One Step Visual Homogeneous Immunoassay of a Rheumatoid Arthritis Biomarker in Serum via Target-Regulated Steric Hindrance and Competitive Recognition

Homogeneous analysis techniques offer several advantages as alternatives to heterogeneous immunoassays, such as simplicity and rapidity. In this study, a visual homogeneous immunoassay without a labeling process was developed based on target-induced steric hindrance to regulate competitive recognition mechanism. Specifically, as the analyte concentration varies, the change of microenvironment based on steric hindrance could affect the recognition of Cu²⁺ by signal probes. Herein, taking anticyclic citrullinated peptide antibody (anti-CCP) as an example, the method was verified. Cu²⁺ can bind to histidine (His), as well as signal probes. Cyclic citrullinated peptide containing His was served as the capture antigen, and the fluorescence of both CdTe quantum dots and calcein can be quenched by Cu²⁺. Then, this quenching effect can be regulated by the change of steric hindrance, so that anti-CCP analysis can be realized. The limit of detection of anti-CCP was as low as 0.002 and 0.01 U/mL in fluorescence mode and red, green, and blue (RGB) mode, respectively. Furthermore, clinical practicality was validated through 46 clinical samples, including rheumatoid arthritis patients (n = 28) and healthy donors (n = 18), with the assay demonstrating a sensitivity and specificity of 96.4% and 88.9%, respectively. Indeed, the results were consistent with those of clinical electrochemiluminescence immunoassays and digital radiography images. Overall, this method shows great potential for clinical application and offers a universal template for a label-free homogeneous immunoassay.