Femtomolar oligonucleotide detection by a one-step gold nanoparticle-based assay

A sequence-specific oligonucleotide detection method based on the tail-to-tail aggregation of functionalized gold nanoparticles in the presence of target analytes is presented together with its optimization and capabilities for detection of single nucleotide polymorphisms (SNPs). In this single-step method, capture probes are freely accessible for hybridization, resulting in an improved assay performance compared to substrate-based assays. The analytes bring the nanoparticles close to each other via hybridization, causing a red shift of the nanoparticle plasmon peak detected by a spectrophotometer or CCD camera coupled to a darkfield imaging system. Optimal conditions for the assay were found to be (i) use of capture probes complementary to the target without any gap, (ii) maximum possible probe density on the gold nanoparticles, and (iii) 1M ionic strength buffer. The optimized assay has a 1 fM limit of detection and fM to 10 pM dynamic range, with detection of perfect match sequences being three orders of magnitude more sensitive than targets with single nucleotide mismatches.