Automated Paper-Based Femtogram Sensing Device for Competitive Enzyme-Linked Immunosorbent Assay of Aflatoxin B₁ Using Submicroliter Samples

Microfluidic paper-based analytical devices (μPADs) are promising biosensors that may be used in a variety of bioanalytical applications. A μPAD for automating the competitive enzyme-linked immunosorbent assay (ELISA) of small-sized target detection at the femtogram level using submicroliter samples is reported in this study. The proposed μPAD was integrated with a sucrose valve to automate the sequential delivery of reagents, providing simple control of reagent delivery time and simple operation. The use of a sample solution dropping location at the zones on the device that had been prepared with an antibody-conjugated enzyme before immersion in a running buffer allowed minimization of sample volume to 0.6 μL, while eliminating the possible loss of a target molecule by adsorption on the membrane, thus improving detection sensitivity. Furthermore, the proposed device was successfully applied to the automation of competitive ELISA for the detection of aflatoxin B₁ (AFB₁), a potent carcinogen that causes substantial health risks to humans worldwide, with a detection limit of 60 femtograms or 0.1 ng/mL. The method developed in this study provides high sensitivity, small sample volume, on-site and equipment-free measurements, low-cost operation, and user-friendliness. This approach could be used to analyze small-sized molecules in the fields of food safety and quality control, environmental monitoring, and clinical diagnostics.