Use of Photopolymerization for Amplification and Detection of a Molecular Recognition Event

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 one aspect, a fluorescent polymer, a magnetic polymer, a radioactive polymer or an electrically conducting polymer can form the basis of detection and amplification. In another aspect, 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 aspect, detectable particles can be included in the polymer formed. In another aspect, sufficient polymer forms to be detectable by visual inspection.

1 . 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; the method comprising the steps of:
 a. contacting the target with the probe array 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, the photoinitiator label comprising a photoinitiator capable of being activated by exposure to ultraviolet (UV) light;   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 UV light, thereby forming a polymer; and   g. detecting the polymer formed,   
       wherein the location of the polymer formed indicates the probe which forms a target-probe complex with the target, thereby identifying the target and the oxygen content of the polymer precursor solution during step f) is limited by contacting the polymer precursor solution with a purge gas prior to step e), during step e), during step f), or combinations thereof. 
     
     
         2 . The method of  claim 1 , wherein the target comprises one of biotin or a biotin-binding protein, the photoinitiator label comprises the other of biotin or a biotin-binding protein, and the photoinitiator label is attached to the target-probe complex by interaction between the biotin and the biotin-binding protein. 
     
     
         3 . The method of  claim 2 , wherein the photoinitiator label comprises a plurality of photoinitiators and at least one biotin-binding protein attached to backbone of a second polymer. 
     
     
         4 . The method of  claim 3 , wherein the average number of initiators attached to the polymer backbone is from 100 to 200. 
     
     
         5 . The method of  claim 3 , wherein the polymer precursor solution is aqueous and comprises a monomer and a crosslinking agent. 
     
     
         6 . The method of  claim 4 , wherein in step c) the target-probe complex is contacted with an aqueous solution comprising the photoinitiator label and the polymer backbone is hydrophilic. 
     
     
         7 . The method of  claim 6 , wherein the polymer backbone is poly (acrylic acid co-acrylamide). 
     
     
         8 . The method of  claim 6 , wherein the photoinitiator is water soluble. 
     
     
         9 . The method of  claim 6  wherein a blocking agent is used to minimize nonspecific adsorption of the photoinitiator label on the substrate. 
     
     
         10 . The method of  claim 9 , 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. 
     
     
         11 . The method of  claim 9 , wherein the target comprises one of an antibody or antigen and the probe comprises the other of an antibody or antigen. 
     
     
         12 . The method of  claim 9 , wherein the target comprises a first protein, the probe comprises a second protein, and the first and second protein are capable of molecular recognition. 
     
     
         13 . The method of  claim 9 , wherein the amount of the target can be determined through comparison of the polymerization conditions to obtain a selected value of a detectable characteristic of the polymer to a reference correlation. 
     
     
         14 . The method of  claim 13 , wherein the minimum exposure time in step f) required to obtain sufficient polymer formation for visual detection is compared to a reference correlation. 
     
     
         15 . The method of  claim 13 , wherein the minimum radiation dose in step f) required to obtain sufficient polymer formation for visual detection is compared to a reference correlation. 
     
     
         16 . The method of  claim 9  wherein the amplification factor is at least 1×10 6 . 
     
     
         17 . 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; the method comprising the steps of:
 a. contacting the target with the probe array 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, the photoinitiator label comprising a photoinitiator capable of being activated by exposure to visible light;   d. removing photoinitiator label not attached to the target-probe complex;   e. contacting the photoinitiator-labeled target-probe complex with a polymer precursor solution comprising a polymer precursor and a co-initiator;   f. exposing the photoinitiator-labeled target-probe complex and the polymer precursor solution to visible light, thereby forming a polymer; and   g. detecting the polymer formed,   
       wherein the location of the polymer formed indicates the probe which forms a target-probe complex with the target, thereby identifying the target and the oxygen content of the polymer precursor solution during step f) is limited by contacting the polymer precursor solution with a purge gas prior to step e), during step e), during step f), or combinations thereof. 
     
     
         18 . The method of  claim 17 , wherein the target comprises one of biotin or a biotin-binding protein, the photoinitiator label comprises the other of biotin or a biotin-binding protein, and the photoinitiator label is attached to the target-probe complex by interaction between the biotin and the biotin-binding protein. 
     
     
         19 . The method of  claim 18 , wherein the photoinitiator label comprises a plurality of photoinitiators attached to a biotin-binding protein. 
     
     
         20 . The method of  claim 19 , wherein the average number of initiators attached to the biotin-binding protein is from 2 to 3. 
     
     
         21 . The method of  claim 20 , wherein the initiator is fluorescein or a fluorescein derivative. 
     
     
         22 . The method of  claim 19 , wherein the polymer precursor solution is an aqueous solution and comprises a water soluble difunctional monomer. 
     
     
         23 . The method of  claim 22  wherein the pH of the polymer precursor solution is of the polymer precursor solution is greater than 7 and less than or equal to 9. 
     
     
         24 . The method of  claim 19  wherein a blocking agent is used to minimize nonspecific adsorption of the photoinitiator label 
     
     
         25 . The method of  claim 24 , wherein the polymer precursor solution further comprises a plurality of detectable particles. 
     
     
         26 . The method of  claim 25 , wherein the detectable particles are nanoparticles encapsulating a fluorescent dye. 
     
     
         27 . The method of  claim 24 , 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. 
     
     
         28 . The method of  claim 24 , wherein the target comprises one of an antibody or antigen and the probe comprises the other of an antibody or antigen. 
     
     
         29 . The method of  claim 24 , wherein the target comprises a first protein, the probe comprises a second protein, and the first and second protein are capable of molecular recognition. 
     
     
         30 . The method of  claim 24 , wherein the amount of the target can be determined through measurement of a detectable characteristic of the polymer formed. 
     
     
         31 . The method of  claim 30 , wherein the thickness of the polymer formed is measured. 
     
     
         32 . The method of  claim 25 , wherein the amount of the target can be determined through measurement of a detectable characteristic of the detectable particles. 
     
     
         33 . The method of  claim 32 , wherein the detectable particles are fluorescent nanoparticles and the fluorescence of the nanoparticles is measured. 
     
     
         34 . The method of  claim 24  wherein the amplification factor is at least 1×10 6 .