Sensor Technology Page 3. 1 Lateral Flow Biosensors How paper, a “plain” material, can be used for producing cheap and efficient diagnostic tools. Introduction After Covid-19 Pandemic (SARS-CoV-2 virus), it is clearer than ever the importance of having rapid diagnostic tools which can be easily deployed and produced on a large scale. These tools must be cheap, portable, provide a fast and real-time response, easy-to-use and, desirably, equipment and battery-free. Only by fulfilling these conditions the tool can reach everyone: people in isolated areas, low resource regions, patients who cannot abandon their home, etc. During the pandemic, the so-called “antigen rapid diagnostic tests” emerged and became a standard for detecting the virus contagion, putting aside the PCR test that, although being more sensitive, is also much more expensive, needs much longer time and requests trained personnel to be performed. However, this technology was not something new, since its principle is also employed in pregnancy tests (that are well known since decades): the lateral flow biosensors (LFBs). LFBs are an evolution of paper chromatography in which the sample to be analysed flows by capillarity across paper and the user can, just by naked eye, understand the results. In early 70s, enzyme-linked immunoassay (ELISA) tests were invented and the use of antibodies for sensing (immunoassays) became a trend. Some years later several patents appeared regarding immunoassays on paper substrate.1 These patents are considered the first LFBs (which later in the 90s would evolve into the well-known pregnancy tests, for example). How does a lateral flow test work?
Sensor Technology Page 3. 2 LFBs are presented as paper strips, composed of different “pads”,2 as it is shown in the figure above (1). The sample (any liquid, e.g. water, urine or blood) is dropped onto the “sample pad”, made of pure paper (i.e. cellulose) pretreated with different salts and surfactants that control the pH across the strip and help the analytes to flow thought the paper pores, respectively.Then, the sample flows to the conjugate pad (2), normally made of polyester or glass fibbers, where the analyte will be captured by the nanoconjugate (a nanomaterial, responsible of producing the colorimetric signal in the assay, and an antibody that specifically recognises the target analyte).The analyte, together with the nanoconjugate, will flow across the detection pad (3), made of nitrocellulose, and form an “immunosandwich” on the test line (where another antibody will also capture the analyte), which will become visible as the nanomaterial accumulates in the area. In the detection pad also a control line will become visible as the nanoconjugate reaches the line (an antibody immobilized on control line will always recognise the antibody conjugated on the nanoparticles, as a control of the fluidic in the strip).The presence of both test and control lines indicates that the response is “positive” (i.e. the analyte is present in the sample). In the case that there is no analyte in the sample (4), only the control line will be visible, thus “negative” response will be considered. If control line does not appear, it may indicate a failure in the process and the response would be considered “null”, indifferently if the test line appears or not.The last pad, the absorbent pad, is made of cellulose and its function is to absorb the excess of liquid avoiding the flow to stop before the nanoconjugate surpasses the control line. What to know to develop a lateral flow test? Paper (cellulose and its derivates) is the main component of LFBs and understanding how it affects the performance of the test is a key factor when designing the assay. For example, the thickness and width of the sample pad will affect the minimum volume of sample that the user must apply on the test for the sample to flow through all the strip. Also, some specially designed sample pads may help retaining components of the sample matrix which may interfere in the test (e.g. blood cells). On another hand, nitrocellulose porosity rate can be controlled during its production, so knowing the average pore dimension of the material the flow can be controlled.At bigger pore dimension, the flow will be faster, which is good if we want to obtain a response in less than a couple of minutes, but if the pores are smaller, thus the flow being slower, the nanoconjugate will have more time for interacting with the sample, so the sensitivity of the assay could be greater, although the time until one can see the response would be longer. Of course, pore dimension must be also considered regarding the analyte dimension, it is not the same detecting a DNA strain or a small protein than a bacterium or a whole cell.
Sensor Technology Page 3. 3 The nitrocellulose manufacturer may also control the amount of nitro groups, making the substrate more affined for proteins.As greater is the amount of nitro groups in the nitrocellulose, lower would be the affinity of proteins for the substrate, so they may flow easily across the paper. However in this last case it would be harder to immobilize biomolecules (e.g. antibodies) in the test and control lines.The affinity of paper can be also modified by treating the paper substrate with other proteins or surfactants. Another important element on a LFBs is the transducer, the materi al which produces the colour that can be observed on the test and control lines. Nowadays, nanomaterials are the standard transducer on LFBs, replacing enzymes (e.g. horseradish peroxidase bound antibodies) and dyed microparticles (e.g. latex beads).The different properties of the nanomaterials increase the sensitivity of the assay,3 allowing the upgrade of the tests from a qualitative measurement (positive vs. negative) to semiquantitative (the colour intensity of test line can be related with the amount on nanoconjugate stopped in the area and, therefore, with the concentration of analyte). Gold nanoparticles (AuNPs) are the most used nanomaterial on LFBs, espe cially in commercial tests.AuNPs of around 20 nm diameter exhibit a strong red colour, good stability and are very easy to conjugate with biomolecules such as antibodies, DNA strains or aptamers. Other nanomaterials such as graphene, metal oxide nanoparticles and quantum dots (the latter with fluorescent properties) start to emerge in academic reports, but not yet in the market. Limitations of LFBs must be also considered.The detection limit and sensitivity of a LFBs will not (or will hardly) surpass the levels achieved by an ELISA test, even when employing the same antibodies. LFBs are an affordable, robust and portable tool that can provide a fast response, being easy-to-use by even non-trained users, but as the response is read without any equipment, by naked eye, it is hard to identify low concentrations of analyte and discern between close concentration values. Future perspectives Nowadays, several LFBs tests for diagnostic applications can be found in the market: pregnancy prediction (human chorionic gonadotropin hormone), Covid-19, Human Immunodeficiency Virus (HIV), parasites (e.g. Malaria), allergens (in food, drink and rooms), bacteria (e.g. Clostridium difficile Toxin A+B), etc. It is important to highlight the fact that, being able to use these biosensors at home (at point-of-care), LFBs grant the patient with a plus of privacy.After pandemics, LFBs have demonstrated their usefulness, so it is to expect many LFBs (rapid diagnostic tests) being launched for uncovered medical needs and new biomarkers (e.g. miRNA or extracellular vesicles, be tween others), but also for applications out of the diagnostic field, as environmental sensing (e.g. detection of heavy metals and pollutants in water).
Sensor Technology Page 3. 4 It is also to be expected that the technology will evolve, seeking for an improved quantification and lower detection limits, integration within the signal readout as part of other techniques rather than optical, such as electrochemical measurements, or even combined approaches that can improve the performance, such as electrophoresis.4 Re garding the bioreceptors immobilized in the test (in the nanoconjugate, test and control lines), it is highly probable that LFBs will move from the immunoreactions (i.e. antibodies) to molecular sensing (i.e. DNA strains, aptamers and derivates), since DNA is more stable and affordable than antibodies. In addition, by using the DNA-based detection the integration of isothermal amplification methods (even inside the paper strip), greatly lowering the detection limit, such as Loop-Mediated Isothermal Amplification (LAMP) or Recombinase Polymerase Amplification (RPA) may be expected.5 References 1. Charlton, D. E. (1988).Test device and method for colored particle immunoassay. Patent: US6485982B1. 2.Wong, R. & Tse, H. (2009). Lateral Flow Immunoassay, Springer Link, ISBN: 978-1-59745240-3. 3. Nguyen,V.T.; Song, S.; Park, S. & Joo, C. (2020). Recent advances in high-sensitivity detection methods for paper-based lateral-flow assay. Biosens. Bioelectron., 152, 112015. 4. Sena-Torralba,A.; Álvarez-Diduk, R.; Parolo, C.; Piper,A. & Merkoçi,A. (2022).Toward Next Generation Lateral Flow Assays: Integration of Nanomaterials. Chem. Rev., 122, 18, 14881–14910. 5. Rubio-Monterde,A.; Quesada-González, D. & Merkoçi,A. (2023).Toward Integrated Molecular Lateral Flow Diagnostic Tests Using Advanced Micro- and Nanotechnology. Anal. Chem., 95, 1, 468–489. Catalan Institute of Nanoscience and Nanotechnology (ICN2)
Sensor Technology Page 3. 5 Contributed by: Daniel Quesada-González Chemistry (PhD) Postdoctoral Researcher at Nanobioelectronics and Biosensors Group Email: daniel.quesada@icn2.cat Google Scholar: https://scholar.google.es/citations?user=RGOUj24AAAAJ&hl=es&oi=ao LinkedIn Profile: https://es.linkedin.com/in/danielquesadagonzalez ORCID: 0000-0003-3064-2146 Tel.: +34 937374610 Arben Merkoçi Chemistry (PhD) ICREA Professor and Group Leader at Nanobioelectronics and Biosensors Group Catalan Institute of Nanoscience and Nanotechnology (ICN2) Email: arben.merkoci@icn2.cat Google Scholar: https://scholar.google.es/citations?user=hF6hPeIAAAAJ&hl=es LinkedIn Profile: https://es.linkedin.com/in/arben-merko%C3%A7i-27439693 ORCID: 0000-0003-2486-8085 Tel.: +34 937374604 Research Group social media and more information at: http://linktr.ee/merkoci.group
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