A competitive immunoassay system for microfluidic paper-based analytical detection of small size molecules

The development of a competitive immunoassay system for colorimetric detection on microfluidic paper-based analytical devices (μPADs) is reported. The μPADs were fabricated via photolithography to define hydrophilic flow channels and consisted of three main elements: the control and test zones, where target detection was performed, the sample introduction zone, and the competitive capture zone located between the sample introduction zone and the test zone. The chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) was deposited at the control and test zones. μPAD surface modification was performed at the capture zone first via chitosan activation, then the BSA-conjugated target compound was immobilized. The sample solution consisting of the target compound, the peroxidase-conjugated antibody, and the hydrogen peroxide oxidizing agent was introduced into the device and competition occurred at the capture zone, allowing only the target-bound peroxidase-conjugated antibody to travel past the capture zone and into the test zone via capillary action. The developed competitive immunoassay system was successfully demonstrated on the μPAD detection of biotin as a model compound with a detection limit of 0.10 μg mL^-1. The applicability of the proposed immunoassay system for point-of-need testing was further demonstrated using aflatoxin B₁, a highly toxic foodborne substance, with a detection limit of 1.31 ng mL^-1. The μPAD with the competitive immunoassay format showed promising results for practical applications in point-of-need testing involving small molecular weight targets in food and water safety and quality monitoring, environmental analysis, and clinical diagnostics.