Electrochemical biosensor for ultrasensitive exosomal miRNA analysis by cascade primer exchange reaction and MOF@Pt@MOF nanozyme

Exosomal miRNAs have been discovered as important and reliable biomarkers for early diagnosis of tumors. However, it is still challenging to achieve accurate determination of trace exosomal miRNAs in real samples. Herein, we report an electrochemical strategy based on the cascade primer exchange reaction (PER) with MOF@Pt@MOF nanozyme for ultrasensitive detection of exosomal miRNA. Target-triggered PER that only includes a gated hairpin, a primer, and DNA polymerase can produce a long single strand in an autonomous and isothermal manner. Then, the nascent strand releases the protector B used to blockade capture probes, resulting in the binding of the nanozyme to sensing interface. Under the catalysis of the nanozyme, hydrogen peroxide (H2O2) is decomposed into H2O and O-2, thus producing an amplified electrochemical signal. Benefiting from the cascade PER and the noticeable catalytic activity of the multiple-layered nanozyme, the established biosensor shows high sensitivity with limit of detection down to 0.29 fM and high specificity that can distinguish homologous miRNAs with single base mismatch. The developed strategy allows discrimination of tumor cells and breast cancer patients by detecting exosomal miRNA-21, and the results of this biosensor are consistent with qRT-PCR. Therefore, the electrochemical strategy has wide potential in the early screening of tumors.