H.O.S.T.: Hemoglobin microbubble-based Oxidative stress Sensing Technology

Abstract

In this work, we discuss the development of H.O.S.T., a novel hemoglobin microbubble-based electrochemical biosensor for label-free detection of Hydrogen peroxide (H₂O₂) towards oxidative stress and cancer diagnostic applications. The novelty of the constructed sensor lies in the use of a sonochemically prepared hemoglobin microbubble capture probe, which allowed for an extended dynamic range, lower detection limit, and enhanced resolution compared to the native hemoglobin based H₂O₂ biosensors. The size of the prepared particles Hemoglobin microbubbles was characterized using Coulter Counter analysis and was found to be 4.4 microns, and the morphology of these spherical microbubbles was shown using Brightfield microscopy. The binding chemistry of the sensor stack elements of HbMbs' and P.A.N.H.S. crosslinker was characterized using Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy and UV-Vis Spectroscopy. The electrochemical biosensor calibration (R² > 0.95) was done using Electrochemical Impedance Spectroscopy, Cyclic Voltammetry, and Square Wave Voltammetry. The electrochemical biosensor calibration (R² > 0.95) was done using Electrochemical Impedance Spectroscopy, Cyclic Voltammetry, and Square Wave Voltammetry. The specificity of the sensor for H₂O₂ was analyzed using cross-reactivity studies using ascorbic acid and glucose as interferents (p < 0.0001 for the highest non-specific dose versus the lowest specific dose). The developed sensor showed good agreement in performance with a commercially available kit for H₂O₂ detection using Bland Altman Analysis (mean bias = 0.37 for E.I.S. and - 24.26 for CV). The diagnostic potential of the biosensor was further tested in cancerous (N.G.P.) and non-cancerous (H.E.K.) cell lysate for H₂O₂ detection (p = 0.0064 for E.I.S. and p = 0.0062 for CV). The Michaelis Menten constant calculated from the linear portion of the sensor was found to be [Formula: see text] of 19.44 µM indicating that our biosensor has a higher affinity to Hydrogen peroxide than other available enzymatic sensors, it is attributed to the unique design of the hemoglobin polymers in microbubble.