Aptamer-based dual-enzyme, amplification-free biosensor integrating CRISPR-Cas12a and Exo III for sensitive detection of ATP

Adenosine triphosphate (ATP) plays a critical role in cellular energy metabolism, acting as the energy source for many biological reactions. Abnormal fluctuations in ATP levels are directly linked to various diseases such as malignancies, bacterial infections, and cardiovascular disorders, making early-stage disease detection through rapid, highly sensitive ATP measurement essential. The current methods for ATP detection suffer from drawbacks such as complex procedures, reliance on expensive instruments, and prolonged reaction times. To this end, we have developed a novel biosensor named ATP Output Sensor Activated by CRISPR (AOSAC).This biosensor employs aptamer-based specific recognition combined with a dual-enzyme, amplification-free system involving Exo III and CRISPR-Cas12a for ATP detection. This sensor operates through an aptamer in a closed duplex structure, upon binding ATP, releases a trigger strand. This strand displaces an activation chain, which in turn interacts with Cas12a's crRNA, initiating trans-cleavage activity that cleaves a single-stranded DNA probe to produce a fluorescent signal. Concurrently, the activation chain's release sites are targeted by Exo III, enhancing signal amplification without the need for complex nucleic acid amplification, thus providing a highly selective, rapid detection method. This technique offers a linear detection range from 0 nM to 20 μM with a detection limit of 44.2 nM, presenting a novel strategy for signal amplification and high specificity in distinguishing ATP from its analogues.