Experiment #2 - Reaction Buffer Screen
Due to the uniqueness of each protein, different reaction buffer formulations can strongly affect how the proteins behave in solution. The protein behavior will determine which lysine residues are presented for amide bond formation with the sulfo-NHS ester on the activated particles.
While activation of NHS esters is most efficient at pH 5, conjugation of sulfo-NHS esters to amine groups on proteins is most efficient at pH 7–8. Upon addition of EDC and sulfo-NHS, the gold nanoshell solution will drop to pH 5. After activation, it is important to transfer the activated particles to a neutral pH for amide bond formation with the protein of interest.
This reaction buffer screen will test three different buffer formulations that are most commonly used during lateral flow development.
Technical Notes
- NHS esters have a half-life of 4–5 hours at pH 7, 1 hour at pH 8, and only 10 minutes at pH 8.6.
- PEG 20 kDa is included in the buffer formulation to help re-disperse the pelleted particles. It is not required but is recommended, especially if you do not have access to a bath sonicator.
- For competitive format assays where it is desirable to limit the number of antibodies per particle, we recommend adding 10 µg of antibody per mL of 20 OD gold nanoshells, and shortening the antibody incubation time to 30 minutes.
- The PBS reaction buffer formulation is supplied at a 50× concentration. Prepare 10 mL of 1× stock for conjugations.
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. There are two means in which antibodies are bound to a reporter particle: passive adsorption and covalent coupling. This module discusses passive adsorption and provides instructions on how to build robust, reliable and optimized passively/physisorbed conjugates.
Materials
We recommend using the following - or similar - materials:
- DI water
- 3 × 1.5 mL LabCon® test tubes
- 3 mL BioReady™ 150 nm Carboxyl Gold Nanoshells, 20 OD
- 1 mL each aliquoted into 3 × 1.5 mL LabCon® tubes (provided)
- Reaction buffers:
- 5 mM potassium phosphate, 0.5% PEG 20 kDa, pH 7.4
- 5 mM sodium phosphate, 0.5% PEG 20 kDa, pH 7.4
- 5× PBS, 25% PEG 20 kDa, pH 7.4 (50× concentrated stock)
Note: Prior to beginning conjugation bring PBS to 1× (0.01× PBS, 0.5% PEG 20 kDa) by diluting 200 µL 50× stock into 10 mL fresh DI water. Check pH to ensure it falls between 7.0–7.6
- EDC: 10 mg aliquot
- Sulfo-NHS: 10 mg aliquot
- Hydroxylamine solution
- Conjugate diluent
- Microcentrifuge
- Rotator or end-over-end mixer for antibody incubation
General Conjugation Flow Chart
Figure 1 shows the methodology overview for the 15 steps required during the reaction buffer screen experiment, with further details from the protocol outlined below:Select Optimal Reaction Buffer Formulation
Observe the visible color of the conjugate and compare it to the stock nanoshell color. Only a slight color change should be visible. For a more precise comparison of color, compare the UV-Vis spectra before and after conjugation. If using lateral flow as a functional readout, run the conjugate on a test strip and measure the performance. Note any particle aggregation at the sample pad/nitrocellulose interface, non-specific binding, and the relative signal intensity strength of the positive signal.
Select the reaction buffer that resulted in the best colloidal stability, lowest non-specific binding, and highest positive signal intensity. If all three reaction buffers perform equally, we recommend moving forward to the next optimization step with 5 mM potassium phosphate 0.5% PEG 20 kDa, pH 7.4.
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