Microscale diffusion immunoassay

• US 20030124619A1
• Filed: 02/18/2003
• Published: 07/03/2003
• Est. Priority Date: 03/29/1996
• Status: Active Grant

• Current Assignee: Andrew Kamholz, Anson Hatch, Bernhard H. Weigl, Paul Yager
• Original Assignee: Andrew Kamholz, Anson Hatch, Bernhard H. Weigl, Paul Yager
• Inventors: Yager, Paul, Weigl, Bernhard H., Hatch, Anson, Kamholz, Andrew
• Granted Patent: US 7,271,007 B2
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: B01F 25/31 in conduits or tubes through which the main component flows
• CPC Class Codes: B01F 33/30 Micromixers
• CPC Class Codes: B01F 33/3039 with mixing achieved by diffusion between layers
• CPC Class Codes: B01J 19/0093 Microreactors, e.g. miniaturised or microfabricated reactors laboratory containers with capillary fluid transport in microfabricated channels or chambers B01L3/5027
• CPC Class Codes: B01L 2200/0636 Focussing flows, e.g. to laminate flows
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502776 specially adapted for focusing or laminating flows
• CPC Class Codes: G01N 15/10 Investigating individual particles
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 30/0005 Field flow fractionation
• CPC Class Codes: G01N 30/38 Flow patterns
• CPC Class Codes: G01N 30/6095 Micromachined or nanomachined, e.g. micro- or nanosize
• CPC Class Codes: G01N 30/74 Optical detectors measurement of intensity, velocity, spectral content, polarisation, or phase of infrared, visible or ultraviolet light G01J
• CPC Class Codes: G01N 33/558 using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387
• CPC Class Codes: G01N 35/085 Flow Injection Analysis
• CPC Class Codes: Y10T 436/117497 with a continuously flowing sample or carrier stream
• CPC Class Codes: Y10T 436/118339 with formation of a segmented stream
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

Methods and apparatuses are provided for determining presence and concentration of analytes by exploiting molecular binding reactions and differential diffusion rates. Analyte particles and binding particles are allowed to diffuse toward each other, and slowing of the diffusion front is detected when they meet. From the position of the diffusion front, presence and concentration of analyte particles can be determined. One embodiment provides a competitive immunoassay in a microfluidic format. This diffusion immunoassay (DIA) relies on measuring the concentration of labeled antigen along one dimension of a microchannel after allowing it to diffuse for a short time into a region containing specific antibodies. A simple microfluidic device, the T-Sensor, was used to implement a DIA to measure the concentration of phenytoin, a small drug molecule. Concentrations of analyte over the range of 50 to 1600 nM can be measured in less than a minute. The assay is homogeneous, rapid, requires only microliter volumes of reagents and sample, and is applicable to a wide range of analytes, including therapeutic drugs, molecular biological markers, and environmental contaminants. Methods for separating particles of similar size in a diffusion separator are also provided.

Claims

1. A method for detecting the presence of analyte particles comprising:

a) providing binding particles capable of binding with said analyte particles;

b) providing a system in which at least one of said binding particles and said analyte particles can diffuse toward the other;

c) providing means for detecting said binding particles or said analyte particles, or complexes thereof, or a diffusion front created by any of said particles, in said system;

d) detecting the position of any of said particles or diffusion front as an indication of the presence of said analyte particles.

2. The method of claim 1 wherein slowing of a diffusion front is detected.

3. The method of claim 1 also comprising providing labeled analyte particles or binding particles.

4. The method of claim 1 wherein said binding particles are labeled.

5. The method of claim 1 wherein said analyte particles are supplemented with labeled analyte particles.

6. The method of claim 1 wherein said detection comprises comparison of the position of said diffusion front with the position of a diffusion front in a calibration system.

7. The method of claim 1 wherein said system is a flowing system.

8. The method of claim 7 wherein said diffusion front is stationary.

9. The method of claim 1 wherein said diffusion front is detected at a predetermined time after initiating diffusion within said system.

10. The method of claim 1 comprising determining the concentration of said analyte particles as a function of the position of said diffusion front.

11. A method for determining the presence or concentration of sample analyte particles in an analyte fluid comprising:

a) adding to an analyte fluid additional analyte particles labeled with a detectable marker to provide a predetermined concentration or amount of labeled analyte particles in said analyte fluid;

b) providing a diffusion fluid containing binding particles capable of binding to said sample analyte particles and said labeled analyte particles;

c) providing a laminar flow channel comprising an analyte stream inlet and a diffusion stream inlet;

d) flowing analyte fluid into said analyte stream inlet as an analyte stream, and flowing diffusion fluid into said diffusion stream inlet as a diffusion stream whereby said streams flow in-adjacent laminar flow;

e) allowing diffusion between said streams of sample analyte particles, labeled analyte particles and binding particles;

f) detecting a diffusion profile in said channel formed by said labeled analyte particles;

g) determining from said diffusion profile the presence or concentration of said sample analyte particles.

12. The method of claim 11 wherein said concentration is determined by comparing said diffusion profile to a calibration profile.

13. The method of claim 11 wherein said concentration is determined by calculation.

14. The method of claim 11 wherein said concentration is continuously determined.

15. The method of claim 11 wherein said analyte fluid is whole blood.

16. The method of claim 11 wherein said binding particles are antibodies.

17. The method of claim 11 also comprising providing a reference fluid containing a known concentration of unlabeled analyte particles and a known concentration of unlabeled analyte particles and flowing said fluid as a reference stream in laminar flow contact with said diffusion stream.

18. The method of claim 11 also comprising providing an inert separation stream between said analyte stream and said diffusion stream.

19. The method of claim 11 wherein the concentration of binding particles is at least about two-fold greater than the concentration of analyte particles.

20. The method of claim 11 wherein the concentration of binding particles is approximately equal to the concentration of analyte particles.

21. A method of determining the presence of concentration of sample analyte particles in an analyte substance comprising:

a) adding to an analyte substance additional analyte particles labeled with a detectable marker to provide a predetermined concentration of labeled analyte particles in said analyte substance;

b) providing a diffusion substance containing binding particles capable of binding to said analyte particles and said labeled analyte particles;

c) contacting said analyte substance with said diffusion substance;

d) allowing diffusion of said analyte particles between said analyte and diffusion substances;

e) detecting a diffusion profile formed by said labeled analyte particles;

f) determining from said diffusion profile the presence or concentration of said sample analyte particles.

22. The method of claim 21 wherein said analyte substance is a fluid.

23. The method of claim 21 wherein said diffusion substance is a gel.

24. A microscale device for determining the presence or concentration of sample analyte particles in an analyte fluid comprising:

a) a laminar flow channel comprising an analyte stream inlet and a diffusion stream inlet;

b) said laminar flow channel comprising, in adjacent laminar flow;

i) an analyte stream containing said analyte fluid to which additional analyte particles labeled with a detectable marker have been added to provide a predetermined concentration of labeled analyte particles in said analyte fluid;

ii) a diffusion stream containing binding particles capable of binding to said analyte particles and said labeled analyte particles;

c) means for detecting a diffusion profile in said channel formed by said labeled analyte particles;

d) means for determining from said diffusion profile the presence or concentration of said sample analyte particles.

25. The device of claim 24 wherein said means for detecting said diffusion profile comprises a CCD camera or scanning laser.

26. The device of claim 24 wherein said means for determining comprise a computer processor programmed to calculate said presence or concentration based on an algorithm utilizing process variables.

27. The device of claim 26 wherein said process variables are selected from the group consisting of flow rates of said fluids, volumes, diffusion coefficients of said binding particles, said sample analyte particles and labeled analyte particles and labeled analyte particle/binding particle complexes, concentrations of said binding particles and labeled analyte particles, channel diffusion dimensions, length of channel (l) to detection zone, and binding kinetics of said analyte and binding particles.

28. The device of claim 24 also comprising a reference stream inlet into said laminar flow channel constructed and arranged such that a reference stream entering the laminar flow channel through said inlet can flow in laminar flow contact with said diffusion stream.

29. The device of claim 24 also comprising a reference stream comprising a known concentration of labeled analyte particles and a known concentration of unlabeled analyte particles in said laminar flow channel.

30. The device of claim 24 also comprising an inert separation stream inlet constructed and arranged such that an inert separation stream entering the laminar flow channel therethrough can flow in laminar flow contact with and between said analyte and diffusion streams.

31. The device of claim 24 also comprising an inert separation stream flowing in said laminar flow channel between said analyte and diffusion streams.

32. A device for determining the presence or concentration of sample analyte particles in a medium comprising:

(a) means for contacting a first medium containing analyte particles with a second medium containing binding particles capable of binding to said analyte particles;

(b) wherein at least one of said analyte or binding particles is capable of diffusing into the medium containing the other of said analyte or binding particles; and

(c) means for detecting the presence of diffused particles.

33. The device of claim 32 wherein at least one of said analyte particles or said binding particles is labeled.

34. The device of claim 32 wherein said first medium contains a known quantity of labeled analyte particles.

35. A method for detecting the presence of at least first and second analyte particles in a first fluid comprising:

(a) providing a second fluid comprising first and second binding particles for said first and second analyte particles, respectively;

(b) flowing said first and second fluids in adjacent laminar flow in a laminar flow channel;

(c) allowing said first analyte particles to diffuse into said second fluid and bind with said first binding particles to form first complexes; and

allowing said second analyte particles to diffuse into said second fluid and bind with said second binding particles to form second complexes; and

(d) detecting the presence of said first and second complexes.

36. The method of claim 35 wherein said first and second complexes have detectably different diffusion coefficients.

37. The method of claim 35 wherein said first and second complexes form detectably different diffusion profiles.

38. The method of claim 35 wherein said first and second complexes are labeled with detectably different labels.

39. The method of claim 35 wherein said first and second complexes are not labeled with detectably different labels.

40. The method of claim 35 wherein a stream comprising a major portion of said first complexes is conducted from said channel as a first outlet stream.

41. The method of claim 40 wherein a stream comprising a major portion of said second complexes is conducted from said channel as a second outlet stream.

42. The method of claim 40 wherein the presence of said first analyte particles is detected in said first outlet stream.

43. The method of claim 41 wherein the presence of said second analyte particles is detected in said second outlet stream.

44. A device for detecting the presence of at least first and second analyte particles in a first fluid comprising:

(a) first inlet means for conducting a first fluid comprising said first and second analyte particles into a laminar flow channel;

(b) second inlet means for conducting a second fluid comprising first and second binding particles for said first and second analyte particles, respectively, into said laminar flow channel;

(c) a laminar flow channel in fluid communication with said first and second inlet means, comprising said first and second fluids in adjacent laminar flow, said flow channel having a length sufficient to allow said first analyte particles to diffuse into said second fluid and bind with said first binding particles to form first complexes; and

to allow said second analyte particles to diffuse into said second fluid and bind with said second binding particles to form second complexes; and

(d) means for detecting the presence of said first and second complexes.

45. The device of claim 44 wherein said first and second complexes have detectably different diffusion coefficients.

46. The device of claim 44 wherein said laminar flow channel displays detectably different diffusion profiles for of said first and second complexes.

47. The device of claim 44 wherein said first and second complexes are labeled with detectably different labels.

48. The device of claim 44 wherein said first and second complexes are not labeled with detectably different labels.

49. The device of claim 44 comprising outlet means spaced along said laminar flow channel for conducting a stream comprising said first complexes from said channel as a first outlet stream.

50. The device of claim 49 comprising outlet means spaced along said laminar flow channel for conducting a stream comprising said first and second complexes from said channel as a second outlet stream.

51. The device of claim 49 comprising means for detecting the presence of said first analyte particles in said first outlet stream.

52. The device of claim 50 comprising means for detecting the presence of said second analyte particles in said second outlet stream.

53. A method for separating first and second particles of similar size contained in a first fluid, in a diffusion separator, said method comprising:

(a) providing a second fluid comprising at least first and second binding particles for said first and second analyte particles, respectively, said first binding particles having a higher diffusion coefficient than said second binding particles;

(b) flowing said first fluid into a channel comprising said second fluid;

(c) allowing said first analyte particles to diffuse into said second fluid and bind with said first binding particles to form first complexes; and

allowing said second analyte particles to diffuse into said second fluid and bind with said second binding particles to form second complexes;

(d) conducting a stream containing said first complexes from said channel through a first outlet; and

(e) conducting a stream containing said first and second complexes from said channel said through a second outlet positioned downstream from said first outlet along said channel.

54. The method of claim 53 wherein said second fluid comprises a plurality of binding particles for a plurality of particles in said first fluid to be separated.

55. The method of claim 53 wherein said first and second binding particles have diffusion coefficients differing by at least about a factor of two.

56. The method of claim 53 wherein said first and second binding particles have diffusion coefficients differing by at least about a factor of ten.

57. The method of claim 53 wherein said second outlet forms the inlet stream to a further first diffusion separator.

58. The method of claim 53 wherein a second diffusion separator is connected to said first diffusion separator.

59. A device for separating first and second particles of similar size contained in a first fluid, in a diffusion separator, said device comprising:

(a) a flow channel comprising a second fluid containing at least first and second binding particles for said first and second analyte particles, respectively, said first binding particles having a higher diffusion coefficient than said second binding particles;

(b) a first inlet into said channel on a first side of said channel, said first inlet containing said first fluid;

(c) a second inlet on the second side of said flow channel containing an acceptor stream;

(d) a first outlet on the second side of said flow channel downstream from said second inlet containing a stream predominantly comprising said first complexes; and

(e) a second outlet on the second side of said flow channel downstream from said first outlet containing a stream containing said first and second complexes.

60. The device of claim 59 also comprising a third outlet on the first side of said flow channel.

61. The device of claim 59 also comprising an additional diffusion separator connected to said first outlet.

62. The device of claim 59 also comprising an additional diffusion separator connected to said second outlet.