Electrochemiluminescence Immunosensor Based on Nanobody and Au/CaCO₃ Synthesized Using Waste Eggshells for Ultrasensitive Detection of Ochratoxin A

A novel ultrasensitive electrochemiluminescence (ECL) immunoassay based on Au/CaCO₃ was proposed for detecting ochratoxin A (OTA) in coffee. Au/CaCO₃ nanocomposites synthesized using waste eggshells as the template with a large surface area and excellent electrochemical properties were applied for immobilizing a large amount of Ru­(bpy)₃²⁺ and conjugating a high-affinity nanobody (prepared by the phage display technique). Coupling of the Au/CaCO₃ nanocomposites and nanobody technologies provided an ultrasensitive and highly selective ECL immunosensor for OTA detection in the range of 10 pg/mL–100 ng/mL with a low detection limit of 5.7 pg/mL. Moreover, the as-prepared ECL immunosensor showed excellent performance and high stability. Finally, the proposed ECL sensor was applied to analyze OTA in coffee samples, confirming the desirable accuracy and practical applicability potential. Overall, this work presents a new nanomaterial for fabricating the sensing interface of immunosensors by harnessing natural waste as the source and a method for detecting toxic OTA in foods.