Surface Plasmon Resonance (SPR) Troubleshooting Guide

Below you will find some starting pionts for troubleshooting issues you are facing during SPR testing.

Surface Plasmon Resonance (SPR) is a powerful analytical technique used in the fields of biochemistry, biophysics, and material science to study interactions between molecules. When working with SPR experiments, various issues and problems can arise. This troubleshooting guide will help you identify and address common problems encountered during SPR experiments.

1. Baseline Issues:

Issue: The baseline (signal in the absence of analyte) is unstable or drifting.

Solution:

• Ensure that the buffer is properly degassed to eliminate bubbles.
• Check for leaks in the fluidic system that may introduce air or bubbles.
• Use a fresh buffer solution to avoid contamination.
• Optimize the flow rate, temperature, and stabilization time settings.
• Calibrate the instrument and sensor chips if needed.

Issue: The baseline exhibits noise or fluctuations.

Solution:

• Confirm that the instrument is located in a stable environment with minimal temperature fluctuations and vibrations.
• Ensure proper grounding to minimize electrical noise.
• Use a clean, filtered buffer solution.
• Check for contamination on the sensor surface and clean or regenerate if necessary.
• Verify the integrity of the reference channel.

 2. Signal Issues:

Issue: There is no significant change in the signal upon analyte injection.

Solution:

• Verify that the analyte concentration is appropriate for the experiment.
• Check the ligand immobilization level, which may be too low.
• Ensure that the analyte and ligand are compatible and the interaction is expected.
• Confirm the functionality and integrity of the ligand.
• Adjust the flow rate, temperature, or running buffer as needed.
• Evaluate the quality of the sensor surface and regenerate if necessary.

Issue: The signal change upon analyte injection is weak.

Solution:

• Increase the analyte concentration, if feasible.
• Optimize the ligand immobilization density.
• Extend the association time or adjust the flow rate.
• Check for possible issues with the ligand's orientation or coupling chemistry.
• Evaluate the stability of the ligand.

Issue: The sensorgram reaches saturation too quickly, making it difficult to determine kinetic parameters.

Solution:

• Reduce the analyte concentration or injection time.
• Optimize ligand immobilization to achieve lower ligand density.
• Increase the flow rate or temperature to decrease mass transport effects.
• Consider using a Steady State Affinity for analysis if applicable.

Issue: High levels of non-specific binding are observed.

Solution:

• Block the sensor surface with a suitable blocking agent (e.g., BSA or ethanolamine) before ligand immobilization.
• Optimize the regeneration step to efficiently remove bound analyte.
• Consider alternative ligand immobilization strategies (e.g., site-directed immobilization).
• Use a lower analyte concentration or modify the running buffer to reduce non-specific binding.

Issue: Regeneration does not completely remove bound analyte, causing carryover effects.

Solution:

• Optimize the regeneration conditions, including pH, ionic strength, and regeneration buffer composition.
• Increase the flow rate or regeneration time.
• Ensure that the sensor surface is properly cleaned and regenerated between runs.Consider using Single-Cycle Kinetics for test.

Issue: Analyte or ligand has solubility problems in the running buffer.

Solution:

• Optimize the sample preparation process to improve solubility.
• Use different buffer conditions or additives to enhance solubility.
• Consider alternative analyte or ligand formats if solubility remains problematic.
  
  

SENSOR SURFACE MAINTENANCE:

Issue: The sensor surface degrades over time.

Solution:

• Follow the manufacturer's guidelines for sensor surface regeneration and maintenance.
• Minimize exposure to harsh chemicals or extreme pH conditions.
• Store and handle sensor chips carefully to avoid physical damage.
• Monitor the sensor surface's condition during experiments and regenerate as needed.

Issue: Data from replicate experiments are inconsistent.

Solution:

• Standardize the immobilization procedure to ensure uniform ligand coverage.
• Use consistent sample handling techniques.
• Verify the stability of the ligand over time.
• Ensure that the instrument is properly calibrated and maintained.
• Review the experimental protocol for any inconsistencies.
• Check the state of the sample (whether it is completely dissolved or precipitation)

Remember that troubleshooting SPR experiments may require a systematic approach, including method optimization, equipment maintenance, and careful experimental design. Consulting instrument manuals, seeking advice from experienced researchers, and collaborating with colleagues can also be valuable resources in resolving issues with your SPR experiments.