Aptamer-Based Lateral Flow Assays: Current Trends in Clinical Diagnostic Rapid Tests

The lateral flow assay (LFA) is an extensively used paper-based platform for the rapid and on-site detection of different analytes. The method is user-friendly with no need for sophisticated operation and only includes adding sample. Generally, antibodies are employed as the biorecognition elements in the LFA. However, antibodies possess several disadvantages including poor stability, high batch-to-batch variation, long development time, high price and need for ethical approval and cold chain. Because of these limitations, aptamers screened by an in vitro process can be a good alternative to antibodies as biorecognition molecules in the LFA. In recent years, aptamer-based LFAs have been investigated for the detection of different analytes in point-of-care diagnostics. In this review, we summarize the applications of aptamer technology in LFAs in clinical diagnostic rapid tests for the detection of biomarkers, microbial analytes, hormones and antibiotics. Performance, advantages and drawbacks of the developed assays are also discussed.

Nanophotonics: An Emerging and Promising Approach for COVID-19 Diagnostic Technology

The public health crisis initiated by the emergence of the COVID-19 pandemic emphasizes the need for rapid and accurate diagnostic tests to monitor large populations through community mass testing. Many testing techniques have been implemented to prevent disease spread, critical to pandemic control. Polymerase chain reaction (PCR) tests for detecting viral RNA and immunoassay tests for detecting SARS-CoV-2 antibodies are currently used to diagnose COVID-19. PCR tests are time-consuming, with a 24–48 hours turnaround time. Samples undergoing PCR detection must also be sent to a laboratory to be processed by highly specialized workers, preventing a point-of-care diagnosis from being provided. Popular immunoassay tests have drawbacks as well. Enzyme-linked immunosorbent assays (ELISAs) are extremely labor-intensive and expensive, whereas lateral flow assays (LFAs) are primarily used for antigen detection. In this work, we propose a photonic SARS-CoV-2 detection method based on a ring resonator. We calculate the sensor performance using the finite-difference eigenmode (FDE) method. The sensor sensitivity in ring resonator resonance frequency is 29 nm/RIU, with an intrinsic detection level (iLOD) of 6.89 × 10-5 RIU. We envision ring resonator-based lab-on-chip devices being widely used for applications such as early diagnosis, with the added benefit of being ultra-compact and easily handled by non-specialists.

Ball pen writing-without-ink: a truly simple and accessible method for sensitivity enhancement in lateral flow assays

A ball pen writing-without-ink method was developed to amplify the detection signal of LFAs through controlling fluid flow rate.

Evaluation of rapid, cassette immunochromatographic tests in the serological diagnosis of COVID-19

Introduction: Lateral flow assays (LFIA) are the technology behind low-cost, simple, rapid and portable detection devices popular in biomedicine. Lately, they are very common used in serodiagnosis of SARS-CoV-2 infections. The aim of the presented study was to assess the usefulness of selected LFIA in serological diagnosis of COVID-19. Methods: The usefulness of seven lateral flow assays in the serodiagnosis of COVID-19 was evaluated (VAZYME, DIAGNOSIS, PCL, INGEZIM, BIOSENSOR, ACCU-TELL, NOVAtest). The study used 107 serum samples obtained from 74 individuals with current SARS-CoV-2 infection confirmed by RT-PCR. The ELISA-IgG (Euroimmun) was used as the reference assay for sensitivity and specificity testing. Results: The highest percentage of positive results was obtained when searching for IgG antibodies with the NOVAtest (40.6%) and DIAGNOSIS (39.2%) sets and the lowest detection for the PCL set - 25.5%. In the case of searching for IgM antibodies in all sets, significantly lower percentages of positive results compared to the IgG class were recorded. In general, all lateral flow assays showed low sensitivity in relation to the Euroimmun ELISA-IgG. The DIAGNOSIS kit (64.5%) was characterized by the highest sensitivity, and the PCL kit was the lowest (38.7%). On the other hand, the specificity of all kits was very high, almost 100% in almost all cases. Conclusions: Lateral flow assays due to their low sensitivity are not suitable for quick diagnosis of the current SARS-CoV-2 infections and cannot be an alternative to genetic or even antigen tests. They may be used only to retrospectively test the presence of IgG antibodies. However, a negative results of LFIA in suspected disease or after vaccination should be confirmed by more sensitive serological tests.

Comparative Study of Four Coloured Nanoparticle Labels in Lateral Flow Immunoassay

The detection limit of lateral flow immunoassay (LFIA) is largely determined by the properties of the label used. We compared four nanoparticle labels differing in their chemical composition and colour: (1) gold nanoparticles (Au NPs), red; (2) Au-core/Pt-shell nanoparticles (Au@Pt NPs), black; (3) latex nanoparticles (LPs), green; and (4) magnetic nanoparticles (MPs), brown. The comparison was carried out using one target analyte—Erwinia amylovora, the causal bacterial agent of fire blight. All nanoparticles were conjugated with antibodies through methods that provide maximum functional coverage like physical adsorption (Au NPs, Au@Pt NPs) and covalent bonding (LPs, MPs). All conjugates demonstrated the same ability to bind with E. amylovora through enzyme-linked immunosorbent assay where optical properties of the nanoparticles do not determine the registered signal. However, half-maximal binding was achieved at different numbers of nanoparticles because they differ in size. All conjugates based on four nanoparticle labels were used for lateral flow assays. As a result, Au@Pt NPs provided the minimal detection limit that corresponded to 103 CFU/mL. Au NPs and LPs detected 104 CFU/mL, and MPs detected 105 CFU/mL. The results highlight that simply choosing a coloured label can significantly affect the detection limit of LFIA.

Highly sensitive interleukin 6 detection by employing commercially ready liposomes in an LFA format

Recent years have confirmed the ubiquitous applicability of lateral flow assays (LFA) in point-of-care testing (POCT). To make this technology available for low abundance analytes, strategies towards lower limits of detections (LOD), while maintaining the LFA’s ease of use, are still being sought. Here, we demonstrate how liposomes can significantly improve the LOD of traditional gold nanoparticle (AuNP)–based assays while fully supporting a ready-to-use system for commercial application. We fine-tuned liposomes towards photometric and fluorescence performance on the synthesis level and applied them in an established interleukin 6 (IL-6) immunoassay normally using commercial AuNP labels. IL-6’s low abundance (< 10 pg mL−1) and increasing relevance as prognostic marker for infections make it an ideal model analyte. It was found that liposomes with a high encapsulant load (150 mmol L−1 sulforhodamine B (SRB)) easily outperform AuNPs in photometric LFAs. Specifically, liposomes with 350 nm in diameter yield a lower LOD even in complex matrices such as human serum below the clinically relevant range (7 pg mL−1) beating AuNP by over an order of magnitude (81 pg mL−1). When dehydrated on the strip, liposomes maintained their signal performance for over a year even when stored at ambient temperature and indicate extraordinary stability of up to 8 years when stored as liquid. Whereas no LOD improvement was obtained by exploiting the liposomes’ fluorescence, an extraordinary gain in signal intensity was achieved upon lysis which is a promising feature for high-resolution and low-cost detection devices. Minimizing the procedural steps by inherently fluorescent liposomes, however, is not feasible. Finally, liposomes are ready for commercial applications as they are easy to mass-produce and can simply be substituted for the ubiquitously used AuNPs in the POCT market. Graphical abstract

Open-source, Adaptable, All-in-one Smartphone-based System for Quantitative Analysis of Point-of-care Diagnostics.

Point-of-care (POC) diagnostics, in particular lateral flow assays (LFA), represent a great opportunity for rapid, precise, low-cost and accessible diagnosis of disease. Especially with the ongoing coronavirus disease 2019 (COVID-19) pandemic, rapid point-of-care tests are becoming everyday tools for identification and prevention. Using smartphones as biosensors can enhance POC devices as portable, low-cost POC platforms for healthcare and medicine, food and environmental monitoring, improving diagnosis and documentation in remote, low-income locations. We present an open-source, all-in-one smartphone-based system for quantitative analysis of LFAs. It consists of a 3D-printed photo box, a smartphone for image acquisition, and an R Shiny software package with modular, customizable analysis workflow for image editing, analysis, data extraction, calibration and quantification of the assays. This system is less expensive than commonly used hardware and software, so it could prove very beneficial for diagnostic testing in the context of pandemics, as well as in low-resource countries.