A Novel Electrochemiluminescence Immunosensor Based on Resonance Energy Transfer between g-CN and NU-1000(Zr) for Ultrasensitive Detection of Ochratoxin A in Coffee

In this study, an electrochemiluminescence (ECL) immunosensor based on nanobody heptamer and resonance energy transfer (RET) between g-C3N4 (g-CN) and NU-1000(Zr) was proposed for ultrasensitive ochratoxin A (OTA) detection. First, OTA heptamer fusion protein was prepared by fusing OTA-specific nanometric (Nb28) with a c-terminal fragment of C4 binding protein (C4bp alpha) (Nb28-C4bp alpha). Then, Nb28-C4bp alpha heptamer with the high affinity used as a molecular recognition probe, of which plenty of binding sites were provided for OTA-Apt-NU-1000(Zr) nanocomposites, thereby improving the immunosensors' sensitivity. In addition, the quantitative analysis of OTA can be achieved by using the signal quenching effect of NU-1000(Zr) on g-CN. As the concentration of OTA increases, the amount of OTA-Apt-NU-1000(Zr) fixed on the electrode surface decreases. RET between g-CN and NU-1000(Zr) is weakened leading to the increase of ECL signal. Thus, OTA content is indirectly proportional to ECL intensity. Based on the above principle, an ultra-sensitive and specific ECL immunosensor for OTA detection was constructed by using heptamer technology and RET between two nanomaterials, with a range from 0.1 pg/mL to 500 ng/mL, and the detection limit of only 33 fg/mL. The prepared ECL-RET immunosensor showed good performance and can be successfully used for the determination of OTA content in real coffee samples, suggesting that the nanobody polymerization strategy and the RET effect between NU-1000(Zr) and g-CN can provide an alternative for improving the sensitivity of important mycotoxin detection.