A Highly Sensitive and Selective Electrochemical Biosensor for Direct Detection of MicroRNAs in Serum

Abstract

On the basis of hybridized target microRNA (miRNA) strand initiated cleavage of hybridized deoxyribonucleic acid (DNA) capture probes (CPs) by a duplex-specific nuclease (DSN), a highly sensitive and selective label-free miRNA biosensor is developed in this article. Briefly, thiolated DNA CPs are immobilized onto a gold electrode through self-assembly. The electrode is then hybridized to a sample solution containing the target miRNA. The hybridized CPs in the miRNA-CP duplexes are simultaneously cleaved by the DSN, releasing the target miRNA strands back to the sample solution. The released target miRNA strands again hybridize with the remaining CPs on the electrode, thus forming an isothermal amplification cycle. The distinct difference in electrochemical impedance between a control and the DSN cleaved biosensor allows label-free detection of miRNA down to femtomolar levels. The mismatch discrimination ability of the DSN permits miRNA expression to be profiled with high selectivity. The exceptional amplification power of the DSN along with the simple assay protocol makes direct miRNA expression profiling possible in real-world samples with minimal or no sample pretreatments. Attempts are made in direct profiling circulating miRNAs in serum and miRNAs in total RNA extracted from cancer cells.

Electrochemical genosensor based on disc and screen printed gold electrodes for detection of specific DNA and RNA sequences derived from Avian Influenza Virus H5N1

“…The literature examples relating to biosensors for a microRNA (miRNA) detection are collected in a Table 2 [32][33][34][35][36][37][38][39][40][41]. Although the examples of sensors given in the Table 2 have lower detection limits, they are able to detect the RNA sequences up to 30-mer.…”

Electrochemical Detection of miRNAs

“…5) [49]. A gold electrode was modified with a mixed monolayer of thioglycolic acid and thiol-labeled DNA probe that captures the complementary miRNA by hybridization.…”

Sensitive detection of microRNAs based on the conversion of colorimetric assay into electrochemical analysis with duplex-specific nuclease-assisted signal amplification

“…More importantly, this enzyme can clearly discriminate among mismatched duplexes, even a one-base mismatch. 11,12 Therefore, analytic specificity has been significantly improved with DSN-mediated analytical strategies, including colorimetric assays, [13][14][15] electrochemistry, [16][17][18][19][20][21][22][23][24] fluorescence,…”

“…17,21,22,[53][54][55][56] Furthermore, we demonstrated that the DSN-assisted colorimetric assay can be converted into an electrochemical analysis with improved sensitivity and specificity. Specifically, the DNA probe immobilized on the electrode surface and that labeled with ferrocene (Fc) (denoted "Fc-DNA-Fc") in the solution triggered the in situ assembly of positively charged AuNPs through electrostatic interaction.…”