Capillary-Driven Nanopaper Assay (c-NAPA) for Sensitive Point-of-Need Biomarker Detection
The lateral flow immunoassay (LFI) is a popular paper-based point-of-need (PON) assay platform. However, sensitivity of current LFIs is still limited, thus hindering its wider application, possibly due to the low sample volume it can wick through (~154 µl/mm2) and its large paper pore size (> 5µm) that may impede efficient target capture. Recently, our group at University of Arizona Center for Applied NanoBioscience and Medicine has developed a vertical flow immunoassay (VFI) that showed 10x sensitivity enhancement over LFI by flowing sample vertically through a nanopored nitrocellulose paper membrane at a much higher sample volume per sensor area. The drawback of the current VFI is that it uses a syringe pump for sample flow through, which is not PON friendly. In this project, to achieve equipment-free sensitive PON detection, we test the idea of a capillary-driven nanopaper assay (c-NAPA). In c-NAPA, a 3D or 2D nanopaper absorbent structure is used to generate high capillary pressure while maintaining a low fluidic resistance to flow sample quickly through a nanopaper sensor. A vertical format c-NAPA was tested first with wax printing or tape masking to reduce the sensor area for low sample/reagent consumption. However, reliable contact for sample wicking turned out to be challenging. A lateral format c-NAPA was then derived with a cantilever design to ensure sample-paper contact. High relative humidity was also found indispensable for sample wicking by nanopaper absorbent material. An LcrV (plague biomarker) sandwich immunoassay was used to test the lateral c-NAPA using a 0.45 µm nitrocellulose membrane. Detection antibody and gold nanoparticle conjugate (dAb-GNP) concentration as high as 150 pM was used without generating background signal. Study of sample and dAb-GNP mixing time then showed that longer mixing time correlated to stronger signal. The limit of detection was measured to be about 15 ng/ml, similar to the traditional LFI, but with only half the sample consumption per sensor area (~70 µl/mm2). Future research will focus on further enhancing the sensitivity of the lateral flow c-NAPA by exploring cantilevered design, nanopaper pore size and 3D wicking structure, as well as solving the contact issues of the vertical format c-NAPA.