Lateral Flow Testing: Principles, Applications and Innovations

An LFT is a simple, low-cost device used to detect a target analyte rapidly in a liquid sample.1 Core components of an LFT include: LFTs are immunoassays that rely on the specific binding between an antibody and its target antigen. Direction of sample flow Analyte Antigen in samples Gold rabbit antigen IgG conjugate Test line: antigen antibody Control line: anti-rabbit control IgG antibody Positive Negative C T C T Nitrocellulose membrane Sample pad Conjugate pad Test line Control line Backing Absorption pad A liquid sample is applied to the sample pad. The sample moves laterally along the strip through capillary action. If the target analyte is present, it binds to labeled reagents and forms a visible line at the test region. In this example, the target is an antigen, so the test line contains immobilized targetspecific antibodies. If the target was an antibody, the test line would contain immobilized antigens. The sample continues along a porous membrane and binding at the control line confirms the test has run correctly. Remaining sample flows into the absorption pad. LFTs can be in a direct (as shown in the example above) or competitive format. Competitive formats are used when testing for small molecules with single antigenic determinants. The absence of a test line indicates a positive result. What is an LFT? How do LFTs work? Sample pad Sample is applied here Conjugate pad Contains labeled antibodies or reagents Membrane (typically nitrocellulose) Houses test and control lines Absorption pad Draws fluid through the device 1 1 4 4 2 2 5 5 3 3 Key applications of LFTs Human health LFTs are widely used in human diagnostics and monitoring: „ Pregnancy testing – detect hCG in urine,3 available since the 1980s „ Infectious diseases – used for detecting malaria,4 TB,5 HIV,6 COVID-197 and others „ Therapeutic drug monitoring – support dose optimization for narrow-margin drugs, e.g., digoxin used for the treatment of tachycardia8 „ Drugs of abuse – detect substances in urine, sweat or fingerprints, e.g., cocaine,9 THC10 Animal health LFTs are used in veterinary medicine for disease surveillance and reproductive management, supporting onfarm decisions and helping to limit disease spread: „ Pathogen detection – e.g., African swine fever,11 bovine diarrhea12 „ Pregnancy testing – for livestock in field settings Food safety LFTs help safeguard consumers by enabling rapid analysis of food: „ Allergen testing – detect gluten,13 casein,14 soy15 and more „ Pathogen monitoring – e.g., for Salmonella16 and mycotoxins like aflatoxins17 „ Consumer empowerment – smartphonecompatible kits for real-time food testing Environmental testing LFTs are used to detect contaminants that threaten ecosystems and public health: „ Heavy metals – mercury,18 cadmium,19 chromium20 „ Organic pollutants – including BPA21 and pesticides „ Water safety22 – LFTs enable rapid analysis in field locations Advances in materials, detection methods and digital integration are enhancing the performance, versatility and usability of lateral flow tests, paving the way for more sensitive, multiplexed and connected diagnostics.23, 24 References: 1. O’Farrell B. Evolution in lateral flow–based immunoassay systems. Lateral Flow Immunoassay. 2008:1-33. doi: 10.1007/978-1-59745-240-3_1 2. Posthuma-Trumpie GA, Korf J, van Amerongen A. Lateral flow (immuno) assay: its strengths, weaknesses, opportunities and threats. A literature survey. Anal Bioanal Chem. 2009;393(2):569-582. doi: 10.1007/s00216- 008-2287-2 3. Leuvering JHW, Goverde BC, Thal PJHM, Schuurs AHWM. A homogeneous sol particle immunoassay for human chorionic gonadotrophin using monoclonal antibodies. J Immunol Meth. 1983;60(1):9-23. doi: 10.1016/0022-1759(83)90330-7 4. How malaria RDTs work. World Health Organization. https://www.who. int/teams/global-malaria-programme/case-management/diagnosis/ rapid-diagnostic-tests/how-malaria-rdts-work. Accessed July 9, 2025. 5. Ariffin N, Yusof NA, Abdullah J, et al. Lateral flow immunoassay for naked eye detection of Mycobacterium tuberculosis. 2020. J Sensors. doi: 10.1155/2020/1365983 6. Turbé V, Herbst C, Mngomezulu T, et al. Deep learning of HIV field-based rapid tests. Nat Med. 2021;27(7):1165-1170. doi: 10.1038/s41591-021- 01384-9 7. Peto T, UK COVID-19 Lateral Flow Oversight Team. COVID-19: Rapid antigen detection for SARS-CoV-2 by lateral flow assay: A national systematic evaluation of sensitivity and specificity for mass-testing. eClinicalMedicine. 2021. doi: 10.1016/j.eclinm.2021.100924 8. Ruppert C, Phogat N, Laufer S, Kohl M, Deigner HP. A smartphone readout system for gold nanoparticle-based lateral flow assays: application to monitoring of digoxigenin. Microchim Acta. 2019;186(2):119. doi: 10.1007/s00604-018-3195-6 9. Guler E, Sengel TY, Gumus P, et al. Mobile phone sensing of cocaine in a lateral flow assay combined with a biomimetic material. Anal Chem. 2017. doi: 10.1021/acs.analchem.7b03017 10. Hudson M, Stuchinskaya T, Ramma S, et al. Drug screening using the sweat of a fingerprint: lateral flow detection of Δ9-tetrahydrocannabinol, cocaine, opiates and amphetamine. J Anal Tox. 2019. doi: 10.1093/jat/ bky068 11. Onyilagha C, Nguyen K, Luka PD, et al. Evaluation of a lateral flow assay for rapid detection of African swine fever virus in multiple sample types. Pathogens. 2022. doi: 10.3390/pathogens11020138 12. Cho Y, Sun D, Cooper V, et al. Evaluation of a commercial rapid test kit for detecting bovine enteric pathogens in feces. J Vet Diagn Invest. 2012. doi: 10.1177/1040638712440997 13. Hnasko RM, Jackson ES, Lin AV, Haff RP, McGarvey JA. A rapid and sensitive lateral flow immunoassay (Lfia) for the detection of gluten in foods. Food Chem. 2021. doi: 10.1016/j.foodchem.2021.129514 14. Galan-Malo P, Pellicer S, Pérez MD, Sánchez L, Razquin P, Mata L. Development of a novel duplex lateral flow test for simultaneous detection of casein and β-lactoglobulin in food. Food Chem. 2019. doi: 10.1016/j.foodchem.2019.04.039 15. Gautam PB, Sharma R, Lata K, Rajput YS, Mann B. Construction of a lateral flow strip for detection of soymilk in milk. J Food Sci Technol. 2017. doi: 10.1007/s13197-017-2890-3 16. Charlermroj R, Makornwattana M, Phuengwas S, Karoonuthaisiri N. A rapid colorimetric lateral flow test strip for detection of live Salmonella Enteritidis using whole phage as a specific binder. Front Microbiol. 2022. doi: 10.3389/fmicb.2022.1008817 17. Anfossi L, Baggiani C, Giovannoli C, Giraudi G. Lateral flow immunoassays for aflatoxins b and g and for aflatoxin m1. In: Aflatoxins - Recent Advances and Future Prospects. IntechOpen; 2013. doi: 10.5772/51777 18. He Y, Zhang X, Zeng K, et al. Visual detection of Hg2+ in aqueous solution using gold nanoparticles and thymine-rich hairpin DNA probes. Biosens Bioelectron. 2011. doi: 10.1016/j.bios.2011.05.003 19. López_Marzo AM, Pons J, Blake DA, Merkoçi A. High sensitive goldnanoparticle based lateral flow immunodevice for Cd2+ detection in drinking waters. Biosens Bioelectron. 2013. doi: 10.1016/j. bios.2013.02.031 20. Liu X, Xiang JJ, Tang Y, et al. Colloidal gold nanoparticle probe-based immunochromatographic assay for the rapid detection of chromium ions in water and serum samples. Analytica Chimica Acta. 2012. doi: 10.1016/j. aca.2012.06.029 21. Mei Z, Qu W, Deng Y, et al. One-step signal amplified lateral flow strip biosensor for ultrasensitive and on-site detection of bisphenol A (BPA) in aqueous samples. Biosens Bioelectron. 2013. doi: 10.1016/j. bios.2013.06.006 22. Guo YR, Liu SY, Gui WJ, Zhu GN. Gold immunochromatographic assay for simultaneous detection of carbofuran and triazophos in water samples. Anal Biochem. 2009. doi: 10.1016/j.ab.2009.03.020 23. Omidfar K, Riahi F, Kashanian S. Lateral flow assay: a summary of recent progress for improving assay performance. Biosensors (Basel). 2023. doi: 10.3390/bios13090837 24. Vealan K, Joseph N, Alimat S, Karumbati AS, Thilakavathy K. Lateral flow assay: a promising rapid point-of-care testing tool for infections and non-communicable diseases. Asian Biomed (Res Rev News). 2023. doi: 10.2478/abm-2023-0068 Lateral flow tests (LFTs) are used across diverse fields, from clinical care to food safety and environmental monitoring, to detect analytes in liquid samples. This infographic explores how LFTs work, their key applications and the latest innovations shaping their future. Recent innovations in lateral flow technology LFTs are evolving into connected, highly adaptable diagnostic tools. In the future, they are likely to: „ Support personalized, at-home diagnostics „ Have expanding use in low-resource settings „ Be integrated with telehealth platforms „ Enable real-time epidemiological monitoring Flow rate optimization Engineered flow control strategies, such as geometric constrictions, porous material tuning and chemical barriers, are being used to slow capillary flow, increase analyte– reagent interaction time and boost assay sensitivity. Signal amplification Enhanced signal output is being achieved using doped gold nanoparticles, nanozymes, silver/copper enhancement and novel carrier particles, delivering stronger visual or fluorescent signals for low-abundance targets. Multiplexing for comprehensive testing Multiplex LFTs can simultaneously detect multiple biomarkers on a single strip – streamlining workflows, increasing clinical insight and reducing cost per test. Improved immobilization Nanomaterials like silica, graphene oxide and electrospun nanofibers increase the effective surface area for capture molecule attachment, enhancing binding efficiency and signal intensity. Smart readers and digital integration Smartphone-enabled readers and portable optical, magnetic or thermal devices are improving result accuracy, enabling quantification and supporting real-time data sharing. AI and image analysis tools further refine result interpretation. LFTs are valued for their speed and simplicity.2 Their affordability and robustness make them especially useful in resource-limited settings and for home diagnostics. Despite their strengths, LFTs come with certain drawbacks.2 Sensitivity and specificity can vary, and results are often qualitative. Advantages and limitations of LFTs Fast – results in less than 30 minutes User-friendly – no specialized training required Portable – ideal for point-of-care and field testing Cost-effective – inexpensive materials and manufacturing Stable – long shelf life without refrigeration Lower sensitivity/specificity than lab-based assays Qualitative results – limits clinical decision-making Short read window – results may fade or overdevelop Limited throughput – not ideal for processing large sample volumes Batch variability and cross-reactivity can affect reliability Added complexity and cost when using digital readers Viscous samples and particulates can be problematic Advantages Limitations LATERAL FLOW TESTING: Principles, Applications and Innovations Written by Anna MacDonald and Karen Steward | Designed by Janette Lee-Latour