Automated Paper-Based Femtogram Sensing Device for Competitive Enzyme-Linked Immunosorbent Assay of Aflatoxin B-1 Using Submicroliter Samples

Microfluidic paper-based analytical devices (mu PADs) are promising biosensors that may be used in a variety of bioanalytical applications. A mu PAD for automating the competitive enzyme-linkedimmunosorbent assay (ELISA) of small-sized target detection at thefemtogram level using submicroliter samples is reported in this study. Theproposed mu PAD was integrated with a sucrose valve to automate thesequential delivery of reagents, providing simple control of reagent delivery time and simple operation. The use of a sample solution dropping location atthe zones on the device that had been prepared with an antibody-conjugatedenzyme before immersion in a running buffer allowed minimization ofsample volume to 0.6 mu L, 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.