(Invited) CRISPR-Based Molecular Diagnostic Platform for Ultrasensitive and Target Amplification-Free Detection

Molecular diagnostics, in particular, nucleic acid testing, is critical in a wide range of fields relevant to the quality of human life. Over the past few decades, quantitative polymerase chain reaction (qPCR) has remained the gold-standard technique for nucleic acid testing. However, qPCR is limited by expensive instruments, the need for operation by well-trained personnel, and long sample-to-results time. Recently, the discovery of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) systems has opened a new path for nucleic acid detection. Despite many advances, the current CRISPR-based biosensors in nucleic acid detection still require target preamplifications. The target amplification process often requires a set of proteins and primers (2-6) and additional sample preparation steps, which dramatically complicates and lengthens the detection process (20 min to 2 hours) as well as increases the contamination risk of patient samples. In addition, the target preamplification process is usually nonlinear and reaches saturation rapidly, limiting the ability to quantitatively measure viral copy numbers for predicting and/or monitoring disease progression. In this presentation, I will present a biosensor, termed CRISPR Cas13a-gFET, for target amplification-free nucleic acid detection with an attomolar sensitivity via harnessing the trans-cleavage mechanism of CRISPR Cas13a and ultrasensitive graphene field-effect transistor (gFET) (Angew. Chem. Int. Ed. 2022, 61(32): e202203826). This CRISPR Cas13a-gFET platform can be adapted to detect a variety of DNA targets for medical diagnostics, environmental monitoring, and food safety (ACS Sensors2023, 8, 4, 1489–1499). I will also present a point-of-care microfluidic device that offers ultrasensitive yet rapid detection of viral RNA from clinical samples (Lab on a Chip 2023, 23, 3862-3873). These CRISPR-based molecular diagnostic platforms significantly advanced our knowledge on the design and development of next-generation biosensors for target amplification-free detection and can be used for ultrasensitive detection of a wide range of nucleic acid targets with clinical, biological, and environmental importance.