Oriented surface imprinted 96-well microplate-based fluorescent biosensor for glycoprotein detection by boronate affinity sandwich assay.

Glycoproteins perform vital functions in numerous biological processes and have important clinical implications. Many glycoproteins have been used as biomarkers and therapeutic targets for disease diagnosis. Due to low concentration of glycoprotein biomarkers and the presence of high-abundance interfering species in biological samples, a selective and sensitive detection method for glycoprotein is essential for real-world applications. In this study, we develop an oriented surface imprinted microplate-based fluorescent biosensor by boronate-affinity sandwich assay (BASA) for the specific, sensitive and high throughput determination of glycoproteins in complex samples. The structure of the BASA is based on sandwich formation between boronate affinity-oriented surface-imprinted microplates, target glycoproteins, and boronate affinity fluorescence probes. The imprinted microplates ensure the high specificity, high affinity and high throughput, while the fluorescence probes, consisting of boronic acid-modified CdTe QDs, provide high sensitivity. The proposed approach could exhibit a wide linear range of 1 ng/mL-10 ng/mL, with a low LOD of 0.528 ng/mL using horseradish peroxidase (HRP) as a model glycoprotein. As compared with traditional "turn off" fluorescent sensor, the developed "turn on" fluorescent sensor provided three orders of magnitude higher sensitivity at least. The fluorescent biosensor achieved average recoveries ranging from 96.8 % to 106.0 % in urine samples.