An Efficient Electrochemical Sandwich ELISA for Urinary Human Serum Albumin-Biomarker Based on Highly Redox-Active Thionine Surface-Confined MWCNT/PEDOT.PSS Platform

ABSTRACT

Human serum albumin (HSA) is an abundant protein in blood serum and an indicator of several illnesses, for instance, Covid-19, wherein, a severe decrement in the concentration level is noticed that leads to a higher risk of mortality. The conventional ELISA-based protocol has been referred for the analysis which is less sensitive, cumbersome and time-consuming (4-6 hours). Herein, we introduce, a new sandwich-type electrochemical immunosensor based on Thionine(Th)-redox immobilized f-MWCNT(f-carboxylic functionalized)+PEDOT.PSS modified glassy carbon/screen-printed electrode (GCE/f-MWCNT+PEDOT@Th) for quick (∼20 min), low-sample volume (1 µL) and low concentration (10-9 g/mL) analysis of HSA. The GCE/f-MWCNT+PEDOT@Th showed a highly stable and fouling-free surface-confined redox feature at an apparent standard electrode potential, Eo’=-0.25 V vs Ag/AgCl with a surface-excess value, ΓTh=14×10-9 mol cm-2. A sandwich-type electrochemical immunosensor was prepared by sequential immobilization of 1 µL respective solutions without any linking agent on the chemically modified electrode followed by incubation at 37°C for 3—5 min and testing its bioelectrocatalytic H2O2 (500 µM) reduction reaction in pH 7 PBS. Interrelated solution phased and experimental conditions were systematically optimized. Under an optimal working condition, the as-prepared bioelectrode, CME-Ab1p-SkM-HSA-Ab1m-Ab2HRP showed a reliable calibration plot of HSA in a concentration window, 10-9—10-4 g/mL. The obtained calibration plot was parallel to the ELISA of HSA in a limited concentration window, 10-7—10-4 g/mL. As a proof of concept, electrochemical immunosensing of urinary HSA has been demonstrated. Since, the new approach is simple, rapid, and efficient over conventional ELISA, it can be extendable to various HSA-real sample analyses.