Passive Nanoparticle Conjugation for Lateral Flow Assays

Introduction to Passive Conjugation

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. A simple and effective method of creating nanoparticle conjugates is to simply mix a "bare" gold nanoparticle with an antibody. The antibody binds to the surface of the particle and can target biomarkers and analytes with high sensitivity and specificity. Though conceptually simple, this passive (or physisorption) process needs to be carefully controlled in order to generate reproducible conjugates.

Passive adsorption is the traditional method for attachment of proteins to lateral flow nanoparticle probes and is still widely used. By taking advantage of intermolecular forces between molecules and surfaces (e.g. van der Waals and ionic forces), antibodies will spontaneously bind to a bare gold nanoparticle surface to form a conjugate. The antibody is typically added in excess to ensure complete coverage of the surface of the nanoparticle. Any free antibody remaining in solution is removed via centrifugation or filtration after the conjugation is complete.

Several important steps need to be performed to produce high quality passive conjugates. During passive conjugation, the antibody orientation on the nanoparticle surface is not well controlled due to the manner in which the antibody and particles are simply mixed together. Antibodies are proteins and as such, can have different conformations (shapes) under different solution conditions. Thus, the pH of the solution plays an important role. It has been found that adjusting the pH of the solution to a value that is close to the isoelectric point (zero charge) of the antibody typically produces the highest performing conjugates. Sweeping the pH of the conjugation is important (e.g. increments of 0.2 from pH 7‒9; see protoco> and section on pH adjustment for more details).

After passively adsorbing antibody to the detector particle, the conjugate stability (i.e. quality) should be tested by exposing the prepared conjugate to a salt gradient. Gold nanoparticles with an incomplete antibody coating will be less stable at high salt concentrations and will aggregate. Since the red color of the gold nanoparticles is due to the plasmon resonance of individual gold spheres, gold nanoparticle aggregates will exhibit a color shift. By monitoring this color shift, an optimal conjugation condition can be determined. The performance of the conjugate on the strip is then measured and further optimization of the conjugation conditions such as the antibody/particle ratio and blocking agents can be varied to maximize performance.

Passive Adsorption vs Covalent Conjugation

Passive adsorption is a common and highly effective method for the conjugation of antibodies to reporter particles, but there are some important considerations to consider when deciding whether passive adsorption is the right fit for your assay. First of all, every antibody requires slightly different conditions for optimal performance. Identifying the right conditions can involve extensive optimization, which can be costly in terms of both time and resources. Secondly, because there is no covalent bond holding the conjugate intact, there is the possibility for antibodies to detach from the nanoparticle surface under certain conditions (e.g. salt and pH changes). Any detachment of antibodies from the particle surface can release free antibody into solution resulting in a signal reduction. That said, there are some unique advantages to using passive adsorption over covalent coupling. For one, the process of passive adsorption is very common and relatively straight forward. Secondly, passive adsorption can result in high antibody loading on the particle surface which can be very useful for maximizing sensitivity.

Bare Surfaces for Passive Conjugation

The self-assembly of antibodies and other proteins on the surface of a gold nanoparticle is difficult to control. Small changes in the pH or salt conditions can alter the charge, hydrophobicity, or structure of the antibody, which can affect the antibody density and orientation on the particle surface. The gold particle surface has exposed crystals with various orientations. Each orientation can have a different affinity for different portions of the antibody. For all physisorption processes, the starting particle should be bare, which is to say that no molecular ligands are bound to the particle surface. Technically, it is not possible to have a stable nanoparticle that is completely bare because the charge of the colloidal double layer keeps the particles from aggregating. Therefore, to produce particles that are as bare as possible, ultra-high purity nanoparticles are fabricated and suspended in a buffer only with components that weakly associate with the surface. In the presence of an antibody or other protein, the protein will displace the weakly associated molecules and bind to the particle surface.

The most common buffer for bare nanoparticles is citrate. Sodium citrate 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. Fortis Life Sciences® offers BioReady™ 40 nm diameter gold nanoparticles suspended in a weak (0.02 mM) sodium citrate buffer. The particles are made in large lots, extensively washed in ultra high purity water to remove residual reactants, and are a drop-in replacement for gold nanoparticles that are currently being used in many commercial assays.

BioReady pH Adjustment for Passive Conjugation

For successful conjugation to occur, pH and salt concentrations typically require adjustments in order to maximize the efficacy of the antibody adsorbed to the particle surface. BioReady bare particles with a citrate surface are provided in a citrate buffer (0.02 mM sodium citrate).

The pH of the starting solution should immediately be tested before conjugation. At Fortis Life Science, we typically adjust the pH of the solution with solutions of 10 mM potassium phosphate monobasic (for pH ranges of 6.3 – 8.2) or 10 mM potassium phosphate dibasic (for pH ranges of 7.2 to 8.6). Due to the age of the solution and exposure to CO2 in the atmosphere, the pH of the BioReady gold solutions can change with time. Titrations must be performed with care to make sure that the addition of the buffers does not overshoot the target pH.

Lot Size & Pricing

BioReady™ nanoparticles are fabricated under the oversight of the Fortis Life Science Quality Management System (ISO 13485:2016 registered) to produce high lot-to-lot consistency of the particle properties.

Lot sizes as large as 400,000 OD-mLs (equivalent to 400 L of 1 OD gold) are possible, where a large lot can generate up to 1,000,000 lateral flow assay strips. Specific lots can be reserved under a supply contract that allows for sampling from the same lot for up to a year as using the same lot reduces downtime from re-optimization when switching to a new lot. The large lot manufacturing also allows the extensively characterized nanoparticles to be available at competitive pricing.

When comparing pricing, it is important to adjust for prices that are supplied for different volumes and concentrations. Consider pricing in terms of OD-mL, which is the price of the product divided by the OD of the solution times the volume.

For example, if 100 mL of gold nanoparticles at a 10 OD costs $600, then the cost per OD-mL is $600/(100×10) = $0.60.

Read more about covalent nanoparticle conjugation ❯