Portable miRNA Detection with Ligation-initiated Transcription and CRISPR/Cas12a

Abstract

Early detection of cancer is important for improving patient prognosis. miRNAs are good biomarkers for cancer screening because they are found in peripheral blood and their dysregulation is associated with cancer development. However, current miRNA detection assays, like RT-PCR, require expensive equipment. We are developing an assay for detecting specific miRNAs using padlock-probe based rolling circle transcription that activates a CRISPR/Cas12a complex, resulting in the cleavage of DNA-conjugated gold nanoparticles. This produces a colorimetric output that is quantified using a 3D printed portable detector. The portable detector is controlled by a Raspberry Pi that utilizes a PCB heater and thermistor; this incubates the assay at the desired temperature, while regularly taking photos to measure the color change. The presence of miRNA successfully initiates ligation, rolling circle transcription, and the activation of Cas12a. However, the conjugation of gold nanoparticles and the aggregation of gold nanoparticles with the linker still need to be optimized. The portable detector can facilitate the reaction and quantify the assay results without extensive equipment or intensive labor, making it efficient and cost-effective.