In situ amplified QCM immunoassay for carcinoembryonic antigen with colorectal cancer using horseradish peroxidase nanospheres and enzymatic biocatalytic precipitation

Abstract

Methods based on enzyme labels or nano labels have been developed for immunoassays, but most of them have low sensitivity and are unsuitable for low-abundance protein diagnostics and biosecurity. Herein, an innovative quartz crystal microbalance (QCM) immunosensing method was designed for high-efficiency detection of carcinoembryonic antigen (CEA) from serum samples with colorectal cancer patients by using horseradish peroxidase (HRP) nanoparticles as the enhancer, accompanying enzymatic biocatalytic precipitation (EBCP) toward 4-chloro-1-naphthol (4-CN) on an anti-CEA capture antibody-conjugated QCM probe. Initially, HRP nanospheres were synthesized on the basis of the reverse micelle method with the assistance of glutaraldehyde cross-linking, and then covalently conjugated onto polyclonal anti-CEA detection antibodies. The QCM immunosensing probe was prepared by immobilizing monoclonal anti-CEA capture antibodies on an L-cysteine-modified gold substrate. In the presence of target CEA, a sandwich-type immunoreaction was carried out between capture antibody and detection antibody, thereby resulting in the attachment of HRP nanospheres on the gold probe. Upon addition of 4-CN in the QCM cell, the carried HRP nanospheres catalyzed the 4-CN oxidation to produce an insoluble precipitate on the gold electrode, thus causing a change in the frequency. Relative to the conventional HRP-labeled strategy and direct antigen–antibody reaction, improved analytical features were obtained with HRP nanospheres. With the HRP nanosphere labeling method, the factors influencing the performance of the immunoassay were also studied. The covalent conjugation of antibodies with HRP nanospheres and the gold substrate achieved a good repeatability and intermediate precision down to 10.7%. Under optimum conditions, the frequency variation of the QCM immunoassay was proportional to the target CEA concentration within a dynamic linear range of 0.01–100 ng mL⁻¹ with a detection limit (LOD) of 7.8 pg mL⁻¹. In addition, the developed QCM immunoassay showed high specificity and long-term storage stability, and could be used for the analysis of human serum samples with consistent results in comparison with those obtained from the commercial enzyme-linked immunosorbent assay (ELISA) method.