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 nanoComposix 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 modules discusses passive adsorption and provides instructions on how to build robust, reliable and optimized passively/physisorbed conjugates. 

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:
  1. 5 mM potassium phosphate, 0.5% PEG 20 kDa, pH 7.4
  2. 5 mM sodium phosphate, 0.5% PEG 20 kDa, pH 7.4
  3. 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

Conjugation Flow Chart

Conjugation Protocol

1. Remove 1 × 10 mg aliquot of EDC and 1 × 10 mg aliquot of sulfo-NHS from cold storage and allow to come to room temperature prior to use (~20 minutes).
2. Thoroughly shake the BioReady™ 150 nm Carboxyl Gold Nanoshells to disperse particles and aliquot 1 mL particles into three separate tubes. Label tubes “A”, “B”, and “C”.

IMPORTANT: Steps 3–6 should be completed within 5 minutes of solubilizing EDC and sulfo-NHS to minimize hydrolysis of the sulfo-NHS ester in water and enhance the efficiency of conjugation.

3. Pipette 1 mL of fresh DI water into the 10 mg aliquot of EDC for a final concentration of 10 mg/mL. Vortex for 10 seconds.
4. Add 8 µL of 10 mg/mL EDC to each of the three 1 mL aliquots of BioReady™ 150 nm Carboxyl Gold Nanoshells.
5. Pipette 1 mL of fresh DI water into the 10 mg aliquot of sulfo-NHS for a final concentration of 10 mg/mL. Vortex for 10 seconds.
6. Add 16 µL of 10 mg/mL sulfo-NHS to each of the three 1 mL aliquots of BioReady™ 150 nm Carboxyl Gold Nanoshells.
7. Vortex each solution for 10 seconds and incubate at room temperature for 30 minutes while mixing.
8. After a 30 minute incubation, balance tubes in a microcentrifuge and spin at 2.0k RCF for 5 minutes.  

9. Carefully remove supernatant (~950 µL) to remove any excess EDC/sulfo-NHS and resuspend each pelleted nanoparticle with 1 mL of reaction buffer that corresponds to the tube label. Vortex and/or sonicate (10 seconds) to fully resuspend.
   1. 5 mM potassium phosphate, 0.5% PEG 20 kDa, pH 7.4
   2. 5 mM sodium phosphate, 0.5% PEG 20 kDa, pH 7.4
   3. 01× PBS, 0.5% PEG 20 kDa, pH 7.4*

*Freshly diluted from 50× stock by user

Note: Observe any differences in the pellet between each tube; plating on the inside of the tube may result in an unstable conjugate.

10. Add 20 µg of antibody to each 1 mL tube.
11. Vortex each solution (10 seconds) and incubate at room temperature for 60 minutes on a rotator or end-over-end mixer.
12. After incubation, add 10 µL of hydroxylamine quencher to deactivate any remaining active NHS-esters. Vortex and incubate at room temperature for 10 minutes using the rotator or end-over-end mixer.

Note: Observe the tube and the color of the conjugates after quenching and compare to the “parent” unconjugated gold nanoshell material. A lighter blue color or the presence of any black “specks” is a sign of colloidal instability.

13. Centrifuge aliquots at 2.0k RCF for 5 minutes and carefully remove supernatant. Resuspend pellet with 1 mL of reaction buffer that corresponds to the tube label. Vortex and/or sonicate to fully resuspend the conjugate.
14. Repeat centrifugation and resuspension step #13 to remove any excess antibody.
15. Centrifuge one final time at 2.0k RCF for 5 minutes. Remove supernatant and bring volume of pellet up to 1 mL in conjugate diluent. Vortex and/or sonicate to fully resuspend conjugate.
16. Store conjugate at 4°C. Do not freeze.

Select Optimal Reaction Buffer Formulation

Observe the visible color of the conjugate and compare 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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