Conjugation (coupling) Techniques for Lateral Flow Assays

By Robert Hudak, Co-founder & COO, Artemis Dx

Lateral Flow Assays (LFAs) are widely used diagnostic tests, due to their simplicity, rapid results, and low cost. They are immunoassays that rely on the interaction between biomolecules and present their results usually by movement along a porous membrane or microfluidic pathway yielding a result. Modern LFAs use colored particles to generate their results, either as a colored line in the case of a non-competitive assay, e.g., an hCG test, or lack of a colored line in a competitive assay, e.g., a THC test. The particle coupling technique therefore is a critical step for the success of an LFA.

One key component that significantly influences the sensitivity, specificity, and overall performance of LFAs is the selection of colored particles (such as gold nanoparticles, microspheres, cellulose nanobeads or other types of nanoparticles) as the visual indicator as well as the coupling method used to prepare these particles.

It seems that all fields have terms that may be misnomers. Lateral flow diagnostics is not immune (pun intended) to this practice. “Conjugates”, according to the chemistry definition, ”are formed by the joining of two or more chemical compounds through Pi bonds”. Thus, a molecule non-covalently coupled to a particle is not a conjugate. However, rather than oppose this current terminology, conjugation and coupling will have an equivalent meaning in this review.

Another misnomer in this field is “latex particles”. Microsphere particles are not latex, they are actually polystyrene spheres. Again, rather than oppose this current terminology, latex particles and microspheres will have an equivalent meaning in this review.

Here’s a cursory review of some common coupling techniques used for particle labeling in lateral flow assays:

Covalent Coupling

Covalent coupling is one of the most reliable and widely used methods in particle conjugation. In this technique, functional groups (like amines, thiols, or carboxyls) are introduced on the particle surface, and these groups form strong covalent bonds with biomolecules such as antibodies, antigens, or enzymes. The strong bond ensures that the biomolecules stay attached to the particles throughout the assay process.

Advantages
- High stability of the conjugate in various solutions with challenging pHs, high surfactant concentrations and buffer salt molarity.
- Strong and irreversible attachment of biomolecules.
- Can be used with various biomolecules, such as proteins or nucleic acids.
Disadvantages
- Requires a well-optimized reaction to avoid over-conjugation, under-conjugation or binding to the active epitope which may alter particle functionality.
- Potential for cross-linking to unintended sites.
- Requires specific buffers and pH to optimally form the covalent bond.
- Technique dependent.
Common Techniques
- Amine coupling (Carboxy particles binding to the amino group on molecule): This is probably the most commonly used covalent coupling method. A one-step carbodiimide only method or a two-step carbodiimide and N-hydroxysuccinimide method covalently couple the carboxylated particles to amino groups on the targeted molecule. The one-step method has the potential to cross-link and form large aggregates. The use of unwanted amino groups in the coupling solution must be precluded. The use of TRIS buffers must be avoided.
- Sulfo coupling (Sulfo particles binding to sulfo groups on the molecule): This method usually is not routinely used, but may be useful if tertiary amino groups are not available on the target molecule or if they are in the active epitope. This technique activates sulfo groups on the particles and on the biomolecules and quickly forms a disulfide linkage.
- Glutaraldehyde cross-linking (Amino particles binding to amino groups on the molecule) : Glutaraldehyde has active carbonyls on each end of the molecule. Thus, in theory, one end of the gluteradehyde binds to an amino group on the particle and the other end binds to an amino group on the protein. However, there is a huge potential for cross linkage since there is no way to specify which amino groups will be bound by the gluteradehyde. Once again, the use of unwanted amino groups in the coupling solution must be precluded. The use of TRIS buffers must be avoided.

Non-Covalent Coupling

Non-covalent coupling methods employ weaker interactions like hydrogen bonding, electrostatic interactions or hydrophobic interactions. These methods are often simpler and less costly than covalent techniques but yield lower coupling rates, un-orientated biomolecules and coupled particles that are less stable.

Advantages
- Easier to prepare than covalent methods and usually don’t require a reagent to propagate coupling.
- Can be used in situations where covalent coupling may alter the particle’s functionality
- Less expensive compared to covalent coupling techniques.
- pH and buffering salt choices less critical.
Disadvantages
- Reduced particle stability leading to potential dissociation of the biomolecules from the particle under some assay conditions.
- May exclude the ability to use surfactants in the assay buffer as surfactants may cause dissociation of the biomolecule from the particle.
- Less assay sensitivity (detection level), as compared to a covalent conjugate.
- Some molecules may not non-covalently attach to the target particle.
Common Techniques
- Hydrogen bonds: This is a special type of an electrostatic attraction between molecules. It is not a covalent bond to a hydrogen atom. It results from the attractive force between a hydrogen and a very electronegative atom.
- Electrostatic interactions: The particles typically have some charge, positive or negative, especially if they have functional group on their surface, and an opposite charge on the biomolecule leads to adsorption of the biomolecule to the particle surface.
- Hydrophobic interactions: This method utilizes the non-polar nature of certain biomolecules making them less “water friendly”. This hydrophobicity drives them to be attached to the particle.

Affinity-Based Coupling

Affinity-based techniques are used when high specificity is required between the particle and the biomolecule, and they typically rely on highly specific binding pairs. Common systems include species specific antibody- primary antibody interactions, receptor-ligand interactions, or biotin-avidin systems.

Advantages
- High specificity thus improving assay performance (minimal non-specific binding, etc.).
- Correct biomolecule orientation, thus maximizing specific binding while minimizing biomolecule usage.
- Higher biomolecule coverage on particle.
Disadvantages
- Requires precise optimization to ensure that all binding sites have been quenched.
- More costly due to the need for high-quality antibodies and another coupling biomolecule.
- Requires that the orientation biomolecule initially be bound to the particle. A covalent bond of the “orientation” biomolecule to the particle is highly recommended.
Common Techniques
- Biotin-Streptavidin System: This system relies on the strong, high affinity, non-covalent binding between biotin and avidin/streptavidin to link biomolecules to particles. In general, streptavidin is covalently bound to the particle and biotin is covalently coupled to the target moiety, for example an antibody. The biotinylated biomolecule is incubated with the streptavidin particle and the biomolecule is bound to the particle through Biotin-Avidin linkage.
- Antigen-Antibody Binding: This system employs a “2nd” antibody, an animal specific anti-IgG, that is usually covalently bound to the particles. The target specific antibody is incubated with the 2nd” antibody particle and the target specific antibody is bound to the particle through the antibody-antibody linkage.
- Protein A/G-antibody Binding: This system also relies on the strong, high affinity, non-covalent binding between Protein A or Protein G, or a mixture of both, to link antibodies to particles. Protein A and Protein G have unique binding targets thus it is important to know the species and subclass of the antibody you wish it to bind. In general, Protein A, G or A/G are covalently bound to the particle. The Target specific antibody is incubated with this coated particle and the antibody is bound to the particle through the Protein A, G or A/G linkage.

Surface Modification

Surface modification involves altering the physical or chemical properties of the particle surface to facilitate coupling. These modifications might be used in conjunction with covalent or non-covalent methods.

Advantages
- Surface modification can help improve particle dispersion, stability, and conjugation efficiency.
- Allows for control over particle size and surface chemistry to optimize the coupling process.
Disadvantages
- Surface modifications can add complexity to the production process.
- Requires high-quality control to ensure consistency.
Common Techniques
- Polymer coating: Coating particles with polymers like polyethylene glycol (PEG) can reduce aggregation, enhance stability, and provide reactive groups for coupling.
- Silica coating: Silica particles are often coated with functionalized silanes to facilitate biomolecule conjugation.

Particles

Gold Nanoparticle Coupling

Gold nanoparticles (AuNPs) are the most widely used particles for lateral flow tests due to their distinctive red color, which is visible even at low concentrations. The surface of these nanoparticles is usually not functionalized, thus binding to these particles is non-covalent. Usually, a 40 nm particle is used as the optimal gold nanoparticle.

Surface Modified Gold Nanoparticle Coupling

Gold nanoparticles are now commercially available with a Carboxy modified surface thus allowing the attachment of biomolecules through covalent bonding or using linker molecules as described above.

Polystyrene Microspheres (Latex Particles) Coupling

Polystyrene microspheres, commonly called latex particles, are used as an alternative to gold nanoparticles. These particles are commercially available with the surface of the microspheres functionalized with amine, sulfo or carboxyl groups, allowing the attachment of biomolecules through covalent bonding or using linker molecules as described above. They are available in a variety of colors offering line differentiation by color. They offer the advantages of covalently coupled particles: high stability, strong and irreversible attachment and the ability to bind various biomolecules. Most often, the particle size of choice is 300 to 400 nm.

Colloidal Silver Particles Coupling

Similar to gold nanoparticles, colloidal silver nanoparticles can be used in LFTs for visual detection. Particles are available as carboxy surface modified or non-surface modified, thus allowing for covalent or non-covalent coupling. Although providing a cost-effective alternative to gold nanoparticles, colloidal silver particles are seldom used in LFAs anymore.

Ceramic or Silica Particles Coupling

Silica-based particles are sometimes used for lateral flow tests, often when specific surface properties or stability is required. These particles can be functionalized using silane chemistry (e.g., aminopropyl silane) to covalently bond with biomolecules like antibodies or enzymes, offering the same advantages of other covalently coupled particles.

Cellulose Nano Beads (CNB) Coupling

These beads are typically not surface modified and thus binding to these particles is non-covalent. More recently, they can also be purchased carboxy surface modified thus allowing for covalent bonding or electrostatic interactions. These particles are usually in the 300-400 nm size, are available in various colors, are very intensely colored, use 5-10 times less biomolecules than gold and are more cost effective, especially with moderate to expensive reagents. The surface modified CNB have all of the advantages of covalently coupled particles: high stability, strong and irreversible attachment and the ability to bind various biomolecules.

Conclusion

The choice of the particle and coupling technique is critical for the success of a lateral flow assay. Therefore, it is essential to understand the sample type and the targeted detection molecule properties to correctly select the particle and coupling technique that will provide the best test signal. Not all particles function well in all sample types. While covalent coupling offers strong and stable conjugates, non-covalent and affinity-based coupling methods provide ease-of-use and specificity. Gold nanoparticles are a popular choice due to their optical properties, though other types of particles such as polystyrene microspheres or cellulose nanobeads also have distinct advantages depending on the specific requirements of the assay. The key to optimizing a lateral flow assay lies in selecting the best particle for your application as well as the right coupling strategy to ensure alignment with the analytical goals, cost constraints, and desired test performance.

Artemis Dx has experience with all the particles and coupling methods described in this review and can support your efforts when needed. Please inquire about our development and consulting services at info@artemisdx.com.