Paper-based Vertical Flow Immunoassay (VFI) for detection of bio-threat pathogens

Abstract

Currently, the standard method for identifying biological agents of potential threats to national security and public health, such as pathogens, virus, and toxins, mainly rely on microbiological cultivation. This method is time-consuming and it requires sophisticated equipment and well-trained personnel, which are often unavailable in remote areas or at point-of-need. Therefore, an alternative rapid, simple, and sensitive method for detecting bio-threat agents is in crucial need. We report a paper-based Vertical Flow Immunoassay (VFI) device that can overcome these limitations. The VFI device utilizes a nanoporous nitrocellulose membrane encapsulated in a stainless steel filter holder. As the sample is pushed through the membrane, which is pre-functionalized with capture antibody, a sandwich assay is formed and colorimetric signal is generated to reflect the presence of target antigens. Through theoretical analyses of antigen-antibody binding process inside a porous membrane, we identified two critical factors - membrane pore size and sample flow rate that can be optimized to improve the assay sensitivity. Then, the effects were demonstrated through experimental studies using Burkholderia pseudomallei (the causative agent of melioidosis) as a model pathogen. The B. pseudomallei VFI was based on an immunoassay targeting the B. pseudomallei surface capsular polysaccharide (CPS). The experimental results agreed well with the theory showing that increasing the flow speed (up to 1.06 mm/s) and reducing the membrane pore size (down to 0.1 µm) could improve the sensitivity by at least 5 times. The VFI's limit-of-detection for CPS spiked in buffer solution was determined to be 0.02 ng/mL. The developed VFI shows great potential as a point-of-care tool for detection of bio-threat agents in a variety of clinical and resource-restricted conditions.

Lateral and Vertical Flow Assays for Point‐of‐Care Diagnostics

“…One of the ways to improve the LFAs is to flow samples vertically rather than parallelly, known as VFAs. A typical VFA device contains a porous membrane with separated immunoreaction spots for multiplexing assays simultaneously ( Figure ) . In the VFAs, the interactions of the specific antigen, capture antibody, and the gold conjugate result in an immediate and permanent red dot that can be detected by eye or a smartphone reader.…”

“…C–E) Detection intensity as a function of pore size, flow speed, and analytes concentration. Reproduced with permission . Copyright 2018, Elsevier B.V.…”

“…This method was able to sense picomolar multiplexed samples within 5 min through colorimetric detection that allowed naked eye interpretation . Nanoporous nitrocellulose membrane encapsulated in a stainless steel filter holder was also utilized to measure colorimetric signal from antigen–antibody binding process inside a porous membrane (Figure ) . Two critical pore size parameters of the membrane and sample flow rate were optimized to increase the sensitivity of immunoassay.…”

A Critical Comparison between Flow-through and Lateral Flow Immunoassay Formats for Visual and Smartphone-Based Multiplex Allergen Detection

“…Selection of NC based on this capillary flow rate is a compromise between assay sensitivity and assay speed with mid-speed membranes (120-150 s/4 cm) offering advantages in both areas [27]. When detection speed is not a constraint, a membrane with a slower flow rate and smaller pore size increases the available binding time between the labeled antibody-analyte and the test line antibody which can result in increased assay sensitivity [27][28][29]. In order to speed up LFIAs, in combination with NC with a good flow rate, antibodies with fast association rates towards their target should be used.…”

“…In order to overcome restrictions typically associated with LFIAs, a flow-through immunoassay format can be used instead [31,32]. Flow-through immunoassays are reported to offer the benefits of increased assay speeds, better sensitivities-owing to the use of larger sample volumes, excellent multiplexing capabilities and the absence of the 'hook-effect' [27,33,34]. The hook-effect is a phenomenon that is commonly encountered in one-step, sandwich format LFIAs.…”