Programmable readout sensor for microRNA: CRISPR/Cas12a-assisted multi-amplification strategy activated photoelectrochemistry-colorimetry detection

Abstract

The microRNA (miRNA) has a unique physiological regulation function in tissues, thus its expression level can reflect the development of various diseases. Nevertheless, due to the low-abundance and high-homologous similarity of miRNA, the precise detection of miRNA remained a challenge. Herein, a CRISPR/Cas12a-assisted multi-amplification strategy-mediated programmable miRNA (Cas-Master) biosensor was constructed based on photoelectrochemistry (PEC) and colorimetry (CM). Initially, by employing the rolling circle amplification (RCA), the miRNA was transformed into the long single-stranded DNA with a large repetitive sequence region, which specifically triggered the Cas12a’s trans-cleavage performance. Afterwards, the activated-Cas12a randomly cleaved the trigger strands of hybridization chain reaction (HCR). Thus, Cas12a was used as the medium of RCA and HCR reaction to form the multi-amplification circuit. Then, the glucose oxidase (GOx) was combined with β-CD@AuNPs and NH2-MIL-88B (Fe) nanozyme to form dual-cascade system for PEC and CM response, respectively. Both the PEC and CM signal showed linear relationship with the logarithm of the miRNA concentrations from 1 fM to 100 nM with the limit of detection of 0.3 fM (PEC) and 0.5 fM (CM). Furthermore, through the programmed design of the RCA reaction, the Cas-Master can be applied to recognize different types of miRNAs. Besides, a portable device was constructed based on Cas-Master, providing a new solution to expand the Cas12a-based platform into areas of point-of-care test (POCT) and early disease diagnosis.