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Principles and Applications of Surface Plasmon Resonance (SPR) Technology
2026-06-18
Methods for Affinity DetectionANTIBODY
Affinity detection is mainly divided into three categories: kinetic and solution-state analysis, thermodynamic analysis, and label-based high-throughput screening. The mainstream methods include the following:
Bio-Layer Interferometry (BLI)
BLI is a real-time detection technology based on optical interference. It is characterized by the absence of a microfluidic chip, making operation simpler and faster, especially suitable for crude sample analysis. It uses a fiber-optic biosensor to measure affinity by detecting the shift in the interference spectrum caused by changes in the bio-layer thickness at the probe tip due to molecular binding.
Isothermal Titration Calorimetry (ITC)
ITC is the only method that can simultaneously obtain all thermodynamic parameters including binding affinity, stoichiometry, enthalpy change (ΔH), and entropy change (ΔS). It characterizes interactions by precisely measuring the heat released or absorbed upon molecular binding.
MicroScale Thermophoresis (MST)
MST is a solution-state method that does not require immobilization. It requires very small sample volumes (microliter scale) and is compatible with complex matrices such as cell lysates, making it particularly suitable for difficult-to-purify samples like membrane proteins.
Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA is the most classic, popular, low-cost, and high-throughput detection method, suitable for qualitative or semi-quantitative analysis of large numbers of samples. It works by immobilizing antigen/antibody on a solid phase, detecting with an enzyme-labeled antibody, and catalyzing substrate color development. The color intensity reflects the amount bound, from which the apparent affinity can be calculated.
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Click for inquiryRadioligand Binding Assay (RLA)
RLA is a highly sensitive heterogeneous detection method. Due to its extremely high sensitivity, it was once the gold standard. It uses radioisotope-labeled ligands and quantifies affinity by detecting the radioactive signal generated upon binding to the receptor.
Surface Plasmon Resonance (SPR)
Surface plasmon resonance (SPR) technology is a label-free, optics-based novel analytical technique used to detect binding interactions between two or more molecules in real time. Its instrumentation platforms offer high throughput, flexibility, and sensitivity, allowing researchers to characterize biomolecular interactions in binding studies. It is broadly applicable to different molecules, such as ions, fragments, small molecules, proteins, viruses, etc., and can provide biophysical data including affinity, kinetics, and thermodynamics.
The working principle of a conventional SPR sensor is: binding of the target to the immobilized receptor causes a time-dependent change in the refractive index near the sensor surface, and the binding effect between the target and the immobilized receptor is determined by measuring the shift in the resonance angle or resonance wavelength.
Types of SPR DetectionANTIBODY
SPR sensors can be classified based on two dimensions: structural coupling mode and signal detection mode, which together determine the sensor's performance and applicable scenarios.
Classification Based on Structural Coupling Mode
This classification is primarily based on how the SPR effect is excited.
Prism-Coupled Type
Prism-coupled type is the most common and commercially successful. It uses prism coupling and attenuated total reflection (ATR) to excite surface plasmon waves. Among these, the Kretschmann configuration is the most widely used due to its simple structure and high sensitivity. Mainstream commercial instruments, such as the Biacore series, employ this design.
Grating-Coupled Type
Grating-coupled type excites SPR by fabricating a diffraction grating on the metal surface. The grating structure facilitates design and fabrication, but requires high machining precision and cost.
Waveguide-Coupled Type
Waveguide-coupled type uses a planar waveguide structure to couple light to the metal surface to excite SPR. It is easy to integrate onto a chip, facilitating system miniaturization and integration.
Classification Based on Signal Detection Mode
This classification focuses on how the SPR effect is converted into a measurable signal, mainly divided into four types:
Angle Modulation
The wavelength of incident light is fixed, the incident angle is scanned, and the reflected light intensity is monitored. Detection is achieved by tracking the shift in the resonance angle (dip). This method offers high sensitivity and a large dynamic range and is the core technology used in mainstream instruments such as Biacore.
Wavelength Modulation
The incident angle is fixed and the wavelength of incident light is scanned. Since the shift in the resonance wavelength is measured directly and reliably, there is no need for mechanical angle scanning, making the system very stable.
Intensity Modulation
The incident light and angle are fixed, so the change in reflected light intensity at a specific angle is measured directly and conveniently. However, it must be noted that this method is very sensitive to light source fluctuations.
Phase Modulation
This method measures the phase jump of the incident light polarization at resonance and is currently the most sensitive method. Consequently, it naturally comes with the disadvantages of system complexity and a limited dynamic range.
Typical SPR Experimental WorkflowANTIBODY
Pre‑Experiment Preparation
Instrument warm-up, buffer and sample preparation, and selection and installation of an appropriate sensor chip. Commonly used chips include: CM5 chip (carboxylated surface, suitable for covalent immobilization of most proteins); SA chip (coated with streptavidin, used to capture biotinylated molecules); NTA chip (captures His‑tagged proteins via nickel chelation); and Protein A/G chip (immobilizes antibodies via Fc capture).
Chip Activation
An EDC/NHS mixture is flowed over the chip surface to convert carboxyl groups to reactive ester groups, preparing for ligand coupling.
Ligand Immobilization
One interactant (ligand) is flowed over the activated surface and covalently immobilized onto the chip. The immobilized ligand protein amount is typically between 1000–5000 RU.
Baseline Calibration
Buffer is continuously flowed through the system until the signal stabilizes and a baseline is established.
Analyte Binding and Dissociation Detection
Different concentrations of analyte solution are injected to measure the signal increase upon analyte-ligand binding (association phase), followed by buffer flow to measure the signal decrease upon complex dissociation (dissociation phase). Typically, at least 8 concentration gradients should be set.
Chip Regeneration
A regeneration solution such as low‑pH buffer or high‑salt solution is used to wash off the bound analyte from the chip, restoring the chip to its initial state for the next run.
Post‑Experiment Processing and Data Fitting
The chip is thoroughly rinsed, the fluidic system is cleaned, the chip is removed and stored as required, and the instrument is shut down. The experimental data are then subjected to model fitting.
Alpha Lifetech provides affinity detection services, including but not limited to SPR, BLI, ITC, MST, etc., to meet different experimental needs of customers and support them on their research journey.
FAQsANTIBODY
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1. What can SPR do and what cannot it do?
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2. What are the specific requirements for samples and ligands?
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3. How should the concentration gradient be designed?
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4. How to choose the appropriate chip?
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5. How to choose the immobilization method?
ReferenceANTIBODY
[1]Puiu M, Bala C. SPR and SPR Imaging: Recent Trends in Developing Nanodevices for Detection and Real-Time Monitoring of Biomolecular Events. Sensors (Basel). 2016 Jun 14;16(6):870.
[2]Sülzen H, Klima M, Duchoslav V, Boura E. SPR is a fast and straightforward method to estimate the binding constants of cyclic dinucleotides to their binding partners, such as STING or poxin. Biophys Chem. 2025 Apr;319:107392.
[3]Linman MJ, Abbas A, Cheng Q. Interface design and multiplexed analysis with surface plasmon resonance (SPR) spectroscopy and SPR imaging. Analyst. 2010 Nov;135(11):2759-67.
[2]Sülzen H, Klima M, Duchoslav V, Boura E. SPR is a fast and straightforward method to estimate the binding constants of cyclic dinucleotides to their binding partners, such as STING or poxin. Biophys Chem. 2025 Apr;319:107392.
[3]Linman MJ, Abbas A, Cheng Q. Interface design and multiplexed analysis with surface plasmon resonance (SPR) spectroscopy and SPR imaging. Analyst. 2010 Nov;135(11):2759-67.