NanoComposix BioReady™ 150 nm Carboxyl Gold Nanoshells can be conjugated to proteins through carbodiimide activation chemistry. Covalent coupling of proteins (e.g. antibodies) to a gold nanoparticle surface yields a robust and stable gold particle conjugate. The BioReady™ 150 nm Carboxyl Gold Nanoshells are functionalized with a tightly bound monolayer containing terminal carboxylic acid functional groups which can be activated through EDC/Sulfo-NHS chemistry to generate gold nanoparticle-antibody amide bonds.
Contact us for inquiries regarding custom conjugation, technical support, or determining which nanoparticle is right for your application.
Material Information & Storage
BioReady™ 150 nm Carboxyl Gold Nanoshells are provided at OD 20 in water. Store at 4 °C. Do not freeze. Thoroughly shake or sonicate contents to disperse particles if settling occurs.
Proper handling and storage of EDC and Sulfo-NHS is critical for successful conjugations. These reagents can be purchased from a number of vendors and the manufacturer guidelines for handling and storage should be followed. EDC and Sulfo-NHS should be stored with desiccant at -20 °C and 4 °C respectively. Ensure that reagents are brought to room temperature before opening to avoid water condensation.
Additional Materials Required
Antibody purification filters/columns
DI Water
Antibody purification buffer
10 mM potassium phosphate, pH 7.4
Reaction buffer
5 mM potassium phosphate, 0.5% 20K MW PEG at pH 7.4
15 mL Labcon SuperClear® conical Cat #3131-335-028
50 mL Labcon SuperClear® conical Cat #3181-345-008
Vortex
Rotator
Important:The reaction buffer used when activating NHS esters and forming covalent bonds with your protein is important. The activation with EDC and Sulfo-NHS is most efficient at pH 5, and the particle solution will self-adjust to this pH after reagent addition. The conjugation of these activated particles with primary amines on the antibody is most efficient at pH 7-7.5. For best results, perform the conjugation step with the suggested reaction buffer or another amine-free, low-salt buffer in the pH 7-7.5 range. Performing the reaction at a higher pH drastically reduces the half-life of the NHS-ester. Once a stable conjugate is formed it can be transferred into the buffer of choice.
Antibody Preparation
The antibody for conjugation should be purified and adjusted to a concentration > 1 mg/mL in a low ionic strength buffer free of additional proteins or free amines, such as 10 mM potassium phosphate. Commercial antibodies may contain protein additives for stabilization (e.g. BSA), preservatives (e.g. sodium azide), or amines in the buffer (e.g. Tris) which all need to be removed before covalent conjugation to nanoparticles. Antibodies can be purified from salt preservatives using spin columns or dialysis tubing with the appropriate molecular weight cutoff, and can be transferred into a non-amine-containing buffer using the same mechanisms. If Tris or another amine-containing buffer is used to elute the antibody from an affinity column during isolation from stabilizing proteins, the antibody will need to be buffer-exchanged to transfer it to a suitable amine-free buffer.
Antibody purification into an amine-free buffer is different from affinity purification of the antibody during development. Even a small concentration of sodium azide (NaN3) will interfere with the efficacy of conjugation.
Antibody Concentration
For covalent conjugation to BioReady™ 150 nm Carboxyl Gold Nanoshells, a typical antibody to gold ratio is 20–30 µg of antibody per 1 mL of gold at OD 20.
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 for 1 mL of OD 20 BioReady™ 150 nm Carboxyl Gold Nanoshells that will result in 1 mL of antibody-gold conjugate at OD 20. For larger or smaller volumes, scale proportionately.
Tubes with specialized treatments (e.g. low-bind) or with residual plasticizer from the manufacture process can cause instability of the particles during activation steps. If using tubes other than those specified in this protocol, thoroughly rinse tubes with isopropyl alcohol and DI water before use.
Important:Steps 2-8 should be completed immediately after solubilizing EDC and Sulfo-NHS to minimize hydrolysis of the Sulfo-NHS ester in water and enhance the efficiency of conjugation.
Prepare EDC and Sulfo-NHS at 10 mg/mL in H2O immediately before conjugation steps.
Hint: Ensure the reagents are at room temperature before opening vials. Weigh out approximately 1-10 mg EDC and Sulfo-NHS in individual microcentrifuge tubes and record mass. Just prior to conjugation, dissolve in the H2O to bring the concentration to 10 mg/mL.
Example: Mass of EDC = 4.3 mg, add 430 µL H2O Mass of Sulfo-NHS = 6.1 mg, add 610 µL H2O
Add 80 µg EDC (20 µL freshly prepared EDC at 10 mg/mL) to 1 mL of BioReady™ 150 nm Carboxyl Gold Nanoshells.
Add 160 µg Sulfo-NHS (40 µL of freshly prepared Sulfo-NHS at 10 mg/mL) to the 1 mL BioReady™ 150 nm Carboxyl Gold Nanoshells.
Vortex solution and incubate at room temperature for 30 minutes while rotating.
Centrifuge at 2000 RCF for 5 minutes.
Carefully remove supernatant to remove any excess EDC/Sulfo-NHS and resuspend pelleted nanoparticles with 1 mL reaction buffer. Vortex and/or sonicate (<30 seconds) to fully re-suspend particles.
Repeat step 6-7 to wash particles of any excess EDC/NHS.
Add 30 µg antibody and vortex.
Incubate at room temperature for 1 hour while rotating (shorter or longer incubation times may yield better results).
After incubation, add 10 µL of quencher to deactivate any remaining active NHS-esters. Vortex and incubate at room temperature for 10 minutes while rotating.
Centrifuge at 2000 RCF for 5 minutes. Carefully remove supernatant and resuspend pellet with 1 mL of reaction buffer. Vortex and/or sonicate to fully resuspend conjugate.
Repeat centrifugation and resuspension (Step #12) to remove any excess antibody.
Centrifuge again at 2000 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.
Store conjugate at 4 °C. Do not freeze.
Frequently Asked Questions
We guarantee our BioReady™ particles for 6 months from date of delivery when our storage and handling guidelines are followed. Longer stability (> 1 year) can be expected.
The shelf life of the conjugate depends on a number of factors including the antibody, storage buffer, and storage conditions. Monitor the stability of your conjugate over time for your specific application. A preservative (e.g. NaN3) can be added to the storage buffer after conjugation. Optimal salt concentrations may differ between conjugates and can affect stability. Proteins such as BSA can also help stabilize the conjugate. Store all conjugates at 4 °C.
BioReady™ Carboxyl Gold can be used to covalently attach any proteins with free primary amines (-NH2) by producing amide bonds.
Lateral flow assays are simple tests for evaluating conjugates. Contact us for preparation of custom test strips that can be used for the validation of your conjugate. For more information regarding lateral flow, refer to our handbook.
Many variables can be adjusted to optimize the conjugate including the antibody/gold ratio, antibody incubation time, blocking steps, conjugate diluent, and reaction buffer. Lower antibody ratios may be required for competitive lateral flow assays. When decreasing the antibody loading, it is also recommended to decrease the antibody incubation time. Optimal incubation time can be as short as 5 minutes.
We recommend optimizing the amount of surfactants, proteins, and adding other components in the conjugate diluent as needed. Addition of sucrose and/or trehalose can also help with the release of the conjugate after drying down into the conjugate pad. Changing the pH or the buffers of the final conjugate diluent may help stabilize larger particle conjugates. Selection of the pad materials and appropriate treatment can also aid in the release and flow of the conjugate.
Based on the amount of reagent used during the surface functionalization step and the surface area available for the ligand to bind, we calculate a maximum of ~2.5 amine groups/nm2 at the particle surface. This is consistent with literature reports, which estimate approximately two amine groups/nm2.
Depending on orientation, packing density, and other factors, only a portion of the amines may be accessible for conjugation. Further, in some cases there are also amine groups that are incorporated into the silica network below the particle surface, and which contribute to the zeta potential of the particle and can be detected using different characterization methods. Because they are embedded within the silica shell, however, these amines are not accessible for conjugation.
By clicking “Acknowledge”, you consent to our website's use of cookies to give you the most relevant experience by remembering your preferences and to analyze our website traffic.
