Microfluidic fluorescence immunosensor using ZnONFs for invasive aspergillosis determination

Galactomannan (GM) is a polysaccharide cell-wall component released by Aspergillus species, used as a biological marker for invasive aspergillosis (IA) screening. A fluorescence microfluidic immunosensor for GM detection was developed by using ZnO nanoflowers (ZnONFs) synthesized by a wet chemical method and characterized by scanning electron microscopy and energy dispersive spectrometry. The ZnONFs were covalently linked to the microfluidic channel through a silanization process with 3-aminopropyl triethoxysilane and then used as a bioaffinity nanoplatform for the immobilization of monoclonal anti-GM antibodies, which react specifically with the GM present in human serum samples. Subsequently, the GM amount that reacted was quantified by an HRP-anti-GM antibody (type sandwich immunoassay), which in the presence of H2O2 catalyzes the oxidation of 10-acetyl-3,7-dihydroxyphenoxacine to a highly fluorescent resorufin, which was measured by a laser-induced fluorescence (LIF) detector (excitation at 561 nm and emission at 585 nm). The measured fluorescence is directly proportional to the concentration of GM present in the patient serum sample. The fluorescence method was compared with a previously published electrochemical method, and a commercial ELISA test, showing outstanding properties. This immunosensor can be applied to the IA early diagnosis since it offers several attractive advantages such as high selectivity and sensitivity.