A critical assessment of aptamer and CRISPR-Cas12a-based biosensors for small molecule detection

Analyte detection through aptamer-induced signal generation by CRISPR-Cas enzymes has rapidly emerged as a popular biosensing approach. Here, we investigated the implementability and analytical performance of this setup for the detection of diverse small molecule analytes. We selected nine aptamers from the literature targeting seven analytes and tested a commonly used assay design whereby analyte binding by the aptamer liberates a short complementary DNA strand, which in turn activates Cas12a to generate a fluorescence signal. After extensive optimization, the assay functioned for only two of the seven analytes, and several previously reported results could not be reproduced. While Cas12a fluorescence detection was robust, the low success rate is likely due to aptamers not functioning reliably, underscoring the need for careful aptamer validation. Overall, our study provides a critical assessment of aptamer-Cas12a assay performances and discusses potential strengths, limitations, and pitfalls of this biosensing strategy.