Q&A of Surface Plasmon Resonance (SPR)

Proteins, small molecules, DNA/RNA, lipids/liposomes/biomembranes, polysaccharides, peptides, whole cells, viruses, and microorganisms.

Samples need to be fresh and active, and should be filtered through a 0.22 μm membrane or centrifuged. The concentration of ligands should be above 90%, and the analyte solution should not contain high refractive index substances such as glycerol, sucrose, and imidazole.

• Presence or absence of binding
• Specificity and selectivity of binding
• Binding strength of two molecules - affinity
• Kinetics of binding and dissociation, and stability of complex formation
• Participants, collaborators, and assembly sequence in the formation of functional complexes
• Temperature and thermodynamic characteristics of molecular binding
• Detection of target molecule activity content

The sample to be tested (undiluted mother liquor), chip, pipette and tips, buffer bottle (500/250 ml), EP tube and other required reagents and consumables.

*Chip selection:

CM5 chip → protein (PI>4), peptide, small molecule compound

CM7 chip → small molecule compound

SA chip → biotin-labeled molecules, such as nucleic acids, sugars, etc.

Biotin CAP chip → reversible biotin capture chip

ProteinA, G, L chip → antibody

NTA chip → His recombinant protein

L1 chip → simulated lipid bilayer environment

HPA chip → analysis of membrane system interaction

Each chip has 4 channels, and channels 1&2 are usually used together (with channel 1 as reference) and channels 3&4 are used together (with channel 3 as reference), with ligands coupled for detection in the non-reference channels. After the experiment, suitable regeneration conditions are chosen to regenerate the chip, and the chip is soaked in a suitable buffer for storage after use to ensure that the coupled ligand is still active. The chip can be used multiple times.

The residual analyte molecules bound to the ligand on the chip surface must be thoroughly removed while maintaining the activity of the ligand molecules on the chip. The binding level can be maintained stably after multiple injections, with changes in the binding level compared to the first injection within 10%.

A sensor chip is a solid surface on which the ligand is immobilized, while a flow cell is a chamber through which the analyte solution is flowed. The flow cell is attached to the sensor chip and allows for the analyte solution to be injected over the immobilized ligand.

The reference surface in SPR analysis is a surface that is identical to the sensor surface except that it is not functionalized with the ligand. It provides a baseline for the signal and allows for the measurement of specific binding events.

The ligand can be immobilized onto the sensor chip surface through various methods, including covalent coupling, physical adsorption, and affinity capture. Covalent coupling is the most commonly used method and involves the formation of a stable, covalent bond between the ligand and the sensor chip surface.

The regeneration step in SPR analysis is used to remove the bound analyte from the sensor chip surface and restore the surface to its original state. This allows for repeated measurements of the same ligand-analyte interaction and reduces the need for sensor chip replacement.

Equilibrium analysis in SPR measures the steady-state binding affinity between the ligand and analyte, while kinetic analysis measures the rate of association and dissociation between the two molecules. Kinetic analysis provides additional information on the binding mechanism and can be used to determine the binding kinetics and thermodynamics.

The concentration of analyte solution in SPR analysis is optimized to ensure that the sensorgram signal is in the linear range of the sensor chip. This is typically achieved by performing a concentration series of the analyte and selecting a concentration that provides a measurable signal without saturation.

The buffer solution in SPR analysis is used to maintain a stable environment for the ligand and analyte interactions. It can also be used to optimize the binding conditions, such as pH and ionic strength, for specific ligand-analyte interactions.

Data analysis in SPR analysis involves fitting the sensorgram data to a mathematical model that describes the binding kinetics and thermodynamics. The resulting parameters, such as the association and dissociation rate constants and equilibrium dissociation constant, can be used to characterize the ligand-analyte interaction.

Temperature is controlled in SPR analysis using a temperature-controlled flow cell, which allows for the analyte solution to be maintained at a constant temperature during the measurement. The temperature can be adjusted to optimize the binding conditions for specific ligand-analyte interactions.

The sensitivity of an SPR assay is determined by the limit of detection (LOD), which is the smallest concentration of analyte that can be reliably detected. The LOD is influenced by factors such as the quality of the sensor chip surface, the affinity and concentration of the ligand, and the background noise in the assay.

The flow rate in SPR analysis can affect the binding kinetics and signal-to-noise ratio of the assay. A slower flow rate can result in more complete binding and higher signal-to-noise ratio, but can also increase the time required for the assay. A faster flow rate can decrease the time required for the assay, but can also result in incomplete binding and lower signal-to-noise ratio.

The orientation of the ligand on the sensor chip surface can be controlled through various methods, including site-directed immobilization and the use of orientation-specific capture agents. Site-directed immobilization involves attaching the ligand to a linker molecule that is oriented in a specific manner, while orientation-specific capture agents are used to selectively capture the ligand in a specific orientation.

The stability of the ligand on the sensor chip surface can be maintained through the use of stabilizing agents, such as bovine serum albumin (BSA), polyethylene glycol (PEG), or surfactants. These agents can prevent non-specific binding of analytes to the sensor chip surface and stabilize the ligand during repeated measurements.

pH can affect the binding kinetics and stability of the ligand-analyte interaction in SPR analysis. It is important to optimize the pH for specific ligand-analyte interactions, as changes in pH can affect the surface charge of the sensor chip surface and alter the binding kinetics.

The non-specific binding of analyte to the sensor chip surface can be minimized through the use of blocking agents, such as BSA or PEG, which cover the remaining surface area of the sensor chip and prevent non-specific binding. The use of surfactants or detergents can also be employed to reduce non-specific binding.

Data quality in SPR analysis can be ensured through the use of appropriate controls, such as blank injections and reference surface subtraction. The use of replicate measurements and statistical analysis can also be employed to ensure the accuracy and precision of the data.

Salt concentration can affect the binding kinetics and stability of the ligand-analyte interaction in SPR analysis. It is important to optimize the salt concentration for specific ligand-analyte interactions, as changes in salt concentration can affect the ionic strength and alter the binding kinetics.

The mass transport limitation of SPR analysis can be overcome by increasing the flow rate or increasing the concentration of the analyte. This can improve the kinetics of the binding reaction and increase the sensitivity of the assay.

The binding affinity of a ligand-analyte interaction can be determined by fitting the binding data to a binding model, such as Langmuir or Biacore T100. The dissociation constant (KD) is a measure of the binding affinity and can be calculated from the rate constants of the binding reaction.

The specificity of a ligand-analyte interaction can be determined through the use of negative controls, such as non-binding analytes or mutated ligands. The specificity of the interaction can be assessed by comparing the binding responses of the negative control to the binding response of the specific ligand-analyte interaction.

The reproducibility of SPR analysis can be ensured through the use of quality control measures, such as regular calibration of the instrument and use of appropriate standards. Replicate measurements can also be employed to assess the variability of the assay and ensure the reproducibility of the results.

Surface chemistry can affect the binding kinetics and specificity of ligand-analyte interactions in SPR analysis. It is important to optimize the surface chemistry for specific ligand-analyte interactions, as changes in surface chemistry can affect the orientation and stability of the ligand on the sensor chip surface.

The effect of mass transport limitation on the kinetic analysis of an interaction can be evaluated by assessing the dependence of the observed rate constants on the flow rate or concentration of the analyte. A correction factor can be applied to the observed rate constants to account for the effect of mass transport limitation.