Colorimetric ultrasensitive detection of DNA based on the intensity of gold nanoparticles with dark-field microscopy

We present an ultrasensitive colorimetric nucleic acid assay based on the intensity of gold nanoparticles (Au NPs) using dark field microscopy. In the absence of target DNA, two hairpin-like DNA strands with protruding single-stranded DNA (ssDNA) can be absorbed onto the Au NP surface via non-covalent interactions between the exposed nitrogen bases of ssDNA and Au NPs, which inhibits Au NPs from aggregating in a high concentration of salt media, while in the presence of target DNA, two hairpin DNA strands hybridize with target DNA to form double-stranded DNA (dsDNA). After hybridization chain reaction (HCR) amplification, rigid dsDNA polymers are formed, which results in serious Au NP aggregation in the salt environment. By measuring the intensity change of yellow and red dots on a dark-field image, the concentration of target DNA can be accurately quantified with a limit of detection (LOD) of 66 fM.