A bidirectional immuno-microfluidic chip for detecting autoantibodies based on an indirect assay

Autoantibodies play a critical role in predicting diseases and monitoring therapeutic outcomes. Limitations of current autoantibody detection techniques include prolonged detection times and operational complexity. This study aims to develop a bidirectional microfluidic platform for the detection of autoantibodies. A "double inlet" and "waste outlet" structure was developed on the bidirectional lateral-flow microfluidic chip (BiLFMC). Using anti-citrullinated peptide antibodies (ACPA) as a model, this study validated the feasibility and performance advantages of the BiLFMC in the diagnosis of autoimmune disease. The BiLFMC was optimized by selecting the optimal distances between the sample inlet and waste outlet, as well as the ideal diameter for the waste outlet. The most suitable absorbent material was chosen for the waste absorbent pad. The detection and anti-interference performance of the unidirectional mode, unidirectional sequential mode, and bidirectional mode were compared, demonstrating that the bidirectional microfluidic chip significantly enhanced anti-interference capabilities. For ACPA detection, a serum sample required only a 1:100 dilution and 10 μL volume, with results available in 8 min. The coefficient of variation (CV) for repeatability ranged from 7.04% to 13.55%, while the CV for intermediate precision ranged from 11.06% to 14.49%. The limit of quantitation was 3.951 RU/mL, and the upper limit of the linear range was 500 RU/mL. When compared to ELISA results, the consistency coefficient was 0.8302. Furthermore, the BiLFMC was able to accurately distinguish samples exceeding the ELISA upper limit (> 196 RU/mL). A bidirectional microfluidic chip has been developed, incorporating indirect detection methods to enhance the anti-interference capability of the microfluidic platform.