Pixel Counting of Fluorescence Spots Triggered by DNA Walkers for Ultrasensitive Quantification of Nucleic Acid

A pixel counting strategy is designed on the basis of DNA walker-triggered fluorescence spots for ultrasensitive detection of nucleic acid. The two-dimensional DNA walker was constructed by hybridization of two types of capture DNAs, which were covalently modified by click chemistry on a glass slide, and dye-labeled hairpin structure (hDNA) as track and swing strand (sDNA) as DNAzyme, respectively. Introduction of target DNA unlocked the sDNA via strand displacement to form the activated DNAzyme, and the latter cut nearby hDNA with Mn²⁺ as cofactor, resulting in fluorescence recovery of dye-labeled hDNA on the substrate due to the separation from the quencher. Meanwhile, the DNAzyme sequence of sDNA was released to cut the next hDNA and, thus, initiated autonomous walking of sDNA for signal amplification. The enhanced fluorescence spots were digitalized as pixels on the basis of DNA walker-built compartments and extracted by a homemade program in MATLAB. The association between fluorescent pixel numbers and DNA concentrations was further proved by a mathematical model and led to an ultrasensitive quantification of nucleic acid with a linear range from 100 fM to 10 pM. The designed pixel counting strategy shows a more sensitive and accurate comparison with conventional methods based on fluorescence intensity or spot counts and provides a new dimension in designing next-type biosensors.