Electrochemical Immunosensor for the Early Detection of Rheumatoid Arthritis Biomarker: Anti-Cyclic Citrullinated Peptide Antibody in Human Serum Based on Avidin-Biotin System

摘要

Rheumatoid arthritis (RA) is a chronic autoimmune disease that produces a progressive inflammatory response that leads to severe pain, swelling, and stiffness in the joints of hands and feet, followed by irreversible damage of the joints. The authors developed a miniaturized, label-free electrochemical impedimetric immunosensor for the sensitive and direct detection of arthritis Anti-CCP-ab biomarker. An interdigitated-chain-shaped microelectrode array (ICE) was fabricated by taking the advantage of microelectromechanical systems. The fabricated ICE was modified with a self-assembled monolayer (SAM) of Mercaptohexanoic acid (MHA) for immobilization of the synthetic peptide bio-receptor (B-CCP). The B-CCP was attached onto the surface of SAM modified ICE through a strong avidin-biotin bio-recognition system. The modified ICE surface with the SAM and bio-molecules (Avidin, B-CCP, Anti-CCP-ab and BSA) was morphologically and electrochemically characterized. The change in the sensor signal upon analyte binding on the electrode surface was probed through the electrochemical impedance spectroscopy (EIS) property of charge-transfer resistance (Rct) of the modified electrodes. EIS measurements were target specific and the sensor response was linearly increased with step wise increase in target analyte (Anti-CCP-ab) concentrations. The developed sensor showed a linear range for the addition of Anti-CCP-ab between 1 IU mL−1 → 800 IU mL−1 in phosphate buffered saline (PBS) and Human serum (HS), respectively. The sensor showed a limit of detection of 0.60 IU mL−1 and 0.82 IU mL−1 in the PBS and HS, respectively. The develop bio-electrode showed a good reproducibility (relative standard deviation (RSD), 1.52%), selectivity and stability (1.5% lost at the end of 20th day) with an acceptable recovery rate (98.0% → 101.18%) and % RSD’s for the detection of Anti-CCP-ab in spiked HS samples.