Methods and apparatus for the detection of an analyte utilizing mesoscale flow systems

Claims

What is claimed is:

1. A device for detecting the presence of an analyte in a fluid sample, the device comprising:

a solid substrate microfabricated to define:

a sample inlet port, said port being dimensioned for connection to a pump means; and

a mesoscale flow system comprising:

a sample flow channel extending from said inlet port; and

an analyte binding region in fluid communication with said flow channel comprising a binding moiety immobilized on a surface in said analyte binding region, wherein said binding moiety specifically binds to analyte in said fluid sample, at least one of said sample flow channel or said analyte binding region having mesoscale cross-sectional dimensions; and

a binding means associated with said analyte binding region or in fluid communication with said analyte binding region for detecting binding of said analyte to said binding moiety.

2. The device of claim 1, wherein said binding moiety is immobilized on a surface of a solid particle in said binding region.

3. The device of claim 1, wherein said analyte and said immobilized binding moiety comprise a ligand/receptor pair.

4. The device of claim 1, wherein said analyte is an intracellular molecular component in a cell-containing liquid biological sample, said device further comprising:

cell lysing means in said mesoscale flow system in fluid communication with said flow channel, said analyte binding region being downstream from said cell lysing means; and

means for engaging cells in said cell-containing sample with said cell lysing means, thereby to release said intracellular molecular component.

5. The device of claim 4, wherein said cell lysing means comprises a portion of said flow channel having cell-membrane piercing protrusions extending from a wall thereof.

6. The device of claim 4, wherein said cell lysing means comprises a region of restricted cross-sectional dimension sufficient to permit passage of intracellular molecules while prohibiting passage of cells.

7. The device of claim 4, wherein said analyte is a cell population in said sample, said device further comprising:

a cell separation region, upstream from said analyte binding region comprising an immobilized cell binding moiety that binds cell surface molecules of said cell population; and

pump means for inducing flow of said sample to said separation region.

8. The device of claim 1, wherein said solid substrate comprises microfabricated silicon.

9. The device of claim 1, wherein said sample flow channel and said binding region are microfabricated in a surface of said solid substrate and enclosed by a cover adhered to said surface.

10. The device of claim 1, wherein said binding means comprises a window for optically probing said analyte binding region, said window being disposed over said analyte binding region on said substrate.

11. The device of claim 10, wherein said analyte binding region comprises particles having said analyte specific binding moiety immobilized on the surface thereof which, in the presence of an analyte, induce particle agglomeration optically detectable through said window.

12. The device of claim 10, wherein said substrate further defines a control region in fluid communication with said sample inlet port and a control region window, disposed over said control region on said substrate, for optically probing said control region whereby data determined optically in said control and analyte binding regions may be compared.

13. The device of claim 10, wherein said flow system further comprises:

a fractal region in fluid communication with said flow channel comprising bifurcations leading to plural secondary flow channels; and

pump means for inducing flow of a biological sample through said flow channel and said fractal region.

14. The device of claim 1, wherein the width and depth of said flow channel each are between 2.0 um and 500 um.

15. A device for detecting the presence of an analyte in a fluid sample, the device comprising:

a solid substrate microfabricated to define:

a sample inlet port; and

a mesoscale flow system comprising:

a sample flow channel extending from said inlet port; and

an analyte binding region in fluid communication with said flow channel comprising a binding moiety immobilized on a surface of said analyte binding region, wherein said binding moiety specifically binds to analyte in said sample, at least one of said sample flow channel or said analyte binding region having mesoscale cross-sectional dimensions;

a pump means for delivering to said analyte binding region a labelled substance which binds analyte bound to said binding moiety to produce an optically detectable signal indicative of the presence of said analyte; and

a binding means associated with said analyte binding region or in fluid communication with said analyte binding region for detecting said optically detectable signal, thereby to determine the presence of the analyte.

16. The device of claim 15, wherein said binding means comprises:

a window for optically probing said analyte binding region, said window being disposed over said analyte binding region on said substrate; and

optical binding means disposed over said window for detecting the presence of said detectable signal through said window.

17. The device of claim 15, wherein said optically detectable signal is a luminescent signal.

18. The device of claim 15, wherein said optically detectable signal is a fluorescent signal.

19. A device for detecting the presence of multiple analytes in a fluid sample, the device comprising:

a solid substrate microfabricated to define:

a sample inlet port; and

a first and at least a second mesoscale flow system, each said flow system comprising:

a sample flow channel extending from said inlet port; and

an analyte binding region in fluid communication with said flow channel comprising binding moiety immobilized on a surface in said analyte binding region, wherein said binding moiety specifically binds to analyte, at least one of said sample flow channel or said analyte binding region having mesoscale cross-sectional dimensions; and

a binding means associated with each analyte binding region or in fluid communication with each analyte binding region for detecting the binding of analyte to the binding moiety in each analyte binding region, thereby to determine the presence of said analyte; and

wherein the analyte binding regions of said flow systems comprise different immobilized binding moieties capable of binding different analytes.

20. A device for detecting the presence of an analyte in a fluid sample, the device comprising:

a solid substrate microfabricated to define:

a sample inlet port; and

a mesoscale flow system comprising:

a tortuous sample flow channel extending from said inlet port, wherein said tortuous flow channel is microfabricated with a length which allows timed mixing of reagents and sample fluid; and

an analyte binding region in fluid communication with said tortuous flow channel comprising a binding moiety immobilized on a surface in said analyte binding region, said binding moiety specifically binds to said analyte, at least one of said sample flow channel or said analyte binding region having mesoscale cross-sectional dimensions; and

a binding means associated with said analyte binding region or in fluid communication with said analyte binding region for detecting the binding of analyte to the binding moiety thereby to determine the presence of the analyte.

21. A method for detecting the presence of an analyte in a fluid sample, the method comprising the steps of:

(i) providing a device comprising:

a solid substrate microfabricated to define:

a sample inlet port, said port being dimensioned for connection to a pump means; and

a mesoscale flow system comprising:

a sample flow channel extending from said inlet port; and

an analyte binding region in fluid communication with said flow channel comprising a binding moiety immobilized on a surface in said analyte binding region, wherein said binding moiety specifically binds to said analyte, at least one of said sample flow channel or said analyte binding region having mesoscale cross-sectional dimensions; and

a binding means associated with said analyte binding region or in fluid communication with said analyte binding region for detecting binding of said analyte to said binding moiety, thereby to determine the presence of said analyte;

(ii) delivering a sample to said inlet port and, by a pump means, through said flow system to said analyte binding region to permit binding of said analyte to said binding moiety; and

(iii) detecting the binding of analyte to said binding moiety with said binding means, thereby to determine the presence of the analyte.

22. The method of claim 21, wherein said binding means in the device provided in step (i) comprises a window disposed over said analyte binding region on said substrate; and

wherein step (iii) includes the step of optically probing said binding region through said window.

23. The method of claim 22, wherein said substrate provided in step (i) further defines a control region in fluid communication with said sample inlet port and a control region window, disposed over said control region on said substrate, for optically probing said control region whereby data determined optically in said control and analyte binding regions may be compared; and

wherein step (iii) includes the step of optically probing and comparing said control region and said binding region.

24. The method of claim 21, wherein the device provided in step (i) further includes:

a pump means for delivering to said analyte binding region a labelled substance which binds to analyte bound to said binding moiety to produce a detectable signal indicative of the presence of said analyte; and

means for detecting the binding of the labelled substance to analyte bound to said binding moiety;

said method further comprising the steps of:

(iv) delivering said labelled substance to said analyte binding region to bind to said bound analyte; and

(v) detecting said detectable signal with said binding means, thereby to determine the presence of the analyte.

25. The method of claim 24, wherein said detectable signal comprises a luminescent signal.

26. The method of claim 21, wherein said sample comprises a cell population, said method comprising the additional step of separating said cell population from other cells within said substrate prior to step (iii).

27. The method of claim 21, wherein the width and depth of said flow channel each are between 2.0 um and 500 um.

28. The method for detecting the presence of a cell population in a sample, the method comprising the steps of:

(i) providing a device comprising:

a sample inlet port, said port being dimensioned for connection to a pump means; and

a mesoscale flow system comprising:

a sample flow channel extending from said inlet port; and

an analyte binding region in fluid communication with said flow channel comprising a binding moiety immobilized on a surface in said analyte binding region, wherein said binding moiety specifically binds a surface molecule on members of said cell population, at least one of said sample flow channel or said analyte binding region having mesoscale cross-sectional dimensions; and

a window disposed over said analyte binding region on said substrate;

(ii) delivering a sample to said inlet port and, by a pump means, through said flow system to said binding region, to permit said binding moiety to bind to a surface molecule on members of said cell population to induce agglutination of said cell population on the surface of said binding region; and

(iii) detecting said agglutination optically in said binding region through said window, thereby to determine the presence of the cell population in the sample.

29. A method for detecting the presence of one or more analytes in a fluid sample, the method comprising the steps of:

(i) providing a device comprising:

a solid substrate microfabricated to define:

a sample inlet port; and

a first and at least a second mesoscale flow system, each said flow system comprising:

a sample flow channel extending from said inlet port; and

an analyte binding region in fluid communication with said flow channel comprising binding moiety immobilized on a surface in said analyte binding region, wherein said binding moiety specifically binds to analyte, at least one of said sample flow channel or said analyte binding region having mesoscale cross-sectional dimensions; and

a detection means associated with each analyte binding region or in fluid communication with each analyte binding region for detecting binding of analyte to the binding moiety in each analyte binding region, thereby to determine the presence of said analyte;

(ii) delivering a sample to said inlet port and, by pump means, through said flow system to a said analyte binding regions; and

(iii) detecting the binding of an analyte to the binding moiety in each of the analyte binding regions in said first and at least second system with said binding means, thereby to determine the presence of the analytes.

30. In combination, a device for determining an analyte in a fluid sample, said device comprising a solid substrate having at least one surface and being microfabricated to define:

a sample inlet port; and

a flow system comprising:

a sample flow channel extending from said inlet port; and

an analyte binding region in fluid communication with said flow channel comprising a binding moiety which specifically binds to said analyte, at least one of said sample flow channel or said analyte binding region having mesoscale cross-sectional dimensions;

detection means associated with said analyte binding region or in fluid communication with said analyte binding region for detecting binding of said analyte to said binding moiety; and

an appliance for use with said device, said appliance comprising a holder for said device, a fluid sample input conduit interfitting with said inlet port of said device and a pump for passing fluid sample through said flow system.

31. The combination of claim 30, wherein the analyte binding region of said device has mesoscale cross-sectional dimensions.

32. The combination of claim 30, wherein the flow channel of said device has mesoscale cross-sectional dimensions.

33. The combination of claim 30, wherein said binding moiety is immobilized on a surface within the analyte binding region of said device.

34. The combination of claim 30, wherein said appliance further comprises a fluid reservoir and means for delivering fluid contained in said reservoir to said flow system.

35. The combination as claimed in claim 34, wherein said reservoir contains a labelled reagent which produces a detectable signal when bound to said analyte.

36. The combination of claim 30, wherein said analyte binding region comprises particles having analyte binding moieties immobilized on the surface thereof which in the presence of said analyte, induce particle agglomeration.

37. The combination of claim 30, wherein the flow system of said device is microfabricated in a surface of said solid substrate and enclosed by a transparent cover adhered to said surface.

38. The combination of claim 30, wherein said analyte is a ligand and said binding moiety is a receptor which binds specifically to said ligand.

39. The combination of claim 30, wherein said analyte is an antigen and said binding moiety is an antigen binding protein which binds specifically to said antigen.

40. The combination of claim 30, wherein said analyte is a polynucleotide and said binding moiety is a complementary polynucleotide that hybridizes to said polynucleotide analyte.

41. A method for determining the presence or amount of an intercellular component in a cell-containing biological fluid sample, said method comprising the steps of:

(i) providing a device comprising a solid substrate microfabricated to define:

a sample inlet port; and

a flow system comprising:

a sample flow channel extending from said inlet port; and

an analyte binding region in fluid communication with said flow channel comprising a binding moiety which specifically binds said analyte, at least one of said sample flow channel or said analyte binding region having mesoscale cross-sectional dimensions;

cell lysing means in said mesoscale flow system, and upstream of said analyte binding region, in fluid communication with said flow channel;

binding means associated with said analyte binding region or in fluid communication with said analyte binding region for detecting binding of said analyte to said binding moiety;

(ii) delivering said sample to said inlet port;

(iii) lysing said cells within said device to release said intercellular component;

(iv) delivering said intercellular component to said analyte binding region; and

(v) detecting binding of said intercellular component to said binding moiety as determinative of the presence or amount of said intercellular component in said sample.

42. The method of claim 41, wherein said intercellular component is in a subpopulation of cells within a mixed cell population in said sample, and said method includes the additional step of separating said cell subpopulation from said mixed cell population within said device prior to said lysing step.

43. The method of claim 41, wherein the device provided in step (i) further includes means for delivering to said bound analyte binding region a labelled reagent which binds to said bound intercellular component to produce a detectable signal indicative of the binding of said intercellular component to said binding moiety, said method further comprising delivering said labelled reagent to said analyte binding region, and, in said detecting step, detecting said signal.

44. The method of claim 41, wherein the analyte binding region in said device provided in step (i) comprises particles having analyte binding sites immobilized on the surface thereof which, in the presence of said intercellular component, induce particle agglomeration, and, as a result of step (iv), said intercellular component binds to said particles to induce agglomeration; and, in said detecting step, detecting said agglomeration.

REFERENCE TO RELATED APPLICATIONS

This application is being filed contemporaneously with the following related co-pending applications: U.S. application Ser. No. 07/887,701, now abandoned; U.S. application Ser. No. 07/887,536, now U.S. Pat. No. 5,304,487, issued Apr. 19, 1994; U.S. application Ser. No. 07/887,662, now abandoned; and U.S. application Ser. No. 07/887,661, now U.S. Pat. No. 5,296,375, issued Mar. 22, 1994.

Continuation applications have been filed based on the above-noted abandoned applications, all of which are currently pending.

BACKGROUND OF THE INVENTION

This invention relates generally to methods and apparatus for conducting analyses. More particularly, the invention relates to the design and construction of small, typically single-use, modules capable of receiving and rapidly conducting a predetermined assay protocol on a fluid sample.

In recent decades the art has developed a very large number of protocols, test kits, and cartridges for conducting analyses on biological samples for various diagnostic and monitoring purposes. Immunoassays, agglutination assays, and analyses based on polymerase chain reaction, various ligand-receptor interactions, and differential migration of species in a complex sample all have been used to determine the presence or concentration of various biological compounds or contaminants, or the presence of particular cell types.

Recently, small, disposable devices have been developed for handling biological samples and for conducting certain clinical tests. Shoji et al. reported the use of a miniature blood gas analyzer fabricated on a silicon wafer. Shoji et al., Sensors and Actuators, 15:101-107 (1988). Sato et al. reported a cell fusion technique using micromechanical silicon devices. Sato et al., Sensors and Actuators, A21-A23:948-953 (1990). Ciba Corning Diagnostics Corp. (USA) has manufactured a microprocessor-controlled laser photometer for detecting blood clotting.

Micromachining technology originated in the microelectronics industry. Angeli et al., Scientific American, 248:44-55 (1983). Micromachining technology has enabled the manufacture of microengineered devices having structural elements with minimal dimensions ranging from tens of microns (the dimensions of biological cells) to nanometers (the dimensions of some biological macromolecules). This scale is referred to herein as "mesoscale". Most experiments involving mesoscale structures have involved studies of micromechanics, i.e., mechanical motion and flow properties. The potential capability of mesoscale structures has not been exploited fully in the life sciences.

Brunette (Exper. Cell Res., 167:203-217 (1986) and 164:11-26 (1986)) studied the behavior of fibroblasts and epithelial cells in grooves in silicon, titanium-coated polymers and the like. McCartney et al. (Cancer Res., 41:3046-3051 (1981)) examined the behavior of tumor cells in grooved plastic substrates. LaCelle (Blood Cells, 12:179-189 (1986)) studied leukocyte and erythrocyte flow in microcapillaries to gain insight into microcirculation. Hung and Weissman reported a study of fluid dynamics in micromachined channels, but did not produce data associated with an analytic device. Hung et al., Med. and Biol. Engineering, 9:237-245 (1971); and Weissman et al., Am. Inst. Chem. Eng. J., 17:25-30 (1971). Columbus et al. utilized a sandwich composed of two orthogonally orientated v-grooved embossed sheets in the control of capillary flow of biological fluids to discrete ion-selective electrodes in an experimental multi-channel test device. Columbus et al., Clin. Chem., 33:1531-1537 (1987). Masuda et al. and Washizu et al. have reported the use of a fluid flow chamber for the manipulation of cells (e.g. cell fusion). Masuda et al., Proceedings IEEE/IAS Meeting, pp. 1549-1553 (1987); and Washizu et al., Proceedings IEEE/IAS Meeting pp. 1735-1740 (1988). The art has not fully explored the potential of using mesoscale devices for the analyses of biological fluids and detection of microorganisms.

The current analytical techniques utilized for the detection of microorganisms are rarely automated, usually require incubation in a suitable medium to increase the number of organisms, and invariably employ visual and/or chemical methods to identify the strain or sub-species. The inherent delay in such methods frequently necessitates medical intervention prior to definitive identification of the nature of an infection. In industrial, public health or clinical environments, such delays may have serious consequences. There is a need for convenient systems for the rapid detection of microorganisms.

An object of the invention is to provide analytical systems with optimal reaction environments that can analyze microvolumes of sample, detect substances present in very low concentrations, and produce analytical results rapidly. Another object is to provide easily mass produced, disposable, small (e.g., less than 1 cc in volume) devices having mesoscale functional elements capable of rapid, automated analyses of preselected molecular or cellular analytes, in a range of biological and other applications. It is a further object of the invention to provide a family of such devices that individually can be used to implement a range of rapid clinical tests, e.g., tests for bacterial contamination, virus infection, sperm motility, blood parameters, contaminants in food, water, or body fluids, and the like.

SUMMARY OF THE INVENTION

The invention provides methods and devices for the detection of a preselected analyte in a fluid sample. The device comprises a solid substrate, typically on the order of a few millimeters thick and approximately 0.2 to 2.0 centimeters square, microfabricated to define a sample inlet port and a mesoscale flow system. The term "mesoscale" is used herein to define chambers and flow passages having cross-sectional dimensions on the order of 0.1 .mu.m to 500 .mu.m. The mesoscale flow channels and fluid handling regions have a preferred depth on the order of 0.1 .mu.m to 100 .mu.m, typically 2-50 .mu.m. The channels have preferred widths on the order of 2.0 .mu.m to 500 .mu.m, more preferably 3-100 .mu.m. For many applications, channels of 5-50 .mu.m widths will be useful. Chambers in the substrates often will have larger dimensions, e.g., a few millimeters.

The mesoscale flow system of the device includes a sample flow channel, extending from the inlet port, and an analyte detection region in fluid communication with the flow channel. The analyte detection region is provided with a binding moiety, optionally immobilized therewithin, for specifically binding the analyte. The mesoscale dimension of the detection region kinetically enhances binding of the binding moiety and the analyte. That is, in the detection region, reactants are brought close together in a confined space so that multiple molecular collisions occur. The devices may be used to implement a variety of automated, sensitive and rapid clinical tests including the analysis of cells or macromolecules, or for monitoring reactions or cell growth.

Generally, as disclosed herein, the solid substrate comprises a chip containing the mesoscale flow system. The chips are designed to exploit a combination of functional geometrical features and generally known types of clinical chemistry to implement the detection of microquantities of an analyte. The mesoscale flow system may be designed and fabricated from silicon and other solid substrates using established micromachining methods, or by molding polymeric materials. The mesoscale flow systems in the devices may be constructed by microfabricating flow channel(s) and detection region(s) into the surface of the substrate, and then adhering a cover, e.g., a transparent glass cover, over the surface. The channels and chambers in cross-section taken through the thickness of the chip may be triangular, truncated conical, square, rectangular, circular, or any other shape. The devices typically are designated on a scale suitable to analyze microvolumes (<5 .mu.L) of sample, introduced into the flow system through an inlet port defined, e.g., by a hole communicating with the flow system through the substrate or through a transparent coverslip. Cells or other analytes present in very low concentrations (e.g. nanogram quantities) in microvolumes of a sample fluid can be rapidly analyzed (e.g., <10 minutes).

The chips typically will be used with an appliance which contains a nesting site for holding the chip, and which mates an input port on the chip with a flow line in the appliance. After biological fluid such as blood, plasma, serum, urine, sputum, saliva, or other fluids suspected to contain a particular analyte, cellular contaminant, or toxin is applied to the inlet port of the substrate, the chip is placed in the appliance and a pump is actuated to force the sample through the flow system. Alternatively, a sample may be injected into the chip by the appliance, or the sample may enter the mesoscale flow system of the chip through the inlet port by capillary action.

In the devices, the binding of an analyte to a binding moiety serves as a positive indication of the presence of the analyte in a sample. The mesoscale detection region is provided with a binding moiety capable of specifically binding to the preselected analyte. The binding moiety may be delivered to the detection region in, e.g., a solution. Alternatively, the binding moiety may be immobilized in the detection region. The internal surfaces of the mesoscale detection region of the device may be coated with an immobilized binding moiety to enable the surface to interact with a fluid sample in order to detect or separate specific fluid sample constituents. Antibodies or polynucleotide probes may be immobilized on the surface of the flow channels, enabling the use of the mesoscale flow systems for immunoassays or polynucleotide hybridization assays. The binding moiety also may comprise a ligand or receptor. A binding moiety capable of binding cells via a cell surface molecule may be utilized, to enable the isolation or detection of a cell population in a biological microsample. The mesoscale flow system may also include protrusions or a section of reduced cross sectional area to enable the sorting or lysis of cells in the microsample upon flow through the flow system.

Analyte binding to a binding moiety in the detection region may detected optically, e.g., through a transparent or translucent window, such as a transparent cover over the detection region or through a translucent section of the substrate itself. Changes in color, fluorescence, luminescence, etc., upon binding