Nucleic acid detection based on single-cluster analysis of cross-linking aggregates of DNA-modified gold...

Gold nanoparticles (AuNPs) have been utilised as colorimetric biosensors because of their optical properties. Single-stranded DNA (ssDNA)-immobilised AuNPs (ssDNA-AuNPs) are widely used for nucleic acid detection. AuNPs strongly scatter light, allowing individual particles to be observed using dark-field microscope (DFM). In this study, we performed target ssDNA detection using the cross-linked aggregation of ssDNA-AuNPs via DFM. The limit of detection (LOD) was determined by evaluating the aggregation ratio of the ssDNA-AuNPs estimated at the single-cluster level using DFM images. The LOD for the target ssDNA detection based on the intensity of each spot in the DFM image was 15 pM. To the best of our knowledge, this study is the first to perform a single-cluster analysis based on the red/gree/blue (RGB) values of individual spots in DFM images, and the LOD was determined to be 13 pM. These LOD values are approximately 50 times lower than those obtained using the colorimetric method. Our results indicate that single-cluster analysis based on the intensity or colour of each spot in DFM images enables sensitive detection of target molecules.