Digital CRISPR-Powered Biosensor Concept without Target Amplification Using Single-Impact Electrochemistry

The rapid and reliable detection and quantification of nucleic acids is crucial for various applications, including infectious disease and cancer diagnostics. While conventional methods, such as the quantitative polymerase chain reaction are widely used, they are limited to the laboratory environment due to their complexity and the requirement for sophisticated equipment. In this study, we present a novel amplification-free digital sensing strategy by combining the collateral cleavage activity of the Cas12a enzyme with single-impact electrochemistry. In doing so, we modified silver nanoparticles using a straightforward temperature-assisted cofunctionalization process to subsequently detect the collision events of particles released by the activated Cas12a as distinct current spikes on a microelectrode array. The functionalization resulted in stable DNA-AgNP conjugates, making them suitable for numerous biosensor applications. Thus, our study demonstrates the potential of clustered regularly interspaced short palindromic repeats-based diagnostics combined with impact-based digital sensing for a rapid and amplification-free quantification of nucleic acids.