A Nucleic Acid-Based Electrochemical Detection Method for Post Hoc Sample Analysis

Conventional detection schemes for nucleic-acid based electrochemical sensors involve a layer of single-stranded DNA (ssDNA) capture probe that is attached to the working electrode at one end and labeled with a redox reporter at the opposite end. Upon electrochemical interrogation, the probe layer produces a current, which is attenuated upon hybridization with the target DNA or RNA biomarker due to conformational change of the ssDNA. The decrease in current, referred to as percent signal suppression (%SS), indicates successful hybridization. Since the probe and target signals need to be successively acquired for accurately determining %SS, this method is not suitable for sample collection at a site without electrochemical analysis capabilities. Here, we introduce a new approach, where the sample can be blotted onto an electrode that is pre-functionalized with probe DNA. In this scheme, post-hybridization current is obtained first, followed by melting the hybridized DNA duplex in warm deionized water for subsequent acquisition of the post-melt current. The increase in cur-rent upon melting, which we refer to as percent signal enhancement (%SE), similarly indicates presence of tar-get DNA with comparable performance to a standard electrochemical detection technique at representative 2 µM and 100 nM target concentrations. The performance of planar and nanoporous gold (np-Au) electrodes are compared for DNA detection in complex biological samples where np-Au exhibits biofouling-resilient sensing. Finally, remote sample collection is simulated by blotting the complex sample on np-Au electrodes for %SE de-termination, where target DNA is successfully detected.