Introduction
Carboxylate particles can be coupled to amine-containing molecules using a number of reaction strategies. The most frequently used method involves an aqueous two-step coupling process using EDC and NHS or sulfo-NHS to form an amide bond with a protein or other molecules.
This protocol describes a general method for covalently conjugating amine-containing biomolecules (e.g., proteins, antibodies, oligonucleotides) to carboxylated particles, utilizing EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide) and Sulfo-NHS (N-hydroxysulfosuccinimide) chemistry.
Figure. Carboxylate particles can be coupled to amine-containing molecules using a number of reaction strategies.
Two-Step EDC/Sulfo-NHS Coupling Protocol
- EDC activates carboxyl groups, which then react with Sulfo-NHS to form NHS esters, creating a more stable and reactive intermediate for coupling to primary amines on the biomolecule.
- This two-step method is preferred for biomolecules containing both amine and carboxyl groups to minimize polymerization.
- Optimization is generally required to achieve optimal activity, stability, and minimal non-specific binding.
I. Materials
- Carboxylated Particles: Microparticles or nanoparticles with carboxylate functional groups.
- Target Biomolecule: Protein, antibody, peptide, oligonucleotide, or other amine-containing ligand.
- Activation Buffer: 50 mM MES (2-(N-morpholino)ethanesulfonic acid), pH 6.0.
- Coupling Buffer: Phosphate-buffered saline (PBS), pH 7.2-8.5.
- EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide): Water-soluble carbodiimide.
- Sulfo-NHS (N-hydroxysulfosuccinimide): Enhances EDC coupling efficiency.
- Quenching Solution: Ethanolamine, Tris, glycine, or other amine-containing molecule.
- Washing Buffer: PBS with a mild detergent such as 0.05% Tween-20.
- Storage Buffer: Appropriate buffer for long-term storage of the bioconjugate, often containing a preservative.
- Deionized Water
- Microcentrifuge tubes
- Centrifuge
- Vortex mixer
- Rotator or shaker
Figure. Carboxyl-modified microspheres can be conjugated to a biomolecule using two-step reaction
II. Protocol
A. Particle Preparation
1. Wash Carboxylate Particles: Wash the particles with coupling buffer or deionized water using centrifugation or magnetic separation (if applicable).
- Purpose: To remove any storage buffer, preservatives, or unbound substances.
Note: Avoid buffers containing carboxylates or amines (e.g., acetate, glycine, Tris, imidazole) during washing.
2. Resuspend Particles: Resuspend the washed particles in activation buffer at a suitable concentration (e.g., 10 mg/mL).
- Optional: Add a dilute detergent (e.g., 0.01% SDS) to increase particle stability and prevent clumping.
B. Activation of Carboxylate Groups
1. Prepare EDC and Sulfo-NHS Solutions: Dissolve EDC and Sulfo-NHS separately in activation buffer immediately before use.
Reason: EDC is prone to hydrolysis, so use fresh solutions.
2. Mix EDC/Sulfo-NHS and Add to Particles: Combine the EDC and Sulfo-NHS solutions, then add this mixture to the particle suspension.
- Typical Concentrations: Optimize for your specific application.
3. React: Incubate the mixture at room temperature for 15-30 minutes with gentle mixing.
- Mechanism: EDC activates carboxyl groups, and Sulfo-NHS enhances the reaction with amines.
C. Biomolecule Coupling
1. Wash Activated Particles: Remove excess EDC and Sulfo-NHS by washing the particles 2-3 times with coupling buffer.
- Method: Use centrifugation or magnetic separation, as appropriate.
2. Prepare Biomolecule Solution: Dissolve the biomolecule in coupling buffer at the desired concentration.
- Concentration Range: Typically, a 1- to 10-fold molar excess of ligand over the maximal calculated carboxylate group concentration is recommended.
3. Combine Biomolecule and Particles: Add the biomolecule solution to the activated particle suspension.
4. React: Incubate the mixture at room temperature or 4°C for 2-4 hours or overnight with gentle mixing or rotation.
D. Quenching and Blocking
1. Quench Unreacted Sites: Add a quenching solution (e.g., ethanolamine, Tris, or glycine) to block any remaining active sites on the particles.
- Concentration: Use a concentration (e.g., 100 mM ethanolamine or 0.2 M glycine) sufficient to block excess reactive sites.
- Incubate: Mix for 30 minutes to 1 hour at room temperature.
2. Wash the Bioconjugate: Remove excess quenching agent and unbound biomolecule by washing the particles multiple times with washing buffer.
E. Storage
1. Resuspend the Bioconjugate: Resuspend the particles in an appropriate storage buffer containing a preservative.
- Example: PBS with 0.02% sodium azide or another suitable preservative.
2. Store the Bioconjugate: Store at 4°C or according to the manufacturer’s instructions.
III. Key Considerations and Optimization
- pH: Maintain the correct pH for activation and coupling.
- Ligand Concentration: Optimize the ligand concentration to achieve desired surface coverage without aggregation.
- Reaction Time and Temperature: Adjust the reaction time and temperature to optimize conjugation efficiency while preserving biomolecule activity.
- Washing: Ensure thorough washing to remove unreacted reagents and prevent nonspecific binding.
- Quenching: Use appropriate quenching agents to block unreacted sites.
- Stability: Add stabilizers (e.g., BSA) to prevent aggregation and maintain activity.
- Solvent Compatibility: Confirm particle stability in the buffers and solvents used.
- Controls: Run proper controls for specificity and to ensure proper results.
- Nanoparticle considerations: Take into account that conjugation reactions performed with nanoparticles may have greater reactivity than those with microparticles. Re-optimize the protocol when using nanoparticles.
- Buffer Considerations: The compatibility of the buffer and ligand (solubility, activity) is important, and should be considered when selecting buffers.
- Literature Search: Prior to beginning, perform a thorough literature search for immobilization protocols specific to your biomolecule of interest.
- Alternative buffers: Alternative coupling buffers for immobilization onto NHS-activated supports include 0.1-M MOPS (pH 7.0), 0.1- to 0.2-M phosphate (pH 7.2–7.5), 0.1 to 0.2-M NaHCO3 (pH 8.0), or 0.1-M sodium borate (pH 8.5), which all may or may not contain NaCl.
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IV. Safety Precautions
- Wear appropriate PPE, including gloves, lab coat, and eye protection.
- Handle EDC and Sulfo-NHS in a fume hood to avoid inhalation.
- Dispose of chemical waste properly according to local regulations.
Reference: Bioconjugate Techniques, 3rd Edition - July 25, 2013, Greg T. Hermanson