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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 to measure the signal.  Most lateral flow assay development companies are more experienced with the assay development than particle manufacturing and surface chemistry.  At nanoComposix, we have a philosophy that the nanoparticle reporter particle is fundamental to achieving success and emphasize the importance of using precisely engineered and highly characterized nanoparticles in lateral flow assays. 

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 attractions between the antibody and the surface of the particle.  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. A pH titration and a sweep of the antibody to gold ratio can be performed to identify conditions where antibody adsorption 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.  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 is typically used in order to ensure dense surface binding and high salt stability post conjugation.  There are two main drawbacks to passive adsorption. Firstly, every antibody requires slightly different conditions. Secondly, some antibodies may detach from the nanoparticle surface which can lead to a decrease in sensitivity and variable results. 

Covalent Binding

Increasingly, LFA developers are covalently binding antibodies to the surface of nanoparticle probes.  Covalent attachment is more stable with less antibody desorption and requires fewer antibodies during conjugation which reduces 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 achieved through an EDC/Sulfo-NHS intermediary generated from a carboxylic acid surfaced 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 carboxylic acid surfaces, as well as an NHS activated surface 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. 

Sulfo-NHS plus EDC (carbodiimide) crosslinking reaction scheme Thermo Fisher

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. It is important to purify and transfer all antibodies to the appropriate buffer before use in a conjugation reaction.  For particles other than gold, passive absorption may not be an option and covalent chemistry must be used to create the particle/antibody conjugates.  Dyed latex spheres and europium fluorescent beads are conjugated to antibodies using covalent methods. 

Reporter Nanoparticle Selection

To help with the selection of nanoparticles for lateral flow assays, we recommend asking the following questions. 

Does my assay require high sensitivity? 

If high sensitivity is required, we recommend 150 nm gold nanoshells using covalent binding chemistry.  The gold nanoshells produce a high contrast green-grey test line that provides the maximum visually read signal. 

Do I just need the advantages of covalent binding?

For users who want the increased stability, the reduced antibody costs, the higher sample matrix stability, and the more reproducible conjugates that can be obtained with covalent binding chemistry, 40 nm and 80 nm diameter carboxyl BioReady gold is recommended.  The 80 nm gold typically provides a higher sensitivity while the 40 nm gold results in assays with the lowest cost. 

Do I want a simple conjugation procedure or to just replace my existing gold nanoparticles?

Our 40 nm and 80 nm gold is available with both citrate and carbonate surfaces .  The carbonate surfaces is our “barest” gold with a surface that allows the highest density of antibodies when using passive adsorption. 

BioReady Gold Nanoparticles

NanoComposix has a line of BioReady products that is specifically tailored for antibody conjugation.  We offer 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 150 NM CARBOXYL GOLD NANOSHELLS FOR INCREASED SENSITIVITY

At nanoComposix 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 compared to 40 nm gold. Because of the dramatic increase in color displayed by the 150 nm gold nanoshells versus the 40 nm gold particles, 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.  Due to 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
  • Up to 65% less antibody is required than for passive adsorption
  • Stable and irreversible amide bond formed
  • Improved control over antibody/particle loading (difficult to accomplish in passive adsorption because of colloidal stability challenges)
  • Up to 20-fold increase in lateral flow sensitivity
  • NHS and COOH based covalent linkage chemistry available
  • More stable compared to passive conjugates in a challenging sample matrices and stable in a wider range of pH and high surfactant and detergent loading

Drawbacks:

  • Requires additional optimization when switching from 40 nm gold nanoparticles
  • Since the extinction of a nanoshell is much larger than a gold nanoparticle, 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 40 NM AND 80 NM CARBOXYL GOLD

NanoComposix 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.

Advantages:

  • Whole antibodies, antibody fragments, and small molecules can be irreversibly bound
  • Up to 65% less antibody is required than for passive adsorption
  • Stable and irreversible amide bond formed
  • Improved control over antibody/particle loading (difficult to accomplish in passive adsorption because of colloidal stability challenges
  • More stable compared to passive conjugates in a challenging sample matrices and stable in a wider range of pH and high surfactant and detergent loading

Drawbacks:

  • Requires additional optimization when switching from 40 nm gold nanoparticles
  • Not as sensitive as gold nanoshells

BIOREADY 40 NM AND 80 NM NHS GOLD

NanoComposix BioReady 40 nm NHS  gold can be covalently conjugated to primary amines (-NH2) of proteins in a simplified procedure. Covalent coupling of proteins (e.g. antibodies) to a gold nanoparticle surface yields robust and reliable gold particle conjugates. The BioReady 40 nm NHS gold 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. 

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 and performing EDC/NHS chemistry immediately before antibody binding may increase assay sensitivity.
  • Moderately lower binding efficiency compared to carboxylic acid surface due to the inherent half-life of the NHS ester intermediate

BIOREADY 40 NM OR 80 NM BARE GOLD 

Our BioReady 40 nm bare gold nanoparticles have a “naked” particle surface with only a weakly associated carbonate or citrate molecule to stabilize the particle, and can be conjugated to proteins 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.  We also offer the “naked” surface in a carbonate buffer, which is a smaller and less complex molecule with a lower affinity to the gold nanoparticle surface than citrate.  The greater displaceability of the carbonate molecules may produce better performing conjugates. 

 Both of the 40 nm and 80 nm bare gold nanoparticles with either the carbonate or citrate surface 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 carbonate gold is provided at an optical density (OD) of 20 at pH 5.5-6, and the 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.

Advantages:

  • Traditional method of conjugate preparation
  • Very little 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 and can desorb
  • Risk of aggregation if conditions are not optimized
  • Binding mechanism is antibody dependent
  • Need to carry out multiple trial conjugations

Other Probes for Lateral Flow

There are many other probes used in lateral flow assays that are not 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 but can also have stability issues in certain matrices and be challenging to optimize.  For higher sensitivity, fluorescent probes can be used and can provide 10 – 100X increases in sensitivity over 40 nm gold.  However, there often no characterization performed on the surface chemistry, and there is significant variation in the number of carboxyl ligands on the surface available for binding.  Fluorescent probes also require a specialized fluorescent reader to analyze and quantify the result.  Europium beads and up-converting nanoparticles are two fluorescent particles that are commonly used in fluorescent LFA assays.    

Selection of the nanoparticle probe will be based on the type of assay, sensitivity requirements, price-point requirements, and the available reader technology. This is an important decision since many of the subsequent steps in the lateral flow development process will require optimization that is dependent on which nanoparticle was used as a probe.  For help determining which probe is best suited for your application, please contact us at info@nanocomposix.com

Nanoparticle Selection FAQs

I have an existing LFA that works with 40 nm gold and I need another gold source or a moderate increase in sensitivity without requiring additional regulatory approvals. 

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, while the BioReady Bare Carbonate has superior binding characteristics without requiring a change to regulatory filings.  Both the citrate and carbonate 40 nm gold are offered at 20 OD, which 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. 

I have a LFA where there the target analyte range is easily achievable with lateral flow (> 1 ng/mL) assays and where quantification is not required.

The most cost-effective option is to use BioReady Bare 40 nm.  For passive absorption we recommend BioReady Bare Carbonate due to its superior absorption characteristics compared to the citrate version. Carboxyl 40 nm gold is also an option as the conjugates will have  increased stability from covalent binding and reduced antibody costs.    

I can’t achieve the required sensitivity with 40 nm gold and I’m looking for an increase in signal strength. 

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 since the per particle absorption is higher, the number of particles 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 40 nm gold). 

I still can’t achieve the required sensitivity I need, even with the gold nanoshells. 

At nanoComposix 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!