Reporter Nanoparticle Selection for Lateral Flow Immunoassays
Reporter probe selection is one of the most important decisions that needs to be made during the planning of a new lateral flow assay. The reporter choice impacts the achievable sensitivity and specificity, the stability in the sample matrix, the cost of the assay, the development time, and whether or not a reader is required for final signal readout. To achieve the best and most reproducible results, the initial particles must be high quality and well characterized. Most lateral flow assay development companies are more experienced with assay development than particle manufacturing and surface chemistry. At Fortis Life Sciences, our philosophy is that the nanoparticle reporter particle is fundamental to achieving success, emphasizing the importance of using precisely engineered and highly characterized nanoparticles in lateral flow assays.
To help with the selection of nanoparticles for lateral flow assays, we ask the following questions:
If high sensitivity is required, we recommend 150 nm Carboxyl Gold Nanoshells using covalent binding chemistry. The gold nanoshells produce a high contrast blue-grey test line that provides the maximum visual signal, and highest possible sensitivity.
Robust and effective binding of an antibody to the surface of a reporter particle is critical for obtaining the target sensitivity and selectivity of the assay. Passive adsorption is the traditional method for attachment of proteins to lateral flow nanoparticle probes and is still widely used due to quick and easy conjugate preparation. For proteins with free amines available for binding, covalent conjugation can be used to ensure robust, permanent protein attachment to the nanoparticle surface. Covalent conjugates often offer increased stability in challenging sample matrices, over a range of pH conditions, and at high surfactant or detergent concentrations.
If the answer is yes, you may consider using BioReady Streptavidin Gold conjugates for your assay development. Streptavidin surfaces are available on our 40 nm Gold Nanospheres and 150 nm Gold Nanoshells. The particles are prepared by covalently attaching the tetrameric protein to the surface of the functionalized nanoparticles to facilitate excellent retention of biotin-binding activity.
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Passive Adsorption
This is the original method for attachment of proteins to lateral flow nanoparticle probes, and is still widely used. The mechanism of passive adsorption is based on van der Waals and other attractive forces between the antibody and the surface of the particle. These attractive forces between the antibody and the nanoparticle probe are reversible and strongly influenced by both the nanoparticle surface and 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, so a pH titration and a sweep of the antibody to gold ratio should be performed to identify the optimal conditions for antibody adsorption. It is recommended that the pH of the adsorption buffer be slightly above the isoelectric point of the protein, which varies from antibody to antibody. The constant region of the antibody (Fc portion) is generally more hydrophobic and therefore 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 may be required to ensure dense surface binding and high salt stability post conjugation. Please keep in mind that every antibody requires slightly different conditions which must be optimized according to the considerations described above.
Covalent Binding
Covalent attachment is an irreversible chemical reaction that minimizes antibody desorption. Covalent binding may require the use of less antibody during conjugation which may reduce the cost per test strip.
Covalent attachment can be accomplished with several different chemistries. For our BioReady products that are optimized for lateral flow, we typically use amide bonds to connect a carboxylic acid functionalized nanoparticle to free amines on the antibody. This covalent bond is created through an EDC/Sulfo-NHS intermediary, as shown in the image below. For antibodies, lysine residues are the primary target sites for EDC/NHS conjugation. A typical IgG antibody commonly used in lateral flow will have 80–100 lysine residues, of which 30–40 will be accessible for EDC/NHS binding. Proteins such as bovine serum albumin (BSA) have similar numbers of surface-accessible lysine groups. Fortis Life Sciences sells BioReady nanoparticles with carboxylic acid (carboxyl) surfaces, as well as an NHS-activated surfaces to allow for simplified conjugation that eliminates 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.
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. Therefore, all antibodies should be purified and transferred to the appropriate buffer before use in a conjugation reaction. For particles other than gold, passive adsorption may not be an option and covalent chemistry must be used to create the particle/antibody conjugates. For example, dyed latex spheres and europium fluorescent beads are conjugated to antibodies using covalent methods.
BioReady Gold Nanoparticles
Fortis Life Sciences offers a line of BioReady products that is specifically tailored for antibody conjugation. We also provide detailed protocols and technical support for conjugation to each particle type. The following sections list the benefits and trade-offs of the different particle sizes, shapes, and surfaces.
BioReady Carboxyl Gold Nanoshells (150 nm) for increased sensitivity
At Fortis Life Sciences we fabricate hundreds of different sizes and shapes of metal nanoparticles that strongly interact with light due to their plasmon resonance. While 40 nm gold has historically been the nanoparticle of choice for lateral flow assays, gold nanoshells, another type of plasmonic nanoparticle, can dramatically increase the sensitivity of lateral flow assays because each particle is 30x more strongly colored than the 40 nm gold. Due to the dramatic increase in color, fewer binding events are required in order to see a result at the test line in a lateral flow assay. The gold nanoshells consist of a 120 nm silica core surrounded by a thin 15 nm shell of gold. The gold nanoshells have a much larger diameter than 40 nm gold nanoparticles but flow unimpeded through the nitrocellulose membrane because of the low-density silica core. The gold nanoshells have the same gold surface as traditional 40 nm spherical gold nanoparticles, so only minor modifications to existing 40 nm gold protocols are required. For increased stability at the larger particle size, covalent binding chemistry is used to link antibodies to the surface of nanoshells.
Advantages
- Whole antibodies, antibody fragments, and small molecules can be irreversibly bound via a stable amide bond.
- May require up to 65% less antibody as compared to passive adsorption.
- Improved control over antibody/particle loading.
- Up to 20-fold increase in lateral flow sensitivity.
- NHS and carboxyl-based covalent linkage chemistry available.
- May offer increased stability in challenging sample matrices, over a range of pH conditions, and at high surfactant or detergent concentrations.
Drawbacks
- Requires optimization when switching from 40 nm gold nanoparticles.
- Nanoshell extinction is much greater than a gold nanoparticle, thus there are fewer particles per OD when purchased in solution. When optimized, a higher OD of particles may be necessary on each strip in order to maximize sensitivity.
BioReady Carboxyl Gold (40 nm or 80 nm)
Fortis Life Science’s BioReady 40 nm and 80 nm carboxyl (-COOH) gold is an effective and economical nanoparticle for covalent conjugations to proteins through carbodiimide crosslinker chemistry. Covalent coupling of proteins (e.g. antibodies) to a gold nanoparticle surface yields robust and stable gold particle conjugates. The nanoparticles are surface functionalized with a tightly bound monolayer that contains terminal carboxylic acid functional groups which can be activated through EDC/Sulfo-NHS chemistry to generate gold nanoparticle-antibody amide bonds. The 80nm variant is more sensitive due to the larger size and increased surface area available for binding.
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Advantages
- Whole antibodies, antibody fragments, and small molecules can be irreversibly bound via a stable amide bond.
- May require up to 65% less antibody as compared to passive adsorption.
- Improved control over antibody/particle loading.
- May offer increased stability in challenging sample matrices, over a range of pH conditions, and at high surfactant or detergent concentrations.
- Contains more particles per mL than gold nanoshells which may lead to reduced costs in optimized assays.
Drawbacks
- Less sensitivity than gold nanoshells.
BioReady NHS Gold (40 nm)
Fortis Life Sciences BioReady 40 nm NHS gold can be covalently conjugated to primary amines (-NH2) of proteins in a simplified procedure compared to the carboxyl surface. These nanoparticles are surface functionalized with an active NHS ester to generate gold nanoparticle-antibody amide bonds, eliminating the need for the user to perform the intermediary EDC/Sulfo-NHS chemistry steps. The particles are supplied as a lyophilized powder that can be resuspended with a buffer to covalently bind to an added antibody. This coupling reaction is rapid, simple, robust, and requires little optimization.
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Advantages
- Fast: stable gold conjugates in as little as 15 minutes hands-on-time.
- Convenient: rapidly screen multiple antibodies for assay development without having to perform pH or salt optimizations for each antibody.
- Economical: reduced antibody loading and minimal pH optimization required.
Drawbacks
- NHS gold solution must be used immediately upon resuspension.
- At larger scales, it is much more cost effective to perform EDC/NHS chemistry with nanoparticles having carboxylic acid surfaces.
- Performing EDC/NHS chemistry immediately before antibody binding may increase assay sensitivity (inherent half-life of the NHS ester intermediate may reduce binding efficiency).
BioReady Bare Gold (40 nm or 80 nm)
Our BioReady 40 nm gold nanoparticles have a "bare" particle surface with only a weakly associated citrate molecule to stabilize the particle, and can have proteins attached through passive adsorption (also referred to as physisorption). The most common buffer for bare nanoparticles is trisodium citrate, which is used as a reductant in many gold nanoparticle fabrication methods and provides a balance between stability during particle formation and displaceability when making particle conjugates. Each of the three carboxylic acids weakly bind to the particle surface but are readily displaced in the presence of a protein.
Both the 40 nm and 80 nm bare gold nanoparticles can be used for passive adsorption to proteins. The mechanism of adsorption is based on van der Waals interactions between the proteins (e.g. antibodies) and the surface of the particles. The resulting forces between the antibody and the nanoparticle are influenced by the coupling environment. The BioReady 40 nm citrate gold is provided at an OD of 20 at pH 6.5-7. A pH titration should be performed to optimize the conjugation efficacy.
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Advantages
- Traditional method of conjugate preparation.
- Minimal chemistry involved.
- Highly reproducible nanoparticle synthesis method with low batch-to-batch variance which is critical for semi-quantitative and quantitative assay development.
- Low cost.
Drawbacks
- pH sweep required for adsorption optimization.
- Whole antibodies or thiolated ligands required.
- Proteins are not covalently attached to particle surface.
- Binding mechanism is antibody dependent.
Other Probes for Lateral Flow
There are many other probes used in lateral flow assays besides gold. Dyed polystyrene particles (typically 200 nm or greater in size) and cellulose beads can be used for increasing visible signatures on strips. Cellulose beads (e.g. Asahi Kasei Fibers Corporation) have large diameters and work well for certain systems. For higher sensitivity, fluorescent probes may perform better than 40 nm gold, though a specialized fluorescent reader is required to analyze and quantify the result. Europium beads and up-converting nanoparticles are two fluorescent particles that are commonly used in fluorescent LFA assays. One common challenge with these particles is significant variation in the number of carboxyl ligands on the surface available for binding between different lots.
Selection of the nanoparticle probe will be based on the type of assay, sensitivity requirements, price-point requirements, and the available reader technology. Particle selection is a particularly important decision because many of the subsequent steps in the lateral flow development process will require optimization that is dependent on which nanoparticle is being used. For help determining which probe is best suited for your application, please contact us.
Nanoparticle Selection FAQs
BioReady Bare Gold (40 nm) is a direct drop-in replacement for many assays that use gold from another source or currently make their own. The BioReady Bare Citrate is a direct replacement for most gold colloid recipes. Provided at 20 OD, it can be diluted with a low molarity buffer or pH adjusted at 20 OD for reduced volume and more efficient binding kinetics, often resulting in superior conjugate performance compared to conjugates prepared at lower OD.
BioReady Citrate or Carboxyl Gold will be the most cost-effective options, depending whether you'd like to use passive or covalent conjugation.
80 nm gold nanoparticles or 150 nm gold nanoshells can provide increases in sensitivity compared to 40 nm gold nanoparticles. The highest sensitivities are achieved with 150 nm gold nanoshells. One important consideration for using 80 nm gold or the gold nanoshells is that because the per particle absorption is higher, the number of particles at a given OD is lower than the 40 nm colloids. This means that a larger volume or higher concentration of nanoshells may be necessary in a fully optimized assay (typically 2–5 times the volume or OD required for gold nanoshells, compared to the 40 nm gold).
At Fortis Life Sciences we use a wide array of ultrasensitive nanoparticle probes including quantum dot composites, magnetic nanoshells, and nucleic acid amplification to increase sensitivity limits beyond traditional visual probes.
Contact us with your challenging sensitivity issues!
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