A self-supply crRNA-mediated CRISPR/Cas12a-driven controlled-release homogeneous biosensor for ultrasensitive detection of microRNA

Abstract

The abnormal expression of microRNAs (miRNAs) will lead to the occurrence of cancer and serious genetic related diseases. In this work, a novel self-supply crRNA-mediated CRISPR/Cas12a-driven controlled-release homogeneous biosensor was developed for ultra-sensitivity detection of miRNA-155, using single stranded DNA1 (ssDNA1, S1) as a bio-gate and UiO-66-NH2 as a nanocontainer. Initially, the exponential amplification reaction (EXPAR) was designed to transform and amplify the target recognition event into the T7 promoter, which could function as a trigger for transcription amplification to generate a large number of crRNA. The abundant crRNA formed a ternary complex with Cas12a and activator, and the trans-cleavage activity of the CRISPR/Cas12a was activated. Then the ssDNA2 completely complementary to ssDNA1 was cleaved, resulting in inability to form double-stranded DNA (dsDNA), which made ssDNA1 inseparable from UiO-66-NH2. Eventually, the stored methylene blue (MB) signal cannot be released from UiO-66-NH2. On the contrary, the ssDNA1 bio-gate was opened, and MB signal molecules escaped from the container, producing an obvious current signal. Through triple signal amplification and precise control and release system, the prepared biosensor could realize an ultralow detection limit of miRNA-155 as low as 31.6 aM. More importantly, the proposed strategy showed excellent sensitivity and specificity in complex serum environments and tumor cell lysates, providing a new approach to develop a robust tool for disease diagnosis.