Countercurrent Microfluidic Immunoaffinity Platform for Continuous Enrichment and Multiplexed Detection of Neuroinflammatory Biomarkers

Abstract

Neuroinflammation plays a major role in the development of numerous neurological disorders, yet conventional immunoassays are restricted by large sample volume requirements, limited multiplexing capability and insufficient analytical sensitivity. To develop and evaluate a continuous, low-volume and high-performance diagnostic platform for multiplex neuroinflammatory biomarker detection. A countercurrent microfluidic immunoaffinity platform was developed incorporating antibody-functionalised serpentine microchannels, nanocomposite-based electrochemical immunosensors and real-time computational analysis. Antigen–antibody interaction was enhanced by countercurrent laminar flow, while enzymatic redox amplification enabled ultrasensitive electrochemical quantification. The system achieved a capture efficiency of 89.2%, representing a 2.3-fold improvement over single-pass flow and an enrichment factor of 18.4 for interleukin-6 in cerebrospinal fluid (CSF). Electrochemical detection reached a limit of detection of 0.05 pg/mL, providing approximately 40-fold higher sensitivity than the enzyme-linked immunosorbent assay (ELISA) with strong linearity ( R > 0.99). Clinical CSF samples demonstrated successful multiplexed detection of 12 biomarkers, all with coefficients of variation < 8% and strong correlation with ELISA ( R = 0.91–0.98). The technology achieved 92.3% accuracy in identifying neuroinflammatory disease subtypes while reducing sample volume ten-fold and total assay time to 22 min. This countercurrent microfluidic immunoaffinity platform offers a low-volume, high-sensitivity and clinically feasible method for multiplex neuroinflammatory biomarker detection. Its integration of continuous-flow enrichment with advanced electrochemical sensing positions it as a promising next-generation diagnostic tool for point-of-care neurological assessment.