US20060263264A1

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Publication number: US20060263264A1
Application number: US11/486,186
Authority: US
Inventors: Sebastian Bohm; John Gilbert
Original assignee: Cytonome Inc
Current assignee: Cytonome ST LLC
Priority date: 2001-06-20
Filing date: 2006-07-13
Publication date: 2006-11-23
Also published as: US20030007898A1; US7179423B2

Abstract

A fluid interface port in a microfluidic system and a method of forming the fluid interface port is provided. The fluid interface port comprises an opening formed in the side wall of a microchannel sized and dimensioned to form a virtual wall when the microchannel is filled with a first liquid. The fluid interface port is utilized to fill the microchannel with a first liquid, to introduce a second liquid into the first liquid and to eject fluid from the microchannel.

Description

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 10/028,852, filed Dec. 21, 2001, which claims priority to U.S. Provisional Patent Application No. 60/299,515 filed Jun. 20, 2001, and is related to Attorney Docket No. CVZ-OOlb, entitled “Microfluidic System Including a Virtual Wall Fluid Interface Port for Interfacing Fluids with the Microfluidic System”, filed herewith; Attorney Docket No. CVZ-001c, entitled “Microfluidic System Including a Virtual Wall Fluid Interface Port for Interfacing Fluids with the Microfluidic System”, filed herewith; Attorney Docket No. CVZ-002, entitled “Microfabricated Two-Pin Liquid Sample Dispensing System”, filed herewith; Attorney Docket No. CVZ-003, entitled “Small Molecule Substrate Based Enzyme Activity Assays”, filed herewith; and Attorney Docket No. CVZ-005, entitled “Droplet Dispensing System”, filed herewith. The contents of the foregoing patent applications are herein incorporated by reference. The contents of all references, issued patents, or published patent applications cited herein are expressly incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to microscale fluid handling devices and systems. More particularly, the present invention relates to fluidic interface ports for providing bi-directional fluid interfacing in a microfluidic system.

BACKGROUND OF THE INVENTION

In the chemical, biomedical, bioscience and pharmaceutical industries, it has become increasingly desirable to perform large numbers of chemical operations, such as reactions, separations and subsequent detection steps, in a highly parallel fashion. The high throughput synthesis, screening and analysis of (bio)chemical compounds, enables the economic discovery of new drugs and drug candidates, and the implementation of sophisticated medical diagnostic equipment. Of key importance for the improvement of the chemical operations required in these applications are an increased speed, enhanced reproducibility, decreased consumption of expensive samples and reagents, and the reduction of waste materials.

Microfluidic devices and systems provide improved methods of performing chemical, biochemical and biological analysis and synthesis. Microfluidic devices and systems allow for the performance of multi-step, multi-species chemical operations in chip-based micro chemical analysis systems. Chip-based microfluidic systems generally comprise conventional ‘microfluidic’ elements, particularly capable of handling and analyzing chemical and biological specimens. Typically, the term microfluidic in the art refers to systems or devices having a network of processing nodes, chambers and reservoirs connected by channels, in which the channels have typical cross-sectional dimensions in the range between about 1.0 μm and about 500 μm. In the art, channels having these cross-sectional dimensions are referred to as ‘microchannels’.

By performing the chemical operations in a microfluidic system, potentially a number of the above-mentioned desirable improvements can be realized. Downscaling dimensions allows for diffusional processes, such as heating, cooling and passive transport of species (diffusional mass-transport), to proceed faster. One example is the thermal processing of liquids, which is typically a required step in chemical synthesis and analysis. In comparison with the heating and cooling of liquids in beakers as performed in a conventional laboratory setting, the thermal processing of liquids is accelerated in a microchannel due to reduced diffusional distances. Another example of the efficiency of microfluidic systems is the mixing of dissolved species in a liquid, a process that is also diffusion limited. Downscaling the typical dimensions of the mixing chamber thereby reduces the typical distance to be overcome by diffusional mass-transport, and consequently results in a reduction of mixing times. Like thermal processing, the mixing of dissolved chemical species, such as reagents, with a sample or precursors for a synthesis step, is an operation that is required in virtually all chemical synthesis and analysis processes. Therefore, the ability to reduce the time involved in mixing provides significant advantages to most chemical synthesis and analysis processes.

Furthermore, reduced dimensions enhance separation operations utilized in chemical synthesis and analysis processes. One example is capillary electrophoresis, which is a separation technology based on the migration of dissolved charged species through a liquid filled capillary by the application of a longitudinal electric field. By reducing the cross-sectional size of the capillaries, the separation efficiency can greatly be improved, thereby resulting in rapid separations. For examples, see Effenhauser et al., Anal. Chem. 65:2637-2642 October (1993), Effenhauser et al., Anal. Chem. 66:2949-2953 September (1994), Jacobson et al., Anal. Chem. 66:4127-4132 December (1994) and Jacobson et al., Anal. Chem. 66:1114-1118 April (1994).

Another aspect of the reduction of dimensions is the reduction of required volumes of sample, reagents, precursors and other often very expensive chemical substances. Milliliter-sized systems typically require milliliter volumes of these substances, while microliter sized microfluidic systems only require microliters volumes. The ability to perform these processes using smaller volumes results in significant cost savings, allowing the economic operation of chemical synthesis and analysis operations. As a consequence of the reduced volume requirement, the amount of chemical waste produced during the chemical operations is correspondingly reduced.

It can be concluded that due to the reduced dimensions associated with microfluidic systems, important chemical operations can be accelerated whilst at the same instance lead to a reduction of consumption of chemicals and chemical waste.

Applications of microfluidic systems are myriad. For example U.S. Pat. No. 5,922,591 describes a miniaturized integrated nucleic acid diagnostic device and system. This device is capable of performing one or more sample acquisition and preparation operations, in combination with one or more sample analysis operations. Useful applications for microfluidic systems are in nucleic acid based diagnostics and de novo sequencing applications. International Patent Appln. WO 96/04547, published Feb. 15, 1996, describes the use of electro-kinetic operated microfluidic systems for performing electrophoretic separations, flow injection analysis and chemical reactions and synthesis steps. U.S. Pat. No. 5,942,443 discloses a range of microfluidic devices and methods for performing high-throughput synthesis and analysis, especially useful in screening a large number of different chemical compounds for their effect on a variety of chemical and biochemical systems. U.S. Pat. No. 5,858,804 provides a method of performing an immunological assay in a micro-laboratory array comprising a plurality of microchannels and chambers disposed in a solid substrate. U.S. Pat. No. 6,176,991 B1 discloses a serpentine electrophoresis channel in microchip format providing efficient, high-speed analysis of the composition of chemical samples, especially for nucleic acid sequencing.

Many methods have been described for the interfacing of fluids, e.g., samples, analytes, reagents, precursors for synthesis and buffers, towards, within or between microfluidic systems. In conventional microfluidic systems, the structures and methods used to introduce samples and other fluids into microfluidic substrates limit the capabilities of the microfluidic systems. For example, conventional microfluidic systems may include a separate sample introduction channels for introducing a sample to a microchannel for processing. The sample is first introduced into the sample channel and transported through the sample channel to the microchannel. Another method for introducing a fluid involves the use of sample wells or reservoirs in communication with the microchannel for holding a relatively larger supply of the sample. Reservoirs are structures which accommodate a significantly greater volume of fluid than the microfluidic channel. A relatively small portion of the sample supply in the sample well or reservoir is introduced into the microchannel.

The total number of samples and other fluids that can be processed on a microfluidic substrate is currently limited by the size and/or the number of reservoirs through which these fluids are introduced to the microfluidic system. A disadvantage of known structures and methods for introducing fluids into a microfluidic system is the use and transfer of a much greater volume of fluid than is needed for microfluidic analysis due to significant size inefficiencies and sample loss. Furthermore, with conventional methods of introducing fluids into microfluidic systems, it is difficult to control the amount of sample introduces that is eventually introduces into the microchannel after passing through a sample channel or a reservoir.

One method of fluidic communication with microfluidic systems is by mechanical micropumps and valves, see U.S. Pat. No. 6,033,191 and U.S. Pat. No. 5,529,465. A major disadvantage of this approach for fluidic interfacing is the complex construction and operation of these micropumps and valves. Another disadvantage is there relatively large size and internal volume when compared to the internal volume of the microchannels. Often there are multiple orders of magnitude between these two volumes and the resulting discrepancy renders micropumps unattractive to interface with a large number of small dimensional microchannels.

U.S. Pat. No. 5,173,163 describes a method and device for introducing a fluidic sample in a micro-capillary for electrophoretic separation. In this method liquid is brought into a separation capillary by introducing one end of this capillary into a vessel containing the liquid to be introduced. A combination of applied external pressures and voltages results in the transport of liquid from the vessel into the capillary. The proposed method has disadvantages. The size of the device does not allow the interfacing with a large number of microchannels, and between consecutive injections, the device needs to be cleaned, thereby considerably reducing throughput.

Another method for introducing materials in a microfluidic device is disclosed in U.S. Pat. No. 6,042,709. In this approach electrokinetic forces are employed to move a charged compound through a receiving inlet opening of the microfluidic device. A disadvantage is that the precise amount of injected liquids and substances depends upon a large number of factors which are difficult to control. One important parameter is the surface potential of the microchannel wall, which together with the applied voltage determines the liquid flow. This surface potential depends on pH of the liquid to be pumped as well as its ionic composition and even the type of ions present in the liquid. It is also a disadvantage that it does not allow the efficient interfacing with a large number of different liquids as for every injection port, a separate high voltage supply is required, together with the associated liquid channels for providing a closed electrical circuit.

A method and apparatus for performing electrophoretic experiments in a highly parallel fashion is disclosed in U.S. Pat. No. 6,103,199. Here, a plurality of separation capillaries with associated wells for receiving chemical substances in fluid form are disposed in the form of a two dimensional array. The chemical substances are dispensed from a micro titer plate into these wells by an interfacing methodology employing pressurized chambers associated with the wells to be filled. A disadvantage is that only a very small fraction of the applied liquid is actually introduced in the target microchannel, the bulk of the applied liquid drop remains behind in the well by capillary forces. As a result, most of the liquid is wasted and is not available for a consecutive chemical processing step. The effect that only a small portion of the liquid transported actually is introduced in a targeted part of a microfluidic system, can be referred to as ‘injection efficiency’, i.e. the ratio between the volume of liquid required for a particular chemical operation in a part of the microfluidic system, and the total volume of liquid required for the introductory operation.

In this particular disclosure, only sub-nanoliter amounts are required for the electrophoretic separation (i.e. the chemical operation), whilst many microliters of sample are drawn from the micro titer plate (i.e. introductory operation), yielding an injection efficiency much less than 0.001. A low injection efficiency is disadvantageous because it indicates inefficient use of chemical substances and an increased production of waste.

U.S. Pat. No. 6,090,251 provides micro-fabricated structures for facilitating fluid introduction into microfluidic devices. Fluid is introduced into a plurality of receiving wells in direct communication with associated microchannels, by the dropping of liquids into these receiving wells using pressurized gas. Besides the complexity of the required fluidic manifolds and pressurizing system, also here a disadvantage is the inherently low injection efficiency as only a very small fraction of the applied liquid is actually used in the experiment.

For the introduction of liquids in capillary electrophoresis columns implemented on chip-like devices, generally electrokinetic injection is applied. See Woolley et al., Anal. Chem. 70:684-688 February (1998), Jacobson et al., Anal. Chem. 68:720-723 March (1996), Jacobson et al., Anal. Chem. 66:2369-2373 July (1994) and Effenhauser et al., Anal. Chem. 67:2284-2287 July (1995). In this method, liquid is pumped from a first well towards the microchannel for electrophoretic separation by the application of a high driving voltage between this well and a second well located downstream. Due to the charged inner surfaces of the microchannel walls, an electroosmotic liquid flow is induced pumping liquid out of the first into the targeted microchannel. This method is referred to as ‘electrokinetic injection’ and has some specific disadvantages. One disadvantage is that if a large number of liquids need to be handled, for instance in high-throughput synthesis and screening applications, a large number of wells need to be integrated on the microfluidic device. The relatively large footprint of a typical well (about 5 mm diameter) when compared to the microchannels in which the actual chemical operation is performed (about 50 μm diameter), takes up a dominating portion of the chip surface (see U.S. Pat. No. 6,143,152 and U.S. Pat. No. 6,159,353). As the costs of microfluidic chips strongly depends on the chip surface, the required integration of wells renders this liquid injection scheme unattractive for high-throughput synthesis and screening applications.

Another disadvantage of conventional systems is that for every well, a separate electrode is required together with electronic circuitry for the application of the driving voltages. This requirement results in a complex and expensive apparatus.

Another specific disadvantage with electrokinetic injection is the fact that during the application of the driving voltage on the electrode the electrolysis of water results in the generation of hydroxyl ions (OH⁻), hydrogen ions (H⁺), hydrogen gas (H₂) and oxygen gas (O₂). The generated ions will affect the acidity (i.e. pH) of the liquid pumped from the first well, whilst the produced gasses potentially give rise to the formation of gas bubbles in the microfluidic system thereby destroying the experiment and eventually the microfluidic device. Besides electrolysis of the aqueous medium, any present electroactive species can degrade due to electrochemical reactions at the electrode surface. For instance, the presence of chloride ions, an ion present in most liquid media, will result in the formation of chlorine gas, which on turn can interact and potentially destroy vulnerable (biochemical) compounds present in the liquid to be injected. Also the liquid to be injected can contain electroactive constituents which can be degraded by the electrochemical processes associated with electrokinetic injection. These disadvantages can be grouped and referred to as ‘electrochemical pollution’.

Another disadvantage of electrokinetic injection is that in between consecutive experiments, the well need to be thoroughly cleaned in order to prevent cross-contamination. This required cleaning step results in a reduction of throughput and makes it difficult to implement on-line monitoring. Another disadvantage is that the liquid to be injected is subjected to a high voltage. This aspect of the absence of galvanic separation makes it virtually impossible to use electrokinetic injection for in-vivo or near-vivo applications due to the danger of electrocution. Another disadvantage is that the electrokinetic injection methods referred to, are only applicable in chip like systems produced via microfabrication technology, i.e. via the use of expensive equipment and processes also applied for the fabrication of computer chips. These methods are known to have high costs. It is desirable to provide a n interfacing methodology which is also applicable in current non chip-based capillary systems. Still another disadvantage is the low injection efficiency of electrokinetic injection. To fill a typical well, about 10-50 μl of liquid is required, whilst for a particular chemical operation only sub nanoliter amounts are required.

U.S. Pat. No. 6,130,098 discloses the movement of liquid volumes into and through microchannels by employing pressures generated by heating a volume of air in direct connection with these microchannels. A disadvantage of this fluidic interfacing method is that for a correct and efficient operation the pressure generating air chamber together with electronic heater components need to be integrated with the microfluidic system. This results in a complex device with associated large costs.

It can be concluded based on the above that current methods and systems for fluidic interfacing with microfluidic devices have particular disadvantages regarding the difficulty of integration of a large number of chemical operation nodes to interface with (i.e. electrochromatography columns, reactors etc.), relatively large required liquid volumes, low injection efficiency, electrochemical sample pollution, long rinse time between analysis steps, galvanic separation and required microfabrication technologies. Besides these disadvantages, none of the above mentioned interfacing methods allow bi-directional fluidic interfacing, i.e. transporting liquids to and from microfluidic systems. There has arisen a need in the art for providing suitable bi-directional fluidic interfacing structure that allows for the implementation of a much wider range of chemical operation steps in microfluidic systems.

SUMMARY OF THE INVENTION

The present invention provides methods, devices and systems for interfacing fluids in microfluidic systems. A fluid interface port for directly interfacing a microfluidic channel network and the surrounding environment is provided in a microchannel in a microfluidic system by forming an opening in a sidewall of the microchannel. The aperture forms a virtual wall when the microchannel is filled with a liquid. The aperture has suitable cross sectional dimensions such that capillary forces retain liquid within the microchannel. The virtual wall is defined by the meniscus of the liquid in the opening, which essentially replaces the sidewall of the microchannel so as to not substantially affect or influence fluid flow through the microchannel.

Filling of a microchannel may be accomplished via the opening in the side wall of the microchannel. To fill the microchannel, a user forms droplets of a first liquid and directs the droplet towards the opening, such that the droplets of the first liquid enter and fill the microchannel.

Fluid introduction and/or fluid removal is accomplished through a virtual wall formed in the side wall of a microchannel. The process of introducing a fluid sample to the microchannel comprises forming a droplet of the fluid and propelling the droplet towards a virtual wall. The droplet traverses the virtual wall and enters the microchannel directly, without requiring intermediate reservoirs or channels. The virtual wall formed in the sidewall of the microchannel may also define a fluid ejection port for ejecting a fluid from the microchannel in the form of a droplet.

According to one aspect, a microfluidic device is provided. The microfluidic device comprises a microchannel having an interior bounded by a side wall and a fluid interface port formed in the side wall of the microchannel to provide access to the interior of the microchannel. The fluid interface port has a diameter between about 25 μm and about 100 μm, such that when a fluid is disposed in the interior of the microchannel, the fluid forms a virtual wall at the fluid interface port.

According to another aspect, the invention provides a microfluidic device comprising a microchannel and a first fluid interface port formed in the side wall of the microchannel to provide access to the interior of the microchannel, wherein the microchannel is free of a second coaxially arranged fluid interface port formed in the side wall at a location opposite to the first fluid interface port. When a fluid is disposed in the interior of the microchannel, the fluid forms a virtual wall at the first fluid interface port.

According to another aspect, a microfluidic device is provided, comprising a microchannel and a fluid interface port having a dead volume that is less than one nanoliter formed in the side wall of the microchannel. The fluid interface port provides access to the interior of the microchannel, such that when a fluid is disposed in the interior of the microchannel, the fluid forms a virtual wall in the fluid interface port.

According to yet another aspect, a microfluidic device is provided comprising a microchannel and a fluid interface port having zero dead volume formed in the side wall of the microchannel. The fluid interface port provides access to the interior of the microchannel, such that when a fluid is disposed in the interior of the microchannel, the fluid forms a virtual wall in the fluid interface port.

According to yet another aspect, a microfluidic device is provided, comprising a microchannel and a fluid interface port formed in the side wall of the microchannel to provide access to the interior of the microchannel, wherein the fluid interface is sized and dimensioned such that when a fluid is disposed in the interior of the microchannel, the fluid forms a virtual wall at the fluid interface port, said virtual wall being employed as an optical window for optically analyzing the fluid in the microchannel.

According to still another aspect of the present invention, a liquid volume injection system for injecting a droplet into a microchannel of a fluidic system is provided. The system comprises a droplet generator system for generating the droplet and a virtual wall formed in a fluid interface port by a fluid disposed in the microchannel for receiving the droplet.

According to another aspect, an electrokinetically operated microfluidic system is provided. The electrokinetically operated microfluidic system comprises a microchannel for housing a fluid having a side wall, and a fluid interface port formed in the side wall, wherein the fluid forms a virtual wall within the fluid interface port, a first reservoir including a first electrode connected to a first end of the microchannel, a second reservoir including a second electrode connected to a second end of the microchannel, and a voltage generator for establishing an electric field between the first electrode and the second electrode, thereby inducing movement of the first fluid through the microchannel.

According to still another aspect, a microfluidic device is provided, comprising a microchannel, one or more sample introduction ports formed in the side wall of the microchannel to provide access to the interior of the microchannel, and a filling aperture formed in the side wall of the microchannel for filling the microchannel with fluid. A fluid disposed in the interior of the microchannel forms a virtual wall at the fluid interface port.

According to another aspect, a microfluidic device is provided, comprising a microchannel having an interior bounded by a side wall and one or more fluid interface ports formed in the side wall of the microchannel to provide access to the interior of the microchannel, such that a fluid disposed in the interior of the microchannel forms a virtual wall at the fluid interface port. The fluid ports are sized and dimensioned for pumping the fluid in the channel when a liquid is introduced to the fluid interface port.

According to another aspect, a microfluidic device is provided, comprising a microchannel having an interior bounded by a side wall, a hydrophobic patch disposed on an interior surface of the side wall of the microchannel and a fluid interface port formed in the side wall opposite the hydrophobic to provide access to the interior of the microchannel. The fluid interface port forms a vent for allowing air to escape the interior of the microchannel.

According to a final aspect of the invention, a microfluidic device is provided, comprising a microchannel, a fluid interface port formed in the side wall of the microchannel to provide access to the interior of the microchannel, and at least one waste channel in communication with and intersecting the microchannel to collect waste from the microchannel. The fluid interface port is sized and dimensioned such that when a fluid is disposed in the interior of the microchannel, the fluid forms a virtual wall at the fluid interface port.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of a microfluidic system suitable for implementing the illustrative embodiment of the invention.

FIG. 2ais a top view of a microchannel structure having a fluid interface port formed in a side wall of the microchannel for filling the microchannel with a first fluid according to the teachings of the invention.

FIG. 2bis a cross-sectional view of the microchannel ofFIG. 2aalong the length of the microchannel.

FIGS. 3aand3billustrate the steps of filling the microchannel ofFIG. 2awith a first fluid by directing droplets of the first fluid through the fluid interface port.

FIG. 4aillustrates the microchannel ofFIGS. 2aand2bfurther including a hydrophobic patch disposed in the microchannel interior according to an alternate embodiment of the present invention.

FIG. 4billustrates an alternate embodiment of a microchannel according to the teachings of the invention, wherein the hydrophobic patch is coaxially arranged with a fluid interface port.

FIG. 5ais a top view of a microchannel according to one embodiment, including a filling aperture and a stopper hole for directing the filling of a first fluid in the microchannel according to the teachings of the invention.

FIG. 5bis a cross-sectional view of the length of the microchannel shown inFIG. 5a, illustrating the process of filling the microchannel.

FIG. 6 illustrates the filling of a microchannel with two different fluids by utilizing a plurality of filling apertures disposed in the side wall of the microchannel.

FIG. 7aillustrates the process of closing the filling aperture ofFIGS. 2athrough6 by forming and dispensing drops of encapsulant onto the filling aperture according to one embodiment of the present invention.

FIG. 7billustrates the process of closing the filling aperture using a covering layer according to an alternate embodiment of the present invention.

FIG. 8 illustrates a microchannel having a filling aperture located adjacent to a closed end of the microchannel, such that the introduction of droplets into the microchannel through the filling aperture induces a flow in the microchannel.

FIG. 9ais a cross-sectional side view of a microchannel including a fluid interface port for receiving a liquid droplet into the microchannel.

FIG. 9bis a cross-sectional view perpendicular through the microchannel ofFIG. 9aat the position of the fluid interface port, illustrating the insertion of a drop using a pin.

FIG. 9cis a cross-sectional view of a microchannel having a virtual wall according to the teachings of the invention, illustrating the composition of the liquid inside the microchannel directly after receiving a droplet.

FIG. 9dillustrates a microchannel having a virtual wall according to the teachings of the invention, immediately after injection of the second liquid in the first liquid, whereby the first and second liquid are immiscible.

FIG. 9eis a cross-sectional view of a microchannel having a virtual wall according to an alternate embodiment of the invention.

FIG. 9fis a cross-sectional view of a microchannel having a virtual wall according to an alternate embodiment of the invention, including a covering layer.

FIG. 9gis a cross-sectional view of a microchannel having an array of apertures forming virtual walls.

FIG. 9hillustrates the introduction of a liquid into the microchannel shown inFIG. 9g.

FIG. 10aillustrates a microchannel having a plurality of virtual walls disposed across the radial width of the microchannel according to one embodiment of the present invention.

FIG. 10billustrates a microchannel having a plurality of virtual walls disposed along the axial length of the microchannel according to one embodiment of the present invention.

FIG. 11ais a schematic block diagram representative of a sample introduction system for electrically guiding a selected droplet into a selected microchannel according to the teachings of the present invention.

FIG. 11bis a cross-sectional view of a microchannel array ofFIG. 4aof the present invention wherein microchannels can selectively be charged to attract or repel a selected droplet according to the teachings of the present invention.

FIG. 11cis a cross-sectional view of the microchannel array ofFIG. 11aemploying an array of ring electrodes for guiding a selected droplet into a selected microchannel according to the teachings of the present invention.

FIG. 12ais a schematic view of one embodiment of the present invention for ejecting liquid from a microchannel through a virtual wall by using electric fields.

FIG. 12bis a schematic view of an alternate embodiment of the present invention for ejecting liquid from a microchannel through a virtual wall by the application of pressure pulses to the liquid.

FIG. 12cis a schematic view of still another embodiment of the present invention for ejecting liquid from a microchannel through a virtual wall by the application of a gas pressure pulse to the liquid.

FIG. 12dis a schematic view of yet another embodiment of the present invention for ejecting liquid from a microchannel through a virtual wall by a gas bubble.

FIG. 13ais a cross-sectional view of a microchannel with a single virtual wall wherein liquid inside the microchannel is optically verified via the virtual wall.

FIG. 13bis a cross-sectional view of a microchannel in which two concentric virtual walls are diametrically opposed, and demonstrates optical detection using both virtual walls.

FIG. 14 is a top view of an embodiment of the invention, illustrating the use of a microchannel having a virtual wall to perform a separation.

FIG. 15 shows a cutaway view of an embodiment of the invention in which a plurality of virtual walls are employed for performing multi-step chemical experiments.

FIG. 16 is a schematic cross-sectional view of an embodiment of the present invention in which a microchannel having a virtual wall part of an electrokinetically operated microfluidic system.

FIG. 17 shows a perspective view of an embodiment according to the invention in which a plurality of microchannels with virtual walls is applied in an electrokinetically operated microfluidic system.

FIG. 18 illustrates an application of the virtual wall interface ports of an illustrative embodiment to interface a microfluidic system with a mass spectrometer.

FIG. 19ais an exploded view of a microfluidic chip manufactured according to the teachings of the invention.

FIG. 19bis a top view of the microfluidic chip ofFIG. 19a.

FIG. 19cis a side view of the microfluidic chip ofFIG. 19a.

FIGS. 20a-cillustrate the steps of manufacturing a microchannel having an opening suitable for forming a virtual wall according to an embodiment of the present invention.

FIGS. 21a-21cillustrate the steps of manufacturing a microchannel with a virtual wall interface port according to another embodiment of the present invention.

FIGS. 22a-22cillustrate the steps of manufacturing a microchannel having a virtual wall as a fluidic interface port according to an alternate embodiment of the present invention.

FIG. 23ais a perspective view and of a microchannel with a virtual wall in which a hydrophobic patch is disposed in the microchannel.

FIG. 23bshows the use of the microchannel with the virtual wall depicted inFIG. 23a.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an improved fluidic interface for introducing fluids to and removing fluids from a microchannel in a microfluidic system. The invention further provides a method of forming the fluidic interface. The present invention significantly improves controllability over fluid samples, increases injection efficiency and reduces waste by utilizing an opening defining a virtual wall in a side wall of a microchannel to introduce and remove fluids.

One or more of the illustrative embodiments allow bi-directional fluidic interfacing to and from microfluidic systems. With the present invention, a large number of chemical operations can be performed on a small chip surface, thereby enabling the cost effective implementation and efficient operation of massively parallel synthesis and analysis systems. The present invention further significantly reduces the required liquid volume for interfacing, resulting in a considerable reduction of consumption of chemicals as well as the reduction of chemical waste. The present invention further provides methods and systems for the injection of liquids with near 100% injection efficiency and provides methods and systems for the injection of liquids which do not electrochemically pollute the handled liquids. The present invention presents methods and systems for the fast repetitive injection of liquids in microfluidic systems, pressure driven as well as electrokinetically driven, allowing high throughput synthesis, screening and analysis applications. The present invention further allows for galvanic separation of the liquid to be injected with the electrokinetically operated microfluidic system. The invention further provides fluidic interfacing with microfluidic systems devices and system that are not necessarily manufactured using standard microfabrication technologies. For example, the invention provides for interfacing with standard fused silica capillaries. The present invention applies to a variety of liquid samples, including solutions of compounds, whole cells or cell lysates, enzymes, proteins or peptides, and particles.

FIG. 1 illustrates a microfluidic system suitable for implementing the illustrative embodiment of the present invention. Theillustrative microfluidic system10 comprises a substrate11 having one ormore microchannels3 disposed therein. The microchannels transport fluid through themicrofluidic system10 for processing, handling, and/or performing any suitable operation on a liquid sample. As used herein, the term “microfluidic” refers to a system or device for handling, processing, ejecting and/or analyzing a fluid sample including at least one channel having microscale dimensions. The term “channel” as used herein refers to a pathway formed in or through a medium that allows for movement of fluids, such as liquids and gases. The term “microchannel” refers to a channel preferably formed in a microfluidic system or device having cross-sectional dimensions in the range between about 1.0 μm and about 250 μm, preferably between about 25 μm and about 150 μm and most preferably between about 50 μm and about 100 μm. One of ordinary skill in the art will be able to determine an appropriate volume and length of the microchannel. The ranges are intended to include the above-recited values as upper or lower limits. The microchannel can have any selected shape or arrangement, examples of which include a linear or non-linear configuration and a U-shaped configuration. Themicrofluidic system10 may comprise any suitable number ofmicrochannels3 for transporting fluids through themicrofluidic system10.

According to one practice, the microchannel of the present invention can include a fluid interface port. As used herein, “fluid interface port” refers to a structure in a microfluidic system, such as an aperture formed in a microchannel, which provides fluid access between the interior and the exterior of a microchannel. The fluid interface port is utilized to introduce fluids and other material to the microchannel and/or to remove fluid and/or other material from the microchannel. The fluid interface port may comprise, among other applications, a filling port for filling the microchannel with a carrier liquid, a sample introduction port for introducing a sample into the microchannel and an ejection port for ejecting fluid from the microchannel.

According to the illustrative embodiment, the microchannel is defined by a side wall having any suitable shape enclosing at least a portion of the interior of the channel. The fluid interface port is formed in the side wall of the microchannel by removing a portion of the side wall to define an opening. The fluid interface port of the illustrative embodiment is formed by an aperture in the side wall of the microchannel having a diameter of between about 0.1 μm and about 200 μm and preferably between about 25 μm and about 125 μm and most preferably between about 50 μm and about 100 μm. The aperture forming the fluid interface port may have any suitable shape, including, but not limited to, a cylinder, a disk, a conical shape, an elliptical shape and a cubic shape. The side wall or wall of the microchannel can be formed by two or more components that bound the entire volume of the microchannel. According to a first aspect of the invention, the fluid interface port is utilized to fill the microchannel with a first liquid. The first liquid comprises a carrier fluid for transporting a sample, reagent or other suitable liquid for performing one or more chemical operations, such as reactions and separations. Fluid flow may be induced in the microchannel via any suitable means, including, but not limited to, pressurizing and electroosmotic means.

As illustrated inFIG. 2athroughFIG. 7, thefluid interface port17 formed in the sidewall of the microchannel forms a filling aperture or microaperture for filling the microchannel with the first liquid. Typically, conventional microchannels are filled by inserting the end of the microchannel into a reservoir containing the first liquid. Capillary forces pull the first liquid into the microchannel and thereby fill the microchannel with the first liquid. In order to avoid this procedure, the present invention employs a fluid interface port formed in the sidewall (not end) of the microchannel. This enables a user to fill the microchannel through the port to provide simple, fast and efficient filling without requiring a large, separate liquid reservoir.

FIG. 2ais a top view of amicrochannel structure3 comprising aside wall16 surrounding a hollow interior. The microchannel includes afluid interface port17 located in theside wall16 of the microchannel structure. Thefluid interface port17 may have any suitable shape based upon the intended use, examples of which include circular, cylindrical, elliptical and conical.FIG. 2bis a cross-sectional view of the microchannel structure taken along the length of themicrochannel3, showing thefluid interface port17 disposed in the top side of the microchannel. As shown, the interior of themicrochannel3 is initially empty. According to the illustrative embodiment, themicrochannel3 is filled with a first liquid via thefluid interface port17 formed in theside wall16 of the microchannel.

FIGS. 3aand3billustrate the process of filling themicrochannel structure3 with afirst liquid4 by directingdroplets4aof thefirst liquid4 toward thefluid interface port17. Thedroplets4aenter themicrochannel3 through thefluid interface port17 and form aplug13 of thefirst liquid4 within themicrochannel3. Asmore droplets4aare introduced via thefluid interface port17, theplug13 expands in both directions through the microchannel, as indicated by the arrows, thereby filling the microchannel along the length of the microchannel with thefirst liquid4. Moreover, capillary forces work to draw theliquid4 along the length of themicrochannel3. According to the illustrative embodiment, thefirst liquid4 forms a meniscus in thefluid interface port17 when the microchannel is filled and the liquid is retained in the microchannel by capillary forces.

Thefluid interface port17 is sized and dimensioned to form avirtual wall15 when the microchannel is filled with thefirst liquid4. As used herein, “virtual wall” refers to the meniscus formed by thefirst liquid4 in theport17 formed in the side wall of the microchannel. The meniscus surface can be, although not required, substantially co-planar with thewall16 of the microchannel in which the meniscus is formed. The meniscus essentially replaces the removed portion of the side wall that defines theaperture17. The word “virtual” is chosen to express the effect that the overall liquid flow through the microchannel of the microfluidic system is not influenced by the virtual wall, i.e. the flow of liquid in the microfluidic system having a virtual wall is substantially identical to the flow of liquid through an identical microfluidic system in which no virtual wall is present. The fluid interface port, according to one practice, has appropriate dimensions and surface properties as to substantially not influence the overall liquid flow and liquid shape when compared to a microfluidic system in which no port or meniscus is formed. The virtual wall forms a direct interface between themicrochannel interior5 and the microchannel exterior, allowing direct access to the liquid inmicrochannel3 without introducing dead or unswept volume in themicrochannel3. The virtual wall also serves to seal liquid inside of the microchannel through a range of pressures in the microchannel. Those of ordinary skill will readily recognize that the surface or wall of the fluid interface port can be formed anywhere along the axial height of the port. One of ordinary skill will recognize that the meniscus may be convex or concave, depending on the appropriate system pressure.

According to an alternate embodiment, shown inFIG. 4a, ahydrophobic patch23 is applied to the side wall or interior of themicrochannel3 prior to introduction of thefirst liquid4. When thefirst liquid4 is introduced to the microchannel, thehydrophobic patch23 forms a barrier, and causes theplug13 ofliquid4 to expand only in one direction, away from thepatch23.

According to one embodiment, shown inFIG. 4b, thehydrophobic patch23 is applied to the interior surface of the microchannel through an aperture forming afluid interface port17 in thesidewall16 of the microchannel. Thehydrophobic patch23 may comprise any suitable material for rendering a surface hydrophobic, such as gold. The hydrophobic material may be sputtered, evaporated, sprayed or deposited through theaperture17 and bound to the interior surface of themicrochannel3 opposite the aperture to form thehydrophobic patch23. One skilled in the art will recognize that any suitable surface treatment for the microchannel may be applied through the aperture, in addition to hydrophobic patches.

The aperture used to apply thehydrophobic patch23 may further serve as a vent for themicrochannel3 to allow air to escape from the microchannel interior. As shown, the aperture is coaxially arranged with the hydrophobic patch. The actual vent, formed by theaperture17 in the side wall, is not hydrophobic. However, the microchannel surface coaxially opposite the vent is rendered hydrophobic by application of thehydrophobic patch23. The hydrophobic region is not flushed by liquid, allowing air to escape the microchannel interior through theaperture17 in theside wall16.

According to yet another embodiment, shown inFIGS. 5aand5b, themicrochannel3 further includes astopper hole28 adjacent to thefluid interface port17. Thestopper hole28 is sized and dimensioned to form a pressure barrier for a liquid disposed in the microchannel. One of ordinary skill in the art will be able to determine a suitable size and dimension of the stopper hole to create the pressure barrier. Thestopper hole28 acts as a stop for the meniscus of liquid filling the microchannel, such that theliquid plug13 formed by thefirst liquid4 extends only in one direction along the length of themicrochannel3, away from thestopper hole28.

According to yet another embodiment, the cross-sectional dimensions of themicrochannel3 may be varied locally to affect the pressure within the microchannel interior. For example, the microchannel may be narrowed or widened at certain locations to increase or decrease the capillary forces acting on a fluid in the microchannel interior. One of ordinary skill in the art will be able to determine a suitable cross-sectional dimension to achieve a desired pressure within the microchannel interior.

Alternatively, as shown inFIG. 6, themicrochannel3′ includes two

fluid interface ports

17aand17b, two stopper holes,28aand28b, and avent hole8 disposed between the two

fluid interface ports

17aand17bto allow for filling of themicrochannel3′ with a plurality of different liquids. Afirst liquid4 is introduced into the interior of the microchannel through the firstfluid interface port17a. Thefirst stopper hole28acauses the first liquid to extend along the microchannel towards thevent hole8. Asecond liquid4′ is introduced into the interior of the microchannel through the secondfluid interface port17band extends along the microchannel towards thevent hole8, due to the presence and location of thesecond stopper hole28b. Air in the microchannel is released through thevent hole8.

After filling the microchannel via afluid interface port17, such as a filling hole disposed in the sidewall of the microchannel, the fluid interface port may be closed to prevent leakage of the fluid from the microchannel. According to the embodiment shown inFIG. 7a, thefluid interface port17 is closed by dispensing drops ofencapsulant21aonto thefluid interface port17. The encapsulant drops form acap21 and effectively close themicrochannel3. Alternatively, as shown inFIG. 7b, themicrochannel3 may be closed after filling by adhering aclosing layer41 to themicrochannel3 to cover thefluid interface port17.

According to another embodiment, as shown inFIG. 8, thefluid interface port17 may be utilized to provide a pumping mechanism to induce movement of liquid through the microchannel. As illustrated, thefluid interface port17 may be disposed adjacent to a closed or open end of themicrochannel3. The introduction of droplets into the microchannel through the fluid interface port results in movement of liquid through the microchannel. This movement corresponds to the frequency of the drops introduced to the hole. The movement of liquid induced by the introduction of droplets through the filling hole can be applied in many microfluidic applications as an accurate pumping and/or dosing mechanism. Those of ordinary skill will readily recognize that the microchannel can employ a hydrophobic patch or a stopper hole to induce liquid movement in a selected direction or any suitable means for conveying liquid through the microchannel.

According to another aspect of the invention, a second fluid sample can be introduced to themicrochannel3 through the same or differentfluid interface port17, after the microchannel is filled with thefirst liquid4.FIG. 9ais a side view of amicrochannel3 according to an illustrative embodiment of the invention including afluid interface port17 formed in theside wall16 of the channel and showing adroplet generating system18 for forming droplets of the second fluid sample to be introduced into themicrochannel3. Thefluid interface port17 provides a direct interface between the microchannel interior and the exterior. According to the present invention, the interface between afirst liquid4 in the microfluidic system and a surrounding gas phase is defined by the local absence of a solid wall in the microchannel, rather than a separate channel or reservoir structure.

According to one aspect, thevirtual wall15 formed in theport17 can be used as a sample introduction port for introducing the second liquid into thefirst liquid4 in the form of liquid droplets. A second liquid19acan be directly injected into thefirst liquid4 withinmicrochannel3 through thevirtual wall15 without requiring an intermediate structure, such as a sample introduction channel or a sample reservoir. According to the illustrative embodiment, the second liquid is introduced by forming adroplet19bof the second liquid19aand directing the droplet towards thevirtual wall15 with an appropriate speed and direction, indicated inFIG. 9aby velocity vector V, so as to traverse thevirtual wall15 and enter the interior of the microchannel.

The liquid droplets may be formed and dispensed using any suitabledroplet forming system18, such as the droplet dispensing systems described in U.S. Provisional Patent Application Attorney Docket Number CVZ-002-1 filed Sep. 25, 2001 and entitled “Two-Pin Liquid Sample Dispensing System”, the contents of which are herein incorporated by reference, and U.S. Provisional Patent Application No. 60/325,040 entitled “Droplet Dispensing System”, the contents of which are herein incorporated by reference.

The size of the aperture and the hydrophobicity of the fluid interface port determine the size of the dead volume. For example, the microchannel shown inFIG. 9ahas zero dead volume i.e. no liquid is retained in thefluid interface port17 and a sample injected through theport17 directly enters the microchannel interior. According to other embodiments, the first liquid may partially or totally fill the aperture, and the dead volume may be a non-zero, but substantially small, value. The dead volume also depends in whether themeniscus15 bulges up or down, a factor that is controlled by the hydrophobicity of theport17, the properties of the liquid filling themicrochannel3 and the size of the aperture forming theport17.

The relatively small dead volume provided by thevirtual wall15 results in a direct fluid interface allowing direct injection of a precise volume of sample into the interior of themicrochannel3 from the exterior of the microchannel. The ability to directly inject sample into the microchannel due to the low dead volume of thefluid interface port17 provides improved control over the amount of sample that is injected into the microchannel, allows efficient use of sample, and significantly reduces waste of the sample. Furthermore, the direct injection provided by the very small dead volume reduces or prevents cross-contamination between different samples and allows a third liquid to be directly injected into the system immediately after a second liquid without requiring flushing of the fluid interface port. Conversely, in conventional microfluidic systems employing a sample introduction channel, sample well or sample reservoir for introducing a fluid sample to a microchannel, the dead volume is significantly large relative to the size of the microchannel. In order to introduce a fluid sample into the microchannel interior, the fluid sample must first pass through the dead volume. A larger dead volume leads to dispersion of the sample, a time delay between the time of injection and the time when the sample enters the microchannel, injection inefficiency, potential cross-contamination between different samples and difficulty controlling the amount of sample that actually reaches the microchannel. These problems are avoided or reduced by the use of thefluid interface port17 forming avirtual wall15 according to the illustrative embodiment.

FIG. 9bshows a cross-sectional view perpendicular to themicrochannel3 at the location offluid interface port17, illustrating the process of introducing the second liquid19ainto thefirst liquid4 through thevirtual wall15. As illustrated inFIG. 9b, thedroplet generating system18 comprises a droplet carrying element for carrying the droplet. According to the illustrative embodiment, thedroplet carrying element180 comprises a pin, as described in U.S. Provisional Patent Application Attorney Docket Number CVZ-002-1 filed Sep. 25, 2001 and entitled “Two-Pin Liquid Sample Dispensing System”, and U.S. Provisional Patent Application No. 60/325,040 entitled “Droplet Dispensing System”, for introducing the droplet to the aperture by contacting thevirtual wall15.

FIG. 9cshows a cross-sectional view of themicrochannel3 immediately after injection of the second liquid19ain thefirst liquid4. As illustrated, the second liquid19aforms a well definedplug14 in thefirst liquid4. According to an alternate embodiment, the second liquid dissolves, merges or mixes into the first liquid. After introduction via the virtual wall, the second liquid19ais transported through the microchannel by thefirst liquid4.

According to one embodiment of the invention, illustrated inFIG. 9d, the second liquid, which is introduced into themicrochannel3 via the virtual wall, is immiscible with the first liquid.FIG. 9dshows a cross-sectional view of themicrochannel3 immediately after injection of the immiscible second liquid19ain thefirst liquid4. After penetrating thevirtual wall15, a substantially spherical, micelle-like liquid volume71 of the second liquid is formed in thefirst liquid4. As both liquids are immiscible with each other, thisliquid volume71 remains confined and can be separately transported along themicrochannel3.

The illustrative mode of liquid injection into a microfluidic system via a virtual wall and consecutive handling is particularly preferable for performing a liquid-liquid extraction. Liquid-liquid extraction is a well-known technique, widely used in many chemical analysis and synthesis steps, especially during drug discovery. In liquid-liquid extraction, two immiscible liquids are brought in intimate contact to allow the extraction of specific components from the liquid containing a specific substance (i.e. the source liquid) into the extraction medium (i.e. the target liquid). A suitable example of liquid-liquid extraction is the extraction of a water-soluble substance synthesized in an organic solvent, whereby said solvent is immiscible with water. The organic solvent containing the water-soluble synthesis product of interest is mixed with water to form small micelle-like droplets of solvent in the water phase. The water soluble substance diffuses out of the solvent and is collected in the water phase. The water phase can be re-collected after a predetermined time, due to the immiscible properties of the two substances. In this manner, the water soluble substance is thus extracted from the organic liquid phase. Of key importance is the total contact surface between both liquids. A larger exchanging surface, provided by the shape of the droplets, results in a faster extraction process.

The microfluidic system of the present invention may be utilized to perform a liquid-liquid extraction between a water soluble substance and an organic liquid phase, which is immiscible with water, as described below. To perform a liquid-liquid extraction between a water soluble substance and an organic liquid phase, theillustrative microchannel3 is filled with a suitablefirst liquid4, which is, according to the illustrative embodiment, an appropriate aqueous solution. Thefirst liquid4 forms avirtual wall15 at theopening17 disposed in the sidewall of the microchannel.Droplets19bof the organic phase containing the substance are formed and injected into thefirst liquid4 through thevirtual wall15. After thedroplets19btraverse the virtual wall, micelle-like liquid volumes71 are formed in themicrochannel3, which, due to their very small size, have a relatively very large exchange surface area. Consequently, the water soluble substance initially present in the micelle-like liquid volumes71 is extracted into thefirst liquid4 and is available for further processing. To concentrate the substance in a consecutive step, the extracted micelle-like liquid volumes71 are separated fromliquid3 by a suitable separating technique as known in the art, including, but not limited to, electrophoresis, dielectrophoresis, gravitational or body forces, capillary forces and special selective sieves.

According to an alternate embodiment, shown inFIG. 9e, thefluid interface port17 disposed in themicrochannel side wall16 and forming avirtual wall15 may have any suitable shape, such as a cylindrical or conical shape, having a suitably low dead volume for providing direct access to the microchannel interior. According to one embodiment, theinner wall63 of thefluid interface port17 is formed of or coated with a material that is repellant for thefirst liquid4 to repel the first liquid from theopening17. According to a preferred embodiment, theinner wall64 of themicrochannel3 is attractive for thefirst liquid4, to retain the first liquid inside the microchannel. The liquid repellent section in thefluid interface port17 prevents liquid from leaking out of the microfluidic system and ensures the repeatable formation of avirtual wall15 in thefluid interface port17 when the microchannel is filled with liquid. The use of aninner wall64 that is attractive for thefirst liquid4 further enhances automatic, passive capillary filling of themicrochannel3 via theport17, as described above, or by providing thefirst liquid4 at one end of themicrochannel3. As a result of capillary forces, themicrochannel3 may be automatically filled without requiring application of external energy or pressure sources, such as pumps or pressure chambers.

FIG. 9fillustrates an embodiment where the microchannels andvirtual wall15 are covered with acovering layer66. According to the illustrative embodiment, the coveringlayer66 comprises a liquid layer that is immiscible with thefirst liquid4 in the microchannel. The covering layer prevents the evaporation of thefirst liquid4 from the microchannel through theopening17, while still allowing the injection of a second liquid, such as liquid19a, into the microchannel through thecovering layer66 and thevirtual wall15.

According to an alternate embodiment, as shown inFIG. 9g, the fluid interface port in the microchannel is formed by anarray72 ofopenings17, each forming avirtual wall15 upon filling of themicrochannel3 with thefirst liquid4. Thevirtual walls15 in thearray72 are disposed in close proximity to each other, thereby allowing the injection of liquid via a wicking process, as illustrated inFIG. 9h. To introduce a second liquid into the microchannel, as shown inFIG. 9h, a selected amount of the second liquid19ais deposited on top of thearray72, such that the capillary forces wick the second liquid into themicrochannel3. According to a preferred embodiment, theinner walls63 of thefluid interface port17 are rendered repellant to thefirst liquid4 whilst theouter surfaces65 of thefluid interface ports17 preferably are rendered attractive to the second liquid19a. The use of an array of openings to form an array of virtual walls reduces the necessity and criticality of precisely targeting thedroplets19btowards a particular virtual wall. The droplets need only to be aimed in the direction of the array, allowing capillary forces to pull droplets into the channel interior. The velocity and direction of the propelled droplets are also not as important to achieve injection of the sample into themicrochannel3.

According to yet another embodiment of the invention, a plurality of openings are disposed in the sidewall of the microchannel to allow for the introduction or ejection of liquid via a virtual wall at a plurality of locations in the microchannel. For example, as shown inFIG. 10a, the microchannel can include multiple

fluid interface ports

17a,17bpositioned across or along the width of themicrochannel3 to define a plurality of virtual walls to allow for simultaneous introduction of a plurality of liquids. In this manner, an increased volume of liquid may be immediately injected into themicrochannel3 via the plurality of virtual walls. The use of a plurality of virtual walls across the microchannel width further allows for simultaneous introduction and mixing of a plurality of different liquids. As shown inFIG. 10a, the microchannel includes afirst liquid4. A second liquid19amay be introduced via a firstvirtual wall15a. Simultaneously, a third liquid190amay be introduced via a secondvirtual wall15b. The second liquid19aand the third liquid190amix together with thefirst liquid4, as illustrated by the diffusion profiles of the liquids.

Alternatively, as shown inFIG. 10b, themicrochannel3 may include a plurality of virtual walls disposed along the length of the microchannel to allow for sequential introduction of liquids into the microchannel, or ejection of a liquid from the microchannel along different locations in the fluid flow path.

Theillustrative microfluidic system10 employing avirtual wall15 in a fluid interface port may be utilized with a sample introduction system for forming and guiding a droplet into a microchannel via a virtual wall.FIG. 11aillustrates themicrofluidic system10 employing a suitablesample introduction system67 for guiding a chargeddroplet19dof a selected sample into amicrochannel3aof selected interest. The illustratedsample introduction system67 includes adroplet generator18 for forming droplets of a selected liquid, adroplet charging circuit53 for selectively charging a droplet, and adroplet guiding system150, comprising aground electrode54 and a plurality of electrically controlleddeflection plates56,57, for establishing an electrostatic field to direct the charged droplets to a selected location. Theillustrative microfluidic system10 can be coupled to electronics for controlling the formation and guidance of the droplet to a selected microchannel.

The droplets are generated by thedroplet generator18 having anozzle assembly70. Thenozzle assembly70 ejects liquid and forms individual droplets of the liquid at a breaking offpoint62. Thedroplet charging circuit53 is pre-programmed and a correspondingelectrode52 is positioned within the nozzle assembly to charge a pre-selected droplet with either a positive or negative charge at the breaking offpoint62. Surrounding the breaking offpoint62 of the droplet formation is aground electrode54. After breaking off and passing theground electrode54, the chargeddroplet19ctravels through an electrostatic field established by the first electrically controlleddeflection plate56 and the second electrically controlled deflection plate57. A plate-chargingcircuit55 is associated with each plate and controls the polarities thereof to provide proper electrical charging to the respective deflection plates. As shown inFIG. 11a, thedroplet19dis charged positively and is deflected towards avirtual wall15 disposed in the selectedmicrochannel3aby negatively charging thefirst plate56 and positively charging the second plate57. By controlling the charge on individual droplets withdroplet charging circuit53 as well as controlling the electric field between thefirst plate56 and the second plate57, droplets can be guided and effectively targeted to a particular channel to effect pre-programmed chemical experiments.

FIG. 11bis a cross-sectional view of a plurality ofmicrochannels3 with associatedfluid interface ports17 employing a different charging technique according to an alternate embodiment of the present invention. In the embodiment shown inFIG. 11b, thedroplet guiding system150 comprises anelectrode58 for each microchannel in the system and achannel charging circuit59 for charging the microchannels. Eachmicrochannel3 includes a correspondingelectrode58 for enhanced targeting of the droplets by selectively charging the corresponding microchannel. Theelectrode58 is connected to thechannel charging circuit59, associated with thedroplet charging circuit53 shown inFIG. 11a, for generating a selected charge in each of themicrochannels3. The channel charge interacts with the charged droplets to guide or steer the chargeddroplet19dtowards arespective microchannel3a. As seen inFIG. 11b, thedroplet19dis positively charged whilst a selectedmicrochannel3ais charged negatively, thereby resulting in an attractive force between the positively chargeddroplet19dand thevirtual wall15 of the negatively chargedmicrochannel3a. Guiding is enhanced by charging neighboring

microchannels

3band3cwith identical sign as the selecteddroplet19d, which consequently repel the positively chargeddroplet19dtowards the selectedmicrochannel3a.

FIG. 11cshows an alternate approach to enhance targeting of a chargeddroplet19dinto a selectedmicrochannel3aof a plurality ofmicrochannels3, according to the present invention. In the embodiment shown inFIG. 11c, thedroplet guiding system150 comprises a plurality of targeting electrodes61, each associated with a corresponding fluid interface port or microchannel. The fluid interface ports and corresponding microchannels are charged by a targeting electrode charging circuit61 to result in a force towards thevirtual wall15 of the selectedmicrochannel3a. As seen inFIG. 11c, the selecteddroplet19dis positively charged whilst the targeting electrode61 associated with the selected correspondingmicrochannel3ais charged negatively to guide the selecteddroplet19dinto a selected correspondingmicrochannel3avia thevirtual wall15. Guiding is enhanced by charging a neighboring targeting electrode61 with an identical sign as the selecteddroplet19dto repel the droplet away from the neighboring

microchannels

3b,3cand towards the selectedmicrochannel3a.

According to an alternate embodiment of the invention, thedroplet guiding system150 may comprise a machine vision system that can employ fiducial marks on one or more components of the system. One skilled in the art will recognize that any suitable droplet guiding system for directing a droplet towards a virtual wall may be utilized in accordance with the teachings of the invention.

According to the present invention, thefluid interface port17 of themicrofluidic system10 can optionally be utilized as a bidirectional fluidic interface for the microchannel. In addition to providing an interface for introducing a sample to a microchannel, the illustrative virtual wall formed in themicrochannel3 filled with thefirst liquid4 may also be utilized as an ejection port for ejecting fluid from the microchannel.FIGS. 12athrough12dshow a cutaway view of amicrochannel3 in which afluid interface port17 disposed in a sidewall of the channel can be used as an ejection port. Theport17 forms a virtual wall to allow the ejection of thefirst liquid4 frommicrochannel3. Asuitable ejector108 is provided in communication with the microchannel to effect ejection of the first liquid through thevirtual wall15. One skilled in the art will recognize that theejector108 may comprise any suitable device or system for ejecting a droplet from the microchannel via the virtual wall fluid interface port.

According to the illustrated embodiment shown inFIG. 12a, the ejector comprises afirst electrode34 disposed in thefirst liquid4 and anelectrospray electrode32 positioned in the vicinity of theport17. Avoltage generator31 is connected to thefirst electrode34 and theelectrospray electrode32 for applying a potential difference and generating an electric field between theelectrospray electrode32 and thefirst liquid4. The electric field generated by thevoltage generator31 between the electrodes results in an attractive force on thefirst liquid4, thereby effectively pulling thefirst liquid4 out of the microchannel through thevirtual wall15 in the form of adroplet33. The virtual wall and microchannel arrangement shown inFIG. 12acan also be used to receive droplets of a second liquid through the virtual wall, as discussed above, by removing the electric field, thereby achieving a bi-directional interface.

FIG. 12bshows another embodiment of themicrofluidic system10 of the present invention suitable for ejecting thefirst liquid4 from themicrochannel3. According to this embodiment, the ejector comprises apressure pulse generator51 in communication with thefirst liquid4 in themicrochannel3. Thepressure pulse generator51 applies a pressure pulse having a selected amplitude, frequency and duration to thefluid4 to effectively eject thefirst liquid4 through the virtual wall to form adroplet33. Thepressure pulse generator51 can be any suitable structure for generating a pressure pulse within themicrochannel3 and can include a piezoelectric element, an electromagnetic actuator, a heater, an electrostatic actuator, comprising electrodes that move under the influence of an applied voltage, an electrophoretic pressure actuator, comprising electrodes in channel on which a voltage pulse is applied, pressurized gas or any other suitable pressure pulse generator.

Thepressure pulse generator51 can either be integrated at least partly within themicrochannel3 or can be fully external and working with the channel, such as a rod-like moving actuator tapping locally on the channel.

According to another embodiment, shown inFIG. 12c, a gas pressurizer51ais utilized to eject the liquid from the microchannel. According to the illustrative embodiment, the gas pressurizer51acomprises a pressurized gas tank, which applies pressure via a fast valve. A secondfluid interface port17bforming a secondvirtual wall15bcan be formed in themicrochannel3. The secondvirtual wall15bis substantially coaxially aligned with the first virtual wall formed in the firstfluid interface port17. The second, coaxialvirtual wall15bis in communication with the gas pressurizer51awhich generates a gas pressure pulse having a selected amplitude, frequency and duration as to effectively eject thefirst liquid4 through the firstvirtual wall15 in the form of adroplet33. During the ejection process, the secondvirtual wall15bis displaced inwards towards thefirst liquid4 so as to form thedroplet33.

According to an alternate embodiment, as shown inFIG. 12d, the ejector comprises a heater51bdisposed in themicrochannel3 along the side wall. The heater51blocally heats thefirst liquid4 in order to form a droplet. The heating of the first liquid results in a quickly growing gas vapor bubble51c, which effectively ejects thedroplet33 from themicrochannel3 via thevirtual wall15. The heater may comprises a heated spot, an electrical heater, an optically induced heater or any other suitable heater.

According to yet another embodiment of the invention, thevirtual wall15 formed in thesidewall16 of themicrochannel3 is utilized to optically analyze the interior of the microchannel.FIG. 13ashows an embodiment of the invention where thefirst liquid4 disposed in the microchannel is optically inspected by alight beam27 focused by anoptical element26. Thelight beam27 is co-axially aligned with thefluid interface port17 in the sidewall as to directly penetrate thefirst liquid4 without impinging upon thechannel wall16 opposite theport17. Theoptical element26 can be any suitable lens or prism. Asuitable detector68 is disposed adjacent to thevirtual wall15 to monitor and analyze the liquid in themicrochannel3. In an alternate embodiment, a plurality of

fluid interface ports

17 and17bare at disposed in the side wall at right angles to each other and scattering, rather than absorption, is analyzed.

According to an alternate embodiment for optically analyzing thefirst liquid4 through thevirtual wall15, a secondvirtual wall15bis disposed in themicrochannel3.FIG. 13bshows an embodiment for optical analysis offirst liquid4 inmicrochannel3, in which a firstfluid interface port17 forming a firstvirtual wall15 is disposed on one side of themicrochannel3 and a secondfluid interface port17bforming a secondvirtual wall15bis disposed on the opposite side ofmicrochannel3, such that the firstvirtual wall15 and the secondvirtual wall15bare substantially co-axially arranged. Thelight beam27 passing through and focused by theoptical element26 is substantially co-axially positioned relative to the first virtual wall such that thelight beam27 directly penetrates thefirst liquid4 through theport17, and after traveling through thefirst liquid4 exits on the opposing side of the microchannel via the secondvirtual wall15b. The light passing through thesecond port17bcan be collected, focused or collimated by a second optical element26ain communication with anoptical detector68 to monitor and analyze the liquid in themicrochannel3. Such optical detection means are ideally suitable for analyzing liquids containing compounds that are intrinsically colored or fluorescent, or that are labeled with an optically detectable moiety as described elsewhere herein. Alternatively, mass spectrometry may be used as a detection means when a microfluidic chip of the present invention is interfaced with a mass spectrometer.

According to one application of the present invention, a microfluidic system employing an aperture sized and dimensioned to form a virtual wall as a fluid interface port may be utilized to purify or filter a sample. For example, the illustrative arrangement may be utilized to purify a DNA sample by separating contaminants from the DNA fragments in the sample. As shown inFIG. 14, amicrochannel3 having afluid interface port17 formed in the sidewall and defining a virtual wall is connected to a plurality ofwaste channels136 and anoutlet channel137 formed at the intersection of thewaste channels136 and the microchannelmain body3. Themicrochannel3 has aninlet138 having a smaller diameter than the main body of the microchannel. Themicrochannel3 is filled with asuitable washing medium4 to effect separation of a sample that is introduced via the virtual wall.

To achieve separation of a sample, such as the separation of a DNA sample from contaminants, a fluid flow is induced in the washing medium through the microchannel and the sample is injected into the flowing washing medium via thevirtual wall15. The arrangement exploits diffusion to separate the different components of the sample. The larger molecules (i.e. the DNA fragments) in the sample and the smaller molecules in the sample (i.e. the contaminants) diffuse in the washing medium with different diffusion rates, which effectively separates the different components of the sample according to size. For example, the smaller molecules diffuse into the washing medium faster than the larger molecules. The two sample streams, the residual sample stream containing the larger particles and the diffused sample stream containing the contaminants, are separated into theoutlet channel137 and thewaste channels136, respectively. The purified sample may pass through theoutlet channel137 for further processing, or analysis. Thewaste channels136 and theoutlet channel137 may be sized and positioned to receive a selected component. For example, thewaste channels136 are positioned a predetermined distance from thevirtual wall15 to receive the diffused smaller molecules and theoutlet channel137 is sized and positioned to receive the larger molecules.

Alternatively, the configuration shown inFIG. 14 may be utilized to perform chemical manipulations, such as a chemical reaction, non-covalent binding, adsorption or absorption, antibody binding, nucleic acid or oligonucleotide binding or hybridization, ion pairing, ion exchange, chromatographic separation, receptor hormone interaction, enzyme activity antagonism or agonism, or other suitable reaction on an analyte. The present invention applies to a variety of liquid samples, including solutions of compounds, whole cells or cell lysates, enzymes, proteins or peptides, and particles. Accordingly, the invention also has applications in proteomics, genomics, chromatography, diagnostics, and drug discovery.

Alternatively, the illustrative configuration may be utilized to perform a labeling operation, where the virtual wall is utilized as an interface port for one of the reactants in the labeling scheme. To perform a labeling operation, a labeling fluid is run through the microchannel and a liquid containing a substance to be labeled is injected through the virtual wall. The mixing of the two liquids is relatively fast, achieving rapid labeling of the substance. According to one embodiment, the labeling can be performed before a separation step, such as described above. According to one embodiment, a filtration device may also be utilized to remove excess unreacted label after the labeling scheme.

Depending on the particular type of detection method employed, a wide variety of detectable labels may be used in applications of the present invention. Labels are commonly detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. The label is coupled directly or indirectly to a molecule to be detected (a product, substrate, enzyme, or the like) according to methods well known in the art, and for example, as reagents introduced onto a chip via a virtual wall formed in a side wall of a microchannel according to the illustrative embodiment of the present invention. For example, useful nucleic acid labels include fluorescent dyes, enzymes (e.g., as commonly used in an ELISA), biotin, dioxigenin, or haptens and proteins for which antibodies, preferably monoclonal, are available. Other suitable labels include fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Still other labeling agents include monoclonal antibodies, polyclonal antibodies, proteins, or other polymers such as affinity matrices, carbohydrates or lipids. Detection of labeled compounds may be by a variety of known methods, including spectrophotometric or optical tracking of fluorescent markers, or other methods which track a molecule based upon size, charge, molecular weight, or affinity. A detectable moiety can be of any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of gel electrophoresis, column chromatograpy, spectroscopic techniques, and the like, and in general, labels useful in such methods can be applied to the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical thermal, or chemical means; and such label may be bound covalently to the molecules of interest (e.g., reaction of amine-containing compounds with ninhydrin) or non-covalently (e.g., reaction of a compound with a labeled antibody). Useful labels in the present invention include fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), ezymes (e.g., LacZ, CAT, horse radish peroxidase, alkaline phosphatase and others, commonly used as detectable enzymes, either as marker products or as in an ELISA), nucleic acid intercalators (e.g., ethidium bromide) and colorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads. Fluorescent labels are particularly preferred labels when optical detection means are employed. Preferred labels are typically characterized by one or more of the following: high sensitivity, high stability, low background, low environmental sensitivity and high specificity in labeling.

Several labels comprising fluorescent moieties are known, including 1- and 2-aminonaphthalene, p,p″-diaminostilbenes, pyrenes, quaternary phenanthridine salts, 9-aminoacridines, p,p″-diaminobenzophenone imines, anthracenes, oxacarbocyanine, merocyanine, 3-aminoequilenin, perylene, bis-benzoxazole, bis-p-oxazolyl benzene, 1,2-benzophenazin, retinol, bis-3-aminopyridinium salts, hellebrigenin, tetracycline, sterophenol, benzimidazolylphenylamine, 2-oxo-3-chromen, indole, xanthen, 7-hydroxycoumarin, phenoxazine, calicylate, strophanthidin, porphyrins, triarylmethanes and flavin. In some cases, the amino acid tryptophan, which is either part of a peptide or protein of interest (i.e., it is endogenous to that peptide or protein) or which is added to said protein or peptide, may be used as a fluorescent label. Individual fluorescent reagent compounds which may be used in accordance with the invention, or which can be modified to incorporate such functionalities include, e.g., dansyl chloride; fluoresceins such as 3,6-dihydroxy-9-phenylxanthhydrol; rhodamineisothiocyanate; N-phenyl 1-amino-8-sulfonatonaphthalene; N-phenyl 2-amino-6-sulfonatonaphthalene; 4-acetamido-4-isothiocyanato-stilbene-2,2′-disulfonic acid; pyrene-3-sulfonic acid; 2-toluidinonaphthalene-6-sulfonate; N-phenyl-N-methyl-2-aminoaphthalene-6-sulfonate; ethidium bromide; stebrine; auromine-0,2-(9′-anthroyl)palmitate; dansyl phosphatidylethanolamine; N,N′-dioctadecyl oxacarbocyanine: N,N′-dihexyl oxacarbocyanine; merocyanine, 4-(3′pyrenyl)stearate; d-3-aminodesoxy-equilenin;12-(9′-anthroyl)stearate; 2-methylanthracene; 9-vinylanthracene;2,2′(vinylene-p-phenylene)bisbenzoxazole; p-bis(2-(4-methyl-5-phenyl-oxazolyl))benzene; 6-dimethylamino-1,2-benzophenazin; retinol; bis(3′-aminopyridinium) 1,10-decandiyl diiodide; sulfonaphthylhydrazone of hellibrienin; chlorotetracycline; N-(7-dimethylamino4-methyl-2-oxo-3-chromenyl)maleimide; N-(p-(2-benzimidazolyl)-phenyl)maleimide; N-(4-fluoranthyl)maleimide; bis(homovanillic acid); resazarin; 4-chloro-7-nitro-2,1,3-benzooxadiazole; merocyanine 540; resorufin; rose bengal; and 2,4-diphenyl-3(2H)-furanone. Many such fluorescent labeling reagents are commercially available from SIGMA chemical company (Saint Louis, Mo.), Molecular Probes, R&D systems (Minneapolis, Minn.), Pharmacia LKB Biotechnology. (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersberg, Md.), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), and Applied Biosystems (Foster City, Calif.) as well as other commercial sources known to one of skill in the art.

Fluorescent labels are one preferred class of detectable labels, in part because by irradiating a fluorescent label with light, one can obtain a plurality of emissions. Thus, a single label can provide for a plurality of measurable events. Detectable signal may also be provided by chemiluminescent and bioluminescent sources. Chemiluminescent sources include a compound which becomes electronically excited by a chemical reaction and may then emit light which serves as the detectible signal or donates energy to a fluorescent acceptor. A diverse number of families of compounds have been found to provide chemiluminescence under a variety or conditions. One family of compounds is 2,3-dihydro-1,4-phthalazinedione. The most popular compound is luminol, which is a 5-amino compound. Other members of the family include the 5-amino-6,7,8-trimethoxy- and the dimethylamino[ca]benz analog. These compounds can be made to luminesce with alkaline hydrogen peroxide or calcium hypochlorite and base. Another family of compounds is the 2,4,5-triphenylimidazoles, with lophine as the common name for the parent product. Chemiluminescent analogs include para-dimethylamino and -methoxy substituents. Chemiluminescence may also be obtained with oxalates, usually oxalyl active esters, e.g., p-nitrophenyl and a peroxide, e.g., hydrogen peroxide, under basic conditions. Other useful chemiluminescent compounds are also known and available, including —N-alkyl acridinum esters (basic H₂O₂) and dioxetanes. Alternatively, luciferins may be used in conjunction with luciferase or lucigenins to provide bioluminescence. An illustrative example of on-chip labeling with a fluorescent moiety may be found in Harrison, et al., Sensors and Actuators B, vol. 33, pp. 105-09 (1996).

Other labeling moieties may be non-covalently bound to molecules of interest. Generally, a ligand molecule (e.g., biotin) is covalently bound to a polymer. The ligand then binds to an anti-ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. A number of ligands and anti-ligands can be used. Where a ligand has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, it can be used in conjunction with labeled, anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody. Labels can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol. Means of detecting labels are well known to those of skill in the art. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence, e.g., by microscopy, visual inspection, via photographic film, by the use of electronic detectors such as digital cameras, charge coupled devices (CCDs) or photomultipliers and phototubes, and the like. Fluorescent labels and detection techniques, particularly microscopy and spectroscopy are preferred. Similarly, enzymatic labels are detected by providing appropriate substrates for the enzyme and detecting the resulting reaction product. Finally, simple colorimetric labels are often detected simply by observing the color associated with the label. For example, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.

FIG. 15 illustrates a microfluidic synthesis/analysis system40 of an embodiment of the present invention for performing a microchemical process of a sample on a chip. The microfluidic synthesis/analysis system40 comprises afirst microchannel3, asecond microchannel30 operating in parallel with the first microchannel, and athird microchannel300 disposed at the end of the first and second microchannels and forming an intersection with the first and second microchannels for combining the output of thefirst microchannel3 and thesecond microchannel30. The

microchannels

3,30 and300 include a plurality offluid interface ports17 formingvirtual walls15a-idisposed in the channel side walls for performing a multi-step chemical synthesis or analysis. The illustrative embodiment of the microfluidic synthesis/analysis system40 serves as an example of the application ofvirtual walls15 in microfluidic systems in which complex reaction schemes for synthesis (e.g. labeling, as described above) and analysis are to be performed in a highly parallel fashion. As shown, a large variety of chemical operations can be implemented by using avirtual wall15 disposed in asidewall16 of amicrochannel3.

The illustratedsystem40 further includes a plurality of sample processors, illustrated as

microreactors

43,44,45 and46, disposed at selected locations in the

microchannels

3,30 and300 for performing one ore more reactions on a sample, such as a microchemical analysis or synthesis. According to the illustrative embodiment, the illustrated system is utilized to perform a separation of a sample. One skilled in the art will recognize that any suitable process may be performed on a sample, including, but not limited to a reaction, filtration, dilution, mixing, binding and transporting, alone or in combination with other reactions. The specific arrangement shown inFIG. 15 allows performing of the following Reactions 1-4:

InReaction 1, substance A (e.g. a labeling reagent) reacts with substance B (e.g., a molecule of interest not otherwise conveniently detectable) to form substance C (e.g a labeled conjugate). In Reaction 2, substance D reacts with substance E to form substance F. InReaction 3, substances C and F react to form substance G. InReaction 4, substances G and H react to form substance I. According to the illustrated embodiment,Reaction 1 and Reaction 2 are carried out in parallel, sequentially followed byReaction 3 andReaction 4.

In operation, acarrier liquid4ais disposed in the microfluidic synthesis/analysis system40 and a number liquids containing a specific chemical substance are interfaced with thecarrier liquid4avia a plurality ofvirtual walls15. The reaction products resulting from Reactions 1-4 are carried through the microfluidic synthesis/analysis system bycarrier liquid4a. Droplets42a-iare formed of liquids respectively containing substances A-I listed in the above reaction scheme. The overall direction of flow of thecarrier liquid4 is indicated byarrows41. The direction of fluidic interfacing via thevirtual walls15 is indicated byarrows47.

To initiate the microfluidic process, afirst liquid A42ais introduced into the microfluidic system via a firstvirtual wall15a, as indicated bydroplet direction47, and carried through the microchannel by thecarrier liquid4a. Asecond liquid B42bis added to thefirst microchannel3 through a secondvirtual wall15bdisposed in the side wall of the microchannel downstream from the firstvirtual wall15a, giving rise to a mixture of substances A and B present incarrier liquid4a. Subsequently, the mixture passes through afirst microreactor43. Thefirst microreactor43 has appropriate conditions (i.e. temperature, residence time, presence of catalytic materials etc.) to effectReaction 1 and produce a third substance, liquid C, from the reaction of substances A and B. Liquid C is then carried downstream from thereactor43 by thecarrier liquid4a. After completion ofReaction 1, portions of liquid C are ejected from thefirst microchannel3 through a thirdvirtual wall15c, disposed downstream of the first and second

virtual walls

15a,15c, in the form of adroplet42c, as indicated bydroplet direction47. The ejected portion of liquid C may be subsequently stored, further processed or analyzed to determine the composition of the reacted liquid C.

In alternate embodiments of the invention, one or more of the

sample processors

43,44,45 or46 may comprise a separation means, rather than a reaction means such as a microreactor. A separation means is typically a chromatography column, preferably a chromatography column. A microfluidic system including a chromatography column may be used to perform a separation of a mixture applied, for example, viavirtual wall17, and then reacted with a labeling reagent inmicroreactor45 with a reagent introduced through thevirtual wall15. In such a manner, compounds are labeled post-chromatographic separation. In like manner, a mixture may be introduced though a firstvirtual wall15aand a labeling reagent through a secondvirtual wall15b, followed by reaction in microreactor and subsequent separation in a chromatography column. Capillary electrophoresis chromatography columns are particularly preferred separation means according to the invention. Such microfluidic CE columns are described in U.S. Pat. Nos. 6,159,353, 5,976,336, and 6,258,263, each of which are incorporated herein by reference. Alternatively, multiple separation means, optionally in parallel, may be employed by the present invention. For example, the microfluidic synthesis/analysis system40 may perform a first separation by capillary electrophoresis, and another separation based on a pH gradient.

In an alternate embodiment, a titration may be performed on-chip when a color change occurs upon reaction. Such a titration may be, for example, a pH titration, or a titration of an enzyme with a chromogenic substrate or inhibitor.

At the point of intersection of thefirst microchannel3 and thesecond microchannel30, the liquid C (42c) leaving thefirst microreactor43 and the liquid F (42f) leaving thesecond microreactor44 mix together and are subsequently carried through thethird microchannel300. The mixture of liquid F and liquid C enters thethird microreactor45, in whichReaction 3 proceeds and produces liquid G. A seventhvirtual wall15gdisposed downstream from the third microreactor to allow for portions of liquid G leaving thethird microreactor45 to be ejected frommicrochannel3 in the form of liquid droplets42g. An eighthvirtual wall15hdisposed further downstream from the seventh virtual wall liquid15gis utilized to introduce substance H into themicrochannel300 in the form ofliquid droplet42h, resulting in a mixture of liquid G and liquid H. The G-H mixture enters afourth microreactor46, in whichReaction 4 ensues to form liquid I. Finally, portions of the resulting liquid142ileaving thefourth microreactor46 are ejected from themicrochannel3 via a ninthvirtual wall15idisposed in the sidewall of themicrochannel300 for analysis, storage and/or further processing of the liquid I.

According to the illustrative embodiment, the required concentrations of reactants A, B, D, E and H are precisely controlled by metering the size and number of droplets (19b). Moreover, by controlling the number ofdroplet19bintroduced incarrier liquid4a, a specific dilution can precisely be obtained allowing the study of reactions 1-4 for different dilutions of reactants A, B, D, E and H. A suitable droplet dispensing system for forming droplets of suitable size is described in ProvisionalU.S. Patent Application 60/325,040. The use of a direct interface port formed by a virtual wall in the sidewall of the microchannel in the illustrative microfluidic synthesis/analysis system40 allows precise control over the concentrations of liquids that are introduced into the system and significantly reduces waste while increasing efficiency.

The illustrative embodiment the microfluidic synthesis/analysis system40 described above serves as an example of the application of avirtual wall15 in microfluidic systems in which complex reaction schemes for synthesis and analysis are to be performed in a highly parallel fashion. As shown, a large variety of chemical operations can be implemented by usingvirtual wall15 disposed in a sidewall of a microchannel. One skilled in the art will recognize that the invention is not limited to the illustrative embodiment and that any suitable size and number of microchannels, virtual walls, reactor types and numbers and sample types may be utilized in accordance with the teachings of the invention.

FIG. 16 illustrates an alternate embodiment of the microfluidic system of the present invention. Thesystem10 includes amicrochannel3 including one or more fluid interface ports forming avirtual wall15 and comprises a part of an electrokinetically operatedsystem22. In the electrokinetically operatedsystem22, an electric field is established to transport fluid through themicrochannel3. As illustrated afirst electrode5 and asecond electrode7 are disposed in or in fluid communication with thefirst liquid4 in themicrochannel3. Thefirst electrode5 is disposed in afirst well6alocated at a first end of themicrochannel3 and thesecond electrode7 is disposed in asecond well6blocated at a second end of themicrochannel3. Thefirst well6aand thesecond well6bare in fluid communication with thefirst liquid4 in themicrochannel3. Avoltage generator12 generates a voltage between thefirst electrode5 and thesecond electrode7, which produces a substantially longitudinal electric field in thefirst liquid4 in themicrochannel3. As shown inFIG. 16, the longitudinal electric field is applied by bringing both ends ofmicrochannel3 in fluidic contact respectively with afirst well4 and a second well6.

The illustratedport17 is disposed in the sidewall of themicrochannel3 and forms avirtual wall15 allowing the introduction of a second liquid into afirst liquid4, according to the teachings of the invention. For example, thedroplet generating system18 forms adroplet19aof thesecond fluid19b. Thedroplet19ais directed towards theport17 via any suitable method, such as those described herein. Depending on the surface properties of the inner walls of themicrochannel3, the longitudinal electric field generated by theelectrokinetic system22 induces an electroosmotic liquid flow of thefirst liquid4 in an axial direction through themicrochannel3, thereby substantially transporting the constituents present in the second liquid19bin the axial direction through the microchannel.

The electrokinetically operatedsystem22 can include anoptional detector68 to provide for electrophoretic analysis of the constituents present in the second liquid19b. Theopening17 disposed in themicrochannel3 has suitable properties, such as a selected diameter, length, and inner wall surface properties, to provide electrophoretic separation of the constituents of a liquid. Asecond liquid19bto be analyzed is introduced to themicrochannel3 via thevirtual wall15 in the form of adroplet19agenerated by adroplet generating system18. Constituents present in the introduced second liquid19bare transported by a combination of electroosmotic flow and migration under the influence of the electric field applied with thevoltage generator12. After traveling a sufficient distance to result in electrophoretic separation of the liquid, thedetector68 detects and analyzes the individual constituents at the end ofmicrochannel3 by thedetector68. Thedetector68 generates an electropherogram from which the composition of second liquid19acan be determined. Those of ordinary skill will understand that the electrokinetic system of the invention can perform electroosmotic, electrophoretic, and dielectrophoretic techniques.

FIG. 17 illustrates another implementation of an electrokinetically operatedmicrofluidic system50 according to the present invention comprising a plurality ofparallel microchannels3a-3hformed in acartridge73 in which asubstrate20 is disposed. The microchannels are formed by forming a network of half-open channel structures in the substrate and covering the half-open channels with acover20ato form the plurality of microchannels. The plurality ofmicrochannels3a-3hare disposed in the substrate in communication with a commonfirst well6aand a commonsecond well6b. Themicrochannels3a-hinclude one or morefluid interface ports17 defined by openings formed in the channel side wall that are sized and dimensioned to form virtual walls when the microchannels are filled with afirst liquid4. Afirst electrode5 connected to avoltage generator12 is disposed in the commonfirst well6aand a second electrode connected to thevoltage generator12 is disposed in the commonsecond well6bto establish a substantially longitudinal electric field in themicrochannel3.Droplets19bare generated by adroplet generator18. Thedroplet generator18 forms and propels the droplets towards a selectedvirtual wall15 formed in a fluid interface port form in the side wall of a selected microchannel in a suitable direction and with a suitable velocity to introduce the droplet to the selectedmicrochannel3 via theopening17. Adetector68 is positioned relative to thesystem50 to monitor and detect the fluid in themicrochannels3a-3h.

In a preferred embodiment, the parallel implementation of an electrokinetically operatedsystem50 is applied as a highly parallel electrophoretic separation platform, capable of performing a large number of analyses per unit of time. Themicrochannels3 disposed in thesubstrate20 have suitable properties for electrophoretic separation (inner wall surface properties, diameter and length). The process of electrophoretic analysis of the constituents ofdroplet19bis identical as described before. The electrokinetically operatedapparatus50 further includes adetector68 for detecting and analyzing the individual constituents at the end ofmicrochannels3. Thedetector68 generates an electropherogram from which the composition of second liquid19acan be determined.

As shown, the illustrative electrokinetically operatedsystem50 comprises a compact structure, which allows a plurality of different reactions and processes to occur on a relativelysmall substrate20. The use of openings forming virtual walls to define fluid interface ports in the side walls of theparallel microchannels3a-3hallows direct interfacing of fluid with the microchannels, improves injection efficiency and provides easy control over the volume of liquid introduced into thesystem50.

According to yet another application, the virtual walls may be utilized to interface amicrofluidic system181 with a mass spectrometer, as shown inFIG. 18. For example, a sample may be injected into amicrochannel3 via avirtual wall15 according to the teachings of the invention and the sample may then be separated into different components. After separation, the different components may be ejected from the microchannel through avirtual wall15 forming a fluid ejection port in the form of droplets. The droplets may be directed onto asuitable plate185 for analysis with amass spectrometer186 or other robotic system. For example, themicrofluidic system181 may inject the separated samples onto a multi-well plate to form a multi-well array of the sample for analysis.

FIGS. 19a,19band19cillustrate a method of manufacturing amicrofluidic chip190 having virtual wall interface ports for processing liquid samples.FIG. 19ais an exploded view of amicrofluidic chip190 employing virtual wall fluid interface ports according to the teachings of the invention.FIG. 19bis a top view of themicrofluidic chip190 ofFIG. 19a.FIG. 19cis a cross-sectional side view of the manufacturedmicrofluidic chip190 ofFIG. 19a. As shown, amicrochannel3 with a virtual wallfluid interface ports17 formed in the side wall of the microchannel may be manufactured with a three-layer “sandwich” construction. As shown, a completemicrofluidic chip190 having amicrochannel3, afirst inlet191 formed at a first end of themicrochannel3, anoutlet192 formed at a second end of themicrochannel3, and afluid interface port17 formed in a side wall along the length of themicrochannel3. The microfluidic chip shown inFIG. 19 comprises a firstplanar sheet193 having arecess194 formed therein, amiddle layer197 including achannel3 and anopening17 and a secondplanar sheet195. Theopening17 has dimensions between about 0.1 μm and about 200 μm and preferably between about 25 μm and about 125 μm and most preferably between about 50 μm and about 100 μm, so that a liquid disposed in the microchannel forms a virtual wall at theopening17.

To manufacture themicrofluidic chip190, a portion of the firstplanar sheet193 is removed to form therecess194. Next, the middle layer is applied on top of the first sheet. Then, a portion of themiddle layer197 is removed to form theopening17. Additional portions of themiddle layer197 are removed to form slits defining the microchannel, which are aligned with the recess. The secondplanar sheet195 is applied to themiddle layer197, and a virtualwall access hole170 is formed in the secondplanar sheet195 prior to application or after application of the second planar sheet to the middle layer by removing a portion of the second planar sheet. The virtualwall access hole170 is aligned with theopening17 formed in the middle layer to provide access to the microchannel. A low temperature bonding process may be utilized to assemble the three layers forming the chip.

According to the illustrative embodiment, the first and second

planar sheets

193 and195 comprise glass plates, though one skilled in the art will recognize that any suitable material may be used. Etching, powderblasting, or any suitable method may form therecess197,channel3,inlet191,outlet192 and the

openings

17 and170 forming the fluid interface port. According to an alternate embodiment, therecess197,channel3,inlet191,outlet192 and the

openings

17 and170 may be pre-formed in the layers, i.e. the layers may be molded to form the structures. According to the illustrative embodiment, themiddle layer197 is comprised of a photo patternable material, such as a photosensitive polymer applied by lamination. According to an alternate embodiment, themiddle layer197 of themicrofluidic chip190 also comprises a glass plate.

The use of glass plates to form themicrofluidic chip190 yields better optical detection of the microchannel3 interior, as the top and bottom of the microchannel are formed by two parallel glass surfaces. In conventional round capillaries and also in etched channels on chip (also partly round surfaces) there is substantial light scattering due to the curved surfaces through which the light should pass. The glass surfaces are very flat and smooth to enhance the flow of fluid through themicrochannel3, and further facilitate and enhance electrophoresis. Furthermore, using glass substrates significantly reduces the cost of manufacturing themicrofluidic chip190.

According to one embodiment, a plurality of microchannels may formed in the microfluidic chip, and can be configured to intersect with each other.

FIGS. 20a-cillustrate the steps of manufacturing a microchannel having afluid interface port17 defined by an opening suitable for forming a virtual wall according to an embodiment of the invention.FIG. 20ais a cross-sectional view of asubstrate28 in which a first portion of anopen channel29 is formed. To form an enclosed microchannel, thesubstrate28 is then covered with acover30, as shown inFIG. 20b. Subsequently, at least a portion of thecover30 is removed to form thefluid interface port17 inmicrochannel3, as shown inFIG. 20c. As discussed, thefluid interface port17 is sized and dimensioned to form a virtual wall when the microchannel is filled with a first liquid. In another embodiment thefluid interface port17 is disposed in thecover30 prior to bonding thecover30 on top ofsubstrate28.

According to the illustrative embodiment, thesubstrate30 and thecover28 are formed of silicon, though one skilled in the art will recognize that any suitable material for forming amicrochannel3 in a microfluidic device or system may be utilized. For example, the microfluidic system may be made out of glass, plastic or any other suitable material. Themicrochannel3 may be fabricated from asilicon wafer substrate30 using a standard photolithography etching process to fabricate the microchannel structures. A photolithography process may also be utilized to etch thefluid interface port17 in thecover28. One skilled in the art will recognize that alternative materials and manufacturing techniques, such as wet chemical etching, controlled vapor deposition, laser drilling, and the like, may be utilized.

FIGS. 21a-21cillustrate the steps of manufacturing a microchannel with a virtual wall interface port according to another embodiment of the invention.FIG. 21ais a cutaway view of asubstrate28 in which a halfopen channel structure29 is disposed. Thesubstrate28 is then covered with afirst cover30aon which asecond cover30bis disposed, as shown inFIG. 21bto form theenclosed microchannel3. Subsequently, at least a portion of thefirst cover30aand a portion of thesecond cover30bare removed as to form thefluid interface port17 inmicrochannel3. As shown, the opening extends through thefirst cover30aand thesecond cover30bto form a fluidic interface between the interior of themicrochannel3 and the exterior of the microchannel. In another embodiment thefluid interface17 is formed infirst cover30aandsecond cover30bprior to bonding of said first and

second cover

30aand30bto the top of thesubstrate28.

According to the illustrative embodiment, thesubstrate30 and the first and second covers30aand30bare formed of silicon, though one skilled in the art will recognize that any suitable material for forming amicrochannel3 in a microfluidic device or system may be utilized. For example, the microfluidic system may be made out of glass, plastic or any other suitable material. Themicrochannel3 may be fabricated from asilicon wafer substrate30 using a standard photolithography etching process to fabricate the microchannel structures. A photolithography process is utilized to etch thefluid interface port17 in the first and second covers30aand30b. One skilled in the art will recognize that alternative materials and manufacturing techniques, such as wet chemical etching, controlled vapor deposition, laser drilling, and the like, may be utilized.

FIGS. 22a-22cillustrate the steps of manufacturing a microchannel having a virtual wall as a fluidic interface port according to an alternate embodiment of the invention. In the embodiment shown inFIGS. 22a-22c, the opening forming the virtual wall is formed in a substrate having a half-open channel structure, rather than a cover for enclosing the half-open channel structure.FIG. 22ashows an exploded view of a substantially planarfirst cartridge part35 in which a halfopen channel structure37 is disposed. A portion of the side wall of is removed from the halfopen channel structure37 in thesubstrate35 to form afluid interface port17. Asecond cartridge part36 is bonded to the top of thefirst cartridge part35 to define amicrochannel3.

According to the illustrative embodiment, thefirst cartridge part35 and thesecond cartridge part36 are formed of silicon, though one skilled in the art will recognize that any suitable material for forming amicrochannel3 in a microfluidic device or system may be utilized. For example, the microfluidic system may be made out of glass, plastic or any other suitable material. Themicrochannel3 may be fabricated in thefirst cartridge part36 using a standard photolithography etching process. A photolithography process is utilized to etch thefluid interface port17 in the side wall of the first cartridge part. One skilled in the art will recognize that alternative materials and manufacturing techniques, such as wet chemical etching, controlled vapor deposition, laser drilling, and the like, may be utilized according to the teachings of the present invention.

FIG. 22bandFIG. 22cshow respectively a perspective view and a cutaway view of the resultingvirtual wall15 formed in the microchannel. As illustrated, thefluid interface port17 formed in the microchannel is sized and dimensioned to form a virtual wall at theopening17 when the microchannel is filled with afirst liquid4. Thevirtual wall15 allows fluidic interfacing with the microchannel as described above and is sized and dimensioned to retain liquid within the microchannel without adversely affecting liquid flow through the microchannel.FIGS. 19band19cillustrate the introduction of adroplet19bof a second liquid19ainto afirst liquid4 present in themicrochannel3 via thevirtual wall15 formed within theopening17. As shown, the droplet is propelled towards the virtual wall and traverses the virtual wall to enter the microchannel.

FIGS. 23aand23billustrate an alternate embodiment of the microchannel system ofFIGS. 19-19c.FIG. 23ashows an exploded view of a substantially planarfirst cartridge part35 in which a halfopen channel structure37 is disposed. A portion of the side wall of thecartridge part35 is removed from the halfopen channel structure37 to form anopening17. Ahydrophobic path38 is disposed substantially opposite theopening17 in the halfopen channel structure37. Asecond cartridge part36 is bonded to the top of the first cartridge part to define themicrochannel3. Upon filling of themicrochannel3, a firstvirtual wall15 is formed at theopening17 and a secondvirtual wall15ais defined by thehydrophobic patch38.

The hydrophobic patch enhances introduction of a second liquid into the microchannel. Thehydrophobic patch38 attractsfirst fluid4, and prevents escape of the fluid through virtual wall. As illustrated inFIG. 23b, a droplet of a second liquid is introduced into the microchannel via the virtual wall and drawn into the microchannel by the hydrophobic patch.

The use of a virtual wall in a microchannel side wall to create a bi-directional fluid interface for a microfluidic system provides significant advantages over conventional fluid interfaces. The fluid interface port comprising a virtual wall is relatively simple to manufacture, is compact, provides high ejection efficiency, does not adversely affect operation/flow, can be made bi-directional and is useful for a variety of applications. The illustrative embodiment eliminates the need for a separate structure, such as a channel or a reservoir and permits direct injection of a sample into a microchannel.

EXEMPLIFICATION OF THE INVENTIONEXAMPLE 1Virtual Wall Microfluidic Chip Manufacturing and Use

Microchannel structures having a fluid interface port were manufactured by isotropic etching half-open channels, 100 micrometer in width, 50 micrometer in height and a length of 20 mm, in a 1.1 mm thick glass wafer. A buffered hydrogen fluoride (HF) solution was used as an etchant and photo patterned etchant resistant silicon nitride mask layers were applied to define the microchannel areas to be etched. Access to the channels was provided by powder blasting 1-mm diameter holes completely through the glass wafer at both ends of each etched half-open microchannel.

A covering 50 micrometer thick layer of dry resist film (LAMINAR® 5000, Shipley, Birkenfeld, Germany) was applied on top of the etched wafer. Fluid interface ports were incorporated by photo patterning circular apertures of 50-150 micrometer in the dry resist film whereby the apertures were aligned with and extended into the underlying microchannels. Finally the wafer was diced in individual chips, which were placed in a holder to connect the microchannel structures to external systems, such as fluid pumps and electric power supplies.

The resulting microchannels were filled with an aqueous buffer solution (50 mM bi-carbonate buffer, pH 9.0). It was observed that the microchannels filled automatically via capillary forces on the application of liquid to the fluid interface port. Liquid drops having a diameter of about 50 micrometer were produced using a piezoelectric-actuated drop dispenser system (MicroDrop GmbH, Norderstedt, Germany). Drops were formed from a buffered solution containing a fluorescent dye (50 mM bi-carbonate buffer, pH 9.0, 1 mM fluoresceine) and were directed to a 100 micrometer diameter interface port. The injection process was monitored using an inverted microscope under UV light irradiation to excite the fluorescent dye and obtain a bright yellow-green color.

The present invention has been described relative to an illustrative embodiment. Since certain changes may be made in the above constructions without departing from the scope of the invention, it is intended that all matter contained in the above description or shown in the accompanying drawings be interpreted as illustrative and not in a limiting sense.

It is also to be understood that the following claims are to cover all generic and specific features of the invention described herein, and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween.

Having described the invention, what is claimed as new and protected by Letters Patent is:

Claims

1. A liquid volume injection system for injecting a droplet into a microchannel of a fluidic system, said system comprising:

a droplet generator system for generating the droplet; and

a virtual wall formed in a fluid interface port by a fluid disposed in the microchannel for receiving the droplet.

2. The system ofclaim 1, further comprising a droplet guiding system for directing the droplet toward the virtual wall.

3. The system ofclaim 1, wherein the droplet generator comprises a nozzle assembly.

4. The system ofclaim 3, wherein the nozzle assembly comprises a breaking off point where the droplet is formed, and the system further comprises a droplet charger comprises a charging circuit and a droplet electrode for charging the droplet at the breaking off point.

5. The system ofclaim 2, wherein the droplet guiding system comprises a droplet charger for charging the droplet and including a droplet electrode.

6. The system ofclaim 5, wherein the droplet charger further comprises a droplet charging circuit connected to the droplet electrode.

7. The system ofclaim 5, wherein the droplet charger further comprises a ground electrode for guiding the charged droplet.

8. The system ofclaim 2, wherein the droplet guiding system comprises one or more electrically controlled deflection plates for establishing an electric field to direct the droplet to the virtual wall.

9. The system ofclaim 1, further comprising an array of fluid interfacing ports forming a virtual wall in each said fluid interface port.

10. The system ofclaim 2, wherein the droplet guiding system comprises a channel charging circuit for selectively providing a charge to the microchannel.

11. The system ofclaim 10, wherein the channel charging circuit charges the fluid channel to attract the charged droplet.

12. The system ofclaim 11, wherein the channel charging circuit charges a neighboring fluid channel to repel the charged droplet.

13. The system ofclaim 2, wherein the droplet guiding system comprises a targeting electrode adjacent to the virtual wall for targeting charged droplets towards the virtual wall.

14. The system ofclaim 13, further comprising a targeting electrode charging circuit for charging the targeting electrode.