DEVELOPMENT OF AN ELECTROCHEMICAL SENSOR FOR MORE RAPID HIF-1Α DETECTION AS AN ALTERNATIVE TO WESTERN BLOT ANALYSIS

Oxygen levels in the body/and organs significantly influence the regulation of metabolic reactions. Hypoxia, a decrease in oxygen levels, can potentially trigger various signals leading to serious health issues. This study aimed to develop an electrochemical immunosensor platform for rapidly and accurately detecting the hypoxia biomarker, HIF-1α protein. In this regard, screen-printed gold electrodes were modified using 11-mercaptoundecanoic acid and 3-mercapto-1-propanol (as a spacer) to generate functional carboxyl groups. Employing EDC-NHS chemistry facilitated the immobilization of HIF-1α antibodies, which were then utilized for the selective and specific recognition of their target. Electrochemical voltametric measurements were conducted using a potassium ferri/ferrocyanide redox couple in both hypoxia-cultured cell lysates and phosphate-buffered saline spiked with HIF-1α protein.In addition to electrochemical measurements, Western blotting (WB) was performed to compare findings with electrochemical results and to confirm the presence of HIF-1α in hypoxic cell lysates. While WB results only exhibited the qualitative presence of the respective antigen in lysates, significant signal decreases were observed in both Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV) measurements due to specific antibody-target binding, emphasizing the electrochemical sensor’s performance for more rapid and quantitative protein detection. The low limit of detection (1.46 nM) suggests the potential of our proposed immunosensor platform for detecting HIF-1α protein within a clinically significant range, which is highly desired for point-of-care applications. This study is one of its kind in the literature to develop an electrochemical immunosensor platform for the rapid detection of HIF-1α. The sensor's ability to provide inexpensive, rapid, and quantitative measurements is a significant advancement for HIF-1 α detection.