A CRISPR/Cas12a electrochemical biosensing to detect pig mtDNA D-loop for ensuring food authenticity

Ensuring safe and accurately labeled food products is crucial for communities worldwide. One significant concern in food safety is the unintended inclusion of pork in products, which impacts food labeling, religious dietary practices, legal standards, and public health. Detecting such contamination is challenging due to complex food processing, requiring advanced methods for accurate identification. A CRISPR/Cas12a-based electrochemical biosensor has garnered attention for its rapidity, portability, high sensitivity, and specificity, leveraging trans-cleavage activity for precise detection. This study utilized a modified electrode, SPCE/Ceria/STV/Biotin-ssDNA-Methylene Blue (MB), characterized by SEM/EDX and voltammetry methods to examine morphology and electrochemical behavior. Optimization using the Box-Behnken Design (BBD) yielded ideal conditions: 30 min STV incubation, 1.5 μM probe concentration, 240 min probe incubation, 0.1 % BSA concentration, 30 min target incubation, and 2 μL target volume. These parameters enabled the development of an ultrasensitive biosensor with detection and quantification limits of 4 fM and 71 fM, respectively. Operating within a range of 10 nM to 100 fM, the biosensor achieved 1.1 % RSD. This CRISPR-based biosensor successfully detected pig mtDNA in raw and processed meat samples, achieving a 100.82 % recovery rate for corned samples and maintaining stability for 14 days. These findings highlight the biosensor's potential as a rapid, stable, ultrasensitive, and ultraspecific alternative for food monitoring. Its robustness makes it particularly suitable for ensuring the authenticity of animal-derived food products susceptible to counterfeiting.