Detection of single-molecule DNA hybridization using enzymatic amplification in an array of femtoliter-sized reaction vessels.

Improving the sensitivity of DNA biosensors is extremely important in clinical diagnostics, gene therapy, and a variety of other biomedical studies. In this regard, we have developed a highly sensitive single molecule DNA assay platform with a 1fM experimental detection limit using enzymatic amplification in an array of femtoliter-sized reaction wells. To validate the utility of this technology in our study, we employed a fiber optic array to create thousands of femtoliter-sized reaction wells, each specifically functionalized with oligonucleotide probes capable of capturing biotinylated target DNA. After hybridization, the fiber was incubated with streptavidin-labeled enzyme solution. The bound single enzyme molecules were confined to individual reaction vessels containing excess fluorogenic substrate and catalyzed the production of a sufficient number of fluorescent product molecules to generate a detectable signal. At low target DNA concentrations with relatively short incubation times, only a small percentage of the capture sites bind target DNA, enabling a binary readout of target concentration from the high-density fiber array. This simple binary readout-based scheme is easy to perform and exhibits a high signal-to-noise ratio in the presence of trace amounts of DNA target. Furthermore, it also should be possible to extend this technology to protein detection by modifying the reaction wells with specific capture antibodies. We expect this assay to be useful in a number of biomedical applications where accurate and highly sensitive target analysis is critical.