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. While 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 various forces between molecules and surfaces (e.g. van der Waals and ionic forces) antibodies will spontaneous bind to a bare gold nanoparticle surface to form a conjugate.The antibody is typically added in excess to ensure that there is 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.
While conceptually simple, there are important steps that need to be performed to produce high quality passive conjugates. Since the antibody and particles are simply mixed together the orientation on the nanoparticle surface is not well controlled. Antibody are proteins and as such, can have different confirmations (shapes) under different solution conditions. Thus, 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 zero charge (isoelectric point) 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 pH Adjustment section below).
After passively adsorbing your antibody to your 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 show 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 optimizations of the conjugation conditions such as the antibody/particle ratio and blocking agents can be varied to maximize performance.
Challenges with Passive Adsoprtion
Even though passive adsorption is a common method for the conjugation of antibodies to reporter particles, the process does have some drawbacks. Firstly, every antibody requires slightly different conditions for optimal performance. Identifying the right conditions can involve extensive optimization, which can be both costly in terms of time and resources. Secondly, without being covalently attached, there is the possibility of the antibodies detaching 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. However, there are some unique advantages to using passive adsorption over covalent coupling. Firstly, 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 is important for maximizing sensitivity.
The Importance of the Nanoparticle Surface on Performance
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 defined as not having any molecular ligands bound to the particle surface. Technically, it is not possible to have a stable bare nanoparticle 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. nanoComposix 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. Additionally, nanoComposix offers gold nanoparticles in a carbonate buffer (Figure 3). Carbonate is a smaller and less complex molecule and has a lower affinity to the gold nanoparticle surface than citrate. The greater displaceability of the carbonate molecules typically produces better performing conjugates, and therefore, carbonate-buffered gold particles are preferable for physisorption experiments.
Comparison Between Citrate and Carbonate Conjugates
To measure the difference in lateral flow assay sensitivity between the carbonate and citrate BioReady Bare particles, an anti-cardiac troponin I (anti-cTnI) antibody was adsorbed onto the surface of carbonate or citrate buffered BioReady Bare particles. After conjugation, the conjugate was washed three times in a purification buffer before concentrating to a final concentration of 20 OD/mL. The conjugates were sprayed down onto conjugate pads and assembled into strips. The assembled strips were run with various concentrations of cTnI and the test line intensity was read out using a Qiagen ESE Quant reader. The results are shown in Figure 6. The carbonate buffered BioReady Bare particles had an increased sensitivity compared to the citrate BioReady Bare particles. A number of our customers have reported similar results where the carbonate buffered particles have an increased sensitivity compared to citrate buffered gold particles.
BioReady pH Adjustment for Passive Adsorption
For successful conjugation pH and salt concentrations are often adjusted 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). BioReady bare particles with a carbonate surface are provided unbuffered in deionized water. To adjust the pH of the starting solution the BioReady bare particles should be pH adjusted right before conjugation. At nanoComposix, 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). Table 3 below provides estimates of the amount of 10 mM poteassium phosphate basic/tribasic to add to a 5 mL sample of BioReady citrate/carbonate gold in order to reach the target pH. 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 made with care to make sure that the addition of the buffers doesn’t overshoot the target pH.
Lot Size and Pricing
BioReady™ nanoparticles are fabricated within the ISO 13485 compliant quality system 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. 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. 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. Contact nanoComposix for a quote or help with comparing costs.
A summary of the conjugation procedure described below is for the nanoComposix BioReady Bare 40 nm gold nanospheres. The protocols for the 80 nm gold is similar – only the antibody/particle ratios are different.
Nanoparticles: BioReady™ 40 nm Bare Gold is provided at an optical density (OD) of 20 at ʎmax (~520 nm), and is available with either a citrate or carbonate stabilizing surface. Colloidal gold nanoparticles are suspended in DI water with a weakly associated surface to stabilize the particles. In the presence of protein, the surface is rapidly displaced to passively conjugate the protein. Citrate is most common surface stabilizer for nanoparticles, however carbonate is a smaller molecule and can be more easily displaced than citrate. The increase in rate of displacement can result in an increase in performance. In order to have reproducible batch to batch results it is essential that the particles are extensively characterized. nanoComposix performs extensive analytical testing with each batch of nanoparticles, and provides these results to the customer with each purchase.
Antibodies: The antibody for conjugation should be purified into a low ionic strength buffer free of additional proteins or salt components, such as 10mM potassium phosphate. Commercial antibodies may contain protein additives for stabilization (e.g. BSA), salt as a preservative (e.g. sodium azide), or salt in the storage buffer (e.g. PBS) that need to be removed before passive adsorption of antibodies to nanoparticles. Antibodies can be purified into a non-salt containing buffer using spin columns or dialysis tubing with the appropriate molecular weight cut-off. We recommend storing purified antibodies at a concentration ≥ 1 mg/mL. It is important to note that antibody stability varies and you should always refer to data sheet provided by the antibody supplier for proper storage and handling.
Tubes and Disposables: If you observe flocculation of your particles to the side of the tube (i.e. particles “sticking” to the walls) after centrifugation, the tube itself may be causing issues. Regular tubes that contain residual plasticizer or specialized tubes (e.g. low-bind tubes) may interfere with the particle stability and may cause this flocculation. If this occurs, use a different type of tube or thoroughly rinse the tube with IPA and flush with water prior to adding the particles for conjugation. Contact us for more information regarding the best tubes and procedures if this issue continues.
Antibody Ratios: For passive adsorption to 40 nm gold, a typical antibody-to-gold ratio is 100 µg of antibody per 1 mL of gold at OD 20. For passive adsorption to 80 nm gold, a typical antibody-to-gold ratio is 15 µg of antibody per 1 mL of gold at OD 5. Adjusting the amount of antibody may improve conjugation results.
Conjugation Protocol: It is important to note that optimal conjugation procedures are antibody-dependent; optimization techniques will differ from antibody to antibody.This conjugation protocol is intended for 5 mL of OD 20 BioReady™ 40 nm Bare Gold that will result in 5 mL of antibody-gold conjugate at OD 20. For larger or smaller volumes, scale proportionately.
- Rinse all glassware with DI H2O to ensure materials are free of impurities/contaminants.
- Aliquot 5 mL gold OD 20 into a glass beaker with stir bar or to a glass test tube (>15 mL tube size).
- Rapidly add 500 µg of purified antibody to the gold solution (refer to section 7 for optimizing antibody concentration).
NOTE: Best results are seen with rapid addition of the antibody. Ensure that the solution is stirring rapidly if using a beaker, or add the antibody solution to the gold while vortexing in a glass test tube.
- Cover beaker or test tube with parafilm and incubate at room temperature for 30 minutes while stirring/rotating.
- Block conjugate by adding 2 mL of conjugate block buffer (10% BSA). Vortex/mix the solution.
- Incubate at room temperature for 30 minutes while stirring/rotating.
- Centrifuge at 3600 RCF for 10 minutes.
- Carefully remove supernatant and resuspend with conjugate diluent to 5 mL final volume for an OD 20 conjugate. Store at 4˚C. Do not freeze.
Antibody Concentration Optimization
It may be beneficial to adjust the antibody-to-gold ratio. The following procedure can be used to determine the minimum amount of antibody needed to protect the conjugate from aggregation:
- Dilute gold to OD 1 with water for 2 mL final volume.
- (e.g. 100 µL 20 OD gold + 190 µL water)
- Aliquot 200 µL of gold into eight tubes.
- Add 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.6 µg of antibody to each of the gold aliquots.
HINT: To improve pipetting accuracy, prepare a working dilution at 100 µg/mL in antibody purification buffer immediately prior to conjugation.
- Incubate at room temperature for 30 minutes while rotating.
- Add 20 µL of 10% NaCl solution to each tube and incubate 20 minutes.
- Observe the samples for loss of colloidal stability, indicated by a change from red to purple/gray.
- Determine the lowest antibody concentration that provides colloidal stability
- Divide this number by the volume (0.2 mL) to determine the appropriate amount of antibody to add to each mL of 40 nm gold at OD 1 (e.g. 0.6 µg /0.2 mL = 3 µg antibody/1 mL of OD 1 gold) then multiply the resulting number by 20 to determine the appropriate amount of antibody to add to each mL of 40 nm gold at OD 20 (e.g. 3 µg antibody/1 OD·mL x 20 OD = 60 µg antibody per 20 OD·mL)
To empirically determine the optimal pH of the gold solution for conjugation to a particular protein, titrate the gold to pH increments of 0.2 but pH 7-9. Compare the conjugate efficacy by observing stability and functionality in your particular application. For use in a lateral flow assay, the conjugate that shows the lowest non-specific binding at the test line with the strongest true positive signal can be an indication that the particular pH is optimal for conjugation. After being titrated, the pH of the gold solution will shift. It is important to measure the pH of the solution before beginning a conjugation. We recommend to titrate an aliquot of the gold being used for conjugation, rather than the entire volume if purchasing in large amounts. Always check the pH of the buffers immediately prior to conjugation to ensure they are in the desired range.