Competitive immunoassay combined with magnetic separation and pulsed LIF system for cefalexin detection

Abstract

In this study, a facile, ultrasensitive and interference-free method to detect cefalexin (CEX) was developed for the first time. This assay was carried out by covalently immobilizing cefalexin–ovalbumin (CEX–OVA) on high specific surface area amorphous nanoparticles of superparamagnetic iron oxide (SPIO). Here, the SPIO–CEX–OVA structure was rich in antibody domains for competitive immunological recognition to anti-CEX antibody and to AlexaFluor 488 labeled goat anti-mouse IgG. Compared with traditional laser induced fluorescence detection, the introduction of SPIO can preconcentrate analytes to reduce the detection limit and greatly shorten the assay time. A pulsed laser with higher peak energy was chosen as the excitation source for generating strong fluorescence signals and to improve sensitivity. The detection limit was 0.34 ng mL⁻¹ with linearity in the range of 0.5 ng mL⁻¹ to 50 ng mL⁻¹, and the IC₅₀ was 1.7 ng mL⁻¹. The accuracy and reproducibility were determined by using spiked milk samples with three different concentrations of CEX (5, 20 and 50 ng mL⁻¹). The recoveries of 85.2–111.4% were obtained with relative standard deviations of 5.3–9.1%, respectively. These results indicate that the method provides a pragmatic platform for convenient detection of small molecular residues due to its high sensitivity, selectivity and short assay time.