CRISPR/Cas12a-Powered EC/FL Dual-Mode Controlled-Release Homogeneous Biosensor for Ultrasensitive and Cross-Validated Detection of Messenger Ribonucleic Acid

Accurate detection of cancer-associated mRNAs is beneficialtoearly diagnosis and potential treatment of cancer. Herein, for thefirst time, we developed a novel CRISPR/Cas12a-powered electrochemical/fluorescent(EC/FL) dual-mode controlled-release homogeneous biosensor for mRNAdetection. A functionalized ssDNA P2-capped Fe3O4-NH2 loaded with methylene blue (P2@MB-Fe3O4-NH2) was synthesized as thesignal probe, while survivin mRNA was chosen as thetarget RNA. In the presence of the target mRNA, the nicking endonuclease-mediatedrolling circle amplification (NEM-RCA) was triggered to produce significantamounts of ssDNA, activating the collateral activity of Cas12a towardthe surrounding single-stranded DNA. Thus, the ssDNA P1 completelycomplementary to ssDNA P2 was cleaved, resulting in that the ssDNAP2 bio-gate on Fe3O4-NH2 couldnot be opened due to electrostatic interactions. As a result, therewas no or only a little MB in the supernatant after magnetic separation,and the measured EC/FL signal was exceedingly weak. On the contrary,the ssDNA P2 bio-gate was opened, enabling MB to be released intothe supernatant, and generating an obvious EC/FL signal. Benefitingfrom the accuracy of EC/FL dual-mode cross-verification, high amplificationefficiency, high specificity of NEM-RCA and CRISPR/Cas12a, and highloading of mesoporous Fe3O4-NH2 on signal molecules, the strategy shows aM-level sensitivity andsingle-base mismatch specificity. More importantly, the practicalapplicability of this dual-mode strategy was confirmed by mRNA quantificationin complex serum environments and tumor cell lysates, providing anew way for developing a powerful disease diagnosis tool.