Microfluidic assay devices and methods for making and using the same

1. A microfluidic device comprising:

a contiguous polymeric medium disposed in at least a portion of a flow path of the microfluidic device comprising:

(a) a first analyte detection domain comprising a first covalently bound capture member that specifically binds to a first analyte, wherein the first covalently bound capture member is covalently bound to the polymeric medium through a linker group comprising a benzophenone functional group;

(b) a second analyte detection domain comprising a second covalently bound capture member that specifically binds to a second analyte, wherein the second covalently bound capture member is covalently bound to the polymeric medium through a linker group comprising a benzophenone functional group; and

(c) spacer domain comprising the contiguous polymeric medium and not including a significant amount of the capture members, wherein the spacer domain is a distinct domain disposed between the first and second analyte detection domains, and the spacer domain is in fluid communication with the first and second analyte detection domains.

2. The microfluidic device of claim 1 , wherein the first and second covalently bound capture members are different.

3. The microfluidic device of claim 1 , wherein the first and second covalently bound capture members are the same.

4. The microfluidic device of claim 1 , wherein the polymeric medium comprises a polyacrylamide gel.

5. The microfluidic device of claim 1 , wherein the linker group comprises N-(3-[(4-benzoylphenyl)formamido]propyl) methacrylamide or 3-benzoyl-N-[3-(2-methyl-acryloylamino)-propyl]-benzamide.

6. The microfluidic device of claim 1 , wherein the first capture member comprises an antigen.

7. The microfluidic device of claim 1 , wherein the second capture member comprises an antigen.

8. The microfluidic device of claim 1 , wherein the microfluidic device comprises:

two or more contiguous polymeric media disposed in at least a portion of a flow path of the microfluidic device, each of which comprises:

(a) a first analyte detection domain comprising a first covalently bound capture member that specifically binds to a first analyte, wherein the first covalently bound capture member is covalently bound to the polymeric medium through a linker group comprising a benzophenone functional group;

(b) a second analyte detection domain comprising a second covalently bound capture member that specifically binds to a second analyte, wherein the second covalently bound capture member is covalently bound to the polymeric medium through a linker group comprising a benzophenone functional group; and

(c) a spacer domain comprising the contiguous polymeric medium and not including a significant amount of the capture members, wherein the spacer domain is a distinct domain disposed between the first and second analyte detection domains, and the spacer domain is in fluid communication with the first and second analyte detection domains.

9. A method of determining whether an analyte is present in a sample, the method comprising:

(a) introducing a sample into a microfluidic device comprising a contiguous polymeric medium media disposed in at least a portion of a flow path of the microfluidic device, wherein the polymeric medium comprises:

(i) a first analyte detection domain comprising a first covalently bound capture member that specifically binds to a first analyte, wherein the first covalently bound capture member is covalently bound to the polymeric medium through a linker group comprising a benzophenone functional group;

(ii) a second analyte detection domain comprising a second covalently bound capture member that specifically binds to a second analyte, wherein the second covalently bound capture member is covalently bound to the polymeric medium through a linker group comprising a benzophenone functional group; and

(iii) a spacer domain comprising the contiguous polymeric medium and not including a significant amount of the capture members, wherein the spacer domain is a distinct domain disposed between the first and second analyte detection domains, and the spacer domain is in fluid communication with the first and second analyte detection domains;

(b) applying a directional electric field to the polymeric medium in a manner sufficient to move components in the sample through the polymeric medium; and

(c) obtaining a signal from one or more of the first and second analyte detection domains to determine whether the analyte is present in the sample.

10. The method of claim 9 , wherein the first capture member comprises a first antigen and the first analyte comprises a first specific binding member that specifically binds to the first antigen.

11. The method of claim 9 , wherein the second capture member comprises a second antigen and the second analyte comprises a second specific binding member that specifically binds to the second antigen.

12. The method of claim 10 , wherein the first specific binding member comprises a fluorescent label.

13. The method of claim 11 , wherein the second specific binding member comprises a fluorescent label.

14. The method of claim 9 , further comprising introducing a label into the microfluidic device after introducing the sample into the microfluidic device.

15. The method of claim 14 , wherein the label comprises a secondary specific binding member that specifically binds to the first analyte.

16. The method of claim 14 , wherein the label comprises a secondary specific binding member that specifically binds to the second analyte.

17. The method of claim 14 , wherein the label comprises a fluorescent moiety.

18. The method of claim 9 , wherein the sample comprises blood or a blood product.

19. A system for determining whether an analyte is present in a sample, the system comprising:

(a) a microfluidic device according to claim 1 ; and

(b) a detector.

20. The system according to claim 19 , further comprising one or more microfluidic components configured to direct a fluid through the microfluidic device.

21. A kit comprising:

(a) a microfluidic device according to claim 1 ;

(b) a packaging configured to contain the microfluidic device.

22. A method of producing a microfluidic assay device, the method comprising:

producing a contiguous polymeric medium disposed in at least a portion of a flow path of the microfluidic device and comprising benzophenone functional groups that covalently bond to a capture member upon application of an applied stimulus;

introducing into the polymeric medium a first capture member that specifically binds to a first analyte;

exposing a first region of the polymeric medium to the applied stimulus to produce a first analyte detection domain that comprises the first capture member covalently bound to the polymeric medium through a linker group comprising a benzophenone functional group;

introducing into the polymeric medium a second capture member that specifically binds to a second analyte; and

exposing a second region of the polymeric medium to the applied stimulus to produce a second analyte detection domain that comprises the second capture member covalently bound to the polymeric medium through a linker group comprising a benzophenone functional group,

to produce the microfluidic assay device,

wherein the contiguous polymeric medium comprises a distinct spacer domain which does not comprise a significant amount of the capture members and which is disposed between the first and second analyte detection domains, and wherein the spacer domain is in fluid communication with the first and second analyte detection domains.

23. The microfluidic device of claim 1 , wherein the spacer domain is adjacent to the first and second analyte domains.