Use of photopolymerization for amplification and detection of a molecular recognition event

• US 20060286570A1
• Filed: 03/09/2006
• Published: 12/21/2006
• Est. Priority Date: 09/09/2003
• Status: Active Grant

• Current Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Original Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Inventors: Rowlen, Kathy L., Bowman, Christopher, Birks, John W., Kuchta, Robert, Hansen, Ryan, Sikes, Hadley
• Granted Patent: US 7,354,706 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2565/401 Signal amplification by chemical polymerisation

Abstract

The invention provides methods to detect molecular recognition events. The invention also provides methods to detect the presence of or identify a target species based on its interaction with one or more probe species. The methods of the invention are based on amplification of the signal due to each molecular recognition event. The amplification is achieved through photopolymerization, with the polymer formed being associated with the molecular recognition event. In an embodiment, a fluorescent polymer, a magnetic polymer, a radioactive polymer or an electrically conducting polymer can form the basis of detection and amplification. In another embodiment, a polymer gel swollen with a fluorescent solution, a magnetic solution, a radioactive solution or an electrically conducting solution can form the basis of detection and amplification. In another embodiment, sufficient polymer forms to be detectable by visual inspection.

Claims

1. A method for amplifying a molecular recognition interaction between a target and a probe comprising the steps of:

a. contacting the target with the probe under conditions effective to form a target-probe complex;

b. removing target not complexed with the probe;

c. contacting the target-probe complex with a photoinitiator label under conditions effective to attach the photoinitiator label to the target-probe complex;

d. removing photoinitiator label not attached to the target-probe complex;

e. contacting the photoinitiator-labeled target-probe complex with a polymer precursor solution;

f. exposing the photoinitiator-labeled target-probe complex and the polymer precursor solution to light, thereby forming a polymer; and

g. detecting the polymer formed, thereby detecting an amplified target-probe interaction.

2. The method of claim 1, wherein the target comprises biotin, the photoinitiator label comprises avidin or streptavidin, and the photoinitiator label is attached to the target-probe complex by interaction between the biotin and the avidin or streptavidin.

3. The method of claim 1, wherein the target comprises single-stranded DNA (ssDNA) or RNA and the probe comprises ssDNA having a sequence complementary to at least a portion of the sequence of the target.

4. The method of claim 3, wherein the target comprises Influenza A, B, or C RNA.

5. The method of claim 1 wherein the target comprises one of an antibody or antigen and the probe comprises the other of an antibody or antigen.

6. The method of claim 1 wherein the target comprises a first protein, the probe comprises a second protein, and the first and second proteins are capable of molecular recognition.

7. The method of claim 1, wherein the photoinitiator is a radical initiator and polymer precursor solution is contacted with a gas to control the solution oxygen content during light exposure.

8. The method of claim 1, wherein the photoinitiator label comprises a radical initiator and the oxygen content of the polymer precursor solution is less than about 1×

10<sup>−

5</sup> moles/liter.

9. The method of claim 8, wherein the photoinitiator label comprises a macroinitiator.

10. The method of claim 1 wherein the photoinitiator label comprises a plurality of initiators.

11. The method of claim 10 wherein the photoinitiator label comprises a macroinitiator.

12. The method of claim 11, wherein the macroinitiator comprises a plurality of initiators attached to the backbone of a second polymer.

13. The method of claim 12, wherein the average number of initiators attached to the second polymer backbone is greater than about 100.

14. The method of claim 13, wherein the average number of initiators attached to the second polymer backbone is less than about 200.

15. The method of claim 11, wherein the time of light exposure is sufficiently long that the polymer forms in sufficient quantities to allow visual detection.

16. The method of claim 15, wherein the polymer forms a layer greater than about 1 micron thick.

17. The method of claim 16, wherein the polymer forms a layer greater than about 5 microns thick.

18. The method of claim 1, wherein the probe is attached to a solid substrate.

19. The method of claim 18, wherein nonspecific interactions between the substrate and the target are limited by prior application of a blocking agent to the substrate prior to step a) or by use of a crowding agent during step a).

20. The method of claim 18, wherein nonspecific interactions between the substrate and the photoinitiator label are limited by prior application of a blocking agent to the substrate prior to step c).

21. The method of claim 1, further comprising the step of removing unpolymerized polymer precursor prior to detecting polymer formation.

22. The method of claim 1 wherein during step e) the photoinitiator-labeled target-probe complex is contacted with the polymer precursor in the presence of a cross-linking agent and the polymer formed in step f) is a hydrogel.

23. The method of claim 22 further comprising the steps of i) contacting the hydrogel with a solution comprising a fluorophore, thereby allowing absorption of the fluorophore solution by the hydrogel and ii) removing excess fluorophore solution, both steps being performed prior to step g).

24. A method for identifying a target comprising the steps of providing a probe array comprising a plurality of different probes, wherein the probes are attached to a solid substrate at known locations;

and amplifying the molecular recognition interaction between the target and the probes by the method of claim 1, wherein the target is contacted with the probe by contacting the probe array with the target and the location of the polymer formed indicates the probe which forms a target-probe complex with the target, thereby identifying the target.

25. The method of claim 24, wherein the probe array contains ssDNA, antigens, and proteins.

26. The method of claim 25, wherein the probe array contains ssDNA complementary to Influenza A, B, or C RNA, Influenza A, B, or C antigens, and proteins which capture proteins which form in the outside of Influenza A, B, or C viral particles.