Recent Advancements in Enzyme-Based Lateral Flow Immunoassays

Abstract

Paper-based lateral-flow immunoassays (LFIAs) have achieved considerable commercial success and their impact in diagnostics is continuously growing. LFIA results are often obtained by visualizing by the naked eye color changes in given areas, providing a qualitative information about the presence/absence of the target analyte in the sample. However, this platform has the potential to provide ultrasensitive quantitative analysis for several applications. Indeed, LFIA is based on well-established immunological techniques, which have known in the last year great advances due to the combination of highly sensitive tracers, innovative signal amplification strategies and last-generation instrumental detectors. All these available progresses can be applied also to the LFIA platform by adapting them to a portable and miniaturized format. This possibility opens countless strategies for definitively turning the LFIA technique into an ultrasensitive quantitative method. Among the different proposals for achieving this goal, the use of enzyme-based immunoassay is very well known and widespread for routine analysis and it can represent a valid approach for improving LFIA performances. Several examples have been recently reported in literature exploiting enzymes properties and features for obtaining significative advances in this field. In this review, we aim to provide a critical overview of the recent progresses in highly sensitive LFIA detection technologies, involving the exploitation of enzyme-based amplification strategies. The features and applications of the technologies, along with future developments and challenges, are also discussed.

Keywords: chemiluminescence; colorimetric; enzyme; lateral flow immunoassay; metal nanoparticles; nanozyme; point-of-care.

Figure 1

Schematic illustration of mechanism for the trapLFI biosensor with (a) low and (b) high concentration of cortisol, and (c) comparison of signal behavior of conventional competitive LFIA and trapLFI biosensor in the presence of different concentrations of cortisol (- negative sample, + positive sample low concentration, +++ positive sample high concentration). Adapted from Ref. [52], Copyright (2018), with permission from The Royal Society of Chemistry.

Figure 2

(A) Schematics of the integrated vertical flow assay device for dual detection. The sample pad consists of two different pore size papers: the upper one has the larger pore size (11 µm) and the lower one has the smaller pore size (0.45 µm). (B) Photograph of the VFA device. Adapted from Ref. [65], Copyright (2018), with permission from Elsevier.

Figure 3

Portable CL-LFIA biosensor for the measurement of salivary cortisol levels used aboard the International Space Station (ISS): schematic drawings and images of (a,d) the LFIA fluidic element, (b,e) the LFIA cartridge and (c,f) the CL reader. A detailed view of the fluidics of the LFIA fluidic element around the sample metering chamber is shown on top of panel (a). Scale checkerboards are 2 × 2 cm. Reprinted from Ref. [69], Copyright (2018), with permission from Elsevier.

Figure 4

(a): (A) Scheme of the LFIA for the detection of HIgG. (B) Detail of the different parts of a LFIA strip and cartoons representing the AuNP modified with the antibody anti-human IgG γ chain specific HRP modified, and the different colors expected for the different substrates (TMB, AEC and DAB) used. Reprinted from Ref [72], Copyright (2012), with permission from Elsevier. (b) Scheme of the CLFAs for the Detection of Targets: Sample is introduced on the sample pad and moves to the absorbent pad. When sample flows through the conjugate pad, the targets are recognized by the detection antibodies on the AuNPs. With sample flowing through the NC membrane, targets and detection antibodies, as well as AuNPs, are immobilized on the NC membrane. After the appearance of the red lines because of the aggregation of AuNPs, the substrate pad containing lyophilized substrate is covered on the LFA strip for the transfer of the CL substrate. Then, the substrate pad is removed, and the CL signal is captured for quantitative detection of targets. Reprinted from Ref [76], Copyright (2018), with permission of the American Chemical Society.

Figure 5

Schematic illustration for the colorimetric LFA based on Au-Pt: (A) preparation of Au@Pt nanozyme; (B) process of the colorimetric LFA based on Au-Pt; (C) typical results of the colorimetric LFA assay based on Au-Pt. Reprinted from Ref [97], Copyright (2020), with permission from Elsevier.

Figure 6

Scheme of smartphone-based dual lateral flow immunoassays for simultaneous detection of S. enteritidis and E. coli O157:H7 using Pd@Pt nanoparticles as signal amplification: (A) Dual lateral flow immunoassays; (B) Pd@Pt nanoparticles for signal amplification; and (C) smartphone-based device. Reprinted from Ref. [101], Copyright (2017), with permission from the American Chemical Society.