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Conjugation of Proteins to Silver Nanoparticles

Background

Silver nanoparticle conjugates are useful for a wide range of biological applications including bio-sensing and bio-imaging. Stable silver nanoparticle conjugates can be prepared by two general approaches, passive adsorption and covalent bonding.


Passive adsorption of a protein molecule onto silver nanoparticles is mediated by the electrostatic and hydrophobic interactions between the protein molecule and the surface layer of the colloidal silver. This process is maximally achieved at a pH close to the pI of the protein to be conjugated. Another important parameter is the amount of protein loading. If surface-adsorbed protein is not sufficient, aggregation occurs upon addition of electrolytes present in standard buffers. Therefore, a titration is required to determine the protein concentration for complete saturation and shielding of the silver nanoparticle surface.


However, some proteins may undergo disturbances to the tertiary structure upon passive adsorption, which may impair their affinity and/or specificity in molecular binding applications. To better preserve the activity of the conjugated protein an alternative approach can be used, i.e., covalently coupling protein molecules onto silver nanoparticles functionalized with carboxyl groups via a PEG-linker. The PEG-linker allows for more flexibility and also better accessibility of the conjugated protein to its antigen/substrate due to the inherent mobility of the PEG-linker and the increased distance from the gold surface. Further, the shielding polyethylene glycol (PEG) layer generally also result in conjugates with superior stability and less non-specific binding. Conjugation to these types of functionalized particles can be performed using straightforward carbodiimide (EDC/NHS) coupling chemistry.


Conjugation of Proteins Using a Passive Adsorption Method


Material and Equipment Required

  • Standard Silver Nanoparticles
  • 10% NaCl (w/v)
  • 10% Tween 20 (w/v)
  • 2mM sodium citrate tribasic dihydrate
  • 10X Phosphate Buffered Saline, 10X PBS
  • Bovine Serum Albumin (BSA)
  • UV-VIS Spectrophotometer

Step 1. Determination of Optimal pH and Protein Concentration for Conjugation

  1. Aliquot 200 µl of silver nanoparticles into 1.5ml Eppendorf tubes (200 µl for each condition to be tested).
  2. Adjust the pH of the silver nanoparticle solution to the desired pH (optimal pH is generally close to the pI of the protein to be conjugated).
  3. Add between 0 and 50 µg of protein in 10 µl to the silver nanoparticles and mix well to determine the amount needed to saturate the silver surface.
  4. Incubate for 10 minutes at room temperature
  5. Add 200 µl of a 10% NaCl stock solution and incubate for 10 minutes at room temperature.
  6. Determine at which protein concentration the silver nanoparticle surface becomes saturated and no aggregation occurs upon addition of 10% NaCl by observing the color change and measuring the sample(s) using a UV-VIS spectrophotometer. Degree of aggregation can be measured by an increase in absorbance at 690nm and a decrease in absorbance at 405-480nm (particle size dependant, see silver nanoparticle properties) compared to that of the non-conjugated control particles.

Note: The amount of protein needed to saturate the silver colloid can also be determined and verified through agarose gel-electroporesis. Binding of protein to the silver nanoparticle surface changes the overall particle charge and size both of which will affect the migration pattern in the agarose gel.


Step 2. Scale-Up and Preparation of Final Silver Nanoparticle Conjugate

  1. Transfer the desired volume of silver nanoparticles to 1.5 ml Eppendorf tubes.
  2. Add Tween 20 to a final concentration of 0.025% (w/v).
  3. Centrifuge the solution to pellet the silver nanoparticles. For more information on appropriate centrifugation settings for silver nanoparticles of different sizes, see table I at the end of this document.
  4. Resuspend the silver nanoparticles with 2mM sodium citrate to the original silver colloid volume and concentration.
  5. Adjust the pH of the silver nanoparticle solution as determined in the titration procedure above.
  6. Add the appropriate amount of protein as determined in the titration procedure above plus an additional 10%.
  7. Incubate for 60 minutes at room temperature on a rotary shaker/rocker.
  8. Centrifuge the vial for 30 minutes at the appropriate speed for the silver nanoparticle size that you are conjugating to pellet the particles and remove the supernatant.
  9. Resuspend the pellet in 1X PBS supplemented with 1% BSA (w/v).
  10. Sonicate briefly in a sonicator bath to aid in dispersion if particles are partially agglomerated.
  11. Validate the functionality of the final silver conjugate. For a suggested protocol for evaluation of functionality using a immuno-dot blot assay please see Tech Note #103-Immunoblotting with Gold Nanoparticles.
  12. Store the silver conjugate at 4 degrees Celsius until use.

Covalent Conjugation of Proteins to Carboxylated Silver Nanoparticles


Materials and Equipment Required

  • Carboxyl Functional Silver Nanoparticles (conc. 125 OD in ddH2O)
  • 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)
  • N-hydroxysulfosuccinimide (Sulfo-NHS)
  • Protein to be conjugated (1mg/ml in 1XPBS)
  • Bovine Serum Albumin (BSA)
  • Activation Buffer: 10mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.5
  • Coupling Buffer: 1X Phosphate Buffered Saline (1XPBS)
  • Washing Buffer: 1X Phosphate Buffered Saline, 0.05% Tween 20 (PBST)

Conjugation Procedure

  1. Prepare a fresh EDC/NHS solution in activation buffer at a concentration of 30 and 60 mg/ml, respectively.
  2. Transfer 10 µl of carboxyl silver nanoparticles from stock solution and mix with 10 µL of EDC/NHS solution.
  3. Incubate for 30 minutes at room temperature to activate the particle surface.
  4. Add 1 ml of washing buffer and mix thoroughly.
  5. Pellet the activated silver nanoparticles by centrifugation for 30 minutes at the appropriate speed for the particular silver nanoparticle size you are conjugating. See table I below for recommended centrifugation speeds.
  6. Remove supernatant taking care not to disturb the pellet.
  7. Add 10 µl of protein to be conjugated (1 mg/ml in 1X PBS)
  8. Incubate for 2-4 hours on a rotary shaker/rocker to conjugate the protein to the activated silver nanoparticle surface.
  9. Add 1 ml of PBST and mix thoroughly.
  10. Pellet the silver conjugate as in step 5 above.
  11. Remove supernatant and resuspend the conjugate with 157 µl of 1X PBS supplemented with 1% BSA (w/v).
  12. Sonicate the conjugate briefly in a sonicator bath to aid in dispersion.
  13. Validate the functionality of the final silver conjugate. For a suggested protocol for evaluation of functionality using a immuno-dot blot assay please see Tech Note #103-Immunoblotting with Gold Nanoparticles for procedure and Figure 1 below for an example result.
  14. Store the silver conjugate at 4 degrees Celsius until use.
Silver Conjugate Dot Blot

Figure 1. Validation of functionality (biotin binding) of a streptavidin silver nanoparticle conjugate using an immuno-dot blot assay.


Table I. Recommended centrifugation speeds for silver nanoparticles of different sizes. Note that centrifugation times and speeds are based on a 1 ml sample volume in 1.5 ml microcentrifuge tubes.


Silver Nanoparticle Diameter (nm) Centrifugation Force Time (min)
10nm 21,000 x g 60
20nm 17,000 x g 30
30nm 11,000 x g 30
40nm 3,000 x g 30
50nm 1,500 x g 30
60nm 900 x g 30
70nm 700 x g 30
80nm 500 x g 30
90nm 400 x g 30
100nm 300 x g 30