A two-colour multiplexed lateral flow immunoassay system to differentially detect human malaria species on a single test line

Abstract

Background Malaria continues to impose a tremendous burden in terms of global morbidity and mortality, yet even today, a large number of diagnoses are presumptive resulting in lack of or inappropriate treatment. Methods In this work, a two-colour lateral flow immunoassay (LFA) system was developed to identify infections by Plasmodium spp. and differentiate Plasmodium falciparum infection from the other three human malaria species (Plasmodium vivax, Plasmodium ovale, Plasmodium malariae). To achieve this goal, red and blue colours were encoded to two markers on a single test line of strips, for simultaneous detection of PfHRP2 (red), a marker specific for P. falciparum infection, and pLDH (blue), a pan-specific marker for infections by all species of Plasmodium. The assay performance was first optimized and evaluated with recombinant malarial proteins spiked in washing buffer at various concentrations from 0 to 1000 ng mL−1. The colour profiles developed on the single test line were discriminated and quantified: colour types corresponded to malaria protein species; colour intensities represented protein concentration levels. Results The limit of detection (the lowest concentrations of malaria antigens that can be distinguished from blank samples) and the limit of colour discrimination (the limit to differentiate pLDH from PfHRP2) were defined for the two-colour assay from the spiked buffer test, and the two limits were 31.2 ng mL−1 and 7.8 ng mL−1, respectively. To further validate the efficacy of the assay, 25 human whole blood frozen samples were tested and successfully validated against ELISA and microscopy results: 15 samples showed malaria negative; 5 samples showed P. falciparum positive; 5 samples showed P. falciparum negative, but contained other malaria species. Conclusions The assay provides a simple method to quickly identify and differentiate infection by different malarial parasites at the point-of-need and overcome the physical limitations of traditional LFAs, improving the multiplexing potential for simultaneous detection of various biomarkers.

Switching from Multiplex to Multimodal Colorimetric Lateral Flow Immunosensor

“…Multiplexing LFIA based on differentiable color probes are conventionally based on using latex microparticles embedding dyes with different adsorption peaks [ 47 , 48 ] and noble metal nanoparticles showing localized plasmon resonance bands at variable wavelengths according to their size and shapes [ 17 , 20 , 21 ].However, these approaches involves aligning several lines along the strip to differentiate the target to be detected and the color of the probe is simply exploited to help simplifying the visual reading of the result. Here, we designed a two-parameter strategy, in which the color and space resolution were combined to expand the number of analytes simultaneously detected by a single device.…”

“…Nevertheless, these approaches need instrumentation that limits on-field applications. Dual-color probes, such as gold nanoparticles (GNP) and gold nanostars (GNS) [ 18 ] or silver nanoparticles (SNP) [ 19 ] functionalized with antibodies were used in previous studies to develop multiplexing LFIA in which the differentiation of the probe enabled distinguishing the target [ 18 , 20 , 21 ].…”

Ten Years of Lateral Flow Immunoassay Technique Applications: Trends, Challenges and Future Perspectives

“…Recently, the use of a single strip with a single Test line has also been applied for multiplexed detection of target molecules, exploiting surface-enhanced Raman scattering [ 80 , 81 ] and colorimetric detection [ 82 , 83 ]. Moreover, most recently, Cavalera et al proposed a two-parameter multiplexing LFIA strategy (x2LFIA), which combines the spatial resolution with colour encoding approach to expand the number of information achievable within a single strip test, obtaining tetravalent information in a two-line and two-colour assay [ 84 ].…”

Infrared Spectroscopy of Blood

“…The most common LFIA assay is the pregnancy tests, although, there are also LFIA assay (also called rapid diagnostic tests) to identify infections to malaria. [4][5][6][7] Most POC rapid diagnostics rely on the LFIA platform due to its low-cost, simplicity, expediency, and portability. Concerns over the low clinical sensitivity of some antigen detection on LFIAs have led an effort to develop other types of POC diagnostics that can provide high sensitivity.…”