Lateral Flow Assay - Gabrielle Berns and Bryanne Zonghi
Introduction and Motivation
Lateral flow assays are paper based microfluidic devices used to detect the presence of a target analyte in a complex mixture. Detecting specific chemical components in liquid mixtures can be done simply by using these cheap paper devices, without needing any costly equipment. These paper devices have dominated rapid diagnostics in the past thirty years due them being compact, light-weight, easy to interpret, and disposable [yetizen]. Lateral flow assay tests do not require any training or previous experience and are designed for use in non-laboratory settings, thus these devices are widely used for a variety of different applications. This technology is widely used in medical fields and law enforcement to test bodily fluids such as urine, blood, sweat, and saliva.[koczula] Biomarkers for organ failure, infection, toxic pathogens, or illicit drugs can be detected with high sensitivity and selectivity due to the fact that each device is specifically designed to interact with the molecule of question.[posthuma] Essentially these devices operate through the flow process called capillary action, which pushes the fluid containing the analyte through various zones, where molecules are bound to interact with and detect only that analyte.[posthuma] Lateral flow assay tests are lucrative because there is a large demand for decentralized diagnostic tests that are easy to use, accurate and provide rapid results [tisone].
History
The technical ideas of these microfluidic paper devices originated in 1956. The ideas for lateral flow assays derived from Charles M. Plotz and Jaques M. Singer when they first discovered Rheumatoid Factor Test based on latex agglutination assays. During this time, plate-based immunoassays and the first radio-immunoassay (RIA) were also newly developed. The enzyme immunoassay was first developed in the 1960s and replaced radioisotopes due to its faster reaction times and longer shelf-life. During the years following this, the fundamental principles of lateral flow technology continued to develop until the 1980s [wong] . During the late 1980s, this technology finally became firmly established within the field. Companies finally began to patent this paper microfluidic technology, specifically in the 1970s with great strides in urine testing for medical diagnosis. Finally, the first lateral flow assay device were put on the market during the 1980s. Since the initial introduction to the market, lateral flow assay products have continued to evolve and become more reliable. As of the mid 2000s, hundred of companies internationally are creating their own testing formats relating to lateral flow assay technology. This technology has already expanded to areas of agriculture, food, environmental health and safety, biowarfare, and most commonly for pregnancy tests [wong].
Competitive Assays vs. Sandwich/Direct Assays
Lateral flow assays can be divided into two major categories: competitive assays and direct (also known as sandwich) assays. Competitive assays are not suited for high molecular weight compounds. In competitive assays, the absence of a color at the test line means that the analyte is present. Color at the test line region means the test is negative. Competitive assays are used for detecting analytes when the antibody pairs are not available, or if the analyte is not large enough for many antibody events. In competitive assays, the test line usually has the analyte molecule and the conjugate pad has the detection antibody and nanoparticle conjugate [8] . The analyte will bind to the conjugate if the target analyte is present. Competitive assays have two different layouts. The first layout consists of a solution that has the target analyte on the application pad and the conjugate is hydrated and flows with the liquid. The test line has previously immobilized antigens that bind to label conjugates. The control line has previously immobilized secondary antibodies that can bind with labeled antibody conjugates. The previously immobilized antigens bind to the labeled conjugates when the liquid gets to the test line. A competition to bind with the conjugate takes place between the immobilized antigen at the test line strip and the antigen within the sample. The second layout consists of a labeled analyte conjugate that is dispensed at the conjugate pad and a primary antibody conjugate at the test line. When the analyte solution is applied, there is a competition between the analyte and the labeled analyte to see which can bind with the primary antibody, which occurs at the test line [4]. Competitive assays are specialized for drugs and toxins, and are usually applied to small molecular weight analytes.
Direct assays, or sandwich assays, are used for larger molecular weight analytes with more than one antigenic sites. A colored line means the test is positive, and a negative test comes from the absence of a line there at all. The most common sandwich assay test used commercially is the over the counter pregnancy test. These sandwich assays are used to detect larger analytes that have two or more binding sites. An antibody is conjugated to the nanoparticle on one binding site, and another antibody to a different binding site is used for the test line. The analyte will bind to the antibody and the nanoparticle conjugate as well as to the antibody on the test line if the sample contains an analyte; this would yield a positive test. In sandwich assays, the intensity of the signal at the test line is directly proportional to the quantity of analyte present [8]. This kind of test would not work for small molecular weight analytes, only for large molecular weight analytes. Direct assays consist of a label coated antibody, which is usually an enzyme, nanoparticle, or fluorescent dyes [4]. Primary antibodies against target analytes are immobilized over the test line. At the control zone, a secondary antibody is immobilized against the conjugate antibody that is labeled. The sample with the analyte is applied to the application sample and then it migrates to the remaining sections of the strip. The target analyte is then caught by the immobilized antibody and finally the labeled antibody/analyte complex is formed. This labeled antibody/analyte duo then gets to a nitrocellulose membrane and then moves using capillary action [4]. The analyte then migrates in between the labeled antibody and the primary antibody in a sandwich fashion, forming a labeled antibody complex of analyte, conjugate, and the primary antibody. The secondary antibody captures the labeled antibody conjugate in excess at the control zone. The absorption pad captures buffer or any excess solution. The amount of target analyte can be seen through the intensity of the color at the test line. This is measured by using an optical strip reader on the device. Color at the control line is a good indication that the strip is functioning accurately [4]. Figure XXX shows the differences between sandwich, or direct, assays and competitive assays.
Components of the Assay
Lateral flow assays are generally carried out on strips which contain 4 major parts: a sample application pad, conjugate pad, nitrocellulose membrane and adsorption pad [sajid]. The application pad, typically made of cellulose or glass fiber, is where the sample is first applied at the start of the test. The application pad treats the sample and modulates any chemical variability. The pad is can also be made of cotton linter, glass fibre, rayon, and filtration materials. These pads are pretreated with a pH buffer, surfactants, and blocking reagents to influence the flow rate and viscosity of the sample.[posthuma] This pad must have properties that allow for a continuous transport of the sample to the next part of the device, the conjugate pad. The conjugate pad couples the analyte in the sample with the conjugate and releases the pairing. These conjugates are generally antibodies that specifically bind to the target molecule. These antibodies are bound to the pad using colloidal nanoparticles, such as gold.[posthuma][martin] This pad is where the labeled molecules for biorecognition are supplied before running the assay [sajid]. It is vital to have a conjugate that can remain stable throughout the entire assay or the results could be inaccurate or unreadable. Depending on the desired level of sensitivity of the assay and the release of the conjugate, the material of this pad is selected; cellulose, polyesters and glass fiber are most commonly used. In addition to the conjugate pad material, the nitrocellulose membrane is a large contributor to the sensitivity of the lateral flow assay. These membranes contain various evenly distributed pore sizes and work as a capturing mechanism for the conjugate-analyte pair. This part also forms visible bands when the analyte is present. These bands are placed further down the device to improve interaction time. Changing the material and pore size influences the capillary flow time, which influences the reaction time of the analyte in binding area. [posthuma] They are used for test and control lines, and they provide effective binding areas and support that are inexpensive and contain high affinity for many biomolecules and proteins. [sajid] The last component of the device is the adsorbent, also known as a waste reservoir, which acts as a sink at the end of the assay. This component captures the excess sample and prevents wicking towards the reaction membrane.[posthuma] It also helps to maintain a continuous flow rate while preventing any backflow within the system. The five main steps to create a lateral flow consist of (i) developing the antibody used to target the analyte (ii) making the label (iii) choosing and labeling the molecules for recognition (iv) preparing all reagents at their appropriate pad and arranging all aspects of the assay (v) applying the sample onto the device. [sajid]
Applications
Pregnancy Tests
Lateral flow assay devices, specifically sandwich assays, are most commonly used for pregnancy tests. Urine is placed in the sample introduction window and then on the sample pad, as shown in Figure 1. When a woman is pregnant, the human chronic gonadotropin (HCG) hormone is present in the urine. Over-the-counter pregnancy tests simply work by detecting HCG, which is made at the beginning of pregnancy. The exposed end of the pregnancy test strip is where the urine is applied. As the fluid travels up the strip, it crosses three separate zones. Antibodies grab onto any HCG as it travels up the strip, with the help of enzymes with the ability to turn on di-molecules. If HCG is in the urine, then it binds to an antibody attached to a gold particle [7]. This then flows to meet another antibody that is also complimentary to HCG and results in the stripe ‘T’. As the urine continues to flow up the strip, the urine picks up all the AB-1 enzymes and carries them to the test zone of the strip. There are more Y shaped antibodies that will also stick to HCG during this process. The reason this form of lateral assay is called sandwich assay is because if HCG is present, then it gets sandwiched between the two different enzymes. The antibody conjugates that don’t bind to HCG flow to a third antibody and results in the stripe ‘C’. A negative test happens when the control line appears alone, and a positive test happens when both the test and control line appear [9].
Environmental Testing
Microfluidic devices such as nucleic acid lateral flow (immuno)assays can test for bacteria or pathogens within the environment. This application works because the analyte in this method is a double stranded nucleic acid sequence specific to the organism using primers with two distinct tags. The analyte is recognized because it binds to a tag-specific antibody. When detecting pathogenic bacteria, the nucleic acid was amplified using two tagged primers. At the test line on the device, a solution was sprayed which contained antibodies raised against the tag. One of these tags will bind to specific nanoparticles, and the other tag will bind to the antitag antibody, which results in a colored signal. This response is proportional to how much analyte there is in the sample [7].
Crop and Animal Health
Pesticides are extensively used in crops in order to keep away insects, diseases and weeds, and they can end up in the bodies of humans and animals which may cause many health issues. There is a lateral flow assay that is useful for simultaneously detecting two common toxins, triazophos and carbofuran, from the pesticides within the water at the crops [4]. This assay uses an immunogold conjugate, and the strip contains two test lines (for each toxin) along with the control line. If these toxins are detected, other studies can be performed to determine the how much is actually present and whether the food could safely be given to humans to consume. In terms of animals, there are many lateral flow test that can detect diseases within animals which can be useful information in terms of the animals health as well as the health of humans interacting with them. Bisphenol A (BPA) has been found to harm the fertility of fishes, reducing their population, so a rapid, one step assay was created to detect this contaminant within the water of oceans and rivers. Humans are now able to determine the reproductive status of most dairy animals using a colloidal gold conjugate to detect the analyte, progesterone, found in their milk. Also, an additive with in the food of livestock, ractopamine, is toxic to humans is now detectable using a lateral flow assay within the urine of pigs [4].
Food Testing
Lateral flow assays can also be used for determining and ensuring the safety of some food products. In addition to testing the water on crop land, food products can be tested for pesticides using gold nanoparticles. A recent lateral flow assay was created for detecting Salmonella at lower concentrations using gold nanoparticles and aptamer, and a nucleic acid lateral flow assay is able to actually quantify the Salmonella within a food sample [4]. Another great use for these assays is to prevent the chance of dealing with food poisoning. Staphylococcol exterotoxin B (SEB) is one of the most common toxins affiliated with food poisoning, and lateral flow tests now are capable of detecting, with high reproducibility, SEB in both food and liquids. Food testing using lateral flow assays is an extremely useful, rapid and easy way to detect a wide range of contaminants that could prevent the body from getting food poisoning, viruses or even diseases [4].
Advantages as a Paper Microfluidic
Lateral flow assay technology is low in cost, simple, and rapid. This specific kind of assay are an attractive technology because it has the potential to give patients an instant diagnosis without any equipment needed. This technology is so useful because it involves only one-step, thus the lateral flow assay technology is a fast process with no previous knowledge or experience needed [5]. The results are qualitative (or semi-qualitative) and entail a very simple test procedure. In these lateral flow assay tests, proteins, haptens, nucleic acids, and amplicons can all be detected. In addition to these tests being rapid and very simple, there is no need to refrigerate them, batches can be prepared ahead of time, and the tests have a long shelf life [5]. Due to long shelf lives and the fact that refrigeration is not necessary, lateral flow assay tests can be used in developing countries [6].
Shortcomings
While these tests are indeed very common and easy to use, lateral flow assay technology is dependent on an accurate sample volume. Small or invalid sample volume results in reduced accuracy. In addition to sample volume, good antibody preparation is required for these tests to confidently work. This technology works very well for fluid samples with no pretreatment needed, but sample pretreatment is indeed a requirement when testing non-fluids [5]; this can be a very time consuming process. Another limitation to lateral flow assays is that the results obtained are generally qualitative which may prevent specific conclusions to be made, and the reproducibility of each test can vary quite significantly. Also most devices are able to detect more than one analyte, so this may also lead to confusion when analyzing the results. [4]
Future Work
The main advances in lateral flow assay technology pertains to improving detection sensitivity in order to obtain results for lower concentrations of the analyte that could be found in saliva or sweat. Various novel reagents have been discovered to reduce the detection limits to a minimum of 0.1 ng/ml. To do this, magnetic particles such as nano-gold microspheres can be used to decrease detection limits [6]. Another aim is to find a way to make the assays to be more reproducible and to improve the data processing and quantification capabilities [4].
References
[1] Yetisen, A. K.; Akram, M. S.; Lowe, C. R. Lab on a Chip 2013, 13 (12), 2210.
[2] Tisone, T. C.; O’Farrell, B. Lateral Flow Immunoassay 2008, 1–26.
[3] Wong, R. C.; Tse, H. Y. Lateral Flow Immunoassay 2008, 1–19.
[4] Sajid, M.; Kawde, A.-N.; Daud, M. Journal of Saudi Chemical Society 2015, 19(6), 689–705.
[5] Koczuła, Katarzyna & Gallotta, Andrea. (2016). Lateral flow assays. 10.1042/EBC20150012.
[6] Koczula, K. M.; Gallotta, A. Essays In Biochemistry 2016, 60 (1), 111–120.
[7] Posthuma-Trumpie, G. A.; Korf, J.; Amerongen, A. V. Analytical and Bioanalytical Chemistry 2008, 393 (2), 569–582.
[8] Introduction to Lateral Flow Rapid Test Diagnostics. nanoComposix. https://nanocomposix.com/pages/introduction-to-lateral-flow-rapid-test-diagnostics#target (accessed Feb 22, 2019).
[9] Smith, S.; Govindasamy, K.; Govender, U.; Land, K. J. South African Journal of Science 2015 111(11/12), 1-10.
