Multiplex Lateral Flow Immunoassay: An Overview of Strategies towards High-throughput Point-of-Need Testing

Abstract

Simultaneous measurement of different substances from a single sample is an emerging issue for achieving efficient and high-throughput detection in several fields of application. Although immunoanalytical techniques have well-established and prevailing advantages over alternative screening analytical platforms, one of the incoming challenges for immunoassay is exact multiplexing. Lateral flow immunoassay (LFIA) is a leading immunoanalytical technique for onsite analysis, thanks to its simplicity, rapidity, and cost-effectiveness. Moreover, LFIA architecture is adaptable to multiplexing, and is therefore a possible answer to the pressing demand of multiplexing point-of-need analysis. This review presents an overview of diverse approaches for multiplex LFIA, with a special focus on strategies based on new types of magnetic, fluorescent, and colored labels.

Keywords: immunoassay; immunochromatographic test; point-of-care testing; rapid test.

Figure 1

A typical lateral flow immunoassay (LFIA) strip: the strip architecture includes the detection membrane, where selective antibodies are aligned to form the test and control lines, additional membranes (i.e., the sample probe and absorbent pads), a rigid structure and the probe.

Figure 2

Strategies for multiplexing LFIA by: (a) spatially separate detection sites in a single strip; (b) aligning several strips in an array format; (c) exploiting signal reporters that provide different signals; and (d) using broad-selective antibodies able to bind to several compounds in a class.

Figure 3

Approaches to multiplexing LFIA that can be exploited separately (single ‘x’ multiplexing level) or combining two (x²), three (x³) or even four (x⁴) of them. Combinations highlighted in colors have been reported in literature, while multiplexing strategies theoretically realizable but not yet reported are shown in grey.

Figure 4

Schematic representation and image of the lateral flow microarray test strip. Each of the 32 dots represents a different capturing agent; the validity of the test is assured by the control line as usual [17].

Figure 5

(a) Individual strip for the identification of a single bacterium by means of an antibody linked to the up-converting phosphorous probe. (b) The 10-channel up-converting phosphor technology-based lateral flow (TC-UPT-LF) disc holds 10 detection channels (T1 to T10), each comprising a single strip for the target bacteria [23].

Figure 6

The xLFIA realized through the use of a single test line and multicolor gold nanoparticles (GNPs) as signal reporters. Red and blue GNPs were linked to antibodies directed towards two food contaminants and the test line was formed by the mixture of the two corresponding antigens. As a result of the immunoreactions occurring at the test zone, the test line assumed different colors accordingly to which contaminant was present in the sample. (a) Color code for contamination detection and contaminant identification. (b) Images of strips obtained by the color-encoded xLFIA [36].