Highly Sensitive Immunoassay Based on Controlled Rehydration of Patterned Reagents in a 2-Dimensional Paper Network

Abstract

We have demonstrated a multistep 2-dimensional paper network immunoassay based on controlled rehydration of patterned, dried reagents. Previous work has shown that signal enhancement improves the limit of detection in 2-dimensional paper network assays, but until now, reagents have only been included as wet or dried in separate conjugate pads placed at the upstream end of the assay device. Wet reagents are not ideal for point-of-care because they must be refrigerated and typically limit automation and require more user steps. Conjugate pads allow drying but do not offer any control of the reagent distribution upon rehydration and can be a source of error when pads do not contact the assay membrane uniformly. Furthermore, each reagent is dried on a separate pad, increasing the fabrication complexity when implementing multistep assays that require several different reagents. Conversely, our novel method allows for consistent, controlled rehydration from patterned reagent storage depots directly within the paper membrane. In this assay demonstration, four separate reagents were patterned in different regions of the assay device: a gold-antibody conjugate used for antigen detection and three different signal enhancement components that must not be mixed until immediately before use. To show the viability of patterning and drying reagents directly onto a paper device for dry reagent storage and subsequent controlled release, we tested this device with the malaria antigen Plasmodium falciparum histidine-rich protein 2 (PfHRP2) as an example of target analyte. In this demonstration, the signal enhancement step increases the visible signal by roughly 3-fold and decreases the analytical limit of detection by 2.75-fold.

Highly Sensitive Two-Dimensional Paper Network Incorporating Biotin–Streptavidin for the Detection of Malaria

“…Improvements for this device could be made by incorporating dry reagent storage 7,8 and a plasma separating membrane, allowing for whole blood analysis. Because of the ease of biotinylation, the device can be readily modified for the detection of other antigens that require a highly sensitive point-of-care test.…”

Recent Advances of Fluid Manipulation Technologies in Microfluidic Paper-Based Analytical Devices (μPADs) toward Multi-Step Assays

“…Fluid velocity control Signal enhancement assay (C-reactive protein) [69] Source pad of different sizes ( # 2DPNs) Fluid velocity control Signal enhancement assay (BSA-biotin) [20] Source pad of different sizes (2DPNs) Fluid velocity control Signal enhancement assay (malaria protein PfHRP2) [70,71] Three-dimensional device Fluid velocity control Enzymatic assay (glucose and protein) [72] Three-dimensional device Fluid velocity control Colorimetric-enzymatic assay (protein) [73] Three-dimensional device Fluid velocity control Immunoassay (malaria protein PfHRP2) [74] Three-dimensional device Fluid velocity control Colorimetric-enzymatic assay (glucose) [75] Vertical paper legs of different lengths (2DPNs)…”

Two Orders of Magnitude Improvement in Detection Limit of Lateral Flow Assays Using Isotachophoresis

“…We performed ITP experiments and measured the ITP zone velocity, VITP, width of the ITP plug, δITP, and the effective preconcentration ratio, p. For each ITP experiment we obtained images of the ITP plug prior to reaching the test zone, then converted the intensity values to target concentration using our calibration curve. We fit a Gaussian distribution in the form of 22 exp( (…”