How to start a SPRi experiment - Horiba · SPRi-Biochips and SPRi-Slides are especially designed to...
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Which biochip sensor to select? Microarrays are usually prepared with robotic spotters which deposit nanoliter droplets of ligand solutions on a solid surface in a pre-defined pattern. Manual spotting is also a potential solution. Self Assembled Mono or Multilayer (SAM) surface chemistries on gold sensors are commercially available and allow the immobilization of spots of native or conjugated ligands directly on the surface. Ligands of interest can be proteins (antibody, antigen, peptides), nucleic acids (DNA, RNA), saccharides, in their native states or conjugated with biotin or GST-tag. Immobilization by direct coupling of ligands on a functionalized «ready-to-use» surface is available: - via ligands’ amino group on CS or CO biochips - via biotinylated ligands on extravidin CSe, COe surfaces - via GST-tagged ligands on anti-GST antibody derivatized CTg surface How to immobilize ligands in an array format? Working in an array format allows multiplexed analysis by multiplying the number of ligands and also by using various spotting conditions (concentration, dilution buffer, incubation time…). Usually, preliminary experiments are needed to adjust the immobilization parameters based upon the nature and stability of the ligands. When conditions are optimized, selected ligands can be immobilized as mentioned above by direct spotting on the sensor chip. A control (or negative control) spot has to be part of the array and must be a molecule with a nature similar to that of the ligand of interest. Thus, refractive index changes or non-specific interaction signal on the control spots will be subtracted from the other ligands signal to quantify only the specific interaction. A SPRi-Arrayer TM , adapted for the SPRi-sensor, creates a defined array pattern directly on the functionalized biochip surface before introducing the chip into the SPRi systems (OpenPlex TM or XelPlex TM ). Figure 1: Biochip preparation Table 1: Surface chemistries available Figure 2: Example of SPRi image of 100 ligands spotted on a ready-to-use CO SPRi-Biochip™ To prepare a SPRi (Surface Plasmon Resonance imaging) experiment, there are several questions to consider beforehand: - Which biochip sensor to select? - How to immobilize ligands in an array format? - Which running buffer is the most appropriate? - How to prepare analyte samples for injection? - Is it possible to regenerate the ligands? - Is it possible to re-use a biochip? - How to analyze interaction data? A SPR experiment is composed of several steps, from ligands immobilization to data analysis. The choice of biochip surface chemistry, running buffer and regeneration solution depends on the nature of the ligands and potential analyte. Surface Plasmon Resonance imaging How to start a SPRi experiment Application Note Biology AN SPRi 35
Transcript of How to start a SPRi experiment - Horiba · SPRi-Biochips and SPRi-Slides are especially designed to...
Which biochip sensor to select?
Microarrays are usually prepared with robotic spotters which deposit nanoliter droplets of ligand solutions on a solid surface in a pre-defined pattern. Manual spotting is also a potential solution. Self Assembled Mono or Multilayer (SAM) surface chemistries on gold sensors are commercially available and allow the immobilization of spots of native or conjugated ligands directly on the surface.
Ligands of interest can be proteins (antibody, antigen, peptides), nucleic acids (DNA, RNA), saccharides, in their native states or conjugated with biotin or GST-tag. Immobilization by direct coupling of ligands on a functionalized «ready-to-use» surface is available:
- via ligands’ amino group on CS or CO biochips- via biotinylated ligands on extravidin CSe, COe surfaces- via GST-tagged ligands on anti-GST antibody derivatized CTg
surface
How to immobilize ligands in an array format?
Working in an array format allows multiplexed analysis by multiplying the number of ligands and also by using various spotting conditions (concentration, dilution buffer, incubation time…).Usually, preliminary experiments are needed to adjust the immobilization parameters based upon the nature and stability of the ligands. When conditions are optimized, selected ligands can be immobilized as mentioned above by direct spotting on the sensor chip. A control (or negative control) spot has to be part of the array and must be a molecule with a nature similar to that of the ligand of interest. Thus, refractive index changes or non-specific interaction signal on the control spots will be subtracted from the other ligands signal to quantify only the specific interaction.
A SPRi-ArrayerTM, adapted for the SPRi-sensor, creates a defined array pattern directly on the functionalized biochip
surface before introducing the chip into the SPRi systems (OpenPlexTM or XelPlexTM).
Figure 1: Biochip preparation
Table 1: Surface chemistries available
Figure 2: Example of SPRi image of 100 ligands spotted on a ready-to-use CO SPRi-Biochip™
To prepare a SPRi (Surface Plasmon Resonance imaging) experiment, there are several questions to consider beforehand: - Which biochip sensor to select? - How to immobilize ligands in an array format? - Which running buffer is the most appropriate? - How to prepare analyte samples for injection? - Is it possible to regenerate the ligands? - Is it possible to re-use a biochip? - How to analyze interaction data?A SPR experiment is composed of several steps, from ligands immobilization to data analysis. The choice of biochip surface chemistry, running buffer and regeneration solution depends on the nature of the ligands and potential analyte.
Surface Plasmon
Resonance imaging
How to start a SPRi experiment
Application NoteBiology
AN SPRi 35
Which running buffer is the most appropriate?
According to the ligand/analyte interaction study, running buffer composition is expected to improve specific binding. Salted buffers like PBS, HEPES, or citrate are recommended. Buffer composition, concentration or pH must be adjusted.Binding experiment performance may be temperature dependant. By using the XelPlexTM system equipped with a temperature-controlled flow cell, measurements can be performed at a controlled temperature between 15-40°C.
How to prepare samples for injection?
It is essential to dissolve or dilute the analyte molecule solution in the running buffer to avoid refractive index changes. For a first interaction analysis study, it is recommended to inject different analyte concentrations from the lowest to the highest.Complex mixtures like serum, blood or others can also be injected through the SPRi system without pre-treatment.The injection of a control sample like running buffer or a known analyte without any interaction with the ligand is advised.The injection of the solution containing the analyte molecule must run long enough to increase the ligand analyte association and reach equilibrium. Similarly, enough time must be allowed during the dissociation step for a good determination of affinity constants.
Is it possible to regenerate the ligands?
Most of immobilized ligands are able to be regenerated. This means breaking the specific binding between ligand and analyte, using different proposed solutions:- Cation chelators: EDTA- High ionic strength: NaCl 1 M, MgCl2 2 M- Low pH: Glycine-HCl 100 mM pH 2, HCl 10-100 mM- High pH: Glycine-NaOH 100 mM pH 9.5, NaOH 10-200 mM
Our SAM surfaces are designed to tolerate these regeneration solutions and with ligands being properly anchored to the sensor surface, several successive interaction-regeneration steps are made possible.
Is it possible to re-use a biochip?
If a biochip is stored in good conditions (in buffer, coated with a conservation solution or dry, at 4 °C or at room temperature
according to the nature of the ligands immobilized on the chip), the sensor is suitable for re-use for a new interaction study. Appropriate storage allows the use of the sensor chip several times without any loss of activity.
How to analyse interaction data?
SPRi quantification of analyte binding on the biochip
The SPRi-Analysis software quantifies the surface coverage within the regions of interest and automatically generates reports to facilitate data interpretation.
Determination of kinetic and equilibrium constants
Dedicated software can also compute data from biomolecular interactions for kinetic parameter determination. Affinity constants including association and dissociation constants or equilibrium constants are available using the ScrubberGen software.
Complementary analysis
SPRi-Biochips and SPRi-Slides are especially designed to be used in the XelPlexTM and the OpenPlexTM apparatus. However their design opens the door to use them with other techniques such as MALDI-mass spectrometry, ellipsometry, fluorescence, AFM, etc.
Conclusion
The preparation of a SPRi experiment using new immobilized ligands for analyte interaction studies may be facilitated by using multiplex format sensors to establish the best interaction conditions. We present here advice for the users and a short overview of the results which can be easily reached using a SPRi system, for real time and label-free, specific and sensitive interactions.
HORIBA Scientific functionalized SPRi sensor and SPRi analysis technology enable easy, rapid and precise studies of ligand/analyte interactions, within a multiplex format, in real time and without labeling.
[email protected] www.horiba.com/scientific
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Figure 3: Example of kinetic curve
Figure 4: Data fitting with ScrubberGen
