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.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation-in-part of U.S. application Ser. No. 11 / 372,485, filed Mar. 9, 2006. This application also claims the benefit of U.S. provisional applications 60 / 988,563, filed Nov. 16, 2007 and 60 / 982,992, filed Oct. 26, 2007. U.S. application Ser. No. 11 / 372,485 is a continuation-in-part of International Application serial number PCT / US2004 / 029733, filed Sep. 9, 2004, and claims the benefit of U.S. provisional application Ser. No. 60 / 662,313, filed Mar. 16, 2005; International Application PCT / US2004 / 029733 claims the benefit of U.S. provisional application Ser. No. 60 / 501,755, filed on Sep. 9, 2003. All of these applications are hereby incorporated by reference to the extent not inconsistent with the disclosure herein.ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT[0002]This invention was made at least in part with support from the National Institutes of Health under grant numbers NIH HG003100, R41 AI060057 and SGER 0442047, th...

AI Technical Summary

Benefits of technology

[0018]In an embodiment, the invention provides methods to detect molecular recognition events, in particular a relatively small number of molecular recognition events. The methods of the invention are based on amplification of the signal due to each molecular recognition event, rather than amplification of the number of molecular recognition events taking place. The present invention can limit or eliminate the need for techniques which increase the number of recognition events taking place, including PCR and techniques involving culturing of bacteria. The present invention can replace PCR and RT-PCR techniques for microarray applications as a means to achieve acceptable signals.

[0024]In another embodiment, the quantity of polymer formed is sufficient to allow visual detection of polymer formation. In this embodiment, the polymer need not be fluorescent, magnetic, radioactive or electrically conducting. This embodiment can be achieved through a synergistic combination of reduction of oxygen content in the polymer precursor solution by purging, utilization of a photoinitiator label with an appropriate ratio of initiator to molecular recognition agent, and the identification of the appropriate exposure time. Without wishing to be bound by any particular belief, it is believed that a process having all these attributes can yield much higher degrees of amplification and enable better contrast than is possible with a process having only one of these attributes.

[0041]In another embodiment, the invention provides methods for identification of a target species based on its molecular interaction with an array of different probe species, each probe species being attached to a solid substrate at known locations. In the methods of the invention, if the target species undergoes a molecular recognition reaction with a probe, the probe will be labeled with a polymer. Detection of the polymer-labeled probes allows identification of which probes have undergone the molecular recognition reaction and therefore identification of the target.

[0081]In an embodiment, the methods of the invention provide sufficient amplification that molecular recognition can be detected without instrumentation. In another embodiment, the methods of the invention provide sufficient amplification that molecular recognition can be detected using a relatively inexpensive microarray reader or scanner which may not have the highest instrument sensitivity or resolution.

Problems solved by technology

Detection of molecular recognition events such as DNA hybridization, antibody-antigen interactions, and protein-protein interactions becomes increasingly difficult as the number of recognition events to be detected decreases.

The main drawback to RCA is the necessity of DNA polymerase.

In both the DeRisi and Evans work PCR technology was used to amplify the genetic material for capture and relatively expensive fluorescent labels (˜$50 in labels per chip) were used to generate signals from positive spots.

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