A Surface Plasmon Resonance (SPR) sensorgram, which is a plot of SPR response vs. time generated from an SPR instrument, can reveal whether there is a binding event between an analyte (e.g. antibody) and a ligand (e.g. protein) and whether the binding is specific. Furthermore, it contains valuable kinetic, affinity, and concentration information pertaining to the analyte and ligand of interest (Figure 1). The analyte is the biomolecule or molecule being investigated and the ligand is the recognition element being immobilized on the SPR sensor. This blog will discuss how a sensorgram is obtained and how it is generated from Affinité’s P4SPRTM. Please see our Affinite Technote – Sensorgram for more details on how to qualitatively interpret a sensorgram, features to look for in a good quality sensorgram, and sensorgrams generated by Affinité’s P4SPR.
Figure 1. Schematic of an SPR sensor and the wealth of information that can be obtained. The reverse setup can be done as well (i.e. immobilized antibody as ligand with protein as analyte). The SPR effect generated by the reflected incident light produces a sensitive region responsive to refractive index changes.
In an SPR experiment, a dip or minimum in the reflectance spectrum (intensity of reflection vs. angle or wavelength) signifies the angle or wavelength at which the light has been absorbed by the surface plasmons. Once there is a change in the refractive index at the sensing layer caused by bound molecules, causing surface structural changes, the dip would shift, indicating a change in the angle or wavelength of resonance. Figure 2 is an example of wavelength interrogation. The increase in refractive index causes an increase in wavelength shift in resonance.
Figure 2. Reflectance spectra (light polarizing ratio) of solutions with different RI on an Au SPR sensor in wavelength interrogation mode .
The detected change in the reflected angle or wavelength is proportional to the amount of newly bound analytes at the surface due to an increase in mass. When this change in SPR response is plotted with time, a sensorgram is produced (Figure 3). The relative SPR response in a sensorgram is proportional to the amount of bound analytes.
Figure 3. A typical sensorgram. On/off rates (kon and koff) can be determined from various parts of a sensorgram and dissociation constant (KD) can be determined by the ratio of koff/kon. (1) Baseline, (2) Association, (3) Steady-state, (4) Dissociation, (5) Regeneration. RU represents resonance units.
A sensorgram is composed of five phases (see Figure 3): baseline, association, steady-state, dissociation, and regeneration. The initial phase is the baseline. A running or flow buffer is used to condition the sensor surface and check for any sensor system instability. It is crucial to have a flat baseline because any drift, injection spike, and high buffer response is an indication that the system should be checked and cleaned . Standard running buffers include phosphate-buffered saline and HEPES-NaCl  .
The second phase is association, where analytes (A) begin to bind to immobilized ligands. It is indicated by the initial sharp rise of the SPR signal in the sensorgram and it is ideally a single exponential curve .
The steady-state phase occurs at the top flat portion of the sensorgram where the net rate of bound analytes is zero.
The dissociation phase begins when the analyte solution is replaced by a wash buffer, which causes the specific bonds between the analytes and ligands to break. It is represented by the downward sloped curve after the steady-state phase.
Lastly, the regeneration phase consists of flowing a low pH buffer such as glycine in order to obtain the same SPR baseline signal as the beginning of the experiment . It is important to establish a steady baseline signal to indicate that the sensor system is free of bound analytes and non-specifically adsorbed molecules, has stability (having intact and functional receptors), and that no other effects such as temperature or surface chemistry changes would affect the next set of SPR measurement, as SPR sensing surfaces can be reused numerous times.
Once the sensorgram data are obtained, the rate of association (kon), dissociation (koff), and dissociation constant (KD) can be determined by fitting the data into a suitable binding model. Therefore, kinetic and affinity data can be obtained readily from an SPR experiment.
For an SPR instrument with wavelength interrogation mode such as the P4SPRTM, the detector detects the change in wavelength of the absorption band minimum (or dip) upon introduction of a sample which could change the refractive index near the sensing layer. As the sample continues to flow across the sensing surface, the wavelength position is continuously detected and recorded in the SPR instrument in real-time. The resulting sensorgram is a plot of delta wavelength (referenced against a solution at a specific time) vs. time. Then, any kinetic and affinity data can be calculated by fitting the sensorgram data into a suitable binding model, as mentioned above. Binding models are already integrated with the P4SPRTM system so there is no need to obtain a separate modelling software. Figure 4 summarizes how P4SPRTM converts the SPR signal into a sensorgram.
Figure 4. (1) The P4SPRTM detects a change in refractive index (RI) in terms of change in wavelength (delta lamda) or shift in absorption band minimum; (2) Raw data can be saved as the change in wavelength over time. (3) The P4SPRTM plots the SPR shifts (delta lamda) with respect to time to provide a sensorgram .
From this blog, there is hopefully a deeper sense of how SPR sensorgrams are generated and what occurs in each phase of the sensorgram. Affinité Instruments’ P4SPR’s portable wavelength-mode instrument provides dependable data acquisition and fitting software to meet your research needs.
Thank you for reading and watch out for additional blogs discussing SPR related topics such as SPR vs ELISA, wavelength vs. angle interrogation, planar SPR vs. nanoparticle localized SPR, multiple channel SPR, and integrating pumps with SPR.
Contact us to learn more about SPR at firstname.lastname@example.org
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