Blind Spot: A Braille Patterned Novel Multiplex Lateral Flow Immunoassay Sensor Array for the Detection of Biothreat Agents

Abstract

Lateral flow immunoassays (LFIs) are simple, point-of-care diagnostic devices used for detecting biological agents or other analytes of interest in a sample. LFIs are predominantly singleplex assays, interrogating one target analyte at a time. There is a need for multiplex LFI devices, e.g., a syndromic panel to differentiate pathogens causing diseases exhibiting similar symptoms. Multiplex LFI devices would be especially valuable in instances where sample quantity is limiting and reducing assay time and costs is critical. There are limitations to the design parameters and performance characteristics of a multiplex LFI assay with many horizontal test lines due to constraints in capillary flow dynamics. To address some of the performance issues, we have developed a spot array multiplex LFI using Braille format (hence called Blind Spot) and a sensor, MACAW (Modular Automated Colorimetric Analyses Widget), that can analyze and interpret the results. As a proof of concept, we created a multiplex toxin panel, for detecting three toxins, using two letter codes for each. The results indicated that the six-plex, triple toxin assay performs as well as singleplex assays. The sensor-based calls are better compared to human interpretation in discriminating and interpreting ambiguous test results correctly especially at lower antigen concentrations and from strips with blemishes.

Overlaid Lateral Flow Immunoassay for the Simultaneous Detection of Two Variant-Specific SARS-CoV-2 Neutralizing Antibodies

“…The manual method of preparing the T-line makes it challenging to ensure repeatability and uniformity in cutting from one device to another. To address this issue and facilitate upscale production, we propose the use of an automated system, such as a NanoDrop instrument (AD1520 Aspirate AD1520 Aspirate Dispense System), 36 for creating the T-line. This machine can precisely control the volume and position of each drop.…”

Lateral flow test engineering and lessons learned from COVID-19

“…Large-scale manufacturing of xLFTs requires the printing of multiple test spots in a single disposable strip, and thus, multiple dispensing nozzles with different quality-control measures, potentially increasing production costs. Furthermore, additional test lines complicate result interpretation, although this can be mitigated through use of digital interpretation or test spot array patterns that are recognizable to users 166,167 . The development and commercialization of xLFTs are further limited by the need to validate multiple biomarkers with clinical importance, low market demand and a complicated regulatory approval pathway.…”

Assessment of Urinary Biomarkers for Infectious Diseases Using Lateral Flow Assays: A Comprehensive Overview

“…Kazushi Misawa et al demonstrated the semiquantitative, visual result readout in the form of “text”. This intuitive signal method offered potential for an alternative and suitable POC testing application . Traditional LFAs use colloidal AuNPs as a signal for qualitative diagnostic detection.…”