Sensitive and portable electrochemical immunoassay for lipoprotein-associated phospholipase A2 using BSA-doped CaCO3 nanospheres to regulate pH readout

Abstract

A simple and sensitive electrochemical immunoassay method was developed for the quantitative monitoring of lipoprotein-associated phospholipase A₂ (Lp-PLA₂) on a low-cost microtiter plate by using a portable handheld pH meter. Bovine serum albumin (BSA)-doped CaCO₃ nanospheres were utilized to regulate the pH value of an acidic solution on the basis of classical chemical reactions. To construct such an immunosensing system, the synthesized BSA-CaCO₃ nanospheres were initially conjugated covalently to an anti-Lp-PLA₂ detection antibody, and then a sandwich-type immunoreaction was carried out on the anti-Lp-PLA₂ capture antibody-coated microplate in the presence of the target analyte. Accompanying the sandwiched immunocomplexes, the labeled CaCO₃ nanospheres were dissolved in the presence of acid (CaCO₃ + 2H⁺ → Ca²⁺ + CO₂ + H₂O), thus resulting in the pH change of the detection solution. Introduction of CaCO₃ nanospheres including numerous carbonate ions was expected to enhance the sensitivity of the electrochemical immunoassay. Under optimum conditions, BSA-CaCO₃-based immunoassay displayed good pH responses for the determination of target Lp-PLA₂ within the dynamic linear range from 0.1 ng mL⁻¹ to 300 ng mL⁻¹. The limit of experimental detection achieved was ∼78 pg mL⁻¹ Lp-PLA₂ with good coefficients of variation for the inter- and intra-assays. Also, this system gave good anti-interfering capacity toward other biomarkers and enzyme proteins. Importantly, our strategy was applied for the analysis of human serum specimens with satisfactory results in comparison with commercialized Lp-PLA₂ ELISA kits.