Label-free electrochemical immunosensor for porcine gelatin using a boron-doped diamond electrode <i>via</i> diazonium salt electrografting

Porcine gelatin is widely used in the food and pharmaceutical industries due to its favorable functional properties and low cost. However, its presence in consumer products raises serious concerns for individuals with dietary restrictions based on religious, ethical, or health considerations. In this study, a label-free electrochemical immunosensor was developed using a boron-doped diamond electrode modified with aryl diazonium salt for the selective and sensitive detection of porcine gelatin. The diazonium electrografting enabled stable covalent immobilization of anti-porcine gelatin antibodies via protein A, preserving anti­body orientation and activity. Experimental parameters were optimized using the Box-Behnken design, yielding ideal conditions of 500× antibody concentration, 60 min antibody incubation, and 15 min gelatin incubation. Detection was performed using differential pulse voltammetry with [Fe(CN)₆]3-/4- as a redox probe, allowing label-free monitoring of anti­body-antigen interactions based on changes in current. The immunosensor demonstrated excellent analytical performance, with a detection limit of 142.15 pg mL-1. Specificity testing confirmed that the sensor responds exclusively to porcine gelatin, showing no cross-reactivity with bovine gelatin. These results demonstrate that the proposed immunosensor provides a rapid, highly sensitive, and specific platform for porcine gelatin detection, offering great potential for food authentication and halal verification.