrificial valve835 is released, allowing from 100 μL to about 500 μL of column preparation buffer to flow at rotational speed f₂throughcapillary830 and intoboronate affinity matrix822. Continued or discontinuous rotation motivates column preparation buffer throughboronate affinity matrix822, capillary832, and intowaste reservoir834.
After the column preparation buffer is applied to boronate[0181]affinity matrix chamber822, capillary orsacrificial valve823 is released, allowing the metered lysed blood sample from the first metered section ofsecondary metering structure819 through capillary821 and intoboronate affinity matrix822 and allowed to incubate in the affinity matrix chamber for from about 0.5 to about 5 min. Capillary orsacrificial valve836 is then released, allowing from 100 μL to about 500 μL of column wash buffer to flow at rotational speed f_? throughcapillary830 and intoboronate affinity matrix822. Continued or discontinuous rotation motivates column preparation buffer throughboronate affinity matrix822, capillary732 and intowaste reservoir834. During fluid flow of wash buffer through the boronate affinity matrix chamber, the chamber is preferably illuminated by light at a wavelength of 430 nm through the translucent portion of the chamber. The concentration of glycated hemoglobin in the sample is determined thereby.
An alternative embodiment of the glycated hemoglobin assay microsystem platform of the invention is shown in FIG. 11. Construction of the disk embodiments of the platforms of the invention were as described above. The blood application and metering components and their dimensions and relationships to one another are identical to those described above, comprising sample[0182]entry port chamber901,metering capillary902,overflow capillary903, andoverflow chamber905. As in Example 1, each of the overflow and fluid chambers is also connected with air ports or air channels, such as954, and capillary junction(s)955, that permit venting of air displaced by fluid movement on the platform.
Metered[0183]capillary902 is fluidly connected to capillary910 that is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is connected tosacrificial wax valve911.Sacrificial wax valve911 is further fluidly connected withcapillary912 that is from about 0.03 mm to about 2.2 mm.Capillary912 is further fluidly connected toblood lysis chamber915 that is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm, is positioned from about 1.2 cm to about 14 cm from the center of rotation, and contains from about 25 μL to about 90 μL of blood lysis solution (0.1% Triton-X100 in 50 mM Tris, pH 9.5).Blood lysis chamber915 is further fluidly connected withcapillary918 that is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is connected tosacrificial wax valve913.Sacrificial wax valve913 is further fluidly connected withwax recrystallization chamber914 that is from about 0.02 mm to about 2 mm deep and has a volumetric capacity sufficient to sequester melted wax from a released wax valve and prevent occlusion of the lumen of the capillary918 controlled by the valve.
[0184]Capillary918 is further fluidly connected tosecondary metering structure919.Secondary metering structure919 is from about 0.02 mm to about 3 cm deep, and is positioned from about 1.2 cm to about 14 cm from the center of rotation.Secondary metering structure919 is constructed to comprise three sections. A first section comprises a throwaway section sample having a volumetric capacity of from about 5 μL to about 10 μL because it is thought that by taking the second section a more representative would be obtained. This throwaway section is arranged proximal to the entry position ofcapillary918 and is separated from a metering section by a septum that extends from the distal wall of the structure to a position just short of the proximal wall of the structure. This arrangement produces a fluid connection between the throwaway section and the metering section. The metering section has a volumetric capacity of from about 5 μL to about 10 μL and is fluidly connected to an overflow section having an excess volumetric capacity of from about 15 μL to about 150 μL. The volumetric capacity of the overflow section is sufficient to accommodate the largest blood fluid volume applied to the disk.
[0185]Capillary921 is in fluid connection withsecondary metering structure919 at the distal wall of the metering section.Capillary921 is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is connected to boronateaffinity matrix chamber922. Boronateaffinity matrix chamber922 is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm and is positioned from about 1.2 cm to about 14 cm from the axis of rotation. Boronateaffinity matrix chamber922 further comprises 10-50 μL of boronate-functionalized agarose beads having a mean diameter of about 60 cm; the beads are maintained in thechamber922 using aporous frit927. Fluid flow throughcapillary921 is connected tosacrificial valve923.Sacrificial wax valve923 is further fluidly connected withwax recrystallization chamber924 that is from about 0.03 mm to about 2.2 mm deep and has a volumetric capacity sufficient to sequester melted wax from a released wax valve and prevent occlusion of the lumen of the capillary923 controlled by the valve.
Boronate[0186]affinity matrix chamber922 is further fluidly connected tocapillary928.Capillary928 is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is connected to column washbuffer reservoir929. Columnwash buffer reservoir929 is from about 0.02 mm to about 3 cm deep and has a cross-sectional diameter of from about 0.02 mm to about 10 cm and is positioned from about 1.2 cm to about 14 cm from the axis of rotation, more proximal than boronateaffinity matrix chamber922. Columnwash buffer reservoir929 comprises from about 100 μL to about 500 μL of column wash buffer as described above. Fluid flow throughcapillary928 is connected tosacrificial valve936.Sacrificial wax valve936 is further fluidly connected withwax recrystallization chamber937 that is from about 0.03 mm to about 2.2 mm deep and has a volumetric capacity sufficient to sequester melted wax from a released wax valve and prevent occlusion of the lumen of the capillary928 controlled by the valve.
Boronate[0187]affinity matrix chamber922 is further fluidly connected tocapillary932.Capillary932 is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 10 cm and is connected to samplecollection cuvette array934. Samplecollection cuvette array934 is from about 0.02 mm to about 3 cm deep and has a cross-sectional diameter of from about 0.02 mm to about 10 cm and is positioned from about 1.2 cm to about 14 cm from the axis of rotation. Samplecollection cuvette array934 is separated into a multiplicity of individual chambers, each separated from one another by septa that extend from the distal wall of the cuvettes to a position adjacent to the proximal wall of the cuvettes, so that a fluid passage950 is maintained between each of the cuvettes. The fluid passage950 is formed by the back (proximal wall) of the samplecollection cuvette array934 and the row of septa separating each of the sections of thesample collection cuvettes934.Capillary932 is fluidly connected to samplecollection cuvette array934 at a position adjacent to the proximal wall of the array and directed to the cuvette most proximal to the boronateaffinity matrix chamber922. Alternatively, the septa can be eliminated in samplecollection cuvette array934, wherein the sample cuvette is a single chamber.
[0188]Capillary941 is fluidly connected tosecondary metering structure919.Capillary941 is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is connected tosecondary metering structure919 at a position between the metering section and the overflow section.Capillary941 is further fluidly connected with total hemoglobin readchamber942. Total hemoglobin readchamber942 is from about 0.02 mm to about 3 cm deep, is positioned from about 1.2 cm to about 14 cm from the center of rotation, and has a volumetric capacity of from about 50 μL to about 250 μL. Total hemoglobin readchamber942 is positioned radially more distal from the center of rotation thansecondary metering structure919, and comprises a read window translucent to light having a wavelength of from about 430 nm. In addition, there is no capillary or sacrificial valving controlling fluid flow incapillary941.
The platform also comprises control sample read[0189]cuvettes943 and944, advantageously positioned in proximity to total hemoglobin readchamber942. Control sample readcuvettes943 and944 are each from about 0.02 mm to about 3 cm deep, positioned from about 1.2 cm to about 14 cm from the center of rotation, and have a volumetric capacity of from about 50 μL to about 200 μL. Control sample readcuvettes943 and944 comprise a read window translucent to light having a wavelength of from about 430 nm. Control sample readcuvettes943 and944 are not fluidly connected to any other structure on the platform and contain standards and/or calibration reagents.
As illustrated in FIG. 11, in the use of this platform a volume of blood from about 15 μL to about 150 μL is applied to[0190]metering capillary902, either directly or using the metering components of the platform described above. Release ofsacrificial valve911 and rotation of the platform at a rotational speed f₁of from about 50 rpm to about 1000 rpm motivates blood flow throughcapillary910 and intoblood lysis chamber915. The mixture of blood and blood lysis buffer inblood lysis chamber915 is mixed by agitation, wherein the platform is accelerated repeatedly from about +2000 rpm/sec to −2000 rpm/sec (wherein “+” and “−” indicate rotation in different directions) over a time period of about 30 sec to about 600 sec. Release ofsacrificial valve913 and rotation of the platform at a rotational speed f₂of from about 200 rpm to about 2000 rpm motivates the lysed blood sample to flow throughcapillary918 and intosecondary metering structure919. Continued rotation motivates lysed blood solution to fill the metering section ofsecondary metering structure919; after filling of this section of the structure, excess lysed blood sample flows throughcapillary941 and into total hemoglobin readchamber942. After filling of total hemoglobin readchamber942, any excess lysed blood sample is displaced into the overflow section ofsecondary metering structure919.
Release of[0191]sacrificial valve923 and rotation of the platform at a rotational speed f₃of from about 200 rpm to about 2000 rpm motivates the metered lysed blood sample in the metering section ofsecondary metering structure922 to flow throughcapillary921 and into boronateaffinity matrix chamber922. After incubation of the metered lysed blood sample956 in boronateaffinity matrix chamber922,sacrificial valve936 is released and the platform is rotated at a rotational speed f₄of from about 200 rpm to about 3000 rpm. Column wash buffer flows from column washbuffer reservoir929 throughcapillary921 and into boronateaffinity matrix chamber922, displacing the unbound hemoglobin fraction throughcapillary932 and into samplecollection cuvette array934. Continued rotation of the platform displaces the collected sample sequentially into the separate cuvettes radially away from the position of boronateaffinity matrix chamber922 on the platform. Samplecollection cuvette array934 is then interrogated by illumination with light at a wavelength of 430 nm, and the concentration of non-glycated hemoglobin in the blood sample determined. Additionally, illumination of total hemoglobin readchamber942 with light at a wavelength at 415 nm is performed to determine the concentration of total hemoglobin in the blood sample. The amount of glycated hemoglobin is calculated by subtracting the amount of non-glycated hemoglobin in the sample from the total hemoglobin concentration in the sample. Control sample readcuvettes943 and944 are used to calibrate the spectrophotometric readings.
In alternative embodiments of these microfluidics systems, the boronate affinity matrix is replaced by other substances capable of differentially binding glycated or non-glycated hemoglobin species. In a first embodiment of such an alternative, m-aminophenylboronate polyacrylic acid is used to derivatize a positively-charged nylon 66 membrane (such as Biodyne-B, Pall Biosupport Division, Port Washington, N.Y.). This membrane is used in substitution for the boronate-functionalized agarose beads in the boronate affinity matrix chambers of the invention. In preferred embodiments, the boronate affinity matrix chambers are modified to contain the membrane in contact with or more preferably adhered to the platform surface within the chamber, so that one face of the membrane derivatized with m-aminophenylboronate polyacrylic acid is in contact with the lysed blood sample. Binding of glycated hemoglobin to the membrane is quantitated by visible light reflectance spectroscopy at a wavelength of 415 nm.[0192]
A second alternative embodiment of the glycated hemoglobin microsystems assays of the invention comprises inositol hexaphosphate. In these embodiments, inositol hexaphosphate is attached (covalently or by electrostatic interactions) to a solid support, including but not limited to beads, membranes, pads, etc. The lysed blood sample is treated with sodium dithionite to convert it to the deoxy form. In the Microsystems platforms of the invention, an effective amount of sodium dithionite is provided with the lysis buffer, or as a component of the secondary metering structures, most preferably as a dry powder coating on the walls of one or both of the metering sections thereof. The deoxygenated lysed blood sample is then placed in contact with the solid support comprising inositol hexaphosphate, preferably comprising and in substitution for the boronate affinity matrix chamber of the glycated hemoglobin platforms of the invention. In these embodiments, the portion of hemoglobin that does not bind to the inositol phosphate-containing solid support is the glycated fraction, which can be delivered to a reads chamber, cuvette or other optically-appropriate component of the platform and the amount of glycated hemoglobin determine directly by visible light reflectance spectrophotometry at a wavelength of 415 nm.[0193]
The invention also provides microsystem platforms for performing a multiplicity of reactions including identification of chemical species from solutions or complex mixtures and separations of particular components of a solution or complex mixture. This aspect of the invention is illustrated by a microfluidics array for determining glucose concentration and separating glycated hemoglobin from a blood sample, as shown in FIGS. 12A through 12Q and[0194]13A through13D.
Construction of the disk embodiments of the platforms of the invention were as described above. FIG. 13B shows a detailed description of the microfluidics components of the platform, which are described in additional detail below. FIG. 13C shows the geometry of a screen printed electrical lead layer deposited on a mylar substrate. FIG. 13D shows the positions of screen printed heaters activated by the electrical leads of the lead layer and screen printed on mylar. FIG. 13E shows a overlay of these components in the assembled disc.[0195]
Referring to the microfluidics components of the platform shown in FIG. 13B, an[0196]entry port1 is positioned on the top surface of the disc and is open for the user to apply an unmetered sample.Entry port1 is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm, is positioned from about 1.2 cm to about 14 cm from the center of rotation, and is fluidly connected to capillary channel1A that is from about. 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm. Capillary channel1A is fluidly connected tometering component2, which comprises four sections. The first section is rectangularly-shaped and extends in a direction proximal to the axis of rotation away from its fluid connection with capillary channel1A. About half way up this rectangular section is alateral chamber3, which empties into a blood glucose metering chamber4 and an overflow chamber5. The first section of the metering component is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm, is positioned from about 1.2 cm to about 14 cm from the center of rotation, and has a volumetric capacity of from about 15 μL to about 150 μL. Thelateral chamber3 is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm, and is positioned from about 1.2 cm to about 14 cm from the center of rotation. Blood glucose metering chamber4 is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm, is positioned from about 1.2 cm to about 14 cm from the center of rotation and has a volumetric capacity of from about 1 μL to about 15 μL. Overflow chamber5 is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm, is positioned from about 1.2 cm to about 14 cm from the center of rotation and has a volumetric capacity of from about 5 μL to about 50 μL.
Overflow chamber[0197]5 is fluidly connected to overflow channel8 that is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm and extends from about 10 mm to about 5 cm from overflow chamber5. Overflow channel5 is fluidly connected to shortsample detection cuvette9 that is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm, is positioned from about 1.2 cm to about 10 cm from the center of rotation and has a volumetric capacity of from about 15 μL to about 150 μL Blood glucose metering chamber4 is fluidly connected to capillary15 that is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm and extends from about 1 mm to about 5 cm from blood glucose metering chamber4.Capillary15 is connected tosacrificial wax valve6, which is further fluidly connected with wax recrystallization chamber6A that is from about 0.03 mm to about 2.2 mm deep and has a volumetric capacity sufficient to sequester melted wax from a released wax valve and prevent occlusion of the lumen of the capillary15 controlled by the valve.Capillary15 is further fluidly connected withglucose assay chamber11 that is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm, is positioned from about 1.2 cm to about 14 cm from the center of rotation and has a volumetric capacity of from about 5 μL to about 50 μL.Glucose assay chamber11 comprises a depression11A in the surface of the platform having a depth of from about 0.02 mm to about 3 cm, most preferably comprising a circular or concave depression connected to capillary15 so that blood flows into the chamber through the bottom of depression11A. Depression11A is constructed to have a volumetric capacity of from half to twice the assay volume. Bloodglucose assay chamber11 also comprises a pad ormatrix10 of a hydrophilic substance possessing a pore size of 0.2-2.0 μm, most preferably comprising a positively-charged nylon matrix having a pore size of about 0.8 μm. The upper limit on pore size ofmatrix10 is chosen to inhibit or prevent blood cell entry into the matrix. The matrix is positioned in bloodglucose assay chamber11 to be in fluidic contact with depression11A, more preferably covering depression11A, and most preferably having a surface area greater than the surface area of depression11A. The matrix was further impregnated with immobilized reagents11B which produce a detectable product proportional to the amount or concentration of glucose in a blood sample. Most preferably, the detectable product is a colored product11C, i.e., a product absorbing light at a detectable, most preferably a visible, wavelength.
[0198]Lysis metering chamber2 is fluidly connected to capillary7 controlled by sacrificial valve7A. Sacrificial wax valve7A is further fluidly connected with wax recrystallization chamber7B that is from about 0.03 mm to about 2.2 mm deep and has a volumetric capacity sufficient to sequester melted wax from a released wax valve and prevent occlusion of the lumen of the capillary7 controlled by the valve. Capillary7 is from about 0.02 mm to about 2 mm deep, has a cross-sectional diameter of from about 0.02 mm to about 2 mm, extends from about 1 mm to about 5 cm fromlysis metering chamber2 and is fluidly connected toblood lysis chamber16.Blood lysis chamber16 is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm, is positioned from about 1.2 cm to about 14 cm from the center of rotation, and contains from about 25 μL to about 90 μL of blood lysis solution (0.1% Triton-X100 in 50 mM Tris, pH 9.5).
[0199]Blood lysis chamber16 is fluidly connected at a distal aspect to capillary17 controlled by sacrificial valve18.Capillary17 is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm Sacrificial wax valve18 is further fluidly connected with wax recrystallization chamber18A that is from about 0.03 mm to about 2.2 mm deep and has a volumetric capacity sufficient to sequester melted wax from a released wax valve and prevent occlusion of the lumen of the capillary17 controlled by the valve.Capillary17 is fluidly connected tosecondary metering structure19.Secondary metering structure19 is from about 0.02 mm to about 3 cm deep, and is positioned from about 1.2 cm to about 14 cm from the center of rotation.Secondary metering structure19 is constructed to comprise three sections. Afirst section20 comprises a throwaway section having a volumetric capacity of from about 5 μL to about 10 μL because it is thought that a more representative sample would be obtained thereby.Throwaway section20 is arranged proximal to the entry position ofcapillary17 and is separated from ametering section21 by a septum that extends from the distal wall of the structure to a position just short of the proximal wall of the structure. This arrangement produces a fluid connection betweenthrowaway section20 and themetering section21. Metering section has a volumetric capacity of from about 5 μL to about 10 μL and is fluidly connected to anoverflow section24 having an excess volumetric capacity of from about 15 μL to about 150 μL. The volumetric capacity of the overflow section is sufficient to accommodate the largest blood fluid volume applied to the disk.
Capillary[0200]25 is in fluid connection withsecondary metering structure21 at the distal wall of the metering section. Capillary25 is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is connected to boronateaffinity matrix chamber28. Capillary25 is fluidly connected to sacrificial wax valve26 that is further fluidly connected with wax recrystallization chamber26A Wax recrystallization chamber26A is from about 0.03 mm to about 2.2 mm deep and has a volumetric capacity sufficient to sequester melted wax from a released wax valve and prevent occlusion of the lumen of the capillary25 controlled by the valve.
Boronate[0201]affinity matrix chamber28 is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02 mm to about 10 cm and is positioned from about 1.2 cm to about 14 cm from the axis of rotation. Boronateaffinity matrix chamber28 further comprises boronate-functionalized agarose beads having a mean diameter of about 60 μm; the beads are maintained in thechamber28 using aporous frit29. Boronateaffinity matrix chamber28 is further fluidly connected tocapillary31.Capillary31 is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is connected to column washbuffer reservoir30. Columnwash buffer reservoir30 is from about 0.02 mm to about 3 cm deep and has a cross-sectional diameter of from about 0.02 mm to about 10 cm and is positioned from about 1.2 cm to about 14 cm from the axis of rotation, more proximal than boronateaffinity matrix chamber28. Columnwash buffer reservoir30 comprises from about 250 μL to about 350 μL of column wash buffer as described above. Fluid flow throughcapillary31 is connected tosacrificial valve32.Sacrificial wax valve32 is further fluidly connected with wax recrystallization chamber32A that is from about 0.03 mm to about 2.2 mm deep and has a volumetric capacity sufficient to sequester melted wax from a released wax valve and prevent occlusion of the lumen of the capillary32 controlled by the valve.
Boronate[0202]affinity matrix chamber28 is further fluidly connected to capillary37. Capillary37 is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is connected to samplecollection cuvette array12. Samplecollection cuvette array12 is from about 0.02 mm to about 3 cm deep and has a cross-sectional diameter of from about 0.02 mm to about 10 cm and is positioned from about 1.2 cm to about 14 cm from the axis of rotation. Samplecollection cuvette array12 is separated into a multiplicity of individual chambers, each separated from one another by septa that extend from the distal wall of the cuvettes to a position adjacent to the proximal wall of the cuvettes, so that afluid passage50 is maintained between each of the cuvettes. Thefluid passage50 is formed by the back (proximal wall) of the samplecollection cuvette array12 and the row of septa separating each of the sections of thesample collection cuvettes12.Capillary33 is fluidly connected to samplecollection cuvette array12 at a position adjacent to the proximal wall of the array and directed to the cuvette most proximal to the boronateaffinity matrix chamber28. In alternative embodiments,collection cuvette array12 can be constructed without such septa, and this structure is then just a single collection chamber.
[0203]Capillary22 is fluidly connected tosecondary metering structure19.Capillary22 is from about 0.02 mm to about 2 mm deep and has a cross-sectional diameter of from about 0.02 mm to about 2 mm and is connected tosecondary metering structure19 at a position between the metering section and the overflow section.Capillary22 is further fluidly connected with total hemoglobin readchamber23. Total hemoglobin readchamber23 is from about 0.02 mm to about 3 cm deep, is positioned from about 1.2 cm to about 14 cm from the center of rotation, and has a volumetric capacity of from about 5 μL to about 100 μL. Total hemoglobin readchamber23 is positioned radially more distal from the center of rotation thansecondary metering structure19, and comprises a read window translucent to light having a wavelength of from about 400 nm to about 950 nm. In addition, there is no capillary or sacrificial valving controlling fluid flow incapillary23.
The platform also comprises control sample read[0204]cuvettes13 and14, advantageously positioned in proximity to total hemoglobin readchamber23. Control sample readcuvettes13 and14 are each from about 0.02 mm to about 3 cm deep, positioned from about 1.2 cm to about 14 cm from the center of rotation, and have a volumetric capacity of from about 5 μL to about 100 μL. Control sample readcuvettes13 and14 comprise a read window translucent to light having a wavelength of from about 400 nm to about 950 nm. Control sample readcuvettes13 and14 are not fluidly connected to any other structure on the platform.
[0205]Air displacement channels33 and capillary junction(s)34, that permit venting of air displaced by fluid movement on the platform, are fluidly connected to the components of the platform to permit unimpeded fluid flow.
As illustrated in FIGS. 12A through 12Q, in the use of this platform a volume of blood from about 15 μL to about 150 μL is applied to[0206]entry port1. Blood enterslysis subvolume2 andlateral passageway3 under the influence of gravity and capillary forces in the absence of rotation of the platform, as shown in FIG. 12A. Upon rotation of the platform at a first rotational speed f₁of from about 50 rpm to about 1000 rpm, blood completely fillslysis subvolume2 and also flows throughpassageway3 and into blood glucose metering chamber4 and overflow chamber5, shown in FIG. 12B. Blood is retained in blood glucose chamber4 either due to capillary pressure or by asacrificial valve6, most preferably a wax valve. Similarly, blood is retained inblood lysis subvolume2 by a valve, most preferably a sacrificial valve7. Excess blood flows at rotational speed f₁through overflow channel8 and intooverflow chamber9, shown in FIGS. 12C and 12D. Typical values for the first rotational speed are an acceleration of about 20 to about 60 rpm/sec to a final radial velocity of about 600 rpm.
After blood is metered and excess blood delivered to overflow[0207]chamber9, the rotational speed of the disc is reduced to a rotational speed f_1aof from about 0 rpm to about 500 rpm, typically to about 60 rpm, to perform a blanking measurement on samplecollection cuvette array12, total hemoglobin readchamber23 and bloodglucose assay chamber11. Measurements of blankingcuvettes13 and14 are also advantageously performed.
The disc is then accelerated to a second rotational speed f[0208]₂of about 200 rpm to about 2000 rpm, greater than f₁, and typically in the range of from about 800 rpm to about 1000 rpm. At this speed,capillary valve6 is overcome orsacrificial valve6 is released, and from about 1 μL to about 50 μL of blood from blood glucose metering chamber4 flows throughcapillary15 and into bloodglucose assay chamber11, shown in FIG. 12E. Upon enteringassay chamber11, blood fluid components are forced intoabsorbent matrix10 through depression11A. The blood fluid is incubated inmatrix10 for a time sufficient for the reagents10A to produce a colored product10B in an amount proportional to the amount of glucose in the blood fluid sample. The disc is slowed, typically to a rotational speed f₄of from about 0 rpm to about 500 rpm, typically about 100 rpm, for glucose data acquisition using reflectance spectrometry; data acquisition as the disc is spinning down also enables to instrument to set t=0 for the assay, based on a decrease in reflectance when thematrix10 is wet by the blood fluid components and hence the matrix's scattering decreases. Development of colored product10B is shown in FIG. 12G.
The disc is then accelerated to rotational speed f[0209]₃of about 500 rpm to about 3000 rpm, typically about 1000 rpm, with release of sacrificial valve7A and fluid flow of from about 1 μL to about 50 μL, typically about 5 μL of blood frommetered subvolume2 through capillary7 and intoblood lysis chamber16 containing from about 25 μL to about 90 μL, typically about 45 μL of blood lysis buffer. This is shown in FIG. 12H. The mixture of blood and blood lysis buffer inblood lysis chamber16 is mixed by agitation, wherein the platform is accelerated repeatedly from about +2000 rpm/sec to −2000 rpm/sec (wherein
+
and
−
indicate rotation in different directions), typically from about 250-500 rpm/sec, over a time period of about 30 seconds to about 5 min, typically 1-2 min, as shown in FIGS. 12I and 12J.
The disc then is accelerated to a rotational speed f[0210]₅from about 200 rpm to about 2000 rpm and typically about 750 rpm, and sacrificial valve18 is released. Lysed blood fromblood lysis chamber16 flows throughcapillary17 and intosecondary metering structure19, as shown in FIG. 12K. The lysed blood solution sequentially fillsthrowaway section20, which is used as a trap for cell debris,metering section21 and excess lysed blood then fillsoverflow section24. Filling ofmetering chamber21 is immediately followed by fluid flow throughcapillary22 and filling of total hemoglobin readchamber23. The disc is spun at rotational speed f₅for a time sufficient to substantially completely drainblood lysis chamber16. The configuration of the blood fluids on the disc after this spin is shown in FIG. 12L.
The disc is then accelerated to a rotational speed f[0211]_? from about 200 rpm to about 3000 rpm and typically about 750 rpm, and sacrificial valve26 is released. A metered volume of about 1 μL to about 50 μL, typically about 6 μL of lysed blood frommetering section21 flows throughcapillary27 and into boronate affinity matrix chamber28 (shown in FIG. 12M). The lysed blood solution is allowed to incubate in the chamber for a time from about 30 seconds to about 5 min, typically about 1 min, sufficient for glycated hemoglobin to bind to the matrix. This aspect of the disc is illustrated in FIG. 12N.
The disc is then accelerated to a rotational speed f[0212]_? from about 500 rpm to about 3000 rpm and typically about 1000 rpm, andsacrificial valve32 is released. A volume of about 250 μL to about 350 μL, typically about 290 μL of column wash buffer as described above flows fromwash buffer reservoir30 thoughcapillary31 and into boronate affinity matrix chamber28 (shown in FIG. 12O). The wash buffer displaces the non-glycated hemoglobin and other components of the lysed blood fluid from the affinity column matrix and into samplecollection cuvette array12. FIGS. 12P through 12Q show sequential filling of the individual cuvettes in samplecollection cuvette array12. The rotation speed of the disc is reduced, to from about 0 rpm to about 500 rpm and typically to about 60 rpm for samplecollection cuvette array12 and total hemoglobin readchamber23 to be interrogated spectrophotometrically. The glycated fraction of the blood sample is determine algorithmically by subtracting the non-glycated hemoglobin fraction in samplecollection cuvette array12 from the total hemoglobin detected in total hemoglobin readchamber23.

2. Resistive Heater and Temperature Sensing Components

Temperature control elements are provided to control the temperature of the platform. The invention provides heating elements, specifically resistive heating elements, and elements for detecting temperature at specific positions on the platform. Heating devices are preferably arrayed to control the temperature of the platform over a particular and defined area, and are provided having a steep temperature gradient with distance on the platform from the heater.[0213]
Certain resistors, including commercially-available resistive inks (available from Dupont) exhibit a positive temperature coefficient (PTC), i.e., an increase in resistance with increasing temperature. Applying a fixed voltage across a PTC resistor screen-printed on a plastic substrate results in rapid heating, followed by self-regulation at an elevated temperature defined by the circuit design heat sink and ambient temperature. In such screen-printed resistors, connection to a power source is made by first printing parallel silver conductors followed by printing the PTC ink between the conductors.[0214]
A resistive heating element comprises a conductive ink connected with electrical contacts for activation of the heater, and resistive inks applied between the conductive ink and in electrical contact therewith, wherein application of a voltage (direct or alternating current) between the conductive inks results in current flow through the resistive inks and production of heat. There are two important types of resistive inks used in the resistive heating elements of this invention The first is a standard polymer thick film ink, such as Dupont 7082 or Dupont 7102 ink. These inks produce a surface temperature that is not self-limiting, and the temperature resulting from the use of these inks is dependent primarily on the magnitude of the applied voltage. In contrast, the positive temperature coefficient (PTC) inks show increase resistivity with increasing voltage, so that surface temperature is self-limiting because the amount of heat-producing current goes down as the applied voltage goes up. PTC inks are characterized as having a particular temperature where this self-limiting property is first exhibited; at voltages that produce temperatures less than the critical temperature, the amount of heat is dependent on the magnitude of the applied voltage.[0215]
Resistive inks useful according to the invention include Dupont 7082, 7102, 7271, 7278 and 7285, and other equivalent commercially available polymer thick film ink and PTC inks.[0216]
Conductive inks useful according to the invention include Dupont 5028, 5025, Acheson 423SS, 426SS and SS24890, and other equivalent commercially available conductive inks.[0217]
Additional components of the dielectric layer that serves to insulate the electrical circuit. Dielectric layers advantageously comprise dielectric inks such as Dupont 5018A. Insulation can also be achieved using pressure sensitive transfer adhesive such as 7952MP (3M Co.), or a pressure sensitive transfer adhesive deposited onto a polyester carrier layer such as 7953MP (3M Co.) or thermoplastic bonding films such as[0218]3M 406, 560 or 615.
Resistive heaters of the invention are advantageously used to incubate fluids at a stable temperature and for melting sacrificial valves as described below, and also for thermal cyclic.[0219]
Resistive and conductive inks are preferably screen-printed using methods and techniques well known in the art. See Gilleo, 1995,[0220]Polymer Thick Film(Van Nostrand Reinhold). Inks are typically screen printed to a thickness of about 10 microns; however, repetitive screen printing of resistive inks can be used to deposit thicker layers having reduced resistances. Both conductive and resistive inks are heat cured, typically at between 110° C. and 120° C. for about 10 minutes. The outline of this printing process is shown in FIG. 30. Importantly, each of the layers must be correctly registered with one another for resistive heating to be provided. Heaters can be screen printed to any required size; a minimum area for a screen-printed heater has been determined to be about 0.25 mm²(0.5 mm×0.5 mm).
The ability to tailor the resistance (and hence the temperature profile) of the resistive heaters using choice of ink formulation and reprinting of heater circuits provides control of the final electrical and thermal properties of the resistive heating elements of the invention. The resistance can also be controlled through connection of series and parallel configurations of resistive elements.[0221]

3. Sacrificial Valves

The ability to specifically generate heat at a particular location on a Microsystems platform of the invention also enables the use of sacrificial valves that can be released or dissolved using heat. For the purposes of this invention, the term[0222]
sacrificial valve
is intended to encompass materials comprising waxes, plastics, and other material that can form a solid or semi-solid fluid-tight obstruction in a microchannel, capillary, chamber, reservoir or other microfluidics component of the platforms of the invention, and that can be melted or deformed to remove the obstruction with the application of heat. Sacrificial valves are preferably made of a fungible material that can be removed from the fluid flow path. In preferred embodiments, said sacrificial valves are wax valves and are removed from the fluid flow path by heating, using any of a variety of heating means including infrared illumination and most preferably by activation of resistive heating elements on or embedded in the platform surface as described herein. For the purposes of this invention, the term
wax
is intended to encompass any solid, semi-solid or viscous liquid hydrocarbon, or a plastic. Examples include mondisperse hydrocarbons such as eicosane, tetracosane and octasone, and polydisperse hydrocarbons such as paraffin. In the use of wax sacrificial valves, application of a temperature higher than the melting temperature of the wax melts the valve and removes the occlusion from the microchannel, capillary or other fluidic component of the microsystems platforms of the invention. Particularly when the sacrificial valve is melted on a rotating Microsystems platform of the invention, the melted wax to flow through the microchannel, capillary or other fluidic component of the Microsystems platforms of the invention and away from the original site of the valve.
One drawback, however, is the possibility that the wax will recrystallize as it flows away from the original valve site, and concomitantly, away from the localized heat source. Recrystallization results in re-occlusion of the microchannel, capillary or other fluidic component of the Microsystems platforms of the invention, potentially and most likely at a site other than the site of a localized heat source, and therefore likely to foul fluid movement on the disc. One solution for this problem is the inclusion in the sacrificial wax valves of the invention of a wax recrystallization chamber positioned[0223]
downstream
from the position of the wax valve. Preferably, the wax recrystallization chamber is fluidly connected with the microchannel, capillary or other fluidic component of the Microsystems platforms of the invention that was occluded by the wax sacrificial valve. Typically, the wax recrystallization chamber is a widening of the microchannel, capillary or other fluidic component of the Microsystems platforms of the invention so that recrystallized wax can harden on the walls of the microchannel, capillary or other fluidic component of the Microsystems platforms of the invention with enough distance between said walls that the recrystallized wax does not re-occlude the microchannel, capillary or other fluidic component of the Microsystems platforms of the invention. Preferably, the heating element, most preferably the resistive heating element of the invention, extends past the site of the wax valve and overlaps at least a portion of the wax recrystallization chamber, thereby retarding the propensity of the wax valve to recrystallize.
It is also recognized that this propensity of wax valves to recrystallize can be exploited to create a wax valve at a particular location in a microchannel, capillary or other fluidic component of the microsystems platforms of the invention. In this embodiment, a particular location can be kept below a threshold temperature by failing to apply heat at that location, and a wax valve material can be mobilized from a storage area on a platform by heating and them allowed to flow under centripetal acceleration to a particularly[0224]
cold
site where a wax valve is desired. An advantage of wax valves in this regard is that the proper positioning an activation of resistive heater elements enables flexibility in choosing when and whether a particular microchannel, capillary or other fluidic component of the Microsystems platforms of the invention is to be occluded by a wax sacrificial valve.
In particularly preferred embodiments, the sacrificial valves of the invention comprise a cross-linked polymer that displays thermal recover, most preferably a cross-linked, prestressed, semicrystalline polymer; an example of a commercially available embodiment of such a polymer is heat recoverable tubing (#FP301H, 3M Co., Minneapolis, Minn.). Using these materials, at a temperature less than the[0225]
melting
temperature (T_m), the polymer occludes a microchannel, capillary or other fluidic component of the Microsystems platforms of the invention. At a temperature greater than T_m,, however, the polymer reverts to its pre-stressed dimensions by shrinking. Such shrinking is accompanied by release of the occlusion from the microchannel, capillary or other fluidic component of the microsystems platforms of the invention. Such embodiments are particular preferred because the polymer remains in situ and does not recrystallize or otherwise re-occlude the microchannel, capillary or other fluidic component of the microsystems platforms of the invention. Also, such embodiments do not require the more extensive manipulation in preparing the platforms of the invention that wax valves require.
In another embodiment, the sacrificial valves of the invention comprise a thin polymeric layer or barrier dividing two liquid-containing microchannel, capillary or other fluidic component of the Microsystems platforms of the invention, that can burst when sufficient temperature and/or pressure is applied.[0226]
Another embodiment of the sacrificial valves of the invention are provided wherein a screen-printed resistive heater element is itself a valve. In this embodiment, the resistive heater element is screen-printed on a substrate such as polyester that divides two liquid-containing microchannel, capillary or other fluidic component of the Microsystems platforms of the invention. In these embodiments, localized application of heat using a resistive heating element is used to melt the substrate dividing the liquid-containing microchannel, capillary or other fluidic component of the microsystems platforms of the invention. Preferably, in this embodiment the two liquid-containing microchannel, capillary or other fluidic component of the microsystems platforms of the invention are positioned in adjacent layers through the vertical thickness of the platform.[0227]
As described above, the screen-printed resistive heater elements of this invention provide localized application of heat to a microsystems platform. The degree of localization achieved using these resistive heating elements is sufficient to provide for the placement of two adjacent sacrificial valves separated by a distance of 0.15 cm.[0228]

4. Detectors and Sensors

Detection systems for use on the microsystem platforms of the invention include spectroscopic, electrochemical, physical, light scattering, radioactive, and mass spectroscopic detectors. Spectroscopic methods using these detectors encompass electronic spectroscopy (ultraviolet and visible light absorbance, fluorescence, luminescence, and refractive index), vibrational spectroscopy (IR and Raman), and x-ray spectroscopies (x-ray fluorescence and conventional x-ray analysis using micromachined field emitters, such as those developed by the NASA Jet Propulsion Lab, Pasadena, Calif.).[0229]
General classes of detection and representative examples of each for use with the microsystem platforms of the invention are described below. In addition, the detection implementation systems utilizing the detectors of the invention can be external to the platform, adjacent to it or integral to the disk platform.[0230]

Spectroscopic Methods

1. Fluorescence

Fluorescence detector systems developed for macroscopic uses are known in the prior art and are adapted for use with the microsystem platforms of this invention. For example, an excitation source such as a laser is focused on an optically-transparent section of the disk. Light from any analytically-useful portion of the electromagnetic spectrum can be coupled with a disk material that is specifically transparent to light of a particular wavelength, permitting spectral properties of the light to be determined by the product or reagent occupying the reservoir interrogated by illumination with light. Alternatively, the selection of light at a particular wavelength can be paired with a material having geometries and refractive index properties resulting in total internal reflection of the illuminating light. This enables either detection of material on the surface of the disk through evanescent light propagation, or multiple reflections through the sample itself, which increases the path length considerably.[0231]
Alternative configurations appropriate for evanescent wave systems are provided as understood in the art (see Glass et al., 1987,[0232]Appl. Optics26: 2181-2187). Fluorescence is coupled back into a waveguide on the disk, thereby increasing the efficiency of detection. In these embodiments, the optical component preceding the detector can include a dispersive element to permit spectral resolution. Fluorescence excitation can also be increased through multiple reflections from surfaces in the device whenever noise does not scale with path length in the same way as with signal.
In another type of fluorescence detection configuration, light of both the fluorescence excitation wavelength and the emitted light wavelength are guided through one face of the device. An angle of 90 degrees is used to separate the excitation and collection optical trains. It is also possible to use other angles, including 0 degrees, whereby the excitation and emitted light travels colinearly. As long as the source light can be distinguished from the fluorescence signal, any optical geometry can be used. Optical windows suitable for spectroscopic measurement and transparent to the wavelengths used are included at appropriate positions (i.e., in “read” reservoir embodiments of detecting chambers) on the disk. The use of this type of fluorescence in macroscopic systems has been disclosed by Haab et al. (1995,[0233]Anal. Chem.67: 3253-3260).

2. Absorbance Detection

Absorbance measurements can be used to detect any analyte that changes the intensity of transmitted light by specifically absorbing energy (direct absorbance) or by changing the absorbance of another component in the system (indirect absorbance). Optical path geometry is designed to ensure that the absorbance detector is focused on a light path receiving the maximum amount of transmitted light from the illuminated sample. Both the light source and the detector can be positioned external to the disk, adjacent to the disk and moved in synchrony with it, or integral to the disk itself. The sample chamber on the disk can constitute a cuvette that is illuminated and transmitted light detected in a single pass or in multiple passes, particularly when used with a stroboscopic light signal that illuminates the detection chamber t a frequency equal to the frequency of rotation or multiples thereof. Alternatively, the sample chamber can be a planar waveguide, wherein the analyte interacts on the face of the waveguide and light absorbance is the result of attenuated total internal reflection (i.e., the analyte reduces the intensity source light if the analyte is sequestered at the surface of the sample chamber, using, for example, specific binding to a compound embedded or attached to the chamber surface; see Dessy, 1989,[0234]Anal. Chem.61: 2191).
Indirect absorbance can be used with the same optical design. For indirect absorbance measurements, the analyte does not absorb the source light; instead, a drop in absorbance of a secondary material is measured as the analyte displaces it in the sample chamber. Increased transmittance therefore corresponds to analyte concentration.[0235]

3. Light Scattering

Turbidity can also be measured on the disk. Optics are configured as with absorbance measurements. In this analysis, the intensity of the transmitted light is related to the concentration of the light-scattered particles in a sample. An example of an application of this type of detection method is a particle agglutination assay. Larger particles sediment in a rotating disk more rapidly than smaller particles, and the turbidity of a solution in the sample chamber before and after spinning the disk can be related to the size of the particles in the chamber. If small particles are induced to aggregate only in the presence of an analyte, then turbidity measurements can be used to specifically detect the presence of an analyte in the sample chamber. For example, small particles can be coated with an antibody to an analyte, resulting in aggregation of the particles in the presence of the analyte as antibody from more than one particle bind to the analyte. When the disk is spun after this interaction occurs, sample chambers containing analyte will be less turbid that sample chambers not containing analyte. This system can be calibrated with standard amounts of analyte to provide a gauge of analyte concentration related to the turbidity of the sample under a set of standardized conditions.[0236]
Other types of light scattering detection methods are provided for use with the Microsystems platforms and devices of the invention. Monochromatic light from a light source, advantageously a laser light source, is directed across the cross-sectional area of a flow channel on the disk. Light scattered by particles in a sample, such as cells, is collected at several angles over the illuminated portion of the channel (see Rosenzweig et al., 1994,[0237]Anal. Chem.66: 1771-1776). Data reduction is optimally programmed directly into the device based on standards such as appropriately-sized beads to relate the signal into interpretable results. Using a calibrated set of such beads, fine discrimination between particles of different sizes can be obtained. Another application for this system is flow cytometry, cell counting, cell sorting and cellular biological analysis and testing, including chemotherapeutic sensitivity and toxicology.

Analytic Methods

It will be understood that the interpretation of the optical detection data from performance of analytical assays as provided by the invention may require transformation of “raw” data into information useful for the operator. For example, “reflectance” is determined as the difference between the “signal” and the “dark signal” (i.e., the signal detected in the absence of transmitted light), normalized by the difference between the “reference” and the “dark reference,” wherein each of the reference values is determined for a “blank” cuvette not containing a sample. Similarly, the detection methods used by the invention also include methods for eliminating artifactual signal produced by interfering absorbing or light-scattering components, such as scratches or particulate matter. Advantageously, normalized readings are collected at a wavelength greater than 650 nm, more preferably 660±10 nm as a reference. This reading is then subtracted from the sample reading to as a correction thereof.[0238]
Absorbance from certain components of biological fluid sample, such as hemoglobin in blood, are advantageously removed by using a light source with a wavelength at the absorbance peak of the interfering material; for hemoglobin, this is about 425 nm. In preferred embodiments, light of this wavelength is provided by a blue light-emitting diode (LED) having a wavelength of about 430 nm. The light emission profile of the LED was found to overlap sufficiently with the hemoglobin absorbance profile to effectively quench the hemoglobin absorbance signal in the glucose assays of the invention. The glucose signal was then further treated analytically by subtracting a factor times the (blue-red) signal from the (orange-red) signal, wherein the (orange) signal was the wavelength specific for the glucose assay product. The factor used is dependent on the optics, the reading chamber structure, spectral properties of the light sources and filters, and the spectral absorbance characteristics of hemoglobin and the colored product of the glucose oxidase reaction. The factor can be determined empirically using solutions of known glucose and hemoglobin concentrations, both singly and in combination.[0239]
It will be understood in the art that similar combinations of light sources and interrogated wavelengths can be advantageously used to reduce or eliminate the contribution of other interfering species in optical detection methods used according to the invention.[0240]

5. Chemistries

As described above with regard to the microfluidic components of the Microsystems platforms of the invention, the present invention provides platforms for performing chemical, biochemical, enzymatic, immunological and other assays on fluid samples, most preferably wherein the fluid sample is a biological fluid sample.[0241]

Two exemplary types of assay formats are explicitly set forth herein; one of ordinary skill will recognize that the disclosure is generally applicable to a variety of assay systems as set forth, for example, in[0242]CLINICAL GUIDE TO LABORATORY TESTS,Tietz, ed., W. B. Sanders Co: Philadelphia, 1995. A representative and non-limiting sample of assays advantageously performed using the microsystems platforms of the invention are set forth in Table I below.

TABLE I


Assay for:	Analyte/Detection Type	Components on solid phase:

Acid Phosphatase	enzyme/colorimetric	Alpha-naphthol phosphate, Fast Red TR dye¹
Alanine	enzyme/colorimetric	Alanine, alpha-ketoglutarate, 2,4-
Aminotransferase		dinitrophenylhadrazine dye
Albumin	protein/colorimetric	Bromcresol green dye
Alkaline Phosphatase	enzyme/colorimetric	p-nitrophenyl phosphate
Amylase	enzyme/colorimetric	4,6-ethylidine(G7)-p-nitrophenyl(G1)-alpha,D-
		maltoheptaside, alpha-glucosidase
Apolipoprotein A-1	lipoprotein/immunoturbidimetric	Anti-ApoA1 antibody, polyethylene glycol²
Direct Bilirubin	organic compound/colorimetric	Diazotized sulfanilic acid
Total Bilirubin	organic compound/colorimetric	Caffeine, benzoate, acetate, diazotized sulfanilic
		acid
Calcium	mineral/colorimetric	Cresolphthalein complexone
Cholesterol	lipid/colorimetric	Cholesterol esterase, cholesterol oxidase, 4-
		aminoantipyrine, p-hydroxybenzene sulfonate
Fructosamine	glycated serum	Nitroblue tetrazolium
	protein/colorimetric
Gamma-glutamyl	enzyme/colorimetric	L-gamma-glutamyl-3-carboxy-4-nitroanilide,
transferase		glycylglycine
Iron	mineral/colorimetric	Ferrozine, reducing agent
Microprotein (urine	protein/colorimetric	Pyrogallol red-molybdate complex
or CSF)
Urea	organic compound/colorimetric	Diacetylmonoxime, heat
Specific Protein	immunologically reactive	Antibody reactive with unique species, etc, site;
species, subspecies or	proteins/immunoturbidimetry	precipitation enhancers (e.g., anti-alpha-1-
variant		antitrypsin IgG, polyethylene glycol)³
Antibodies against	immunoreaction to bacteria or	Immunoreactive component from infective
infectious agents	virus infection/heterogeneous	agent, enzyme linked to antibody against the
	enzyme immunoassay	primary immunoglobulin species of reaction to
		the agent, enzyme substrate linked to color
		generation (e.g., Epstein-Barr early antigen,
		anti-human IgG conjugated to horseradish
		peroxidase, 3-3′-5-5′-tetramethylbenzidine)
Drugs	immunologically reactive	Competition between drug and drug-enzyme
	therapeutic drugs or drugs of	reagent for anti-drug antibody binding sites
	abuse/homogeneous enzyme	where antibody binding inhibits enzyme
	immunoassay	activity, enzyme substrate linked to color
		generation (e.g., phenobarbital-glucose-6-
		phosphatase, anti-phenobarbital IgG, NAD)⁴

The requirements for performing such assays on the solid phase (such as the matrices disclosed herein) include: the ability to link, by one or a series of chemical reaction steps, the presence of an analyte of interest in a fluid sample, most preferably a biological fluid sample, to the quantitative generation of a product detectable by optical methods as disclosed herein; stabilization of the necessary reagents (chemicals or biochemicals) onto the solid phase so that the reagents retain their potencies or activities.[0243]
A second general scheme for performing assays on the Microsystems platforms of the invention involve miniaturized versions of affinity chromatography column separations, wherein the analyte specifically binds to a material in a chamber or on a surface, most preferably a derivatized surface, of the platform, or is bound to a material such as a bead, chromatography resin, or membrane on the surface of the platform, so that the remainder of the fluid sample can be washed from the affinity matrix and the analyte separated thereby. In certain preferred embodiments, the analyte is detected indirectly, wherein the biological fluid sample is interrogated after passage of the sample over the chromatograohy matrix. Such detection methods can be subtractive, wherein the interrogated optical property of the biological fluid sample after passage over the chromatography matrix is compared with the same property of a portion of the sample that has not been passed over the matrix; or directly, wherein the analyte is dissociated from the chromatography matrix (either non-specifically, using for example a salt or dielectric gradient, or specifically, using a binding competitor that displaces the analyte from the chromatography matrix).[0244]

Examples of analytes advantageously separated from biological fluid samples and the column affinity material(s) used therefore are set forth in Table II.[0245]

TABLE II


Column Affinity Material	Bound Species

Diatomateous earth	DNA
Protein A-agarose	IgG
Heparin-agarose	coagulation proteins, Protein C, growth factors,
	lipoproteins, steroid receptors
Blue agarose	albumin, coagulation factors, interferon, enzymes
	requiring cofactors with adenyl group
Streptavidin-agarose	biotinylated molecules
Con A-agarose	glycoproteins, polysaccharides with terminal mannose
	or glucose
Lentil lectin-agarose	glycoproteins, polysaccharides with branched mannose
	with fucose linked α(1,6) to N-acetyl-glucosamine
Wheat gern lectin-agarose	glycoproteins, polysaccharides with chitobiose core of
	N-linked oligosaccharides
Peanut lectin-agarose	glycoproteins, polysaccharides with terminal β-galactose
Arginine-agarose	serine proteases
Calmodulin-agarose	ATPases, phosphodiesterases, neurotransmitters,
	protein kinases
Gelatin-agarose	Fibronectin
Glutathione-agarose	S-transferases, glutathione-dependent proteins
Lysine-agarose	plasminogen, plasminogen activator, ribosomal RNA
DNA (denatured)-agarose	DNA polymerase, RNA polymerase, T4 polynucleotide
	kinase, exonuclease, deoxyribonucleases
DNA (native)-cellulose	Glucocorticoid receptor, DNA polymerase,DNA
	binding proteins

2′5′ ADP-agarose	NADP-dependent dehydrogenases
5′ AMP-agarose	NAD-dependent dehydrogenases, ATP-dependent
	kinases
7-Methyl-GTP-agarose	eukaryotic mRNA, cap-binding protein
Poly(U)-agarose	mRNA, reverse transcriptase, interferon, plant nucleic acids
C8-silica	Proteins

It will be appreciated that preparative embodiments of the affinity chromatographic column separations are within the scope of the invention.[0246]
The following Examples are intended to further illustrate certain preferred embodiments of the invention and are not limiting in nature.[0247]

EXAMPLE 1

Blood Glucose Assay Fluidics Structure

A microsystems platform provided by the invention and specifically designed for performing blood glucose assay is illustrated in FIG. 1. Disk embodiments of the platforms of the invention were fashioned from machined acrylic. The overall disc dimensions include an outer radius of about 6 cm and an inner radius of about 0.75 cm, wherein the disk was mounted on the spindle of a rotary device. The thickness of the fluidics disc was 3.2 mm, which was mounted on a conventional compact disc (CD) having a thickness of 1.2 mm. All surfaces coming into contact with blood on the platform are advantageously treated with heparin, EDTA or other anticoagulants to facilitate fluid flow thereupon.[0248]
The components of the blood glucose assay were prepared as follows. Blood sample[0249]entry port chamber101 having a depth in the platform surface of about 0.32 cm and lateral dimensions of about 1 cm was constructed on the platform, and designed to accommodate a volume of 100 μL. This entry port was fluidly connected with ametering capillary102 having a square cross-sectional diameter of about 0.1 cm deep×0.5 cm wideand proximal ends rounded with respect toentry port101; the length of this metering capillary array was sufficient to contain a total volume of about 16 μL. The entry port was also fluidly connected with anoverflow capillary103 having a cross-sectional diameter of about 0.05 cm×0.075 cm and proximal ends rounded with respect toentry port101. The overflow capillary was fluidly connected with a two-layeredoverflow chamber105 having a first depth in the platform of about 0.025 cm and a second depth in the platform of about 0.25 cm, greater than the depth of theoverflow capillary103.Metering capillary102 was fluidly connected to meteredblood fluid chamber104 having a depth in the platform surface of 1 mm and greater than the depth of themetering capillary102. Each of the overflow and fluid chambers was also connected with air ports or air channels, such as114, that have dimensions of 0.025 cm deep and permitted venting of air displaced by fluid movement on the platform. Acapillary junction115 that was 0.051 cm deep was present in the air channel to prevent fluid flow into the air channel.
[0250]Entry port101 was positioned on the platform about 2 cm from the center of rotation.Metering chamber102 extended about 1 cm fromentry port101. The extent of the length ofoverflow capillary103 was 300% greater than the extent of the length ofmetering capillary102. The position ofblood fluid chamber104 was about 4.6 cm from the center of rotation, and the position ofoverflow chamber105 was about 4 cm from the axis of rotation.
[0251]Blood fluid chamber104 acted as a capillary barrier that prevented fluid flow frommetering chamber102 at a first, non-zero rotational speed f₁of about 3000 rpm that was sufficient to permit fluid flow comprising overflow from theentry port101 throughoverflow capillary103 and intooverflow chamber105. This capillary boundary was constructed to be overcome at a second rotational speed f₂of about 600 rpm (so that f₂>f₁).Blood fluid chamber104 was fluidly connected to capillary110 that was about 0.025 cm deep and had a cross-sectional diameter of about 0.025 cm and was connected to capillary orsacrificial valve111.Sacrificial valve111 was further fluidly connected withcapillary112 that was about 0.25 mm deep and had a cross-sectional diameter of about 0.05 cm, andcapillary112 was fluidly connected toassay chamber107.Sacrificial valve chamber111 was positioned to sequester melted wax produced by release of the sacrificial valve.
[0252]Assay chamber107 comprised a depression in the surface of the platform having a depth of about 0.1 cm, and further comprised a circular or rectangularconcave depression113 connected tocapillary112.Assay chamber107 also comprised a pad ormatrix106 comprising a positively-charged nylon matrix having a pore size of about 0.8 μm. The pore size ofmatrix106 was chosen to inhibit or prevent blood cell entry into the matrix. The matrix was positioned inassay chamber107 to be in fluidic contact withdepression113, coveringdepression113 and having a surface area greater than the surface area ofdepression113. The matrix was impregnated with immobilizedreagents108 which produce a detectable product proportional to the concentration of glucose in a blood sample. The detectable product was acolored product109, i.e., a product absorbing light at a detectable, visible wavelength.
As illustrated in FIGS. 2A through 2E, in the use of this platform an imprecise volume (about 30 μL of fluid) of blood was applied to the[0253]entry port101. The fluid wicked intoair channel114 and was stopped bycapillary junction115. Fluid also wicked intometering capillary102 andoverflow capillary103. Fluid flowed through themetering capillary102 andoverflow capillary103 at no rotational speed until the fluid reached capillary junctions at the junction betweenmetering chamber102 andblood fluid chamber104 andoverflow capillary103 andoverflow chamber105.Metering capillary102 was constructed to define a precise volume of about 15 μL of blood betweenentry port101 and the capillary junction atfluid chamber104, which was designed to be at least the amount of the fluid placed by the user inentry port101.
After sample loading by a user and filling of[0254]metering chamber102 andoverflow capillary103 at zero rotational speed, the platform was spun at a first rotational speed f₁of about 300 rpm, which was sufficient to motivate fluid flow through theoverflow capillary103 in this microfluidics array, whereinentry port101 had a depth of about 0.3 cm,metering chamber102 had dimensions of about 0.1 cm deep×0.4 cm wide in cross-section and about 0.5 cm in length from the center of rotation, and overflow capillary103 had dimensions of about 0.05 cm×0.075 cm in cross-section and about 2.7 cm in length from the center of rotation.
Due to the greater distance from the center of rotation of the end of[0255]overflow capillary103 than the end ofmetering chamber102, at rotational speed f₁fluid flowed throughoverflow capillary103 intooverflow chamber105. The platform was spun until all excess fluid was evacuated fromentry port101 and intooverflow chamber105, except the fluid contained inmetering capillary102.
At a second rotational speed f[0256]₂of about 1000 rpm, the precise amount of fluid (16 μL) contained inmetering capillary102 was delivered intofluid chamber104. Fluid movement intofluid chamber104 was accompanied by filling ofcapillary110.
In embodiments comprising a[0257]sacrificial valve111 in-line withcapillary110 at a position betweencapillary110 and112 shown in FIG. 2A, release of the sacrificial valve resulted in fluid flow throughcapillary112 and intoassay chamber107. In said embodiments, fluid flow was achieved at rotational speed f₂with removal of the sacrificial valve. In embodiments of the platforms of the invention comprisingcapillary valve111 at a position betweencapillary110 and112 shown in FIG. 2B, capillary110 filled along with filling ofblood fluid chamber104 until blood reachedcapillary junction111 at the junction betweencapillary110 andcapillary112; in such embodiments, the capillary junction had a depth of about 0.05 cm. At a third rotational speed f₃of about 500 rpm, the fluid contained inblood fluid chamber104 was delivered into assay chamber107 (FIG. 2B). Blood flowing intoassay chamber107 was preferentially directed todepression113 in the assay chamber; the dimensions ofdepression113 are conveniently chosen to be able to contain substantially all of the blood fluid of the sample metered throughmetering chamber102 into assay chamber107 (FIG. 2C). Displaced air flows throughair channel114, and was vented to the surface of the disc.
As blood flowed into[0258]depression113, the fluid component of the blood is driven by pressure and hydrophilic forces intomatrix106, comprising a positively-charged nylon matrix having a pore size of about 0.8 μm; this pore size was chosen to prevent the cellular components of the blood from entering the matrix (FIG. 2D). The cellular blood components were retained indepression113 and the fluid component was efficiently distributed intomatrix106. As the fluid component of the blood enteredmatrix106, driedreagents108 were solubilized and the reaction of the blood component catalyzed by said reagents proceeded. This reaction(s) went to completion within about 1 min. Reaction of the blood component(s) withreagents108 produce coloredproduct109 which was then detected (FIG. 2E), as described below in Example 4.

EXAMPLE 2

Glycated Hemoglobin Assay—Fluidics Structure

A Microsystems platform provided by the invention and specifically designed for performing a glycated hemoglobin assay is illustrated in FIG. 11.[0259]
Construction of the disk embodiments of the platforms of the invention are as described in Example 1. The blood application and metering components and their dimensions and relationships to one another are identical to those described above, comprising sample entry port chamber[0260]801,metering capillary802, overflow capillary803, metered blood fluid chamber804 and overflow chamber805. As in Example 1, each of the overflow and fluid chambers is also connected with air ports or air channels, such as814, and capillary junction(s)815, that permit venting of air displaced by fluid movement on the platform.
Blood fluid chamber[0261]804 was fluidly connected to capillary810 that was about 0.25 mm deep and had a cross-sectional diameter of about 0.25 mm and was connected to sacrificial valve811. Sacrificial valve811 was further fluidly connected with capillary812 that was about 0.25 mm deep and had a cross-sectional diameter of about 0.25 mm. Capillary812 was further fluidly connected to mixingchamber815 that was about 0.25 cmcm deep, had a cross-sectional diameter of about 1 cm, and was positioned about 2.5 cm from the center of rotation. Lysis buffer was loaded directly onto mixingchamber815 in this embodiment and did not use capillary818 orlysis buffer chamber816 as shown in the Figure. 45 μL of lysis buffer was applied to the mixing chamber as a solution of 0.1% Triton X100 in 50 mM Tris pH 9.5.
Mixing[0262]chamber815 was fluidly connected to capillary817 that was about 0.25 mm deep and had a cross-sectional diameter of about 0.25 mm, and was connected tosecondary metering structure819.Secondary metering structure819 was about 0.1 cm deep and was positioned about 3.5 cm from the center of rotation.Secondary metering structure819 was constructed to comprise two sections. A metering section was arranged proximal to the entry position ofcapillary817 and was separated from an overflow section by a septum that extended from the distal wall of the structure to a position just short of the proximal wall of the structure. This arrangement produced a fluid connection between the first metering section having a volumetric capacity of about 6.4μL and the overflow section having an excess volumetric capacity of 90 μL. The volumetric capacity of the overflow section was sufficient to accommodate the largest blood fluid volume applied to the disk.
Capillary[0263]821 was in fluid connection withsecondary metering structure819 at the distal wall of the metering section. Capillary821 was about 0.15 cm deep and had a cross-sectional diameter of about 0.05 cm and was connected to boronateaffinity matrix chamber822. Boronateaffinity matrix chamber822 was about 0.15 cm deep, had a cross-sectional diameter of about 0.3 cm and was positioned about 4.8 cm from the axis of rotation. Boronateaffinity matrix chamber822 was filled with boronate-functionalized agarose beads having a mean diameter of about 60 μm; the beads were maintained in thechamber822 using aporous frit827. Fluid flow through capillary821 was connected to capillary orsacrificial valve823. Boronateaffinity matrix chamber822 was further comprised of a translucent window that permitted reflective spectrophotometry of the contents of the chamber.
Boronate[0264]affinity matrix chamber822 was further fluidly connected tocapillary828.Capillary828 was about 0.25 mm deep and had a cross-sectional diameter of about 0.25 mmand was connected to column washbuffer reservoir829. Columnwash buffer reservoir829 was about 0.25 cm deep and had a cross-sectional diameter of about 2 cm and was positioned about 3.6 cm from the axis of rotation, more proximal to the axis of rotation than boronateaffinity matrix chamber822. Columnwash buffer reservoir829 comprises 290 μL of column preparation buffer that was a solution of magnesium chloride, taurine, D,L-methionine, sodium hydroxide, antibiotics and stabilizers constituted according to the manufacturer's instructions (Isolab Inc. #SG-6220). Fluid flow throughcapillary828 was connected to capillary orsacrificial valve836.
Boronate[0265]affinity matrix chamber822 was further fluidly connected tocapillary832.Capillary832 was about 0.5 m deep and had a cross-sectional diameter of about 0.5 mm and was connected to non-glycated hemoglobin readchamber834.Chamber834 was about 0.25 cm deep and had a cross-sectional diameter of about 2 cm and was positioned about 5 cm from the axis of rotation and was further comprised of a translucent window that permitted reflective spectrophotometry of the contents of the chamber at 430 nm.
As illustrated in FIG. 1, in the use of this platform about a 6.4 μL volume of blood was applied to blood fluid chamber[0266]804, either directly or using the metering components of the platform described above. Blood flowing throughcapillary810 and lysis buffer contained in mixingchamber815 were mixed in the mixing chamber. A 45 μL volume of lysis buffer was mixed with the blood sample. Fluid flow within mixingchamber815 was turbulent, in contrast to fluid flow throughcapillaries810 or818, which was primarily laminar, so that mixing occurred predominantly in mixingchamber815. Fluid flow proceeded throughchannel817 and intosecondary metering structure819. The mixture of lysis buffer and blood, comprising a lysed blood sample841, flowed at a rotational speed f_? of about 750 rpm intosecondary metering structure819 with release of a sacrificial valve853. Lysed blood sample841 entered and filled the metering section ofsecondary metering structure819. Any additional lysed blood sample then emptied into the overflow chamber ofsecondary metering structure819 and filled the total hemoglobin read chamber. Most preferably, a sufficient volume of lysis buffer and blood sample was applied to the disc to fill at least the metering sections ofsecondary metering structure819 and the total hemoglobin read chamber.
After the lysed blood sample[0267]841 was completely transferred tosecondary metering structure819, capillary orsacrificial valve823 was released, allowing the metered lysed blood sample from the metering section ofsecondary metering structure819 through capillary821 and intoboronate affinity matrix822. Capillary orsacrificial valve836 was then released, allowing about 290 μL of column wash buffer843 to flow at rotational speed f_? throughcapillary830 and intoboronate affinity matrix822. Continued or discontinuous rotation motivates column preparation buffer throughboronate affinity matrix822 and into non-glycated hemoglobin readchamber834. The control sample read cuvettes8− and8˜ were then then illuminated by light at a wavelength of 430 nm and the blank reading, i.e. reflectance from cuvettes containing only buffer, was determined. The non-glycated hemoglobin read window was then illuminated by light at a wavelength of 430 nm and the concentration of non-glycated hemoglobin in the sample that has eluted from the column was determined by reflectance spectroscopy. The total hemoglobin read window was also illuminated by light at a wavelength of 430 nm and the concentration of total hemoglobin in the lysed sample was determined by reflectance spectroscopy.
The amount of non-glycated hemoglobin in the sample is determined by dividing the amount of hemoglobin obtained by illuminating the eluted fraction by the amount of hemoglobin obtained in the total fraction.[0268]

EXAMPLE 3

Combination Glucose Concentration—Glycated Hemoglobin Assay Platform

A microsystems platform provided by the invention and specifically designed for performing both a determination of blood glucose concentration and a glycated hemoglobin assay is illustrated in FIGS. 12A through 12Q and[0269]13A through13E.
Construction of the disk embodiments of the platforms of the invention were as described above. FIG. 13B shows a detailed description of the microfluidics components of the platform, which are described in additional detail below. FIG. 13C shows the geometry of a screen printed electrical lead layer deposited on a mylar substrate. FIG. 13D shows the positions of screen printed heaters activated by the electrical leads of the lead layer and screen printed on mylar. FIG. 13E shows a overlay of these components in the assembled disc.[0270]
Referring to the microfluidics components of the platform shown in FIG. 13B, an[0271]entry port1 is positioned on the top surface of the disc and is open for the user to apply an unmetered sample.Entry port1 is about 0.32 cm deep, has a cross-sectional diameter of about 1 cm, is positioned about 2 cm from the center of rotation, and is fluidly connected to capillary channel1A that is about ‘1 mm deep and has a cross-sectional diameter of about 1 mm. Capillary channel1A is fluidly connected tometering component2, which comprises four sections. The first section is rectangularly-shaped and extends in a direction proximal to the axis of rotation away from its fluid connection with capillary channel1A. About half way up this rectangular section is alateral chamber3, which empties into a blood glucose metering chamber4 and an overflow chamber5. The first section of the metering component is about 0.1 cm deep, has a cross-sectional diameter of about 0.4 cm, is positioned about 2.2 cm from the center of rotation, and has a volumetric capacity of about 5 microliters for Hb assay. Thelateral chamber3 is 1 mm deep, has a cross-sectional diameter of 1 mm, and is positioned 2.3 cm from the center of rotation. Blood glucose metering chamber4 is 1 mm deep, has a cross-sectional diameter of 4.5 mm, is positioned 2.6 cm from the center of rotation and has a volumetric capacity of 16 μL. Overflow chamber5 is 1 mm deep, has a cross-sectional diameter of 2 mm, is positioned 2.6 cm from the center of rotation and has a volumetric capacity of 7 μL.
Overflow chamber[0272]5 is fluidly connected to overflow channel8 that is 0.75 mm deep, has a cross-sectional diameter of 0.75 mm and extends 3.8 cm from overflow chamber5. Overflow channel5 is fluidly connected to shortsample detection cuvette9 that is two depths: 0.25 mm and 2.2 mm(inner) deep, has a cross-sectional diameter of 2.5 mm, is positioned 4-5.8 cm from the center of rotation and has a volumetric capacity of 50 μL.
Blood glucose metering chamber[0273]4 is fluidly connected to capillary15 that is about 0.25 mm deep, has a cross-sectional diameter of about 0.25 mm and extends about 0.1 cm from blood glucose metering chamber4.Capillary15 is connected tosacrificial wax valve6, which is further fluidly connected with wax recrystallization chamber6A that is about 0.5 mm deep and has a volumetric capacity sufficient to sequester melted wax from a released wax valve and prevent occlusion of the lumen of the capillary15 controlled by the valve.Capillary15 is further fluidly connected withglucose assay chamber11 that is about 0.1 cm deep, has a cross-sectional diameter of about 0.5 mm, is positioned about 5 cm from the center of rotation and has a volumetric capacity of about 10 μL.Glucose assay chamber11 comprises a depression11A in the surface of the platform having a depth of about 0.1 cm, most preferably comprising a circular or rectangular depression connected to capillary15 so that blood flows into the chamber through the bottom of depression11A. Depression11A is constructed to have a volumetric capacity of from half to twice the assay volume. Bloodglucose assay chamber11 also comprises a pad ormatrix10 of a positively-charged nylon matrix having a pore size of about 0.8 μm. The upper limit on pore size ofmatrix10 is chosen to inhibit or prevent blood cell entry into the matrix. The matrix is positioned in bloodglucose assay chamber11 to be in fluidic contact with depression11A, more preferably covering depression11A, and most preferably having a surface area greater than the surface area of depression11A. The matrix was further impregnated with immobilized reagents11B which produce a detectable product proportional to the amount or concentration of glucose in a blood sample. Most preferably, the detectable product is a colored product11C, i.e., a product absorbing light at a detectable, most preferably a visible, wavelength.
[0274]Lysis metering chamber2 is fluidly connected to capillary7 controlled by sacrificial valve7A. Sacrificial wax valve7A is further fluidly connected with wax recrystallization chamber7B that is 1 mm deep and has a volumetric capacity sufficient to sequester melted wax from a released wax valve and prevent occlusion of the lumen of the capillary7 controlled by the valve. Capillary7 is 0.25 mm deep, has a cross-sectional diameter of 0.25 mm, extends 0.1 cm fromlysis metering chamber2 and is fluidly connected toblood lysis chamber16. Blood lysis is contained within the mixing chamberBlood lysis chamber16 is 2.3 mm deep, has a cross-sectional diameter of 1 cm, is positioned 3 cm from the center of rotation, and contains 45 μL of blood lysis solution (0.1% Triton-X100 in 50 mM Tris, pH 9.5).
[0275]Blood lysis chamber16 is fluidly connected at a distal aspect to capillary17 controlled by sacrificial valve18.Capillary17 is about 0.25 mm deep and has a cross-sectional diameter of about 0.25 mm Sacrificial wax valve18 is further fluidly connected with wax recrystallization chamber18A that is about 0.5 mm deep and has a volumetric capacity sufficient to sequester melted wax from a released wax valve and prevent occlusion of the lumen of the capillary17 controlled by the valve.Capillary17 is fluidly connected tosecondary metering structure19.Secondary metering structure19 is 1.5 mm deep, and is positioned 3.8 cm from the center of rotation.Secondary metering structure19 is constructed to comprise three sections. Afirst section20 comprises a throwaway section that is used to discard the first sample and taking the second sample to provide a better sample for analysis, having a volumetric capacity of about 6.4 μL. Throw awaysection20 is arranged proximal to the entry position ofcapillary17 and is separated from ametering section21 by a septum that extends from the distal wall of the structure to a position just short of the proximal wall of the structure. This arrangement produces a fluid connection betweenthrowaway section20 and themetering section21. Metering section has a volumetric capacity of about 6.4 μL and is fluidly connected to anoverflow section24 having an excess volumetric capacity of about 90 μL. The volumetric capacity of the overflow section is sufficient to accommodate the largest blood fluid volume applied to the disk.
Capillary[0276]25 is in fluid connection withsecondary metering structure21 at the distal wall of the metering section. Capillary25 is about 0.1 5 cm deep and has a cross-sectional diameter of about 0.5 mm and is connected to boronateaffinity matrix chamber28. Capillary25 is fluidly connected to sacrificial wax valve26 that is further fluidly connected with wax recrystallization chamber26A Wax recrystallization chamber26A is about 0.3 cm deep and has a volumetric capacity sufficient to sequester melted wax from a released wax valve and prevent occlusion of the lumen of the capillary25 controlled by the valve.
Boronate[0277]affinity matrix chamber28 is about 0.15 cm deep, has a cross-sectional diameter of about 0.3 cm and is positioned about 4.8 cm from the axis of rotation. Boronateaffinity matrix chamber28 is filled with boronate-functionalized agarose beads (Isolab, $SG-6220) having a mean diameter of about 60 μm; the beads are maintained in thechamber28 using aporous frits29. Boronateaffinity matrix chamber28 is further fluidly connected tocapillary31.Capillary31 is about 0.25 mm deep and has a cross-sectional diameter of about 0.25 mm and is connected to column washbuffer reservoir30. Columnwash buffer reservoir30 is about 0.25 cm deep and has a cross-sectional diameter of about 2 cm and is positioned about 3.6 cm from the axis of rotation, more proximal than boronateaffinity matrix chamber28. Columnwash buffer reservoir30 comprises about 290 μL of column wash buffer comprising a solution of asparagine, magnesium chloride, taurine and D,L-methionine (Isolab, #SG-6220). Fluid flow throughcapillary31 is connected tosacrificial valve32.Sacrificial wax valve32 is further fluidly connected with wax recrystallization chamber32A that is about 0.5 mm deep and has a volumetric capacity sufficient to sequester melted wax from a released wax valve and prevent occlusion of the lumen of the capillary32 controlled by the valve.
Boronate[0278]affinity matrix chamber28 is further fluidly connected to capillary37. Capillary37 is about 0.5 mm deep and has a cross-sectional diameter of about 0.5 mm and is connected to samplecollection cuvette array12. Samplecollection cuvette array12 is about 0.25 cm deep and has a cross-sectional diameter of about 2.1 cm and is positioned about 5 cm from the axis of rotation. Samplecollection cuvette array12 is separated into a multiplicity of individual chambers, each separated from one another by septa that extend from the distal wall of the cuvettes to a position adjacent to the proximal wall of the cuvettes, so that afluid passage50 is maintained between each of the cuvettes. Thefluid passage50 is formed by the back (proximal wall) of the samplecollection cuvette array12 and the row of septa separating each of the sections of thesample collection cuvettes12.Capillary33 is fluidly connected to samplecollection cuvette array12 at a position adjacent to the proximal wall of the array and directed to the cuvette most proximal to the boronateaffinity matrix chamber28.
[0279]Capillary22 is fluidly connected tosecondary metering structure19.Capillary22 is about 0.25 mm deep and has a cross-sectional diameter of about 0.25 mm and is connected tosecondary metering structure19 at a position between the metering section and the overflow section.Capillary22 is further fluidly connected with total hemoglobin readchamber23. Total hemoglobin readchamber23 is about 0.25 cm deep, is positioned about 5 cm from the center of rotation, and has a volumetric capacity of about 50 μL. Total hemoglobin readchamber23 is positioned radially more distal from the center of rotation thansecondary metering structure19, and comprises a read window translucent to light having a wavelength of 300-1000 nm. In addition, there is no capillary or sacrificial valving controlling fluid flow incapillary23.
The platform also comprises control sample read[0280]cuvettes13 and14, advantageously positioned in proximity to total hemoglobin readchamber23. Control sample readcuvettes13 and14 are each about 0.25 cm deep, positioned about 5 cm from the center of rotation, and have a volumetric capacity of about 50 μL. Control sample readcuvettes13 and14 comprise a read window translucent to light having a wavelength of 410 nm. Control sample readcuvettes13 and14 are not fluidly connected to any other structure on the platform.
[0281]Air displacement channels33 and capillary junction(s)34, that permit venting of air displaced by fluid movement on the platform, are fluidly connected to the components of the platform to permit unimpeded fluid flow.
As illustrated in FIGS. 12 A through 12Q, in the use of this platform a volume of blood about 30 μL is applied to[0282]entry port1. Blood enterslysis subvolume2 andlateral passageway3 under the influence of gravity and capillary forces in the absence of rotation of the platform, as shown in FIG. 12A. Upon rotation of the platform at a first rotational speed f₁of 400-600 rpm, blood completely fillslysis subvolume2 and also flows throughpassageway3 and into blood glucose metering chamber4 and overflow chamber5, shown in FIG. 12B. Blood is retained in blood glucose chamber4 either due to capillary pressure or by asacrificial valve6, most preferably a wax valve. Similarly, blood is retained inblood lysis subvolume2 by a valve, most preferably a sacrificial valve7. Excess blood flows at rotational speed f₁through overflow channel8 and intooverflow chamber9, shown in FIGS. 12C and 12D. Typical values for the first rotational speed are an acceleration of about 20 to 60 rpm/sec to a final radial velocity of about 600 rpm.
After blood is metered and excess blood delivered to overflow[0283]chamber9, the rotational speed of the disc is reduced to a rotational speed f_1aof 600 to 100 rpm, typically 60 rpm, to perform a blanking measurement on samplecollection cuvette array12, total hemoglobin readchamber23 and bloodglucose assay chamber11. Measurements of blankingcuvettes13 and14 are also advantageously performed.
The disc is then accelerated to a second rotational speed f[0284]₂greater than f₁, and typically in the range of 800 rpm to about 1000 rpm. At this speed,capillary valve6 is overcome orsacrificial valve6 is released, and about 16 μL of blood from blood glucose metering chamber4 flows throughcapillary15 and into bloodglucose assay chamber11, shown in FIG. 12E. Upon enteringassay chamber11, blood fluid components are forced intoabsorbent matrix10 through depression11A. The blood fluid is incubated inmatrix10 for a time sufficient for the reagents10A to produce a colored product10B in an amount proportional to the amount of glucose in the blood fluid sample. The disc is slowed, typically to a rotational speed f₄of about 100 rpm, for glucose data acquisition using reflectance spectrometry; data acquisition as the disc is spinning down also enables to instrument to set t=0 for the assay, based on a decrease in reflectance when thematrix10 is wet by the blood fluid components and hence the matrix's scattering decreases. Development of colored product10B is shown in FIG. 12G.
The disc is then accelerated to rotational speed f[0285]₃of about 1000 rpm, with release of sacrificial valve7A and fluid flow of about 5-6.4 μL of blood frommetered subvolume2 through capillary7 and intoblood lysis chamber16 containing about 40 μL of blood lysis buffer. This is shown in FIG. 12H. The mixture of blood and blood lysis buffer inblood lysis chamber16 is mixed by agitation, wherein the platform is accelerated repeatedly +250 rpm/sec to −250 rpm/sec (wherein “+” and “−” indicate acceleration in different directions), typically 250-500 rpm/sec, over a time period of about 2 min, typically 1-3 min, as shown in FIGS. 12I and 12J.
The disc then is accelerated to a rotational speed f[0286]₅of 1000 rpm and sacrificial valve18 is released. Lysed blood fromblood lysis chamber16 flows throughcapillary17 and intosecondary metering structure19, as shown in FIG. 12K. The lysed blood solution sequentially fillsthrowaway section20, which is used as a trap for cell debris,metering section21 and excess lysed blood then fillsoverflow section24. Filling ofmetering chamber21 is immediately followed by fluid flow throughcapillary22 and filling of total hemoglobin readchamber23. The disc is spun at rotational speed f₅for a time sufficient to substantially completely drainblood lysis chamber16. The configuration of the blood fluids on the disc after this spin is shown in FIG. 12L.
The disc is then decelerated to a rotational speed of 750 rpm, and sacrificial valve[0287]26 is released. A metered volume of about 6.4 μL of lysed blood frommetering section21 flows throughcapillary27 and into boronate affinity matrix chamber28 (shown in FIG. 12M). The lysed blood solution is allowed to incubate in the chamber for a time of about 1 min, sufficient for glycated hemoglobin to bind to the matrix. This aspect of the disc is illustrated in FIG. 12N.
The disc is then accelerated to a rotational speed of 1000 rpm, and[0288]sacrificial valve32 is released. A volume of about 290 μL of column wash buffer (Isolab #SG-6220) flows fromwash buffer reservoir30 thoughcapillary31 and into boronate affinity matrix chamber28 (shown in FIG. 12O). The wash buffer displaces the non-glycated hemoglobin and other components of the lysed blood fluid from the affinity column matrix and into samplecollection cuvette array12. FIGS. 12P through 12Q show sequential filling of the individual cuvettes in samplecollection cuvette array12. The rotation speed of the disc is reduced, to 60 rpm for samplecollection cuvette array12 and total hemoglobin readchamber23 to be interrogated spectrophotometrically. The glycated fraction of the blood sample is determine algorithmically by subtracting the non-glycated hemoglobin fraction in samplecollection cuvette array12 from the total hemoglobin detected in total hemoglobin readchamber23.

EXAMPLE 4

Blood Glucose Assay—Chemistries

Microsystems platforms as provided by the invention were used to perform blood glucose and glycated hemoglobin assays as described herein.[0289]

Blood Glucose Assay

A series of blood glucose assays were performed to determine the precision of repeated glucose assays using the Microsystems platforms of the invention.[0290]
The assays were performed on a Microsystems platform according to Examine 3 using a round reagent cuvette ([0291]113). 60-70 μL of human whole blood was applied to the disk, and 15 μL metered into the metering capillary. A glucose reagent pad obtained from LifeScan was used for performing the glucose determination. Optical absorbance readings were obtained at 430 nm, 590 nm and 660 nm on the unreacted reagent pad as a control. Blood was released into the reaction chamber and optical data gathered at one-second intervals for about 1 min. Absorbance at 590 nm was specific for the amount of glucose in the sample; absorbance at 430 nm was specific for hemoglobin in the sample; and absorbance at 660 nm was used to detect non-specific background absorbance, as described above.
A total of seven assays were performed for each of two samples and the amount of glucose (in mg/dL) determined as follows:[0292]
Sample 1 Sample 2
Glucose mg/dL 136.0 113.2
% CV 4.4% 4.5%
These assays were repeated as above using a Microsystems platform according to Example 1 comprising a rectangular reagent cuvette ([0293]113). A Biodyne-B membrane (Pall) was impregnated with glucose oxidase reagents as described above and used for performing the glucose determination. In these embodiments,chamber113 had dimensions of 4 mm×5 mm.
The glucose determination was performed exactly as described in the first series of experiments. Standard blood glucose assays were run on these samples in parallel to compare the results obtained with the Microsystems platforms of the invention with conventional assays. These results were as follows:[0294]
mg/dL Reference
Method mg/dL Invention
46.8 61.9
85.9 76.8
107.0 115.8
149.0 144.2
These results established that the microsystems platforms of the invention were capable of determining blood glucose concentrations.[0295]

Correction for Interfering Substances

In addition, assays were performed on a Microsystems platform to Example 3 using a round reagent cuvette ([0296]113) and the glucose results obtained were corrected for hemoglobin interferences as discussed herein. In these experiments, the spectrophotometric results obtained were used to calculate blood glucose with and without correction for the hemoglobin absorbance signal at 590 nm. The correction factor was determined empirically using samples comprised of phosphate buffered saline to which known qualities of glucose and/or hemoglobin were added. The resulting samples were assayed on the Microsystems platform according to Example 3 using a round reagent cuvette (113) to give the following data:
Glucose Hemoglobin
Concentration Concentration Reflectance Reflectance
Sample (mg/dL) (mg/dL) (590 nm) (430 nm)
A 0 0 0.0152 −0.0204
B 0 1.25 0.2116 0.3041
C 75 5 1.2767 0.3452
D 78 0 0.9576 0.1311
For glucose chromagen alone, Sample D, the Reflectance(430 nm) is 16.1% of the Reflectance(590 nm) [(0.131-(-0.0204)/(0.9576-0.152)]. For hemoglobin alone, Sample B, the Reflectance(590 nm) is 60.5% of the Reflectance (430) [(0.2116-0.0152)/(0.3041-(-0.0204))]. For the combination of glucose and hemoglobin, Sample C, the Reflectance(430 nm) is the sum of the Reflectance (430 nm) due to the hemoglobin plus 16.1% of the Reflectance(590 nm) due to the glucose chromagen; similarly, the Reflectance(590 nm) is the sum of the Reflectance(590 nm) due to the glucose chromagen plus 60.5% of the Reflectance(430 nm) due to the hemoglobin. Using standard algebraic methods, the factor to be used was calculated to be:[0297]
Reflectance(590 nm) due to glucose chromagen=Reflectance(590 nm)−F×Reflectance(430 nm) where F=0.274[0298]
The results shown in FIGS. 14A and 14B illustrate the difference in the accuracy of the data obtained with and without correcting for the hemoglobin absorbance signal.[0299]

Glycated Hemoglobin Assay

A series of glycated hemoglobin assays were performed to determine the precision of repeated binding of glycated hemoglobin by measuring the amount of hemoglobin put on a phenyl boronate column and the amount of (non-glycated) hemoglobin that could be eluted from the column.[0300]
These experiments were performed using a microsystems platform of the invention as disclosed in Example 3; the phenyl boronate column material was from a kit obtained from IsoLab Glyc-Affin GHb. 60-70 μL of human whole blood was applied to the disk, and 5 μL metered into and mixed with 45 μL of lysis buffer in the mixing chamber of the platform. 5 μL of the lysed blood was metered onto the phenyl boronate column and non-glycated hemoglobin eluted with 290 μL of elution buffer. The reflective absorbance of the lysed blood and the eluted non-glycated hemoglobin was determined spectrophotometrically at 430 nm and used to calculate the amount of non-glycated hemoglobin in the sample.[0301]
Seven separate assays were performed on each of 2 difference whole blood samples. These results are as follows:[0302]
Sample 1 Sample 2
% NGHb, reference method 93 90
% NGHb, invention 93 92
% CV 1.2% 1.7%

DNA Binding Assays

These experiments were performed to show that the microsystems platforms of the invention could efficiently isolate plasmid DNA from a sample. In these experiments, diatomeceous earth was used to specifically bind plasmid DNA, and the results were compared with a standard plasmid DNA extraction kit (Qiagen).[0303]
In these experiments, the Qiagen assay was performed until the bacterial culture had been disrupted with chaotropic salts. At this point, the solution was split in half, and one half assayed using the Qiagen reagents and columns, and the[0304]other half 300 μL assayed using a microsystem platform adapted from the one described in Example 2. In this embodiment, the diatomeceous earth column was loaded onto the platform and the sample applied thereto by centrifugation at about 800 rpm. The column was washed twice with 100 μL of Qiagen buffer PE at about 850 rpm, and the plasmid DNA was then eluted twice, with 50 μL of Qiagen Buffer EB, once at 600 rpm and again at at 900 rpm. The eluted plasmid DNA from each preparation was precipitated by the addition of ice cold ethanol to 70% and the DNA pellet collected by centrifugation and resuspended in 50 μL of 10 mM Tris-HCl, pH 8. 15 μL were analyzed by conventional agarose gel electrophoresis, stained with ethidium bromide and osverved under ultraviolet light illumination.
The results of these assays are shown in FIG. 15, and illustrate the use of the Microsystems platforms of the invention for isolating plasmid DNA from a biological fluid sample.[0305]
It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and that all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims.[0306]

Claims

We claim:

1. A microsystem platform for separating an analyte from a fluid sample, comprising

a) a rotatable platform, comprising a substrate having a first flat, planar surface and a second flat, planar surface opposite thereto, each surface comprising a center about which the platform is rotated, wherein the first surface comprises in combination

b) an entry port comprising a depression in the first surface having a volumetric capacity of about 1 to about 150 μL, that is fluidly connected by a first microchannel with

c) a mixing chamber positioned on the platform farther from the center of rotation than the entry port; the platform further comprising

i) a reagent reservoir comprising liquid reagents for preparing the fluid sample for the analyte separation assay, wherein the reagent reservoir if fluidly connected to the mixing chamber by a second microchannel, the platform further comprising

d) a secondary metering chamber comprising a first metering portion and a second metering portion each defining a volume of the fluid and separated by a septum that extends from a position in the chamber farthest from the center of rotation to a position just short of a chamber wall closest to the center of rotation, wherein the end of the septum and the chamber wall define a fluid connection between the first and second metering portions, the metering chamber further comprising an overflow portion that is separated from the second metering portion by a septum that extends from a position in the chamber farthest from the center of rotation to a position just short of a chamber wall closest to the center of rotation, and wherein the first portion of the metering chamber is fluidly connected by a third microchannel to

e) an analyte separation assay chamber further comprising

i) an analyte binding matrix, wherein the analyte specifically binds to the matrix and is retained in the separation chamber thereby, wherein the analyte separation assay chamber is further fluidly connected by a fourth microchannel with a separation matrix preparation buffer reservoir containing a preparation buffer and the analyte separation assay chamber is further fluidly connected by a fifth microchannel with a separation matrix wash buffer reservoir containing a separation matrix wash buffer, wherein each of the preparation buffer and wash buffer reservoirs are positioned closer to the center of rotation than the analyte separation assay chamber;

and wherein the second metering portion of the secondary metering chamber is fluidly connected by a sixth microchannel with

f) a read window that is further fluidly connected to the analyte separation assay chamber by a seventh microchannel and is further fluidly connected by a eighth microchannel to

g) a waste reservoir

wherein rotation of the platform motivates the fluid sample from the entry port into the mixing chamber and the reagents from the reagent reservoir into the mixing chamber to provide a sample reagent mixture, and wherein rotation of the platform motivates the sample reagent mixture from the mixing chamber into the secondary metering chamber, wherein the first metering portion is filled before the second metering portion and the second metering portion is filled before the overflow portion;

and wherein rotation of the platform motivates separation matrix preparation buffer through the analyte separation assay chamber, through the read chamber and into the waste reservoir,

and wherein rotation of the platform motivates the volume of the sample reagent mixture from the first metering portion of the secondary metering chamber through the analyte separation assay chamber whereby analyte in the sample reagent mixture binds to the analyte binding matrix;

and wherein rotation of the platform motivates analyte separation matrix wash buffer through the analyte separation assay chamber, thereby displacing the sample reagent mixture through the read window and into the waste reservoir;

and wherein rotation of the platform motivates the volume of the sample reagent mixture in the second metering portion of the secondary metering chamber into the read window and displaces the eluate from the analyte separation assay chamber from the read window.

2. The microsystem platform ofclaim 1 wherein the analyte binding matrix is an inositol phosphate-derived membrane.

3. The microsystem platform ofclaim 1 further comprising

e) a metering capillary and an overflow capillary, each being fluidly connected with the entry port, wherein each capillary defines a cross-sectional area of about 0.02 mm to about 1 mm in diameter, and wherein each capillary extends radially from the center of the platform and defines a first end proximally arrayed towards the center of the platform and a second end distally arrayed from the center of the platform, wherein the proximal end of each capillary defines a curved opening; wherein the metering capillary defines a volume of the fluid and wherein the metering capillary is fluidly connected with the mixing chamber and wherein the overflow capillary is fluidly connected with

f) an overflow chamber having a depth equal to or greater than the overflow capillary and positioned radially more distant from the center of the platform than the mixing chamber and the entry port,

wherein a capillary junction is formed at the junction of the metering capillary and the mixing chamber and at the junction of the overflow capillary and the overflow chamber, whereby fluid placed in the entry port flows by capillary action to the junction of the metering capillary and the mixing chamber, and excess fluid flows by capillary action to the junction of the overflow capillary and the overflow chamber; and wherein rotation of the platform at a first rotation speed motivates fluid displacement in the overflow capillary into the overflow chamber but not fluid displacement in the metering capillary, whereby rotation of the platform at the first rotational speed drains the fluid from the entry port into the overflow chamber; and

wherein rotation of the platform at a second rotation speed that is greater than the first rotational speed motivates fluid displacement of the volume of the fluid in the metering capillary into the mixing chamber; and wherein each of the assay chamber and overflow chamber also comprise air displacement channels whereby air displaced by fluid movement is vented to the surface of the platform.

4. The microsystem platform ofclaim 1, further comprising

j) a sacrificial valve in the fourth, fifth, sixth, or seventh microchannels, wherein release of the sacrificial valve permits fluid flow through the microchannel when the platform is rotated at a non-zero rotational speed.

5. The microsystem platform ofclaim 4 wherein the sacrificial valve is a solid, semi-solid or viscous liquid hydrocarbon, or a plastic.

6. The microsystem platform ofclaim 5 further comprising a heating element in the platform in thermal contact with the sacrificial valve, wherein heating the heating element releases the sacrificial valve.

7. The microsystem platform ofclaim 1, wherein the read chamber comprises a top surface that is translucent.

8. The microsystem platform ofclaim 1 wherein the fluid sample is blood.

9. A microsystem platform for separating an analyte from a fluid sample, comprising

c) a mixing chamber positioned on the platform farther from the center of rotation than the entry port and comprising liquid reagents for preparing the fluid sample for the analyte separation assay, wherein the mixing chamber is fluidly connected by a second microchannel with

d) a secondary metering chamber comprising a first metering portion and a second overflow portion wherein the first metering portion defines a volume of the sample reagent mixture, wherein the first metering portion and the second overflow portion are separated by a septum that extends from a position in the chamber farthest from the center of rotation to a position just short of a chamber wall closest to the center of rotation, wherein the end of the septum and the chamber wall define a fluid connection between the first metering portion and the overflow portion, and wherein the fluid connection between the first metering portion and the overflow portion is fluidly connected by a third microchannel to a read chamber positioned radially more distant from the center of rotation than the secondary metering chamber, and wherein the first metering portion of the secondary metering chamber is fluidly connected by a fourth microchannel to

e) an analyte separation assay chamber further comprising

i) an analyte binding matrix, wherein the analyte specifically binds to the matrix and is retained in the separation chamber thereby, wherein the analyte separation assay chamber is further fluidly connected by a fifth microchannel with a separation matrix buffer reservoir containing a buffer, wherein the matrix buffer reservoir is positioned closer to the center of rotation than the analyte separation assay chamber;

and wherein the analyte separation assay chamber is further fluidly connected by a sixth microchannel with

f) a read window manifold comprising a series of chambers separated by septa and arranged linearly and adjacently on the surface of the platform away from the position of the fluid connection of the manifold with the fifth microchannel;

wherein rotation of the platform motivates the fluid sample from the entry port into the mixing chamber to a sample reagent mixture, and wherein rotation of the platform motivates the sample reagent mixture from the mixing chamber into the secondary metering chamber, wherein the first metering portion is filled before the overflow portion and the read chamber is filled by fluid flow through the second microchannel before the overflow portion is filled;

and wherein rotation of the platform motivates separation matrix preparation buffer through the analyte separation assay chamber and into the read chamber manifold,

and wherein rotation of the platform motivates analyte separation matrix wash buffer through the analyte separation assay chamber, thereby displacing the sample reagent mixture into the read manifold.

10. The microsystem platform ofclaim 9 further comprising

g) metering capillary and an overflow capillary, each being fluidly connected with the entry port, wherein each capillary defines a cross-sectional area of about 0.02 mm to about 1 mm in diameter, and wherein each capillary extends radially from the center of the platform and defines a first end proximally arrayed towards the center of the platform and a second end distally arrayed from the center of the platform, wherein the proximal end of each capillary defines a curved opening; wherein the metering capillary defines a volume of the fluid and wherein the metering capillary is fluidly connected with the mixing chamber and wherein the overflow capillary is fluidly connected with

h) overflow chamber having a depth in the platform equal to or greater than the overflow capillary and positioned radially more distant from the center of the platform than the mixing chamber and the entry port,

11. The microsystem platform ofclaim 9, further comprising

j) a sacrificial valve in the third, fourth, fifth, or sixth microchannels, wherein release of the sacrificial valve permits fluid flow through the microchannel when the platform is rotated at a non-zero rotational speed.

12. The microsystem platform ofclaim 11 wherein the sacrificial valve is a solid, semi-solid or viscous liquid hydrocarbon, or a plastic.

13. The microsystem platform ofclaim 12 further comprising a heating element in the platform in thermal contact with the sacrificial valve, wherein heating the heating element releases the sacrificial valve.

14. The microsystem platform ofclaim 9, wherein the read chamber comprises a top surface that is translucent.

15. The microsystem platform ofclaim 9 wherein the fluid sample is blood.

16. A microsystem platform for separating an analyte from a fluid sample, comprising

c) an assay chamber fluidly connected with the entry port, the reaction chamber further comprising

i) a porous matrix comprising reagents for performing an analyte detection assay

wherein a fluid sample applied to the entry port is delivered to the assay chamber through the capillary microchannel by rotation of the platform, and wherein delivery of the fluid sample to the assay chamber initiates the analyte detection assay; wherein the entry port is further fluidly connected with

d) a mixing chamber positioned on the platform farther from the center of rotation than the entry port and comprising liquid reagents for preparing the fluid sample for the analyte separation assay, wherein the mixing chamber is fluidly connected by a second microchannel with

e) a secondary metering chamber comprising a first metering portion and a second overflow portion wherein the first metering portion defines a volume of the sample reagent mixture, wherein the first metering portion and the second overflow portion are separated by a septum that extends from a position in the chamber farthest from the center of rotation to a position just short of a chamber wall closest to the center of rotation, wherein the end of the septum and the chamber wall define a fluid connection between the first metering portion and the overflow portion, and wherein the fluid connection between the first metering portion and the overflow portion is fluidly connected by a third microchannel to a read chamber positioned radially more distant from the center of rotation than the secondary metering chamber, and wherein the first metering portion of the secondary metering chamber is fluidly connected by a fourth microchannel to

f) an analyte separation assay chamber further comprising

g) a read window manifold comprising a series of chambers separated by septa and arranged linearly and adjacently on the platform away from the position of the fluid connection of the manifold with the fifth microchannel;

17. The microsystem platform ofclaim 16 wherein the analyte binding matrix is an inositol phosphate-derived membrane.

18. The microsystem platform ofclaim 16, further comprising

h) a sacrificial valve in the fourth, fifth, sixth, or seventh microchannels, wherein release of the sacrificial valve permits fluid flow through the microchannel when the platform is rotated at a non-zero rotational speed.

19. The microsystem platform ofclaim 18 wherein the sacrificial valve is a solid, semi-solid or viscous liquid hydrocarbon, or a plastic.

20. The microsystem platform ofclaim 19 further comprising a heating element in the platform in thermal contact with the sacrificial valve, wherein heating the heating element releases the sacrificial valve.

21. The microsystem platform ofclaim 16, wherein the read chamber comprises a top surface that is translucent.

22. The microsystem platform ofclaim 16 wherein the fluid sample is blood.