Paper-based cascade cationic isotachophoresis: Multiplex detection of cardiac markers

Paper-based analytical devices (PADs) are widely used in point-of-care testing (POCT) as they are cost-effective, simple and straightforward. However, poor sensitivity hinders their use in detecting diseases with low abundance biomarkers. The poor detection limit of PADs is mainly attributed to the low concentration of analytes, and the complexity of biological fluid, leading to insufficient interactions between analytes and capture antibodies. This study aims to overcome these difficulties by developing a paper-based cationic isotachophoresis (ITP) approach for simultaneously detecting pico-molar levels of two essential cardiac protein markers: acidic troponin T (cTnT) and basic troponin I (cTnI) spiked into human serum samples. The approach utilizes 3-aminopropyltrimethoxysilane (APTMS) treated glass fiber papers with decreasing cross-sectional area assembled on a 3D printed cartridge device. Our results showed that in the presence of cTnT monoclonal antibody (mAb), fluorescently labeled cTnI and cTnT could be effectively enriched in cationic ITP. Each individual target was captured subsequently by a test line in the detection zone where the capture mAb was immobilized. Detailed analysis suggests that the technology is capable of simultaneous on-board depletion of abundant plasma proteins and enrichment of cTnI/cTnT by ~1300-fold with a sensitivity of 0.6 pmol/L for cTnT and a sensitivity of 1.5 pmol/L for cTnI in less than 6 min. The results demonstrate the potential of this technology for rapid, ultra-sensitive and cost-effective analysis of multiplex protein markers in clinical serum samples at point of care.