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

Abstract

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 Mn 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.

Induction of Cytokines by Nucleic Acid Nanoparticles (NANPs) Depends on the Type of Delivery Carrier

“…A growing library of nucleic acid nanoparticles (NANPs), the design of which takes advantage of natural RNA (and DNA) motifs and canonical Watson-Crick base pairings, have been demonstrated to assemble into precise nanoscaffolds exemplified by hexagonal rings [12], various polygons [13], and fibrous structures [14], to name a few [15]. A variety of NANPs are now being investigated for broad applications in detection and diagnostics [16][17][18], targeting specific disease sites [19], and as therapeutic approaches [9,[20][21][22] for various illnesses. As the technology approaches the stage of preclinical development and clinical translations, many researchers in the field have consolidated their efforts to overcome translational gaps and accelerate the transition of DNA and RNA nanoassemblies from bench to clinic [23][24][25][26][27][28].…”

DNA Assembly‐Based Stimuli‐Responsive Systems

“…[139] Nucleic Acids Sensors: A series of nucleic acid sensors based on DNAzymes have been developed by promoting or inhibiting the formation of DNAzymes with nucleic acid. [150,162,[316][317][318][319][320] For example, the fluorescence detection of target DNA was achieved through the activation of Mg 2+ -dependent DNAzyme systems by target DNA. [316] Zhang's group achieved fluorescence detection of target RNA through the inhibiting effect of target RNA on the formation of G-quadruplex/hemin DNAzyme and the photoinduced electron transfer (PET) process with oligonucleotidetemplated silver nanoclusters (DNA-AgNCs).…”

DNA‐Based Chemical Reaction Networks for Biosensing Applications

“…However, it requires thermocycling. Due to the rapid development of molecular biotechnology, a variety of isothermal amplification methods using specific enzymes have been developed, including restriction enzyme‐dependent amplification, [ 40,45,75,76 ] helicase‐dependent amplification (HDA), [ 44,46,77,78 ] and rolling circle amplification (RCA). [ 48–51 ]…”

“…The same group recently reported another system based on a DNAzyme‐dependent signal amplification DNA walker for ultrasensitive detection of nucleic acid through a pixel counting strategy. [ 76 ]…”