Development of Multiplexed Infectious Disease Lateral Flow Assays: Challenges and Opportunities

Abstract

Lateral flow assays (LFAs) are the mainstay of rapid point-of-care diagnostics, with the potential to enable early case management and transform the epidemiology of infectious disease. However, most LFAs only detect single biomarkers. Recognizing the complex nature of human disease, overlapping symptoms and states of co-infections, there is increasing demand for multiplexed systems that can detect multiple biomarkers simultaneously. Due to innate limitations in the design of traditional membrane-based LFAs, multiplexing is arguably limited to a small number of biomarkers. Here, we summarize the need for multiplexed LFA, key technical and operational challenges for multiplexing, inherent in the design and production of multiplexed LFAs, as well as emerging enabling technologies that may be able to address these challenges. We further identify important areas for research in efforts towards developing multiplexed LFAs for more impactful diagnosis of infectious diseases.

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

“…The benefits of LFAs include cost‐effectiveness and quick response time at POC settings . The simplicity of the lateral flow readout has been demonstrated to be favorable; however, portable, user‐friendly POC platforms require higher sensitivity, accuracy, and multiplexing capability . Multiplexing capability of microarray‐based LFAs requires accurate positioning for flow between upstream and downstream spots .…”

“…Therefore, there has been a growing demand for multiplexed LFAs that could simultaneously measure multiple biomarkers, particularly in resource‐limited POC settings . Additionally, some strains of infections such as tuberculosis, could only be detected by combining an array of antibodies with antigens which required multiple biomarkers at high sensitivities …”

“…Physical limitation of multiplexing test lines on a single lateral flow strip is another issue, since it can cause crosstalk which becomes difficult for operators to interpret multiple results on one strip. Therefore, it has been particularly challenging to develop multiplexed LFAs on a manufacturing scale into commercial products . Furthermore, there are opportunities for developing VFAs for POC testing to provide fast response and multiplexing capability.…”

Development of a Lateral Flow Highway: Ultra-Rapid Multitracking Immunosensor for Cardiac Markers

“…As a result, the conjugate pad is excluded from the test system, along with the need for its impregnation, the dissolution of the conjugate, and the conjugate's passage through the juncture of the pad and the working membrane. Conventionally, multiplexing of the test strip is achieved by the sequential deposit of lines of reagents of different specificities on the working membrane (which increases the duration of analysis), or by combining independent test strips in one device (which increases the consumption of the materials and reagents) [7,8]. In the format proposed here, an ordered array of points on the membrane is used for the independent formation of several specific complexes ( Figure 1C).…”

Development of a smartphone-based rapid dual fluorescent diagnostic system for the simultaneous detection of influenza A and H5 subtype in avian influenza A-infected patients

“…Multiplex detection has recently received great attention, and microfluidic devices have emerged as a promising diagnostic platform because of their favorable material transport process, rapid turnaround time, and low sample/reagent consumption [8,9]. However, several challenges in mass production, particularly sensor fabrication costs, limit their clinical application for POCT [10]. Given the demand for multiplex testing and the major advantages of the lateral flow assay over alternative rapid diagnostic platforms, this study aimed to adapt the simplicity of the membrane-based lateral flow assay and multiplex techniques.…”