Detection of Candida albicans by a sandwich assay using boronate affinity microspheres coupled with amphotericin B-time-resolved fluorescent microspheres.

Abstract

Candida albicans (C. albicans) is an opportunistic pathogenic fungus that poses a threat to public health and food safety, while conventional detection methods suffer from high costs, complicated procedures, and insufficient sensitivity. Here, a novel biomolecule-free sandwich detection platform was constructed to quantitatively detect C. albicans by integrating phenylboronic acid-functionalized magnetic microspheres (PFSEPD) and amphotericin B-labeled time-resolved fluorescent microspheres (ATFM). The stepwise synthesis of PFSEPD was verified through multiple characterizations, and the as-prepared PFSEPD exhibited favorable monodispersity, rapid magnetic responsiveness, and low preparation cost, with adsorption data well fitted by the Langmuir isotherm and pseudo-second-order kinetic models. Meanwhile, the ATFM signal probe was synthesized by covalently grafting amphotericin B (AmB) onto carboxylated time-resolved fluorescent microspheres (TFM). In the established dual-chemical recognition sandwich system, PFSEPD rapidly captured the fungus through boronate-cis-diol specific affinity, while ATFM selectively labeled the captured C. albicans through AmB-ergosterol interactions. After elution with SDS buffer, the fluorescent signal of the released ATFM was quantitatively measured. The PFSEPD-ATFM detection strategy achieved limits of detection (LODs) of 46 and 40 CFU mL^-1 in milk and honey, respectively, with recoveries of 96.2%-101.8% and relative standard deviations (RSDs) less than 5.0%. This dual-chemical recognition sandwich system provides a facile, sensitive alternative for the rapid and quantitative detection of C. albicans.