Generation of a nanobody-alkaline phosphatase fusion and its application in an enzyme cascade-amplified immunoassay for colorimetric detection of alpha fetoprotein in human serum

Sensitive detection of liver disease biomarkers can facilitate the diagnosis of primary hepatoma and other benign liver diseases, and the alpha fetoprotein (AFP) was selected as the model macromolecule in this work. Herein an enzyme cascade-amplified immunoassay (ECAIA) based on the nanobody-alkaline phosphatase fusion (Nb-ALP) and MnO 2 nanoflakes was developed for detecting AFP. The bifunctional biological macromolecule Nb-ALP serves as the detection antibody and the reporter molecule. The MnO 2 nanoflakes mimic the oxidase for catalyzing the 3,3′,5,5′-tetramethylbenzidine (TMB) into the blue oxidized TMB, which has a quantitative signal at the wavelength of 650 nm. Moreover, the Nb-ALP could dephosphorylate the ascorbic acid-2-phosphate (AAP) to form the ascorbic acid (AA) that can disintegrate the nanoflakes to reduce their oxidation capacity with the content decrease of the oxidized TMB. Using the constructed TMB-MnO 2 colorimetric sensing system for Nb-ALP and the optimized experimental parameters, the ECAIA has a limit of detection (LOD) of 0.148 ng/mL which is 18.7-fold lower than that of the p-nitrophenylphosphate (pNPP)-based method (LOD = 2.776 ng/mL). The ECAIA showed good selectivity for AFP with observed negligible cross-reactions with several common cancer biomarkers. The recovery rate for AFP spiked in human serum ranged from 94.8% to 113% with the relative standard deviation from 0.3% to 6.5%. For analysis of the actual human serum samples, a good linear correlation was found between the results tested by the ECAIA and the automatic chemiluminescence analyzer. Thus, the ECAIA was demonstrated to be a promising tool for highly sensitive and selective detection of AFP, providing a reference for analysis of other macromolecule biomarkers.