US20070020147A1 - Miniaturized fluid delivery and analysis system - Google Patents

Abstract

The present invention provides a method for combining a fluid delivery system with an analysis system for performing immunological or other chemical of biological assays. The method comprises a miniature plastic fluidic cartridge containing a reaction chamber with a plurality of immobilized species, a capillary channel, and a pump structure along with an external linear actuator corresponding to the pump structure to provide force for the fluid delivery. The plastic fluidic cartridge can be configured in a variety of ways to affect the performance and complexity of the assay performed.

Description

CROSS-REFERENCE TO RELATED APPLICATION
• This application claims priority to U.S. patent application Ser. No. 10/437,046, filed May 14, 2003, which is hereby incorporated by reference herein in its entirety.
BACKGROUND OF THE INVENTIONField of the Invention
• This invention relates to a system comprising a fluid delivery and analysis cartridge and an external linear actuator. More particularly, the invention relates to a system for carrying out various processes, including screening, immunological diagnostics, DNA diagnostics, in a miniature fluid delivery and analysis cartridge.
• Recently, highly parallel processes have been developed for the analysis of biological substances such as, for example, proteins and DNA. Large numbers of different binding moieties can be immobilized on solid surfaces and interactions between such moieties and other compounds can be measured in a highly parallel fashion. While the sizes of the solid surfaces have been remarkably reduced over recent years and the density of immobilized species has also dramatically increased, typically such assays require a number of liquid handling steps that can be difficult to automate without liquid handling robots or similar apparatuses.
• A number of microfluidic platforms have recently been developed to solve such problems in liquid handling, reduce reagent consumptions, and to increase the speed of such processes. Examples of such platforms are described in U.S. Pat. Nos. 5,856,174 and 5,922,591. Such a device was later shown to perform nucleic acid extraction, amplification and hybridization on HIV viral samples as described by Anderson et al, “Microfluidic Biochemical Analysis System”, Proceeding of the 1997 International Conference on Solid-State Sensors and Actuators, Tranducers '97, 1997, pp. 477-480. Through the use of pneumatically controlled valves, hydrophobic vents, and differential pressure sources, fluid reagents were manipulated in a miniature fluidic cartridge to perform nucleic acid analysis.
• Another example of such a microfluidic platform is described in U.S. Pat. No. 6,063,589 where the use of centripetal force is used to pump liquid samples through a capillary network contained on compact-disc liquid fluidic cartridge. Passive burst valves are used to control fluid motion according to the disc spin speed. Such a platform has been used to perform biological assays as described by Kellog et al, “Centrifugal Microfluidics: Applications,” Micro Total Analysis System 2000, Proceedings of the uTas 2000 Symposium, 2000, pp. 239-242. The further use of passive surfaces in such miniature and microfluidic devices has been described in U.S. Pat. No. 6,296,020 for the control of fluid in micro-scale devices.
• An alternative to pressure driven liquid handling devices is through the use of electric fields to control liquid and molecule motion. Much work in miniaturized fluid delivery and analysis has been done using these electro-kinetic methods for pumping reagents through a liquid medium and using electrophoretic methods for separating and perform specific assays in such systems. Devices using such methods have been described in U.S. Pat. No. 4,908,112, U.S. Pat. No. 6,033,544, and U.S. Pat. No. 5,858,804.
• Other miniaturized liquid handling devices have also been decribed using electrostatic valve arrays (U.S. Pat. No. 6,240,944), Ferrofluid micropumps (U.S. Pat. No. 6,318,970), and a Fluid Flow regulator (U.S. Pat. No. 5,839,467).
• The use of such miniaturized liquid handling devices has the potential to increase assay throughput, reduce reagent consumption, simplify diagnostic instrumentation, and reduce assay costs.
SUMMARY OF THE INVENTION
• The system of the invention comprises a plastic fluidic device having at least one reaction chamber connected to pumping structures through capillary channels and external linear actuators. The device comprises two plastic substrates, a top substrate and a bottom substrate containing capillary channel(s), reaction chamber(s), and pump/valve chamber(s)—and a flexible intermediate interlayer between the top and bottom substrate which provides providing a sealing interface for the fluidic structures as well as valve and pump diaphragms. Passive check valve structures are formed in the three layer device by providing a means for a gas or liquid to flow from a channel in the lower substrate to a channel in the upper substrate by the bending of the interlayer diaphragm. Furthermore flow in the opposite direction is controlled by restricting the diaphragm bending motion with the lower substrate. Alternatively check valve structures can be constructed to allow flow from the top substrate to the bottom substrate by flipping the device structure. Pump structures are formed in the device by combining a pump chamber with two check valve structures operating in the same direction. A hole is also constructed in the lower substrate corresponding to the pump chamber. A linear actuator—external to the plastic fluidic device—can then be placed in the hole to bend the pump interlayer diaphragm and therefore provide pumping action to fluids within the device. Such pumping structures are inherently unidirectional.
• In one embodiment the above system can be used to perform immunoassays by pumping various reagents from an inlet reservoir, through a reaction chamber containing a plurality of immobilized antibodies or antigens, and finally to an outlet port. In another embodiment the system can be used to perform assays for DNA analysis such as hybridization to DNA probes immobilized in the reaction chamber. In still another embodiment the device can be used to synthesize a series of oligonucleotides within the reaction chamber. While the system of the invention is well suited to perform solid-phase reactions within the reaction chamber and provide the means of distributing various reagents to and from the reaction chamber, it is not intended to be limited to performing solid-phase reactions only.
• The system of the invention is also well suited for disposable diagnostic applications. The use of the system can reduce the consumables to only the plastic fluidic cartridge and eliminate any cross contamination issues of using fixed-tipped robotic pipettes common in high-throughput applications.
BRIEF DESCRIPTION OF THE DRAWINGS
• FIG. 1A is a top view of a pump structure within the plastic fluidic device of the invention.
• FIG. 1B is a cross section view of the pump structure within the plastic fluidic device of the invention.
• FIG. 2 is a top view of a plastic fluidic device of the invention configured as a single-fluid delivery and analysis device.
• FIG. 3 is a top view of a plastic fluidic device of the invention configured as a 5-fluid delivery and analysis device.
• FIG. 4 is a top view of a plastic fluidic device of the invention configured as a re-circulating 3-fluid delivery and analysis device.
DETAILED DESCRIPTION OF THE INVENTION
• The system of the invention comprises a plastic fluidic cartridge and a linear actuator system external to the fluidic cartridge.FIG. 1A shows a cross-sectional view of a pump structure formed within the fluidic cartridge of the invention. The plastic fluidic cartridge comprises three primary layers: anupper substrate21, alower substrate22, and a flexibleintermediate interlayer23, as shown inFIG. 1B. The three layers can be assembled by various plastic assembly methods such as, for example, screw assembly, heat staking, ultrasonic bonding, clamping, or suitable reactive/adhesive bonding methods. The upper and lower substrates, depicted as21 and22 inFIG. 1B, both contain a variety of features that define channels of capillary dimensions as well as pump chambers, valve chambers, reaction chambers, reservoirs, and inlet/outlet ports within the cartridge.FIG. 1B shows a top view of the pump structure ofFIG. 1A. The pump is defined by apump chamber14 and twopassive check valves15 that provide a high resistance to flow in one direction only.Passive check valves15 comprise alower substrate channel13 and anupper substrate channel11 separated byinterlayer23 such that holes throughinterlayer23, depicted asholes12 inFIG. 1B, are contained withinupper substrate channel11 but not withinlower substrate channel13. Such check valve structures provide a low resistance to a gas/liquid flowing fromlower substrate channel13 toupper substrate channel11 and likewise provide a high resistance to a gas/liquid flowing fromupper substrate channel11 tolower substrate channel13.Pump chamber14 comprises an upper substrate chamber and ahole141 inlower substrate22 tofree interlayer23 to act as adiaphragm25, as depicted inFIG. 1B. Alinear actuator24 external to the fluidic cartridge can then be placed in the hole131 to benddiaphragm25 and therefore provide the necessary force to deform the diaphragm.
• FIG. 2 shows a top view of a plastic fluidic cartridge of the invention configured as a single-fluid delivery and analysis device. Fluid is first placed into thereservoir31 manually or automated using a pipette or similar apparatus. Apump structure32 similar to that ofFIG. 1B is contained within the device. By repeatedly actuating an external linear actuator, fluid inreservoir31 is pumped through thepump structure32, thecapillary channel33 and into thereaction chamber34.Reaction chamber34 contains a plurality of immobilizedbio-molecules35 for specific solid-phase reactions with said fluid. After a specified reaction time, the fluid is pumped throughreaction chamber34 and out theexit port36.
• Upper substrate21 andlower substrate22 of the plastic fluidic cartridge of the invention can be constructed using a variety of plastic materials such as, for example, polymethyl-methacrylate (PMMA), polystyrene (PS), polycarbonate (PC), Polypropylene (PP), polyvinylchloride (PVC). In the case of optical characterization of reaction results within a reaction chamber,upper substrate21 is preferably constructed out of a transparent plastic material. Capillaries, reaction chambers, and pump chambers can be formed inupper substrate21 andlower substrate22 using methods such as injection molding, compression molding, hot embossing, or machining. Thicknesses ofupper substrate21 andlower substrate22 are suitably in, but not limited to, the range of 1 millimeter to 3 millimeter in thickness.Flexible interlayer23 can be formed by a variety of polymer and rubber materials such as latex, silicone elastomers, polyvinylchloride (PVC), or fluoroelastomers. Methods for forming the features ininterlayer23 include die cutting, rotary die cutting, laser etching, injection molding, and reaction injection molding.
• Linear actuator24 of the present invention, as depicted inFIG. 1B, is preferred to be, but not limited to, an electromagnetic solenoid. Other suitable linear actuators include a motor/cam/piston configuration, a piezoelectric linear actuator, or motor/linear gear configuration.
EXAMPLE 1Immunological Assay
• The plastic fluidic cartridge, as shown inFIG. 2, can be utilized to perform immunological assays withinreaction chamber34 by immobilizing a plurality of bio-molecules such asdifferent antibodies35. In one exemplary embodiment, a sample containing an unknown concentration of a plurality of antigens or antibodies is first placed withinreservoir31. The external linear actuator is then repeatedly actuated to pump the sample fromreservoir31 toreaction chamber34. The sample is then allowed to react with the immobilizedantibodies35 for a set reaction time. At the end of the set reaction time, the sample is then excluded fromreaction chamber34 throughexit port36. A wash buffer is then placed inreservoir31 and the external linear actuator is repeatedly actuated to pump the wash buffer throughreaction chamber34 and out theexit port36. Such wash steps can be repeated as necessary. A solution containing a specific secondary antibody conjugated with a detectable molecule such as a peroxidase enzyme, alkaline phosphatase enzyme, or fluorescent tag is placed intoreservoir31. The secondary antibody solution is then pumped intoreaction chamber34 by repeatedly actuating the linear actuator. After a predetermined reaction time, the solution is pumped out throughexit port36.Reaction chamber34 is then washed in a similar manner as previously describe. In the case of an enzyme conjugate, a substrate solution is placed intoreservoir31 and pumped intoreaction chamber34. The substrate will then react with any enzyme captured by the previous reactions with the immobilizedantibodies35 providing a detectable signal. For improved assay performance,reaction chamber34 can be maintained at a constant 37° C.
• According to the present invention, the plastic fluidic cartridge need not be configured as a single-fluid delivery and analysis device.FIG. 3 shows a plastic cartridge configured as a five fluid delivery and analysis device. Such a device can perform immunological assays, such as competitive immunoassay, immunosorbent immunoassay, immunometric immunoassay, sandwich immunoassay and indirect immunoassay, by providing immobilized antibodies inreaction chamber46. Herereaction chamber46 is not configured as a wide rectangular area, but a serpentine channel of dimensions similar to capillary dimension. This configuration provides more uniform flow through the reaction chamber at the expense of wasted space. For example, during immunoassays, a sample containing unknown concentrations of a plurality of antigens or antibodies is placed inreservoir41. A wash buffer is placed inreservoir42.Reservoir43 remains empty to provide air purging. A substrate solution specific to the secondary antibody conjugate is placed inreservoir44. The secondary antibody conjugate is placed inreservoir45. Each reservoir is connected to apump structure1′ similar to that ofFIG. 1.Pump structures1′ provide pumping fromreservoirs41,42,43,44, and45 throughreaction chamber46 to awaste reservoir49. Asecondary reaction chamber47 is provided for negative control and is isolated from the sample ofreservoir41 bycheck valve48. The protocol for performing immunoassays in this device is equivalent to that described previously for the single-fluid configuration with the distinct difference that each separated reagent is contained in a separate reservoir and pumped with a separate pump structure using a separate external linear actuator. First, an external linear actuator corresponding to a pump connected toreservoir41 is repeatedly actuated until a sample fluid fillsreaction chamber46. After a predetermined reaction time, the sample fluid is pumped to wastereservoir49 using either a pump connected to samplereservoir41 or a pump connected toair purge reservoir43. Next the wash buffer is pumped intoreaction chamber46 by repeatedly actuating the external actuator corresponding to a pump structure connected to washreservoir42. The wash and/or air purge cycle can be repeated as necessary. A secondary antibody solution is then pumped intoreaction chamber46 by repeatedly actuating the external linear actuator corresponding to a pump structure connected toreservoir45. After a predetermined reaction time, the secondary antibody solution is excluded fromreaction chamber46 either by a pump connected toreservoir45 or a pump connected toair purge reservoir43.Reaction chamber46 is then washed as before. The substrate is pumped intoreaction chamber46 by repeatedly actuating a linear actuator corresponding to a pump connected toreservoir44. After a predetermined reaction time, the substrate is excluded fromreaction chamber46 and replaced with wash buffer fromreservoir42. Results of the immunoassay can then be confirmed by optical measurements throughupper substrate21.
• Furthermore, the reactions performed with the plastic fluidic cartridge of the invention need not be limited to reactions performed in stationary liquids.FIG. 4 shows a plastic fluidic cartridge according to the invention, configured to provide continuous fluid motion throughreaction chamber55. In this configuration,reservoirs51,52, and53 are connected to separate pump structures similar to those of the five fluid configuration ofFIG. 3, but in this case the pump structures are connected to anintermediate circulation reservoir56. For example,pump structure57 is connected tocirculation reservoir56 to provide continuous circulation of fluid fromcirculation reservoir56 throughreaction chamber55 and returning tocirculation reservoir56. In this manner, a fluid can be circulated throughreaction chamber55 without stopping. Such a fluid motion can provide better mixing, faster reactions times, and complete sample reaction with immobilized species inreaction chamber55.Pump structure58 is connected such that it provides pumping of fluids fromcirculation reservoir56 to wastereservoir54. Immunological assays similar to those described above can be performed in this device by immobilizing antibodies inreaction chamber55 placing the sample containing unknown concentrations of antigens or antibodies in thecirculation reservoir56, placing a solution of secondary antibody conjugate inreservoir52, placing a substrate solution inreservoir53, and placing a wash buffer inreservoir51. The remaining protocol is identical to the above method with the addition of transferring fluids to and from thecirculation reservoir56 and continuously circulating during all reaction times.
EXAMPLE 2DNA Hybridization
• The system of the present invention can also be used to perform DNA hybridization analysis. Using the plastic cartridge ofFIG. 4, a plurality of DNA probes are immobilized inreaction chamber55. A sample containing one or more populations of fluorescently tagged, amplified DNA of unknown sequence is placed inreservoir52. A first stringency wash buffer is placed inreservoir51. A second stringency wash buffer is placed inreservoir53.Reaction chamber55 is maintained at a constant temperature of 52° C. The sample is transferred tocirculation reservoir56 by repeatedly actuating a linear actuator corresponding to a pump structure connected toreservoir52. The sample is then circulated throughreaction chamber55 by repeatedly actuating a linear actuator corresponding to pumpstructure57. The sample is circulated continuously for a predetermined hybridization time typically from 30 minutes to 2 hours. The sample is then excluded from thecirculation reservoir56 andreaction chamber55 by actuatingpump structures57 and58 in opposing fashion. The first stringency wash buffer is then transferred tocirculation reservoir56 by repeatedly actuating the linear actuator corresponding to the pump structure connected toreservoir51. The first stringency wash buffer is then circulated throughreaction chamber55 in the same manner described above. After a predetermined wash time, the first stringency wash buffer is excluded fromreaction chamber55 andcirculation reservoir56 as described above. A second stringency wash buffer is then transferred tocirculation reservoir56 and circulated throughreaction chamber55 in a manner similar to that previously described. After the second wash buffer is excluded, the DNA hybridization results can be read by fluorescent imaging.
• The invention being thus described, it will be obvious that the invention may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Claims (7)

1-11. (canceled)

12. A method of performing immunological assays of a fluid sample comprising a plurality of bio-molecules at unknown concentrations, comprising the steps of:

(a) placing said fluid sample into a sample reservoir defined in a fluidic cartridge;

(b) placing a wash buffer in a wash buffer reservoir defined in said fluidic cartridge;

(c) providing an air purge reservoir in said fluidic cartridge that remains empty to provide air purging;

(d) placing a secondary antibody conjugate in an antibody reservoir defined in said fluidic cartridge;

(e) placing a substrate solution specific to a secondary antibody conjugate in a substrate reservoir defined in said fluidic cartridge;

(f) pumping, by a pump structure, each of said fluid sample, said wash buffer, said substrate solution, and said secondary antibody conjugate to a waste reservoir through a first reaction chamber;

(g) providing a second reaction chamber for negative control and isolating said second reaction chamber from said fluid sample of said first reservoir by a check valve; and

(h) confirming results of immunoassay by optical measurements.

13. The method, as recited inclaim 12, wherein the step (f) further comprises the steps of:

(f-1) pumping said fluid sample from said sample reservoir to said first reaction chamber until said fluid sample at least partially fills said first reaction chamber;

(f-2) pumping said fluid sample from said first reaction chamber to said waste reservoir after a first predetermined reaction time;

(f-3) pumping, one or more times, said wash buffer from said wash buffer reservoir into said waste reservoir via said first reaction chamber;

(f-4) pumping said secondary antibody conjugate from said antibody reservoir into said first reaction chamber;

(f-5) pumping said secondary antibody conjugate from said first reaction chamber to said waste reservoir after a second predetermined reaction time;

(f-6) pumping said substrate solution from said substrate reservoir into said first reaction chamber; and

(f-7) pumping said substrate solution from said first reaction chamber to said waste reservoir after a third predetermined reaction time and at least partially filling said first reaction chamber with said wash buffer from said wash buffer reservoir.

14. The method, as recited inclaim 13, wherein said pump structure comprises a plurality of pumps and wherein said sample reservoir, said wash buffer reservoir, said air purge reservoir, said substrate reservoir, and said antibody reservoir is each connected to at least one pump in said plurality of pumps for pumping said fluid sample, said wash buffer, said substrate solution, and said secondary antibody conjugate through said first reaction chamber to said waste reservoir respectively.

15. The method, as recited inclaim 13, wherein said sample reservoir is a circulation reservoir and said pump structure is connected to said circulation reservoir to provide a continuous and repeated circulation of fluid, circulating from said circulation reservoir to said first reaction chamber and returning to said circulation reservoir, so that said fluid sample, said wash buffer, said substrate solution and said secondary antibody conjugate are capable of being circulated through said first reaction chamber without stopping.

16. The method, as recited in claims13,14 or15, wherein said reservoir, said wash buffer reservoir, said air purge reservoir, said substrate reservoir, and said antibody reservoir is each connected to said first reaction chamber by one or more channels of capillary dimensions, wherein said fluidic cartridge includes a first substrate, a second substrate and an flexible intermediate interlayer sealedly interfaced between said first substrate and said second substrate to form therein said sample reservoir, said wash buffer reservoir, said air purge reservoir, said substrate reservoir, said antibody reservoir, said one or more channels, and said reaction chamber, and wherein said fluidic cartridge further provides a fluid flow controlling structure therein to restrict a flow of said fluid sample, said wash buffer, said substrate solution, and said second antibody conjugate through said first reaction chamber via said one or more channels in one direction only.

17. The method, as recited inclaim 16, wherein in said pumping step (f), said fluid sample, said wash buffer, said substrate solution, and said secondary antibody conjugate are respectively pumped by pumping actions in a plurality of pump chambers, wherein said plurality of pump chambers are defined in said fluidic cartridge and said pumping actions are provided by a plurality of linear actuators so as to pump said fluid sample, said wash buffer, said substrate solution, and said secondary antibody conjugate to flow from said sample reservoir, said wash buffer reservoir, said air purge reservoir, said substrate reservoir, and said antibody reservoir through said first reaction chamber via said one or more channels.

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