Electrochemical biosensor for the simultaneous determination of rheumatoid factor and anti-cyclic citrullinated peptide antibodies in human serum

Abstract

This paper reports a dual electrochemical biosensor involving carboxylated- or neutravidin-functionalized magnetic microbeads and dual screen-printed carbon electrodes for the simultaneous determination of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCPA) autoantibodies used as biomarkers for the detection of rheumatoid arthritis autoimmune disease. Sandwich-type biosensors involving Fc fragments of IgG Fc(IgG) and biotinylated cyclic cytrullinated peptide (CCP-biotin) to form CCP-biotin-Neutr-MBs for the specific immobilization of RF and CCPA, respectively, as well as conjugation with HRP-IgM and HRP-IgG for RF and CCPA, respectively, were prepared. Amperometric detection was performed at −0.20 V vs. Ag pseudo-reference electrode using the H₂O₂/hydroquinone (HQ) system upon capturing the bioconjugates onto the corresponding working electrode (WE1 or WE2) of SPCdEs. The dual biosensor exhibits high sensitivity for RF and CCPA with LOD values of 0.8 and 2.5 IU mL⁻¹, respectively. The simultaneous determination can be completed in about two hours using a simple protocol and a sample volume (25 μL) four times smaller than that required by the ELISA method. The dual electrochemical biosensor was used for the determination of both target biomarkers in human serum.