Simultaneous multiple target detection platform based on vertical flow immunoassay.

In general, vertical flow assay (VFA) has a disadvantage of requiring a complex analysis process that involves manually injecting various reagents (target analyte, washing buffer, detection conjugate, etc.) sequentially. However, in this study, we have developed an innovative paper-based VFA device that replaces the complex analysis process with one-step and enables the detection of multiple targets. The fabrication process of the multi-target detection VFA device is as follows: preparation and pre-treatment of the strip materials, design of strip cartridge, design of the multiple detection VFA device, optimization experiments for strip sample flow rates, determination of device analysis time, determination of device limit of detection (LOD), multiple target signal uniformity experiment, immunoglobulin G (IgG) and C-reactive protein (CRP) antigen-antibody multiple detection experiment, and data extraction and analysis method. The use of paper-based materials enables the device to be produced at cost-effective, and cartridge production allowed for uniform array formation. IgG and CRP are used to evaluate the performance of the device as common biomarkers. The device proposed in this study is currently under research. To validate multiple target detection capability of the VFA device proposed in this study, two types of antigens-antibodies (Human IgG and Human CRP) were employed. The detection limit is 0.15 μg/mL for IgG and 0.19 μg/mL for CRP in naked eye. Furthermore, it was confirmed that there is no cross-reactivity caused by the device structure through IgG and CRP antigens. In conclusion, the VFA device proposed in this study consists of a one-step analysis process, and it has been confirmed that it can detect multiple targets simultaneously.