Visual Detection of Labeled Oligonucleotides Using Visible-Light-Polymerization-Based Amplification

Abstract

DNA biochip technology holds potential for highly parallel, rapid, and sensitive genetic diagnostic screening of target pathogens and disease biomarkers. A primary limitation involves a simultaneous, sequence-specific identification of low copy number target polynucleotides using a clinically appropriate detection methodology that implements only inexpensive detection instrumentation. Here, a rapid (20 min), nonenzymatic method of signal amplification utilizing surface-initiated photopolymerization is presented in glass microarray format. Visible light photoinitiators covalently coupled to streptavidin were used to bind biotin-labeled capture sequences. Amplification was achieved through subsequent contact with a monomer solution and the appropriate light exposure to generate 20-240-nm-thick hydrogel layers exclusively from spots containing the biotin-labeled DNA. An amplification factor of 10(6) to 10(7) was observed as well as a detectable response generated from as low as approximately 10(4) labeled oligonucleotides using minimal instrumentation, such as an optical microscope or CCD camera.