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Published on: July 21, 2023
A label-free cyclic amplification strategy for microRNA detection by coupling graphene oxide-controlled adsorption
Fengzhou Xu1, Zhenzhen Qiao2, Lan Luo2
1Fujian Provincial Key Laboratory of Ecology-toxicological Effects & Control for Emerging Contaminants, Key Laboratory of Ecological Environment and Information Atlas (Putian University) Fujian Provincial University, College of Environmental and Biological Engineering, Putian University, Putian, 351100, China; State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Hunan University, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Changsha, 410082, China.
Abstract:
Herein, based on a terminal deoxynucleotidyl transferase (TdT)-mediated superlong poly-T-templated-copper nanoparticles (poly T-CuNPs) strategy, a simple, universal and label-free fluorescent biosensor for the detection of miRNA was constructed by employing graphene oxide (GO) and DNase I. In this strategy, GO and DNase I were used as a switch and amplifier of the signal generation pathway, respectively, and the fluorescence of poly T-CuNPs was used as the signal output. In the presence of target miRNA, the DNA dissociated from the GO surface by forming a miRNA/DNA duplex and was degraded by DNase I. The short oligos with 3'-OH, the product of DNase I degradation, could be recognized by the TdT and added to a long poly-T tail. Finally, the fluorescence signal was output through the synthesis of poly T-CuNPs. As a proof of concept, let-7a was analyzed. The method showed good sensitivity and selectivity with a linear response in the 50 pM-10,000 pM let-7a concentration range and a 30 pM limit of detection (LOD = 30 pM, R2 = 0.9954, the relative standard deviation were 2.79%-5.30%). It was also successfully applied to the determination of miRNA in spiked human serum samples. It showed good linearity in the range of 500-10000 pM (R2 = 0.9969, the relative standard deviation were 1.61%-3.85%). Moreover, both the adsorption of GO and the degradation of DNase I are DNA sequence-independent; thus, this method can be applied to the detection of any miRNA simply by changing the assisted-DNA sequence.
