Generation of a nanobody-alkaline phosphatase heptamer fusion for ratiometric fluorescence immunodetection of trace alpha fetoprotein in serum

Alpha fetoprotein (AFP) is an important biomarker for diagnosis of hepatocellular carcinoma (HCC). Whereas, it is always a challenge to detect trace AFP in serum. In this work, a ratiometric fluorescence enzyme immunoassay (RFEIA) was developed using nanobody-alkaline phosphatase (Nb-AP) heptamer and MnFe layered double hydroxides nanoflakes (MnFe LDH) for ultrasensitive detection of AFP. The Nb-AP heptamer (Nb-C4bpα-AP) was constructed by fusion expression of Nb, AP, and α-chain of C4 binding protein (C4bpα), where the C4bpα contributed to multimerization through self-assembly. The dual functional Nb-C4bpα-AP can recognize AFP, dephosphorylate ascorbic acid-2-phosphate (AAP) into ascorbic acid (AA), and thus tune the MnFe LDH-mediated ratiometric fluorescence, which was generated from the oxidization of MnFe LDH on o-phenylenediamine (OPD) and the catalyzation of MnFe LDH on the cyclization reaction between AA and OPD. By integration of Nb-C4bpα-AP, MnFe LDH, AAP, and OPD, the RFEIA showed a limit of detection of 0.013 ng/mL with good selectivity, accuracy and precision. Furthermore, results of clinical serum samples tested by the RFEIA were well confirmed by the automated chemiluminescence immunoassay analyzer. Thus, this work demonstrated that the Nb-C4bpα-AP is a robust immunoreagent and the developed RFEIA could be a very promising tool for diagnosis of HCC.