CRISPR/Cas12a antifouling nanocomposite electrochemical biosensors enable amplification-free detection of Monkeypox virus in complex biological fluids

초록

The escalating global threat of infectious diseases including monkeypox virus (MPXV) necessitates advancements in point-of-care diagnostics moving beyond the constraints of conventional methods tethered to centralized laboratories. Here we introduce multiple CRISPR RNA (crRNA)-based biosensors that can directly detect MPXV within 35 minutes without pre-amplification leveraging the enhanced sensitivity and antifouling attributes of the BSA-based nanocomposite. Multiple crRNAs strategically targeting diverse regions of the F3L gene of MPXV are designed and combined to amplify Cas12a activation and its collateral cleavage of reporter probes. Notably our electrochemical sensors exhibit the detection limit of 669 fM F3L gene without amplification which is approximately a 15-fold improvement compared to fluorescence detection. This sensor also shows negligible changes in peak current after exposure to complex biological fluids such as whole blood and serum maintaining its sensitivity at 682 fM. This sensitivity is nearly identical to the conditions when only the F3L gene was present in PBS. In summary our CRISPR-based electrochemical biosensors can be utilized as a high-performance diagnostic tool in resource-limited settings representing a transformative leap forward in point-of-care testing. Beyond infectious diseases the implications of this technology extend to various molecular diagnostics establishing itself as a rapid accurate and versatile platform for detection of target analytes. © 2024 The Royal Society of Chemistry