Lateral Flow PDF
Lateral Flow PDF
Lateral Flow PDF
DOI: 10.1042/EBC20150012
Lateral flow assays (LFAs) are the technology behind low-cost, simple, rapid and portable
detection devices popular in biomedicine, agriculture, food and environmental sciences.
This review presents an overview of the principle of the method and the critical compo-
nents of the assay, focusing on lateral flow immunoassays. This type of assay has recently
attracted considerable interest because of its potential to provide instantaneous diagnosis
directly to patients. The range and interpretation of results and parameters used for evalu-
ation of the assay will also be discussed. The main advantages and disadvantages of LFAs
will be summarized and relevant future improvements to testing devices and strategies will
be proposed. Finally, the major recent advances and future diagnostic applications in the
LFA field will be explored.
Introduction
The lateral flow assay (LFA) is a paper-based platform for the detection and quantification of analytes
in complex mixtures, where the sample is placed on a test device and the results are displayed within
5–30 min. Low development costs and ease of production of LFAs have resulted in the expansion of its
applications to multiple fields in which rapid tests are required. LFA-based tests are widely used in hos-
pitals, physician’s offices and clinical laboratories for the qualitative and quantitative detection of specific
antigens [1] and antibodies [2], as well as products of gene amplification [3,4]. A variety of biological sam-
ples can be tested using LFAs, including urine [5], saliva [6], sweat [7,8], serum [9], plasma [10], whole
blood [10,11] and other fluids. Further industries in which LFA-based tests are employed include vet-
erinary medicine [12], quality control [13], product safety in food production [14], and environmental
health and safety [15]. In these areas of utilization, rapid tests are used to screen for animal diseases [16],
pathogens [17,18], chemicals [19], toxins [20] and water pollutants [21,22], among others.
In recent years there has been an increasing demand for point-of-care multiple diagnostic assays with
multiple test lines allowing the rapid and simultaneous detection of multiple analytes present in samples.
Such assays (potentially a single LFA) should be easy to perform without the use of laboratory investi-
gation, or individuals trained in chemical analysis. LFAs are very good candidates as they are cheap to
produce, easy to use and, importantly, widely accepted by users and regulatory authorities. As the path-
way for the development and introduction of novel technologies to the clinical diagnostics market requires
hundreds of millions of dollars and decades of work, the improvement and further development of already
established LFA technologies is a favourable alternative. This process has the potential to produce devices
that may become powerful tools for new challenging applications such as early cancer detection. More-
over, because of the long shelf life and the fact that refrigeration is not required for their storage, LFA
are very well adapted for use in developing countries, small ambulatory care settings, remote regions and
battlefields.
Depending on the elements of recognition used, LFAs can be categorized into different types (Figure 1).
This review focuses on ‘lateral flow immunoassays’ (LFIAs), in which antibodies are exclusively used as
Version of Record published recognition elements. Nucleic acid LFA are used for the detection of amplicons which can be formed
30 June 2016 during the polymerase chain reaction (PCR) [23].
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with the coloured conjugate. Therefore, a positive result is indicated by the lack of signal in the test line, while the
control line should be visible independently of the test result.
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Antibody
Although the physical components of the test strip, construction techniques and buffers play the major role in opti-
mizing the test, the heart of these processes are the antibodies, which need to be carefully designed and highly purified.
It is very important to ensure a consistent antibody supply with proven affinity and specificity. Use of monoclonal
antibodies (mostly derived from mouse hybridomas) is preferable, as it allows the production of specific antibodies
in large quantities.
Label
The most important requirements of the nanoparticle label include:
• colloidal stability in solution under various conditions and temperatures
• susceptibility for detection over a large (and useful) dynamic range
• efficiency and reproducibility of conjugation (without the loss of chemical and biological integrity and activity)
• lack of or very low non-specific binding characteristics (ensuring a high signal-to-noise ratio)
• commercial availability at low cost
• easy and scalable conjugation procedure.
Nowadays colloidal gold is the most widely used label in commercial LFIA. Although it can be prepared in the
laboratory at low cost, there are many commercial sources available. It has an intense colour and no development
process is needed for visualization. Moreover, it has high stability in both liquid and dried forms. Another popular
label is latex, which can be tagged with a variety of detector reagents such as coloured or fluorescent dyes, and magnetic
or paramagnetic components. As latex can be produced in multiple colours, it has an application in multiplex assays,
which require discrimination between numerous lines. Carbon and fluorescent labels, or enzymatic modification
[32,33] of the labels, are also used to improve the sensitivity of the assay. Carbon nanotubes have been shown to
exhibit a limit of detection that is 10-fold lower than that of gold [34]. Fluorescent nanoparticles such as quantum
dots may result in a high background noise which has been shown to be overcome by polymer encapsulation and
surface blocking [35].
Membrane
The membrane is considered the most critical element in LFA strips and nitrocellulose is by far the most commonly
used material. Moreover, there are also ‘pillar-based’ capillary LFA devices used for deoxyribonucleic acid (DNA)
hybridization detection (where micropillar arrays replace the membrane), which have the advantage of more precise
control of the capillary flow [36]. Important parameters characterizing a good membrane material are the capillary
forces, as well as the ease of binding and immobilizing proteins necessary for subsequent selection, reaction and
detection. A range of nitrocellulose pore sizes are available, from 0.05 to 12 μm. However, as the pores are not equally
distributed (because of the manufacturing process), capillary flow time is a more accurate parameter and it should
be used when selecting the most effective strip material. The capillary flow time is the time required for the liquid to
travel to and completely fill the strip of the membrane.
Sample pad
The sample pad can have multiple roles, the most important of which is to evenly distribute the sample and to direct it
to the conjugate pad. The sample pad is usually impregnated with buffer salts, proteins, surfactants and other liquids
to control the flow rate of the sample and to make it suitable for the interaction with the detection system. Moreover,
the pores of the sample pad can act as a filter in order to remove redundant materials, e.g. red blood cells (Figure 4).
Conjugate pad
The main role of the conjugate pad is to hold the detector particles and keep them functionally stable until the test is
performed. This is ensured by the composition of the conjugate buffer, containing carbohydrates (such as sucrose),
which serve as a preservative and a resolubilization agent. When the conjugate particles are dried in the presence of
sugar, the sugar molecules form a layer around them stabilizing their biological structures [37]. When the sample
enters the conjugate pad, the sugar molecules rapidly dissolve carrying the particles into the fluid stream. It is crucial
that the release is consistent between individual test strips.
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Figure 4. An example of an integrated sample collection device combined with a cassette and strip reader
The device is designed for easy collection of a whole blood sample and running of a quantitative test strip. Red blood cells are mechanically
separated on the sample pad. (Pictures adapted from http://www.dcndx.com and https://www.youtube.com/watch?v=FvIIozN58gw).
Absorbent pad
The role of the absorbent pad is to wick the fluid through the membrane and to collect the processed liquid. The
absorbent pad allows the use of larger sample volumes, which results in increased test sensitivity. The most popular
absorbent pads are made of cellulose filters.
Detection methods
Since the LFIA is an antibody-based technique, specificity and sensitivity may be affected by other chemicals with
similar structures, leading to false positive results. The sensitivity of assays is limited by the K d (dissociation constant)
of the antibody–antigen conjugate and by the colorimetric read-out. In order to overcome these limitations, both read-
ers and novel biochemical techniques have been developed to improve product quality and customer convenience.
The selection of a detection system is mainly determined by the label employed in the analysis. Fluorescent dyes or
paramagnetic particles cannot be detected directly by the naked eye and require dedicated readers for quantitative
analysis (Table 1). Moreover, automated detection methods provide advantages over manual imaging and processing
in terms of time consumption, interpretation of results and adjustment of variables.
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Table 1. The most commonly used detection methods employed in lateral flow assays
Examples of applications of these systems can be found in optical readers [45,46], camera readers [47], ladder bars [26],
fluorescent readers [48], chemiluminescent readers [49] and electrochemical readers [50]. Examples of labels include:
fluorescent [51,52], paramagnetic [47,53], enzyme [54,55] and carbon nanoparticles [34].
of LFAs, especially with respect to quantitative results. Data can be digitized using scanners or cameras with dedi-
cated software, which will also allow the documentation of results. However, technological improvements will affect
the cost of apparatus and the duration of analysis.
Discussion
In order to address the criteria demanded by next-generation diagnostic markets, some of the fundamental fea-
tures of LFAs must be improved. First, assays need to be more reproducible and sensitive, easier to manufacture
and operate, and most importantly from a clinical point of view, they should provide relevant results that correlate
with other laboratory-based diagnostic systems. Automation of the manufacturing process and sample application,
as well as improved read-out and data processing, are required to achieve these aims. Moreover, material science
should be applied to bring novel more appropriate custom-designed materials into use, as well as the introduction of
new labelling and reading technologies. The use of new labels such as quantum dots and the upconverting phos-
phors (microscopic ceramic powders that convert infrared light wavelengths into visible coloured light) will im-
prove sensitivity, allowing the use of samples with lower concentrations of the analyte such as sweat or salvia. In
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the Western world, integration of LFA into a lab-on-a-chip design may bring additional advantages, but will also in-
crease costs. For the non-laboratory-based applications, the LFAs should remain simple and affordable; however,
good recognition elements must be available and visual qualitative (on/off) or semi-quantitative results must be
sufficient.
Conclusion
The unique and remarkable properties of LFAs have contributed to the detection of disease biomarkers and infectious
agents in medicine, agriculture, food and environmental safety. Although the principle of the method has remained
unchanged for decades, there have been continuous improvements of LFA techniques leading to increased sensi-
tivity and reproducibility, and the simultaneous detection of several analytes. Importantly, these assays can now be
effectively performed outside the laboratory, providing great advantages for use in developing countries and at the
point-of-care, whether in the field or in more traditional clinical settings.
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Summary
• Lateral flow assays (LFAs) can be used for the detection of proteins, haptens, nucleic acids and amplicons.
• LFAs are well established as a valuable tool in medical, veterinary, food, agricultural and environmental
settings and for use in industrial diagnostics.
• The principle of an LFA is based on the movement of a liquid sample though a polymeric strip with attached
molecules that interact with the analyte, providing a signal that can be visually detected.
• Although the concept behind the LFA is simple, the device has a complex architecture and many critical
elements need to be considered during instrumental design.
• The most critical elements of the assay are the antibodies and the membrane, but attention should be paid
to all of the materials used to ensure the compatibility and consistency of the product.
• An LFA is a fast, low cost, portable and easy-to-use assay; however, the results are mostly qualitative (on/off)
or semi-quantitative.
• An LFA is usually used for initial screening, which can be confirmed later by a fully quantitative method.
• LFA devices can be evaluated using parameters such as sensitivity, specificity and efficiency.
• Recent advances and future goals for improving LFAs are focused on identifying new signal amplification
strategies, nanoparticle labels and quantification systems, as well as improving simultaneous detection.
Abbreviations
GNP, gold nanoparticle; hCG, human chorionic gonadotropin; LFA, lateral flow assay; LFIA, lateral flow immunoassay.
Funding
We acknowledge funding from the European Commission Framework Programme 7 through the Marie Curie Initial Training Net-
work PROSENSE [grant number 317420, 2012–2016].
Competing Interests
The Authors declare that there are no competing interests associated with the manuscript.
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