Handbook of Surface Plasmon Resonance

by Richard B M Schasfoort (Editor)

Overview

Surface plasmon resonance (SPR) plays a dominant role in real-time interaction sensing of biomolecular binding events and with the biosensor field expanding more applications are being found. In response to the market, an update to the original title which was published in 2008 is now appropriate. With over fifty percent of the material being updated, this book provides a total system description including optics, fluidics and sensor surfaces.

Spanning theory, instrumentation and applications, it covers all the relevant issues for the practicing researcher. Unlocking the potential for SPR by showing highly exciting and unique opportunities for unraveling the functional relationships of complex biological processes, it is intended for a wide audience. A comprehensive and accessible source it contains expanded tutorial details to inspire students and guide them in this technology.

Product Details

ISBN-13:	9781788011396
Publisher:	RSC
Publication date:	05/24/2017
Sold by:	Barnes & Noble
Format:	eBook
Pages:	524
File size:	10 MB

About the Author

Richard B.M. Schasfoort is currently head of the Biochip Group at the University of Twente (UT), the Netherlands, as well as director of the BioMultianalyte Detection program, strategic research orientation at MESA+ UT. He earned his MSc in biotechnology from the University of Groningen, the Netherlands and his PhD in biosensors. His research interest is focused on the coupling of lab-on-a-chip liquid handling to bio- and chemical sensors for the determination of multiple analytes in complex matrices. Relevant application areas include disease monitoring, biomarkers and food safety. Richard Schasfoort is founder of IBIS Technologies BV that has committed itself to SPR imaging technology. Richard B.M. Schasfoort has (co-)authored about 60 peer reviewed articles and 15 patent applications and was in 2002 awarded a grant for young scientists (vernieuwingsimpuls) by Netherlands Organisation for Scientific Research (NWO). Dr Tudos was senior scientist at the Biochip Group, University of Twente and now Shell Global Solutions International BV, P.O. Box 38000, 1030 BN Amsterdam, the Netherlands, She earned her PhD at the University of Amsterdam in analytical chemistry. Her post doctoral study at Iowa State University focused on electrochemical detection in miniaturized devices. Her major interest is translating scientific concepts, like SPR-based sensors or lab-on-a-chip devices into products for example for application in the food, health or defence industry. Dr Tudos is member of the editorial board of Food Chemistry (Elsevier) and head of the Analytical Chemistry Section of the Royal Dutch Society of Chemists.

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Handbook of Surface Plasmon Resonance

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By Richard B. M. Schasfoort

RSC Publishing

Copyright © 2017 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-1-78801-139-6

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CHAPTER 1

Introduction to Surface Plasmon Resonance

RICHARD B. M. SCHASFOORT

Medical Cell BioPhysics Group (MCBP), MIRA Institute, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands Email: [e-mail]

1.1 Introduction to Surface Plasmon Resonance

During the years following the introduction of the first commercial surface plasmon resonance (SPR) instrument (Biacore) in 1990, the number of publications that include data collected from commercial biosensors increased to more than 20 000 papers by 2016 (PubMed data), as shown in Figure 1.1.

Not only the number of publications but also advances in the technology led to an improvement in detection sensitivity by roughly 100-fold up to 10-8 RIU (refractive index unit) or 0.01 RU (resonance or response unit). The range of affinity and kinetic data that can be determined has been extended at least 200-fold as a consequence of the increased sensitivity and due to improvements in data analysis. The number of independent channels or spots grew from four channels in 1990 (Biacore) to at least 192 flow-controlled spots in the new IBIS MX96 imaging instrument and more than 10 000 drop-spotted ligands in SPR imaging instruments from various manufacturers (e.g. Plexera). The carboxymethylated dextran surface introduced in 1990, still the first choice for many applications, has been complemented with a range of other surfaces (see Chapter 6). Systems for dedicated applications have been introduced by various manufacturers as complements to all-purpose research instrumentation, and the impact of SPR biosensors on biomolecular interaction studies is growing continuously. With improved experimental design and advanced data analysis methods, high-quality data for the determination of kinetic parameters of biomolecular interaction phenomena can be obtained. These data promise additional insights not only into the affinity of biomolecular pairs but also into the mechanisms of molecular binding events, which will be important for function–regulatory protein interaction studies in order to unravel the exciting processes in living species.

1.2 What is a Biosensor?

The term biosensor was introduced around 1975, relating to the exploitation of transducer principles for the direct detection of biomolecules at surfaces. Currently the most prominent example of a biosensor is the glucose sensor, reporting glucose concentration as an electronic signal, e.g. based on a selective enzymatic process. According to the current definition, in biosensors the recognition element (ligand) of the sensor or the analyte should originate from a biological source.

Biosensors are analytical devices comprising a biological element (tissue, microorganism, organelle, cell receptor, enzyme, antibody) and a physicochemical transducer. Specific interaction between the target analyte and the biological material produces a physicochemical change detected by the transducer. The transducer then yields an analog electronic signal proportional to the amount (concentration) of a specific analyte or group of analytes.

Anthony P. F. Turner (Editor, Biosensors and Bioelectronics)

Application of SPR-based sensors to biomolecular interaction monitoring was first demonstrated in 1983 by Liedberg et al. A historical overview of the use of the phenomenon for biosensor applications is given in Chapter 2. To understand the excitation of surface plasmons, let us start with a simple experiment.

1.2.1 A Simple Experiment

Consider the experimental set-up depicted in Figure 1.2. When polarized light is shone through a prism on a sensor chip with a thin metal film on top, the light will be reflected by the metal film acting as a mirror. On changing the angle of incidence and monitoring the intensity of the reflected light, one observes that the intensity of the reflected light passes through a minimum (Figure 1.2, line A). At this angle of incidence, the light will excite surface plasmons, inducing surface plasmon resonance, causing a dip in the intensity of the reflected light. Photons of p-polarized light can interact with the free electrons of the metal layer; inducing a wavelike oscillation of the free electrons, thereby reducing the reflected light intensity.

The angle at which the maximum loss of the reflected light intensity occurs is called resonance angle or SPR-dip. The SPR-dip angle is dependent on the optical characteristics of the system, e.g. on the refractive indices of the media on both sides of the metal, usually gold, and are explained in detail in Chapter 2. Whereas the refractive index at the prism side does not change, the refractive index in the immediate vicinity of the metal surface will change when accumulated mass (e.g. proteins) adsorb on the thin gold layer. Hence the SPR conditions are changing and the real-time shift of the SPR angle is suited to provide information on the kinetics of, e.g., protein adsorption on the surface.

1.2.2 From Dip to Real-time Measurement

Surface plasmon resonance is an excellent method for monitoring changes in the refractive index in the near vicinity of the metal surface. When the refractive index changes, the angle at which the intensity minimum is observed will shift as indicated in Figure 1.2, where line A depicts the original plot of reflected light intensity versus incident angle, and B is the plot after the change in refractive index. SPR is not only suited to measure the difference between these two states, it can also monitor the change in time, if one follows in time the shift of the resonance angle at which the dip is observed. Figure 1.3 depicts the shift of the dip in time, a so-called sensorgram. If this change is due to a biomolecular interaction, the kinetics of the interaction can be studied in real time.

SPR sensors investigate only in a very limited vicinity in a fixed volume at the metal surface. The penetration depth of the electromagnetic field (so-called evanescent field) at which a signal is observed typically does not exceed a few hundred nanometers, decaying exponentially with the distance from the metal layer at the sensor surface. The penetration depth of the evanescent field is a function of the wavelength of the incident light, as explained in Chapter 2.

SPR sensors lack intrinsic selectivity: all refractive index changes in the evanescent field will result in a change of the signal. These changes can be due to a refractive index difference of the medium, e.g. a change in the buffer composition or concentration, or temperature effects; non-specific and specific adsorption of material on the sensor surface can also cause refractive index changes. The amount of adsorbed species can be determined after injection of the original baseline buffer as shown in Figure 1.3. To allow selective detection at an SPR sensor, its surface needs to be modified with ligands suited for selective capturing of the target compounds (the analyte) but which are not prone to adsorb any other components present in the sample or buffer media.

1.3 How to Construct an SPR Assay

Now we have a basic understanding of the SPR signal and how to measure it in time. We know that the sensor surface needs to be modified to permit selective capturing and thus selective measurement of a target compound or analyte. In the following, we will learn more about an SPR measurement. First, the steps of an SPR assay will be discussed from immobilization through analysis to regeneration in a measurement cycle. Next, we become acquainted with standard 1 : 1 interaction models, including kinetics, followed by examples of assay formats. Finally, a short outlook is provided on the basics of the instrumentation.

1.3.1 The Steps of an Assay

In the simplest case of an SPR measurement, a target component in solution or analyte is captured by the capturing element or so-called ligand. The ligand is permanently or temporarily immobilized on the sensor surface (Figure 1.4) prior to the measurement of the analyte interaction. Various sensor surfaces with immobilized ligands are commercially available, and many more can be custom made, as explained in Chapter 6 by Erk Gedig.

In the simplest case, the event of capturing the analyte by the ligand gives rise to a measurable signal; this is called direct label free detection. Figure 1.5 shows the sensor signal step-by-step in the measurement cycle with direct detection.

Each measurement starts with conditioning of the sensor surface with a suitable buffer solution (1). It is vital to have a stable baseline before the capturing event starts. Then common mode effects such as temperature or hydrogel swelling no longer fluctuate. At this point, the sensor surface contains the active ligands, ready to capture the target analytes. On injecting the solution containing the analyte (2), these molecules are captured on the surface. Other components of the sample might also adhere to the sensor surface; without the selection of a suitable ligand, this adherence will be non-specific. In this step, the adsorption kinetics of the analyte molecule can be determined in a real-time measurement. Next, buffer is injected onto the sensor and the nonspecifically bound components are flushed off during the so-called dissociation phase (3). As indicated in Figure 1.5, the accumulated mass can be obtained from the SPR response (ΔR). The double-headed arrow represents the specific response (ΔR). Also in this step, dissociation of the analyte starts, enabling the kinetics of the dissociation process to be studied. Finally, a regeneration solution is injected, which breaks the specific binding between analyte and ligand (4). If properly anchored to the sensor surface, the ligands remain on the sensor, whereas the target analytes are quantitatively removed. In order to perform many tests with the same sensor surface, it is vital to use a regeneration solution that leaves the activity of the ligands intact, as the analysis cycle is required to take place repeatedly hundreds, sometimes even thousands, of times. Again, buffer is injected to condition the surface for the next analysis cycle. If the regeneration is incomplete, remaining accumulated mass causes the baseline level to be increased. A typical example of a repeated injection of the same analyte (8×) over eight channels/spots is shown in Figure 1.6. The instrument is generating raw data, which should be zeroed and referenced to obtain a "clean" sensorgram of the biomolecular interaction only [to "scrub" the data that can be performed in Scrubber 2.0 software (see Chapter 9)].

A typical referenced and zeroed sensorgram is shown in Figure 1.7 with the phases of an analysis cycle of Figure 1.5.

Referencing means that at least two channels/spots are measured, one with ligand and the other without ligand as a reference channel. The referenced (= subtracted) signal is shown in Figure 1.7 and quality features are given in Figure 1.8.

1. Baseline phase: Initially, baseline buffer is in contact with the sensor surface to establish the baseline. For sensor calibration purposes, the injection of a calibration liquid (e.g. a tuned glycerol percentage spiked in baseline buffer) can be incorporated in this phase (not shown) in order to compensate for the RI bulk shift of the analyte buffer.

2. Association phase: Sample containing the target compound is injected; the capturing elements on the sensor surface bind the target compound, resulting in complex formation.

3. Dissociation phase: Upon injection of baseline=system buffer, target compounds (and also nonspecifically bound molecules) dissociate from the surface.

4. Regeneration phase: The regeneration solution (e.g. low-pH buffer) is injected to remove the remaining bound target compounds (not shown in Figure 1.8).

After this phase, the cycle is completed and a new experiment can start by establishing the baseline again. If remaining accumulated mass is present, the baseline level will increase. Because of the different refractive indices of regeneration liquids and the difficulty of referencing caused by swelling or shrinking of the protein-loaded hydrogel (often by the pH shift–salt step), it is not necessary to show the real-time data of the regeneration process. It is sufficient to measure the baseline shift in the system buffer after the regeneration phase. Regeneration buffer scouting protocols as explained in Section 1.5.2 can be applied to find the optimal regeneration conditions.

Often SPR measurements are carried out to determine the kinetics of a binding process. For realistic results, it is vital to avoid immobilization changing the ligand in such a way that would influence its strength or affinity toward the target (analyte) compound. In addition, kinetic experiments can provide information on the equilibrium dissociation constants and the rate constants (on- and off-rate) and on the thermodynamics, e.g. on the binding energy of processes. Examples of kinetic binding curves are provided in Chapter 4 by Arnoud Marquart and the effects of distributed ligand immobilization and kinetic theory can be found in Chapter 5 by Huaying Zhao and Peter Schuck.

1.3.2 Concentration Determination

Apart from kinetic and thermodynamic studies, SPR measurements can also be used for the determination of the concentration of an analyte in a sample (quantitative analysis). In this case, first different concentrations of the analyte are applied in separate analysis cycles. The sensorgrams measured at different concentrations give an overlay plot similar to that depicted in Figure 1.9, with the plateaus of the association step increasing at increasing analyte concentration. However, at a certain high analyte concentration the SPR response saturates.

A calibration curve can be constructed by simply plotting the response (ΔR) after a certain time interval (t) versus the concentration, but it depends on the affinity of the interaction. For example, if the concentration of the analyte in the sample is very high, the undiluted sample will yield results on the upper plateau range of the calibration curve. However, diluted solutions might yield points along the lower, concentration-dependent sections of the calibration curve and the concentration of the target compound can be determined.

A calibration-free concentration analysis (CFCA) can be carried out if the binding of the molecules is partly mass transport limited. This is explained briefly in Section 1.4.2 and in Chapter 7 by Robert Karlsson et al. (Section 7.3.3 and Figure 7.12).

As mentioned above, SPR sensing means detection of refractive index changes at the sensor surface, which in practice translates to the amount of mass deposited at the sensor surface. Direct detection is only possible if the capturing event of the analyte brings about measurable refractive index changes. This is easier to achieve if the molecular weight (MW) of the analyte is large (i.e. around 1000 Da or higher). However, for small molecules to produce a measurable refractive index change, large numbers would be required, making the analysis intrinsically less sensitive. If the analyte is a small molecule (MW<1000 Da), the response will be close to the system noise of the instrument (see Chapter 7). Only if high ligand densities are applied in combination with a highly sensitive instrument will the low-MW compound shift the refractive index sufficiently for SPR detection.

Detection of small molecules can also be carried out using a different strategy. Most often, small molecules are detected in a competition or inhibition assay format. In all immune assay formats, not only is the lower detectable concentration limited, but also the physical number of immobilized elements on the sensor surface, which provide a maximum limiting value. Discussions of the different assay formats can be found in Section 1.5 and detection levels in Chapter 7.

(Continues...)

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Table of Contents

Introduction to Surface Plasmon Resonance;
History and Physics of Surface Plasmon Resonance;
Surface Plasmon Resonance Instruments;
SPRpages- Getting a Feeling for the Curves;
Detailed Analysis of Kinetic Binding Traces with Distributions of Surface Sites;
Surface Chemistry in SPR Technology;
Fragment and Low Molecular Weight Compound Analysis;
Combined Antibody Characterization: High-throughput Ranking, Binning and Mapping;
Treating Raw Data: Software for SPR Applications;
Biolayer Interferometry (Octet) for Label-free Biomolecular Interaction Sensing;
Strategies for Building Protein-Glycosaminoglycan Interaction Networks Combining SPRi, SPR and BLI;
Future Trends in SPR Technology