Enzyme-Free Photoelectrochemical Biosensor Based on the Co-Sensitization Effect Coupled with Dual Cascade Toehold-Mediated Strand Displacement Amplification for the Sensitive Detection of MicroRNA-21

An ultrasensitive photoelectrochemical (PEC) biosensor was developed based on cosensitization of biocompatible CuInS2/ZnS quantum dots (ZCIS QDs) and N-doped carbon dots (N-CDs) coupled with dual cascade toehold-mediated strand displacement amplification (dual cascade TSDA) for microRNA-21 (miRNA-21) detection. On the one hand, the TiO2/Au hybrid structure was used to immobilize double stranded DNA (thiolated capture strand and carboxylated signal strand), which could capture glutathione stabilized ZCIS QDs and N-CDs. The original TiO2/Au/ZCIS/N-CDs structure formed a cascade band gap arrangement, which provided a good band position for effective charge carrier separation, thus improving PEC performance and resulting in an evident decrease in photocurrent signal after the release of signal strands (SIG). On the other hand, the sensitivity of the biosensor was further enhanced by enzyme-free dual cascade TSDA, which was initiated by the target miRNA-21, like a molecular machine, and consumed the substrates and fuels, repeatedly used the target miRNA-21, and released a large number of reporter strands (RS). Subsequently, the released RS replaced SIG to prevent ZCIS QDs and N-CDs from sensitizing the electrode, which remarkably suppressed the photocurrent signal. The introduction of TSDA could produce high amplification capacity and specificity for the target miRNA-21 with advantages of simple primer design and mild reaction conditions. Impressively, with the cascade band gap arrangement for enhanced PEC performance and enzyme-free dual cascade TSDA for amplification capacity and specificity, the PEC biosensor exhibited excellent application in miRNA-21 analysis with a linear range from 1 pM to 100 nM and a low detection limit of 0.31 pM. This PEC biosensor retained good specificity, stability, and reproducibility and provided an effective method for PEC biosensor construction for microRNA. Moreover, the designed PEC biosensor was environmentally friendly, green manufactured, and self-powered and therefore compatible with the purpose of sustainable chemistry.