Lateral flow assays (LFAs) are simple to use, disposable diagnostic devices that can test for biomarkers in samples such as saliva, blood, urine, and food. The tests have a number of advantages over other diagnostic technologies including:
Simplicity: The simplicity of using these tests is unmatched – simply add a few drops to the sample port and read your results by eye a few minutes later.
Economic: The tests are inexpensive - typically less than one dollar per test to manufacture at scale.
Robust: The tests can be stored at ambient temperature and have a multi-year shelf life
Billions of test strips are produced each year for the diagnosis of sexually transmitted diseases, mosquito-borne diseases, tuberculosis, hepatitis, pregnancy and fertility testing, cardiac markers, cholesterol/lipid testing, drugs of abuse, veterinary diagnostics, and food safety, amongst others.
A LFA is made up of a sample pad, a conjugate pad, a nitrocellulose strip that contains test and control lines, and a wicking pad. Each component overlaps by at least 1–2 mm which enables unimpeded capillary flow of the sample.
To use the device, a liquid sample such as blood, serum, plasma, urine, saliva, or solubilized solids, is added directly to the sample pad and is wicked through the lateral flow device. The sample pad neutralizes the sample and filters unwanted particulates such as red blood cells. The sample can then flow unimpeded to the conjugate pad that contains strongly colored or fluorescent nanoparticles that have an antibody on their surface. When the liquid reaches the conjugate pad, these dried nanoparticles are released and mix with the sample. If there are any target analytes in the sample that the antibody recognizes, these will bind to the antibody. The analyte-bound nanoparticles then flow through a nitrocellulose membrane and across one or more test lines and a control line. The test line (labeled T in the image below) is the primary read-out of the diagnostic and consists of immobilized proteins that can bind the nanoparticle to generate a signal that is correlated to the presence of the analyte in the sample. The fluid continues to flow across the strip until it reaches the control line. The control line (labelled C in the image below) contains affinity ligands that will bind the nanoparticle conjugate with or without the analyte present in solution to confirm that the assay is working properly. After the control line, the fluid flows into the wicking pad which is needed to absorb all of the sample liquid to ensure that there is consistent flow across the test and control lines. In some tests, a chase buffer is applied to the sample port after sample introduction to ensure that all of the sample is transported across the strip Once all the sample has passed across the test and control lines, the assay is complete and the user can read the results.
The analysis time is dependent on the type of membrane used in the lateral flow assay (large more membranes flow faster but are generally less sensitive) and is typically complete in less than 15 minutes.
Lateral Flow Formats
The two common assay formats are called “sandwich” and “competitive”. The sandwich assay format is typically used for detecting larger analytes that have at least two binding sites, or epitopes. Usually, an antibody to one binding site is conjugated to the nanoparticle, and an antibody to another binding site is used for the assay’s test line. If there is analyte present in the sample, the analyte will bind to both the antibody-nanoparticle conjugate and to the antibody on the test line, yielding a positive signal. The sandwich format results in a signal intensity at the test line that is directly proportional to the amount of analyte present in the sample. Regardless of the quantity of analyte in the sample, an anti-species antibody at the control line will bind the nanoparticle, yielding a strong control line signal that demonstrates that the assay is functioning correctly.
The competitive format is used for detecting analytes when antibody pairs are unavailable or if the analyte is too small for multiple antibody binding events, such as steroids and drugs. In this format, the test line typically contains the analyte molecule, usually a protein-analyte complex, and the conjugate pad contains the detection antibody-nanoparticle conjugate. If the target analyte is present, the analyte will bind to the conjugate and prevent it from binding to the analyte at the test line. If the analyte is not present, the conjugates will bind to the analyte at the test line, yielding a signal. In the competitive format, the signal intensity is inversely proportional to the amount of analyte present in the sample. As in the sandwich format, the control line will bind the nanoparticle conjugate with or without the analyte providing confidence that the assay is working correctly.
Nanoparticles as Reporters in Lateral Flow
Standard lateral flow tests generate an optical signal that arises from strongly colored particles bound to test lines on a white nitrocellulose strip. This signal can be read by eye (qualitative or semi-quantitative) or by an instrument (quantitative). To maximize the sensitivity of the device, each binding event should produce the strongest signal possible. While larger particles will provide a stronger signal per binding event and be easier to see, particles that are too large will not easily flow through the membrane, and will therefore have limited opportunity to bind to the test line. Thus, particles with sizes between 20 nm and 500 nm are typically selected for use lateral flow assay. Fluorescent particles can also be employed, and the same general rules apply: the more potent the fluorescence per binding event, the stronger the signal.
One of the most common reporter particles used in lateral flow assays are 40 nm diameter gold nanoparticles. Gold nanoparticles have unusual optical properties that make them exceptionally strong absorbers of light. 40 nm diameter gold has a peak absorbance at ~520 nm, resulting in a strong ruby red colored test line. The gold surface strongly binds antibodies and other proteins, allowing for the simple fabrication of robust nanoparticle-antibody conjugates. Other sizes and shapes of nanoparticles have also been used as lateral flow probes. Gold nanoshells with a 150 nm diameter provide a higher contrast per binding event and typically provide a 3 – 20 fold increase in sensitivity when compared to 40 nm gold particles. Due to the silica core, gold nanoshells are less dense than a solid gold particle and are able to flow unimpeded through the nitrocellulose membrane. Since the gold nanoshell has the same gold surface as smaller solid gold nanoparticles, only minor protocol modifications are required to switch from solid gold nanospheres to gold nanoshells. At nanoComposix, the BioReady line of gold nanoparticles are specifically designed for fabricating high quality conjugates that can be used for lateral flow.
Lateral Flow Assay Analysis
The results from a lateral flow test can be either qualitative (“yes/no”), semi-quantitative or quantitative. The pregnancy test is an example of a qualitative yes/no assay, where a positive test line signal correlates to elevated levels of the hCG hormone in urine, indicating that the user is pregnant. For quantitative diagnostics, the test line intensities are compared to a calibration standard and converted to an analyte concentration value. To accurately measure the test line intensity, the LFA result must be analyzed by a strip reader. The recent commercialization of small form factor, inexpensive, mobile-centric readers is poised to transform the LFA industry, as simple methods of quantifying the output of LFAs without the use of stand-alone bench top readers opens up a tremendous opportunity for home use of point-of-care diagnostics. The reader shown below is a new product from Lumos Diagnostics where a strip is inserted into a cartridge, the signal intensity on the strip is analyzed with a photodiode and the results are sent back to a mobile phone to display the result. The calibration curve for any given assay is built in to the software for that test, so that the test line readout can be automatically converted to a mass concentration for the analyte, and a final result is presented to the user.