Abstract
DNA microarrays are invaluable tools for the detection and identification of nucleic acids in biosensing applications. The sensitivity and selectivity of multiplexed single-stranded DNA (ssDNA) surface bioaffinity sensing can be greatly enhanced when coupled to a surface enzymatic reaction. Herein we describe a novel method where the specific sequence-dependent adsorption of a target ssDNA template molecule onto an ssDNA-modified gold microarray is followed with the generation of multiple copies of ssRNA via in situ surface transcription by RNA polymerase. The RNA created on this "generator" element is then detected by specific adsorption onto a second adjacent "detector" element of ssDNA that is complementary to one end of the ssRNA transcript. SPR imaging is then used to detect the subsequent hybridization of cDNA-coated gold nanoparticles with the surface-bound RNA. This RNA transcription-based, dual element amplification method is used to detect ssDNA down to a concentration of 1 fM in a volume of 25 μL (25 zeptomoles).