An efficient electrochemical sandwich ELISA for urinary human serum albumin-biomarker based on highly redox-active thionine surface-confined MWCNT/PEDOT.PSS platform

Abstract

• A new electrochemical immunosensor platform for urinary human albumin serum. • An improved electrochemical immune sensor better than the conventional ELISA. • A surface-confined redox probe-based heterogeneous immune-sensor platform. • 1 uL of test samples, 3 min of incubation time & EDC/NHS linker-free approach. • Methodology validated with conventional ELISA. 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 h). 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 -WCNT+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, E o ’=-0.25 V vs Ag/AgCl with a surface-excess value, Γ Th = 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 H 2 O 2 (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-Ab1 p -SkM-HSA-Ab1 m -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.