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Performance of nanoprobes can often determine the detection level of Lateral immunochromatography. Traditional probes were limited by the quantity and orientation of antibodies, immune activity of the Fab region or binding strength between protein and substrate. This study developed a new efficient and robust technology to construct fluorescent nanoprobes with oriented modified antibodies, based on specific binding of the Fc region of antibody with streptococcal protein G (SPG) on the surface of polystyrene microspheres (MS) and subsequent covalent cross-linking at binding sites to firm them. Lateral flow immunoassay using these probes was applied for the detection of cardiac troponin I (cTnI). The significantly improved detection sensitivity demonstrated that antibody orientation on MS surfaces effectively enhanced immunological activities of probes compared with random immobilizing methods. Furthermore, performance evaluation results of lateral flow test strips met clinical requirements perfectly, including limit of detection (0.032 ng/mL), linearity ( R > 0.99), repeatability (CV < 10%), correlation ( R > 0.99), and heat aging stability. This research also employed heterophilic blocking reagent (HBR) to actively block redundant binding sites of SPG for the first time in order to eliminate false positive interferences, improving the sensitivity and precision of test results further.

No abstract

Nanoprobes have been widely used in biomedical engineering. However, antibodies are generally conjugated onto nanoparticles disorderly, which reduces their antigen recognition ability. The existing antibody orientation approaches are usually complex. Here, we developed and demonstrated a simple antibody-oriented strategy for the lateral flow immunoassay of cardiac troponin I by conjugating antibodies onto polystyrene nanospheres at the optimal pH. The binding amount and orientation of antibodies as well as the detection sensitivity were significantly improved. Although pH regulation is commonly used to optimize antibody conjugation, this paper illustrates the mechanism of its antibody orientation enhancement ability for the first time and reveals the important influences of the density, the charge distribution and hydrophilicity of the antibody, the control of the velocities of physical adsorption and chemical coupling, and other factors on antibody orientation. It is of great significance to understand and regulate antibody conjugation on the surface of micro-or nanospheres to construct high-performance probes for in vitro diagnosis applications.