An amplification-free CRISPR/Cas12a-nanopipette electrochemical sensor for in situ discrimination of Salmo salar and Oncorhynchus mykiss
Abstract
Background: Salmo salar (S. salar) and Oncorhynchus mykiss (O. mykiss) exhibit highly similar morphological characteristics, which frequently leads to market adulteration. This phenotypic resemblance poses significant challenges for accurate on-site species identification using conventional analytical methods.
Results: In this study, an amplification-free electrochemical biosensor was constructed by integrating the CRISPR/Cas12a recognition system with a single nanopipette, enabling precise identification of S. salar DNA without the need for complex pretreatment. This strategy is based on target DNA-induced activation of the trans-cleavage activity of Cas12a, which cleaves the DNA reporter molecules immobilized on the inner wall of the nanopipette, leading to alterations in surface charge and enabling electrochemical readout via ion current rectification (ICR). Under optimized conditions, the sensor exhibited a detection limit as low as 0.11 pM, effectively distinguished O. mykiss DNA, and demonstrated favorable reproducibility and stability.
Significance: This work provides a low-waste, on-site analytical tool for the authentication of aquatic species, enabling direct in situ detection within salmon tissue sections without the need for nucleic acid amplification or complex sample pretreatment, thereby effectively filling a technical gap in rapid and accurate species identification.
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