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

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. [Display omitted] • A split DNAzyme sensor based on NaA43 was designed to use in plasma biopsy. • The sensor achieved detection limit of aM grade in nucleic acid measurement. • The ultra-sensitivity ascribed to the key procedure of butanol dehydration. [ABSTRACT FROM AUTHOR]