Study of Binding with SPR
The P4SPR is based on surface plasmon resonance (SPR), which allows the study of label-free interactions between a surface capture molecule and a target molecule in real time. A capture molecule is immobilized on the sensor chip surface, and the target molecule is flowed over it. The capture and target molecules can range from small to large biomolecules, including nucleic acids (e.g., DNA) and peptides (e.g., proteins). Binding generates a specific SPR response that translates into refractive index changes near the sensor surface.
Overview of Sensor Functionalization
There are many strategies to functionalize an SPR sensor depending on the capture molecule. While antibodies and peptides are the most common, biosensors with DNA/RNA, lipids, and carbohydrates are also widely used.
Most strategies can be broken down into three steps:
Why Surface Functionalization Matters
Selecting the right surface chemistry is key to properly immobilizing the capture molecule, retaining high activity, and obtaining SPR data specific to the interaction of interest. Unlike ELISA — where antibodies passively adsorb to the well wall — SPR immobilization is an active process requiring careful selection of linker, activation reagents, and immobilization conditions (buffer, time, pH). This increased complexity provides more strategy options to optimize the interaction.
P4SPR Sensor Chip Design
The sensor chip is a glass prism coated with a thin layer of gold. Inserting it into the P4SPR cavity creates the optimal condition for SPR signal generation. The surface can then be modified with different chemistries for immobilizing various capture molecules.
Three essential features:
Glass Chip
A miniature dove prism with long edges for the optical path and short edges for handling. Uncoated chips are available for users preparing their own metallic surfaces or patterned substrates.
Metallic Thin Film
Consistent metal deposition by evaporation ensures batch-to-batch reproducibility. A certificate of analysis for film thickness is provided. Custom compositions and thicknesses are possible.
Surface Functionalization
Consistent surface coating with minimal non-specific interactions and pinhole defects. Specificity is determined by the chemical properties of the linker molecules on the surface.
Available Sensor Surfaces
| Sensor Type | Surface | Recommended For |
|---|---|---|
| Au + Ni-NTA | Metal ion–NTA chelation | Recombinant His-tagged proteins |
| Au + AffiCoat™ | Zwitterionic peptide (proprietary) | Covalent coupling in complex matrices (serum, cell lysate) |
| Au + 16-MHA | Long-chain alkanethiol SAM | Covalent coupling; hydrophilic (proteins, DNA/RNA) and hydrophobic (lipids, membrane proteins) capture molecules |
| Au + Streptavidin | Covalently attached streptavidin | High-affinity non-covalent binding of biotinylated molecules (DNA, aptamers, proteins, carbohydrates) |
| Bare Au | Unmodified gold | Thiolated capture molecules (DNA/RNA, protein); custom SAM; molecular imprinted polymers |
Surface Chemistry Details
Au + Metal Ion–NTA
NTA chelates Ni²⁺ or Co²⁺ ions, providing a binding site for His-tagged proteins. Simple and convenient — no activation steps required. Surface can be regenerated with EDTA to release the chelated protein.
Au + AffiCoat™
A zwitterionic peptide surface with high hydrophilicity that resists non-specific adsorption. The α-helical conformation drives biofouling away from the surface, providing increased sensitivity and low background noise in complex biological samples.
Au + 16-MHA
Long-chain alkanethiol molecules attached directly to gold via Au–S bonds. Provides a hydrophobic, uncharged surface. Useful for reduced non-specific interaction of highly charged biomolecules and for work with lipids and membrane-associated molecules.
Au + Streptavidin
Streptavidin covalently attached to linker molecules on the surface. Provides high-affinity non-covalent binding of biotinylated molecules (Kd ~10−15 M). The interaction is essentially irreversible — biotinylated molecules cannot normally be removed to regenerate the surface.
Bare Au
Unmodified gold for users preparing their own surface chemistry. Compatible with thiolated DNA/RNA, thiolated proteins, custom SAMs, and novel materials. Maximum flexibility for advanced applications.
Coupling Approaches
The coupling method should allow sufficient immobilization while preserving the activity of the capture molecule. Three main approaches are used:
1. Direct Covalent Coupling (EDC/NHS)
The capture molecule is coupled via EDC/NHS chemistry to form a covalent amide bond with the thiolated linker molecule. Requires activation and blocking steps during the SPR run. The most common approach. See Protocol #1 →
2. His-Tag Metal Chelation
NTA linker molecules chelate metal ions (Ni²⁺/Co²⁺). His-tagged proteins bind as soon as the solution flows over the surface — no additional activation steps required. See Protocol #2 →
3. Streptavidin–Biotin Binding
Streptavidin is linked to the surface; biotinylated capture molecules bind with extremely high affinity as soon as the solution flows through. See Protocol #3 →
Coupling Method Selection Guide
| Capture Molecule | Direct Covalent (EDC/NHS) | His-Tag Chelation | Streptavidin–Biotin |
|---|---|---|---|
| Antibody / Antigen | ✓ | ✓ | ✓ |
| Antibody fragments | ✓ | ✓ | ✓ |
| Other proteins | ✓ | ✓ | ✓ |
| Peptides | ✓ | ✓ | |
| Small molecules | ✓ | ✓ | |
| DNA / RNA | ✓ | ✓ | |
| Oligonucleotides | ✓ | ||
| Aptamers | ✓ | ✓ | |
| Carbohydrates | ✓ | ||
| Nanoparticles | ✓ | ✓ | ✓ |
Related Resources
- Sensors & Kits — browse available sensor chips and order online
- Surface Chemistry Explained — interactive guide to choosing the right surface
- Protocol #1 — Covalent Immobilization (EDC/NHS)
- Protocol #2 — Metal Chelation (Ni-NTA / His-Tag)
- Protocol #3 — Streptavidin Capture
- TN-03: The SPR Sensorgram Explained — sensorgram phases, shapes, and quality features
- TN-02: DNA Hybridization & Aptamer Biosensor Development — nucleic acid biosensor applications using SPR
- Static vs. Kinetic SPR — P4SPR 2.0 and P4PRO compared
Conclusion
The variations in functionalized P4SPR sensors — in surface chemistry and coupling approach — dictate analytical performance and can be engineered for optimization for any application of interest. In addition to studying biomolecular interactions, the sensor variety also accommodates uses in assay development, clinical analysis, environmental monitoring, and materials science.
TN-01 — Affinité Instruments Technical Note Series