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Lateral Flow Introduction

Figure 1: A commercial pregnancy test which uses 40 nm gold nanospheres as the detection label. The faint red line indicates that the subject is pregnant. The darker red line indicates that the test was valid.

Lateral flow assays (LFAs) are rapid and inexpensive diagnostic tests with long shelf lives that don’t require refrigerated storage. Because of their low cost, billions of strips are produced each year to test for a variety of different conditions and diseases. In the most common configuration, a colored line at the test location indicates a positive test (Figure 1). A second line at the control location indicates that the test was valid.

The lateral flow test is assembled from a few key components:

In most embodiments, the LFA’s components are held together on a plastic adhesive backing card with a carefully controlled overlap, allowing for unimpeded capillary flow of the sample from the sample pad, through the conjugate pad, nitrocellulose and into the wick. Often the strip components are housed inside of a plastic cassette so that only sample pad and test and control lines are visible. A few drops of fluid are applied to the sample pad, and the presence or absence of a test line after a short amount of time (2–15 minutes) indicates the presence or absence of the target analyte (Figure 1). One of the main advantages of this assay format is that a result can be obtained without additional processing of the sample or external equipment, and with minimal training. This simplicity is especially important for point of care or field based diagnostics.

At nanoComposix, we are interested in lateral flow because the colored particles used to indicate whether a test is positive or negative are nanoparticles. The ruby red colored line in many lateral flow strips arises from binding of 40 nm spherical gold nanoparticles via a conjugated antibody. The ruby red color stems from an optical plasmon resonance of the 40 nm gold nanoparticles that strongly absorb green and blue light. While 40 nm gold nanoparticles have found widespread use, many other types of plasmonic particles can be fabricated, each with their own unique optical properties. NanoComposix specializes in the production of a wide variety of plasmonic metal particles with tailored optical signatures that are a function of the size, shape, and material of the nanoparticle. One plasmonic nanoparticle of interest is 150 nm diameter gold nanoshells that consist of a 120 nm silica core coated with a 15 nm thick gold shell. Gold nanoshells have demonstrated dramatic increases in the sensitivity of lateral flow assays when compared to 40 nm gold spheres.

Figure 2: Lateral flow assay with half log dilutions of analyte showing the increase in sensitivity when using gold nanoshells (blue) as the probe over 40 nm spherical gold (red).

Our goal is to help others utilize these new probes and our covalent binding techniques to create next generation lateral flow tests with increased sensitivity and reproducibility. These ultra-sensitive diagnostics can be quantified with inexpensive digital readers (e.g. cell phones); we believe that this combination has the potential to revolutionize the point of care diagnostics industry.

Lateral Flow Components

A finished lateral flow assay contains a sample pad, conjugate pad, nitrocellulose membrane, and a wick/absorbent pad that are all applied to an adhesive backing card. Each component overlaps with the next by 1–2 mm so that the sample can move through the test strip via capillary action.

Figure 3: Schematic of a generic lateral flow test. The sample pad absorbs the sample and transport the sample to the conjugate pad. The conjugate pad contains the dried down antibody-nanoparticle conjugate. The nitrocellulose membrane has test and control lines that show the assay results. The wick pad continues to pull the sample through the strip at an even rate. All components are assembled onto a backing card.

At the core of a lateral flow assay are the “conjugates”, also called detector nanoparticles or probes. These are brightly colored nanoparticles connected to antibodies that recognize the target test compound analyte (e.g. the hormone hCG in the case of a pregnancy test). The readout of the assay occurs on a nitrocellulose strip that has two lines striped on the surface: a capture line, which contains an immobilized protein that either binds to the target analyte or competes with the target analyte for binding, and a control line that contains an affinity ligand for the surface of the particle regardless of the presence or absence of analyte, confirming that the assay is working correctly (Figure 3). The sample to be analyzed (blood, serum, plasma, urine, saliva, or solubilized solids) is added to the sample pad and is drawn through the lateral flow device by capillary action. The sample pad can filter unwanted portions of the sample (such as red blood cells or solid particulates) and neutralize the sample. The liquid wicks to the conjugate pad which contains the dried nanoparticle conjugates. The conjugate is rapidly solubilized on contact with the aqueous sample and can bind to the analyte of interest (if present). The nanoparticles and sample continue to flow through the nitrocellulose membrane until they reach the test line and control line. Binding events to the test line provide a visual indication of whether the analyte was detected or not.

Lateral Flow Immunoassay Formats

Lateral flow assays (LFA) can detect a wide range of targets. One of the first steps in the design of a lateral flow assay is to understand which LFA format is right for the target analyte. The two common formats are “sandwich” and “competitive” which are described below:

Lateral Flow Sandwich Format

The sandwich assay format is typically employed for detecting relatively large analytes. If the analyte has at least two distinct binding sites, a “sandwich” assay can be developed where an antibody to one binding site is conjugated to the nanoparticle and an antibody to another binding site is used for the test line. If the analyte is present in the sample, the analyte will become the “meat” of the sandwich binding the nanoparticle conjugate to the test line, yielding a positive signal. The sandwich format results in a signal intensity that is proportional to the amount of analyte present in the sample.

Lateral Flow Competitive Format

A competitive format is used for detecting analytes where the analyte is too small for two antibodies to bind simultaneously, such as steroids and drugs of abuse. In a competitive assay, the test line of the LFA contains the analyte molecule (usually a protein-analyte complex). The nanoparticles are conjugated to an antibody that recognizes the analyte. If the analyte is not present in the sample, the nanoparticle antibody conjugates will bind to the analyte at the test line, yielding high signal intensity. If the target analyte is present in the sample, the analyte will bind to the antibodies on the nanoparticle surface and prevent the nanoparticle from binding to the test line. This will reduce the signal at the test line resulting in a signal intensity that is inversely proportional to the amount of analyte present in the sample.

Both assay formats utilize a second “control” line immobilized on the nitrocellulose membrane to allow the user to verify that the assay has not been compromised and the result can be trusted. The control line is typically a species-specific anti-IgG, and will bind to the antibody that is conjugated to the nanoparticle probe. For both assay types the control line should be clearly visible regardless of the presence or absence of analyte in the sample. The lack of a control line indicates problems with the test itself and therefore invalidates the results.

Nanoparticles for Lateral Flow

nanoComposix has extensive expertise in the synthesis, characterization and surface modification of nanoparticles. We have been making highly engineered inorganic particles for more than ten years and have developed particles specifically engineered for both their optical properties and the conjugation to affinity ligands such as antibodies.

The methods and techniques used to conjugate antibodies to the surface of nanoparticles are critical for optimizing the performance of lateral flow assay tests. For gold nanoparticles, antibodies can either be physisorbed to the surface, referred to as passive adsorption, or they can be covalently coupled. In both passive and covalent coupling reactions, the purity, affinity, and cross-reactivity of an antibody or other ligand is important for developing sensitive and specific tests. Typically we purify all antibodies before use in a coupling reaction.

It is important to note that the guidance provided here is specific to conjugation procedures for binding antibodies to gold nanoparticles. While antibodies are the most common affinity ligand used in lateral flow tests other molecules can also be attached to nanoparticles, such as small peptides and other proteins (BSA, streptavidin, etc.).

Passive Adsorption

This is the original method for attachment of proteins to lateral flow nanoparticle probes and is still widely used. The mechanism of adsorption is based on Van der Waals and other attractions between the targeting ligand and the surface of the particles. The resulting forces between the antibody and the nanoparticle probe are influenced both by the nanoparticle surface and by the coupling environment. In the case of less hydrophobic antibodies or a more hydrophilic surface (i.e. –COOH modified), attachment by both ionic interactions and hydrophobic interactions can occur. Small changes in pH can alter the association dynamics and affect the efficiency of conjugation. Typically, a pH titration and an antibody loading sweep are performed to identify conditions where antibody absorption is optimal. It is recommended that the pH of the adsorption buffer is slightly above the isoelectric point of the protein (which varies from antibody to antibody). When working with antibodies, the Fc portion is generally more hydrophobic and more likely to be adsorbed as compared to the Fab portion offering some control over binding orientation. A large excess of antibody with respect to nanoparticle surface area is typically used in order to ensure dense surface binding and high salt stability post conjugation. There are two main drawbacks to the passive adsorption. Firstly, every antibody requires slightly different conditions and secondly there is a certain amount of lability of physisorbed antibodies allowing for some antibodies to be released from the nanoparticle surface which can lead to a decrease in sensitivity and variable results.

Covalent Coupling

Increasingly, LFA developers are covalently binding antibodies to the surface of nanoparticle lateral flow probes. Covalent attachment is more stable with less antibody desorption and requires fewer antibodies during conjugation. Covalent attachment can be accomplished with several different chemistries. For our BioReady products that are optimized for lateral flow, we typically utilize amide bonds to connect a carboxylic acid functionalized nanoparticle to free amines on the antibody. This covalent bond is achieved through an EDC/Sulfo-NHS intermediary (see Figure 5) generated from a carboxylic acid surface particle. For antibodies, lysine residues are the primary target sites for EDC/NHS conjugation. A typical IgG antibody will have 80–100 lysine residues of which 30–40 will be accessible for EDC/NHS binding. Proteins such as bovine serum albumin have similar numbers of surface accessible lysine groups. NanoComposix sells BioReady nanoparticles with both carboxylic acid surfaces and also an NHS activated surface to allow for simplified conjugation eliminating the need for the user to perform the intermediary EDC/NHS chemistry steps. In addition to its use in lateral flow, the same particle surface chemistry can be used to bind many other amine containing targeting ligands to the particle surface.

Reader Technology

For many lateral flow diagnostics, such as those that detect pregnancy, a simple “yes” or “no” is all that is necessary. For other lateral flow assays, the strength of the test line can provide “semi-quantitative” results, where the result is reported as falling within bins of a particular range (e.g., low, medium or high) or “quantitative” results, where a number that correlates to the concentration of the analyte is reported. Quantitative lateral flow assays require more stringent fabrication conditions and in most cases, a digital reader. Recent advancements in the development of inexpensive readers based on cell phone technologies have recently become available (see Figure 4) and provide a simple and inexpensive solution for quantifying lateral flow assay results.

A i-calQ smartphone reader with strip holder attachment.B Novarum hardware-free reader.