A sodium-dependent split DNAzyme fluorescent sensor under butanol acceleration for ultrasensitive detection of PIWI-interacting RNAs and microRNAs

Abstract

Split DNAzymes have been designed as multifunctional biosensors. However, their limited sensitivity and low resistance to nuclease hinder their applications in biological samples. Herein, we developed a sodium-dependent and butanol accelerated split DNAzyme fluorescent sensor named Na+-BAS DNAzyme which has an ultra-sensitivity for quantifying PIWI-interacting RNAs/microRNAs and can be directly applied in blood biopsy to detect nucleic acids within the extracellular vesicles (EVs). The Na+-BAS DNAzyme consists of two split DNA strands based on NaA43 DNAzyme, a starting hairpin DNA for binding target to initiate the combining of the split enzymes, and a hairpin RNA-embedded DNA substrate labeled a quencher and a fluorophore for signal readout after its cleavage by the assembled DNAzyme complex. To achieve ultra-sensitivity, we employed a simple but key approach of butanol dehydration to highly accelerate the measuring reaction. The sensor has achieved an ultra-low detection limit of 12 × 10−18 M miR-21 after 30 min reaction, and 3.98 or 2.69 particles μL−1 MCF-7 EVs through miR-21 or piR-20365 detection. With these high performances, the sensor can detect extremely trace abundance of miR-21 in a single MCF-10a or MCF-7 EV, quantifying it as 5.93 × 10−3 or 0.12 copies per EV. Without any chemical structure modification, the sensor can be directly used in the plasma sample for breast cancer diagnosis which has obtained both 100% sensitivity and specificity using the biomarker of piR-20365. The Na+-BAS DNAzyme sensor’s ultra-sensitivity, high efficiency, universality, and simplicity make it a great potential in the clinical diagnostic application.