Ultrasensitive Electrochemical Immunosensor for Multiplex Sandwich Bioassaying Based on the Functional Antibodies

In vitro diagnostics (IVDs) based on electrochemical immunosensors are crucial for disease screening, diagnosis, prognosis, and treatment monitoring. However, label-free electrochemical immunosensors commonly suffer from poor specificity, leading to false positives. To address this issue, we propose a highly sensitive and precise electrochemical immunosensor for protein marker detection. This approach involves directly labeling the detection antibodies (Ab₂) with thionine (Thi). The Ab₂ labeled by Thi exhibits a distinct redox peak upon targeted voltage stimulation, enabling accurate quantification of protein biomarkers. Thi-modified antibodies provide significant advantages over traditional antibody modification methods, such as enhanced detection sensitivity, improved accuracy, and specificity in protein marker identification. The method is straightforward and efficient, ensuring specific analyte detection while minimizing interference from other substances in the sample. Additionally, a multielectrode detection method was employed, achieving remarkably low limits of detection (LoDs) for tumor necrosis factor-alpha (TNF-alpha), cardiac troponin I (cTnI), and interleukin-6 (IL-6), with LoDs of 9.38 fg/mL, 1.70 fg/mL, and 8.14 fg/mL, respectively. The proposed electrochemical immunosensor also exhibited high selectivity and repeatability, with relative standard deviations (RSD) of 6.39% for TNF-alpha, 2.42% for cTnI, and 2.72% for IL-6 (n = 5). Moreover, it demonstrated high sensitivity and was evaluated for serum detection using the standard addition method. The results highlight the great potential of the proposed electrochemical immunosensor for clinical applications, offering a novel approach for future utilization in clinical settings.