CRISPR-Enhanced Colorimetric Aptasensor for Adenosine Triphosphate Detection Based on MoS₂-Based Nanozymes

As the direct energy source in organisms, accurate and simple detection of adenosine triphosphate (ATP) is of great significance. Herein, a colorimetric aptasensor for ATP determination was designed by integrating the CRISPR/Cas12a system with an aptamer, and with Prussian blue nanocube and gold nanoparticle co-functionalized MoS₂ (MoS₂-PBNCs-AuNPs) nanozymes. As expected, the introduced CRISPR/Cas12a system and aptamer could efficiently amplify the detection signal and improve the specific recognition ability, respectively. Meanwhile, the catalytic activity of the MoS₂-PBNCs-AuNPs nanozymes can be regulated with the concentration of ATP. The high-affinity binding of ATP to the aptamer competitively inhibited aptamer-crRNA hybridization, causing fewer Cas12 proteins to be activated. As a result, the uncleaved single-stranded DNA (ssDNA) adsorbed onto the surface of nanozymes to effectively enhance their catalytic oxidation capability toward 3,3',5,5'-tetramethylbenzidine (TMB). According to this phenomenon, this CRISPR-enhanced colorimetric aptasensor can detect down to 0.14 μM ATP with high selectivity, reproducibility, and stability. In addition, acceptable recoveries and low relative standard deviations of the aptasensor for ATP determination suggest that it is promising for application in early detection of clinical-related diseases.