An Ultrasensitive Electrochemical Biosensor Integrated by Nicking Endonuclease-Assisted Primer Exchange Reaction Cascade Amplification and DNA Nanosphere-Mediated Electrochemical Signal-Enhanced System for MicroRNA Detection

Abstract

Specific and sensitive microRNAs (miRNAs) detection is essential to early cancer diagnosis. The development of these technologies including functional nuclease-mediated target amplification and DNA nanotechnology possesses tremendous potential for the high-performance detection of miRNAs in the accurate diagnosis of disease. In this study, we have established an ultrasensitive electrochemical biosensor by combining nicking endonuclease-assisted primer exchange reaction (PER) cascade amplification with a DNA nanosphere (DNS)-mediated electrochemical signal-enhanced system for the detection of miRNA-21 (miR-21). The cascade amplification is initiated by a nicking endonuclease that can cleave specific DNA substrates and highly amplify translation of the target to single-stranded DNA fragments (sDNA). Then, the PER cascade is powered by strand-displacing polymerase and generates a large amount of nascent single-stranded connector DNA (cDNA) via sDNA triggering of the dumbbell probe (DP), thus achieving the cascade amplification of the target. Finally, the DNS loaded with plenty of electroactive substances can be captured on the electrode via cDNA for further enhancing the electrochemical signal and highly sensitive detection of miR-21. The proposed electrochemical biosensor exhibits a wide detection range of 1 aM to 0.1 nM and a low detection limit of 0.58 aM. The excellent selectivity allows the biosensor to discriminate miR-21 from other miRNAs, even the one base-mismatched sequence. Moreover, the practicability of the biosensor is investigated by analyzing miR-21 in human serum and cancer cell lysate. Therefore, our proposed nicking endonuclease-assisted PER cascade amplification strategy provides a powerful platform for the early detection of miRNA-related disease and molecular diagnosis.

Hybridization Chain Reaction-Based Electrochemical Biosensors by Integrating the Advantages of Homogeneous Reaction and Heterogeneous Detection

“…In addition, the anodic peak currents for MCF-7 cells were higher than those for HeLa cells. These results indicated that the level of miRNA-21 in MCF-7 cells is higher than that in HeLa cells, which is consistent with previous reports [30,32,38,67]. Thus, the proposed biosensor shows high practicality for the assays of biological samples, providing an alternate tool for clinical diagnosis and therapeutic assessment of diseases.…”

“…The sequences of initiator DNA, miRNA-21, and hairpin probes used in this work were designed according to the previous reports [1,38]. Their sequences are shown as follows: AGT CTA GGA TTC GGC GTG GGT TAA (initiator DNA), AGT CTA GGA TTG GGC GTG GGT TAA (single-base mismatched), AGT CTT GGA TTG GGC GTG GGA TAA (three-base mismatched), CGT GTA GGT TAT GAC GTG CGT AGC (random), biotin-TTA ACC CAC GCC GAA TCC TAG ACT CAA AGT AGT CTA GGA TTC GGC GTG (H1), biotin-AGT CTA GGA TTC GGC GTG GGT TAA CAC GCC GAA TCC TAG ACT ACT TTG (H2), UAG CUU AUC AGA CUG AUG UUG A (miRNA-21), biotin-CAG ACT GAT GTT GAC GTG AAA CTC AAC ATC AGT CTG ATA AGC TA (H3), and biotin-GTT TCA CGT CAA CAT CAG TCT GTA GCT TAT CAG ACT GAT GTT GA (H4).…”

Smartphone-Assisted Flexible Electrochemical Sensor Platform by a Homology DNA Nanomanager Tailored for Multiple Cancer Markers Field Inspection

“…Deoxyribonucleic acid (DNA) is a kind of biological macromolecule that is the carrier of genetic information in an organism. , The strict base-pairing principles make it an interesting assembly element for the controlled and programmed design and assembly of DNA nanomaterials. DNA nanomanagers have the characteristics of good biocompatibility, stability, high specificity, programmability, etc., adopting DNA as the recognition element, and the structural changes after the combination of DNA and the target are converted into detectable signals is conducive to the strong specific detection of tumor markers, miRNAs. , Nanomachines composed of nanomaterials and performing specific actions have gradually become one of the research hotspots.…”

Irregular DNA Triangular Prism/Triplex Assembly for Duplicate MiRNA Analysis with Nicking Endonuclease-Mediated Amplification

“…For example, Zhu’s group translated target miRNA to single-stranded DNA (ssDNA) by NEase followed by primer exchange reaction cascade amplification. DNA nanospheres were then prepared for electrochemical analysis with enhanced signal responses . Lu et al designed a three-dimensional DNA walker on gold nanoparticles (AuNPs) for an electrochemical miRNA assay.…”

“…DNA nanospheres were then prepared for electrochemical analysis with enhanced signal responses. 21 Lu et al designed a three-dimensional DNA walker on gold nanoparticles (AuNPs) for an electrochemical miRNA assay. After a miRNA-triggered strand displacement reaction, the walking behavior helped the release of a ferrocene-tagged signal probe, which was powered by NEase.…”