If detected early, many illnesses, including cancer, could be effectively treated. The diagnostic process depends on the identification of protein biomarkers, which circulate in body fluids at higher than normal concentrations sometimes even before any other symptoms manifest. As a consequence of this, early diagnosis is crucial. Cell surface carbohydrates are crucial for cell-cell communication, cell-pathogen interactions, and metastasis. Varied patterns of protein glycosylation occur during the diseased states. Since at least more than 50% of human proteins are glycosylated, carbohydrates-based biomarkers are attractive targets. In recent decades, a great effort has been devoted in the field of glycoprotein research for specific and sensitive detection of glycoprotein biomarkers. The electrochemical glycobiosensing approach is appealing as it can be miniaturized, have a short time of analysis, excellent sensitivity, and require basic instrumentation. Enzyme-linked lectin sorbent assays (ELLA) have been used for the analysis of glycoforms in our study wherein lectins such as that from <i>Sambucus Nigra</i> are the natural glycocode readers. Hierarchical bimodal nanoporous gold (hb-NPG) is used as a detection platform in both kinetic and competitive formats, using square-wave voltammetry (SWV). In our study, we demonstrate the differential response seen in SWV assay for the sensitive detection of the glycoprotein fetuin using the hb-NPG with high surface area and large pores for rapid transport of molecules. The kinetic immunoassay is used to monitor the effect of steric hindrance due to varying concentrations of glycoprotein binding, on the rate of enzymatic conversion of the substrate p-aminophenyl phosphate (p-APP) to the product p-AP by the enzyme alkaline phosphatase (ALP). The plot of peak difference current vs. concentration of the glycoprotein is used to quantitatively measure the binding affinity. In the competitive assay, the strategy is to examine the competitive binding of the immobilized glycoprotein with the glycoprotein freely dissolved in the solution containing SNA-ALP conjugate. The difference current is measured after incubating for a fixed time-period and by varying the concentration of glycoprotein in the surrounding solution. The binding specificity is tested by performing another competitive assay wherein the glycoprotein in solution is studied in the presence of a lectin-enzyme conjugate and with glycoprotein immobilized on the surface.

Hierarchical bimodal nanoporous gold (hb-NPG) electrode for the sensitive and specific detection of glycoprotein biomarkers

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