Article pubs.acs.org/jchemeduc
Surface Plasmon Resonance Spectroscopy: A Versatile Technique in a Biochemist’s Toolbox Ray Bakhtiar* Merck Research Laboratories, West Point, Pennsylvania 19486, United States ABSTRACT: Surface plasmon resonance (SPR) spectroscopy is a powerful, label-free technique to monitor noncovalent molecular interactions in real time and in a noninvasive fashion. As a label-free assay, SPR does not require tags, dyes, or specialized reagents (e.g., enzymes− substrate complexes) to elicit a visible or a fluorescence signal. During the last two decades, SPR has been broadly applied to study of noncovalent interactions of protein−DNA, protein−cell, RNA−DNA, DNA−DNA, protein−protein, protein−carbohydrate, small molecule− macromolecule (e.g., receptor−inhibitor complex), protein−peptide, and self-assembled monolayers. In addition, SPR has been successfully applied to drug discovery ligand-fishing and clinical immunogenicity studies (i.e., to monitor an immune response against a therapeutic agent). SPR spectroscopy can address questions such as specificity of an interaction, kinetics, affinity, and concentrations of selected molecules present in a sample of interest. Given the current enhancements in hardware and software capabilities along with its ease of use and maintenance, SPR experiments can be designed for upper-level undergraduate biochemistry, biophysics, and physical chemistry laboratory courses. In this article, an overview of SPR phenomenon, instrumentation, sensor immobilization, and its selected applications is presented. KEYWORDS: Upper-Division Undergraduate, Graduate Education/Research, Biochemistry, Analytical Chemistry, Physical Chemistry, Hands-On Learning/Manipulatives, Bioanalytical Chemistry, Surface Science, Proteins/Peptides, Molecular Recognition
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INTRODUCTION
THEORY When incident light propagates in a medium of relatively higher refractive index to a medium of lower refractive index, the ray of light tends to reflect as opposed to refract. In refraction, the light ray changes direction and bends as it passes through two media. In reflection, the light beam rebounds after impinging on a surface with angles of incident and reflection being the same. When the light beam seizes to cross the boundary and is entirely reflected, a total internal reflection (TIR) occurs. A common example of TIR is in professionally cut diamonds where it renders their maximum sparkle. TIR is also critical in the operation of fiber optics where light travels along the optical fiber, reflecting off its walls, within the core of the cable with minimal loss. During the occurrence of TIR at the interface between the two nonabsorbing media, the fully reflected light beam leaks some electrical field intensity into the medium that has the lower refractive index. The leaked electrical field is referred to as the evanescent field. The evanescent field wave’s amplitude decays with distance from the interface in an exponential fashion. In SPR, the evanescent wave excites electrons within the metal layer of a metal−dielectric (the two nonabsorbing media with different refractive indices) interface, yielding surface plasmons. Surface plasmons or surface plasmon polaritrons are electromagnetic
Surface plasmon resonance (SPR) biosensors have become increasingly popular in real-time in situ investigation of reversible molecular interactions. Typical information that can be obtained from SPR experiments include: specificity of an interaction; binding affinity; binding levels; dissociation and association rate constants; and several key thermodynamic parameters, including entropy, enthalpy, and activation energy. In this regard, SPR spectroscopy has been successfully applied to a wide range of molecular systems such as vaccines,1 DNA-drug binding,2 antibody−antigen interactions,3 carbohydrate−RNA interactions,4 protein conformational changes,5 self-assembly monolayers,6 protein−carbohydrate interactions,7 immunogenicity screening of therapeutic proteins,8 and phospholipid vesicles.9 Because of technological advances, progress in experimental designs, and introduction of user-friendly software platforms by manufacturers of optical-biosensor systems, SPR units have been widely utilized in biochemical research in academic and industrial laboratories.10−12 The objective of this manuscript is to briefly introduce the utility of SPR spectroscopy to macromolecular interactions and promote its incorporation into senior-level undergraduate and beginning graduate biochemistry, biophysics, and physical chemistry laboratory courses. Introduction of theoretical or experimental SPR spectroscopy as a modern instructional tool in understanding noncovalent interactions kinetics has been reported by several academic laboratories.13−16 © 2012 American Chemical Society and Division of Chemical Education, Inc.
Published: November 20, 2012 203
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partners has to be immobilized on the gold layer surface and is referred to as the “ligand”. The second interaction partner, which flows (e.g., 1−100 μL/min) over the chip surface, is referred to as the “analyte”. The analyte is introduced by injection of a sample (e.g., 20−80 μL depending on the application) into a continuous flow of a buffer solution or the “running buffer”. The resonance angle change is proportional to changes in the mass on the sensor surface (i.e., owing to analyte−ligand noncovalent interactions). SPR protocols are label-free, require no wash steps (unlike an immunoassay method), and rely on optical detection of changes in refractive index of the running buffer. The continuous flow of the running buffer delivers the analyte molecules near the sensor’s gold surface and to the close proximity of immobilized ligand molecules. Once an analyte molecule engages in specific noncovalent interactions with the immobilized ligand molecule on the gold side of the sensor surface, a change in molecular weight leading to a change in signal occurs. Therefore, it is imperative that both interaction partners maintain their native conformational states, in order to avoid misleading results.20 The gold surface of the SPR sensor chip is often coated with an alkyl thiol monolayer to minimize nonspecific interactions and provide a structural template for subsequent derivatizations. Following the alkyl thiol monolayer, often a matrix of carboxymethylated dextran is covalently attached at a thickness of about 25−100 nm to promote a hydrophilic environment for interaction with biomolecules. Dextran is an inert moiety with respect to most biomolecules and will not interfere with the SPR signal. Generally, three approaches pertain in SPR sample immobilization and surface preparation.12,21,22 The first approach involves a direct immobilization in which the ligand or binding molecule is covalently attached to the sensor gold surface using one of several established chemistries.23 Some covalent derivatization strategies include using amines, thiols, maleimide, or aldehyde moieties. For example, the amine coupling can be performed using the free primary amine groups in lysine amino acids or the N-terminus of the protein. The immobilization chemistry should be performed in such a way to minimize nonspecific bindings, allow sufficient immobilization density on the chip surface, and most importantly, to retain the native biological and structural attributes of the analyte molecule. During the immobilization step, overcrowding (i.e., very high ligand density), aggregation, and nonspecific bindings need to be avoided. For example, an excess of ligand immobilization can potentially lead to either oligomerization or mass transport issues such as rebinding of analyte molecules to the ligand prior to sensor surface area departure. The direct coupling approach can be used for higher purity ligands (>90%) and poses disadvantages when using heterogeneous ligands. Moreover, protein sample pH and isoelectric point (pI) need to be considered in order to optimize the immobilization chemistry conditions. For example, for the amine coupling protocol, the activated carboxylated dextrans matrix reactivity tends to be higher when negatively charged. The second approach to ligand immobilization uses an indirect noncovalent coupling via a high affinity capture molecule. The affinity ligand must demonstrate sufficient binding to ensure little or no ligand dissociation. Examples of capture molecules are monoclonal antibodies (mAbs), avidin−biotin via biotinylation, or a purified tag commonly used in protein purification, such as histadine (His)-tagged recombinant proteins. In this case, the capture molecule is covalently attached to the sensor chip surface and noncovalently to the ligand of interest. The indirect or
surface waves that propagate parallel to the interface region. As the plasmon waves penetrate into the medium with the lower refractive index, any time-dependent shift in the intensity of the reflected “angle” of polarized light is recorded. The reflected light intensity is calculated as a function of the incident light angle. A shift in the SPR angle by 0.0001 degree corresponds to one unit shift in SPR signal.17
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INSTRUMENTATION Generally, there are three components to an SPR instrument (Figure 1). One critical component is the optical light source
Figure 1. The most common geometrical biosensor setup in SPR instruments (the Kretschmann configuration). The circles and inverted “Y” entities in the flow channel signify analyte and ligand molecules, respectively. In this case, the ligand is a monoclonal antibody (mAb) immobilized on the gold surface. The circle is a protein antigen (Ag) that selectively binds to the mAb. The source of light is a helium−neon (He−Ne) laser. The detector is often a charge-coupled device (CCD). A run using a control surface is highly recommended in each experiment. A good control surface can be done with the same coupling chemistry.
that is often a near-infrared high-efficiency light-emitting diode (LED). The second main component is a sensor chip with a thin layer of gold (∼40−50 nm thick) coupled to a glass layer (glass has a higher refractive index than gold). Gold has a low refractive index, it is inert in physiological buffers, and can be coated chemically to enhance surface immobilization. The gold−glass dielectric layer is coupled to a prism to achieve TIR at the surface rather than dispersion. On the other side of the sensor, facing the gold layer, is the solution side where sample flow and interaction occurs. Flow cell dimensions can vary depending on the model and design, but could be about 2.1 mm × 0.55 mm × 0.05 mm (l × w × h). The third key component is a detection system that can be a position sensing detector (PSD) or a charge-coupled device (CCD). CCD systems have been more common because of their sensitivity and dynamic range owing to their ability to store minute quantities of light until the time of measurement. A CCD uses a linear array of light-sensitive diodes or pixels to cover the range of incident light angles with a resolution of about 10−5 degrees.12,18,19 The SPR excitation scheme shown in Figure 1 is the most widely used design and is referred to as the “Kretschmann” configuration. Theoretical details of SPR spectroscopy have been elegantly described elsewhere.11
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SAMPLE IMMOBILIZATION AND PREPARATION APPROACHES The core of an SPR instrument is its biosensor chip, which serves as a biorecognition transducer (Figure 1). One of the interacting 204
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capture method offers the alternative of using less purified or crude ligand samples because the affinity capture can also serve as a purification step. Another added benefit is the generation of a more homogeneous surface layer, as all analyte molecules tend to be similarly oriented through a common site (e.g., directional or positional capture). Lastly, in affinity-capture immobilization, biological inactivation or loss of structural integrity of the ligand molecule seldom occurs.24 The third approach entails the use of membrane protein anchoring agents. For example, adsorption of lipids from liposomes or micelles to the sensor chip can support interaction studies with membrane-associated ligands.25,26 Alternatively, a bilayer attachment format can be pursued involving on-surface assembly of intact membranes. In addition, a surface-bound vesicles approach can also be used owing to its somewhat fluid nature, which can swell subsequent to analyte binding, rendering a more relevant physiologic condition. This approach is referred to as the “on-surface reconstitution” (OSR), whereby the native environment of a lipid complex on the sensor surface is re-established. The lipid−bilayer environment facilitates to maintain the proper conformation and activity of membranebound proteins once immobilized. The OSR protocol is amenable to SPR experiments on conformational analysis of transmembrane proteins, such as G-protein coupled receptors.27 As SPR measurements are label-free (i.e., do not require a chromophore attachment for signal detection), a myriad of sensor surface immobilization chemistries and molecular orientations can be used to study food components, toxins, veterinary drugs, pathogens, vitamins, therapeutic proteins, cancer biomarkers, diagnostic antibodies, environmental contaminants, cardiovascular disease markers, hormones, and oligonucleotides.11,28 The ability to investigate a diverse set of noncovalent interaction partners makes the choice of SPR sensor and its corresponding surface immobilization chemistry the prerequisite to generation of high quality data.
Figure 2. A generic sensorgram or a binding-progress curve depicting the main stages in an SPR experiment, including baseline establishment, injection of an analyte sample (open circles) into the running buffer, formation of noncovalent complexes between ligand (denoted by a letter “Y”) and analyte molecules where the ligand is immobilized on the gold side of the biosensor, equilibrium occurs between the association and dissociation rates, followed by regeneration. Herein, regeneration is defined as the process in which bound analyte molecules are removed from the sensor chip by mild-to-harsh changes in the pH of the running buffer. The cycle number of the sensor chip’s regeneration depends on the nature of the immobilized ligand. The regeneration conditions are optimized prior to the actual experiment and start with “regeneration scouting”, followed by fine-tuning.
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DATA OUTPUT AND ANALYSIS The underpinning principles of data generation and manipulation that are used in SPR spectroscopy are complex and beyond the scope of this article. Briefly, the graphical output of an SPR experiment is referred to as a sensorgram or a bindingprogress curve (Figure 2). A sensorgram is a plot of the detector response versus time. The detector response, placed on the y-axis, is the SPR angle change expressed as resonance units (RU), where 1000 RU equals to about 0.1° in angle change. Time is placed on the x-axis in a sensorgram and expressed in seconds. The angle change originates from a change in the refractive index of the sensor surface owing to alterations in mass resulting from noncovalent interactions. For the majority of proteins, binding of about 1 ng/mm2 of protein at the dextran sensor surface corresponds to a signal change of about 1000 RU with a time resolution of as low as ∼0.1 s (at the expense of generating larger size acquisition files). However, if the analyte molecule in the running buffer does not bind to the immobilized ligand, the SPR angle change will essentially be zero subsequent to a baseline correction. Figure 3 depicts multiple sets of experimental data showing a global 1:1 fit (a pseudo-first-order approximation) between two different murine monoclonal antibodies (mAb, ligand immobilized on the sensor surface) with a protein antigen (Ag, analyte). In this experiment, a commonly used simple 1:1 Langmuir-binding model was used. Multiple curves signify different concentrations of the protein antigen in order to obtain equilibrium dissociation constants. In this regard, some of the key biophysical parameters
Figure 3. Representative sensorgrams obtained from actual experiments involving two different murine monoclonal antibodies (mAb), denoted as mAb-1 and mAb-2, immobilized on a carboxymethylated dextran. The proprietary protein antigen (Ag) was passed over each immobilized mAb chip in separate runs. In each experiment, different dilutions (typically at least 4−5 concentrations, plus a baseline run) of the Ag were used to determine KD values (32−40 nM). A simple 1:1 interaction model (i.e., mAb + Ag ↔ mAb−Ag) with binding kinetics of pseud-ofirst-order was applied. The time axis in a sensorgram is expressed in seconds.
in SPR experiments include association rate constant (ka) or “onrate” (kon) in M−1 s−1, dissociation rate constant (kd) or “off-rate” (koff) in s−1, equilibrium association constant (KA; KA = ka/kd) 205
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Table 1. Summary of Some Comparative Features of Three Commercial SPR Instruments Surface Plasmon Resonance Spectroscopy Instrument Models Features
Biacore 3000
ProteOn XPR36
Biacore T200
Sample Handling
Autosampler
Auosampler
Automated for 48 h unattended operation
Molecular Weight Detection
>180 Da
>201 Da
No lower limit for organic molecules
Analytical Performance
1 pM
Sub-nM to mM levels (depending on ligand density, affinity constants, and molecular weight of both analyte and ligand)
≥10 pM
Flow Rate Range
1−100 μL/min, through flow cell; steps of 1 μL
20−200 μL
From 1 to 100 μL/min
Required Sample Volume
injected volume + 20−80 μL, depending on application
95 μL (25 μL minimum injected volume + 35 μL system dead volume + 25 μL vial dead volume + 8 μL × number of bubbles used as separators)
Injection volume plus 20−50 μL (application dependent)
Refractive Index Range
1.33−1.40 RIU
1.33−1.37 RIU
1.33−1.40 RIU
Analysis Temperature
4−40 °C (max 20 °C below ambient)
15−40 °C
4−45 °C (maximum 20 °C below ambient)
Number of Flow Cells
4 (used individually, in series, or as 2 pairs)
36 interaction spots; 42 interspot references
4
In-Line Reference Subtraction
Yes
Yes
Automatic
Dimension (L × W × H)
760 × 350 × 610 mm
500 × 950 × 580 mm
690 × 600 × 615 mm
Electric Voltage
100−120 V; 220−240 V
100−240 V
Processing Unit: Autorange 100−240 VAC (±10%), 50−60 Hz
Power Consumption
Max 580 VA
800 W
Processing Unit: max 6.3 A (at 100 VAC)
Net Weight
50 kg/110 lbs
85 kg/187 lbs
60 kg/132 lbs
Comments
Provides interaction analysis with an SPR−MALDI−MS (matrixassisted laser desorption/ ionization mass spectrometry) interface
This protein interaction array (6 × 6) system is applicable to antibody screening, protein−protein interactions, protein interface mapping, protein−nucleic acid interaction, and their kinetic characterization
Low molecular weight drug candidates to high molecular weight proteins (also DNA, RNA, polysaccharides, lipids, cells, and viruses) in various sample environments (e.g., in DMSOcontaining buffers, plasma, and serum)
Vendor Web Address (all accessed Nov 2012)
http://www.biacore.com
http://www.bio-rad.com
http://www.biacore.com
Table 2. Synopsis of Some SPR Commercial Vendors Vendors GE Healthcare Bio-Rad Horiba Scientific BioNavis Biosensing Instrument Plexera Bioscience ́ SensiQ Reichert Technologies Cole-Parmer a
Instrument Modelsa
Web Addresses (all accessed Nov 2012)
Biacore 4000 (array format), 3000, T200, X100, Biacore C, and Biacore Q (dedicated for food product analysis) ProteOn XPR36 (array system) SPRi-Lab+ and SPRi-PlexII SPR Navi 200 and SPR Navi 220A BI-2000, BI-2000G, BI-3000, BI-3000G, with simultaneous electrochemical and SPR analysis option available PlexArray Analyzer ́ Pioneer ST and SensiQ ́ Pioneer SensiQ SR7500DC, SR7000DC, and SR7000 Cole-Parmer Surface Plasmon Resonance Instrument, 115 V
http://www.biacore.com http://www.bio-rad.com http://www.horiba.com http://www.bionavis.com http://www.biosensingusa.com http://www.plexera.com http://www.discoversensiq.com http://www.reichert.com/life_sciences.cfm http://www.coleparmer.com
Source: http://www.sprpages.nl (accessed Nov 2012).
in M−1, and equilibrium dissociation constant (KD; KD = kd/ka) in M. By definition, affinity is directly proportional to ka and inversely proportional to kd. In addition, SPR spectroscopy is capable of extracting a host of thermodynamic parameters such as changes in free energy, enthalpy, and entropy from equilibrium data.12,29 A number of bimolecular interactions may not conform to a pseudo-firstorder binding kinetics or a universal fitting model. Hence, each case may need a distinct mode of analysis to address monovalent or bivalent interactions, two-state reactions, heterogeneous ligand-parallel reactions, competition reactions, bindings
with mass transfer limitations, or drifting baselines.23 All or most of these ka/kd fitting models are available to the user in the instrument software suite by most vendors. For example, the enthalpy can be calculated by determining KD at several temperatures, and plotted against the inverse-oftemperature (1/T). The slope and the y-axis intercept of the resulting line (a van’t Hoff plot) can yield changes in enthalpy and entropy, respectively. Similarly, the Eyring equation can be used with kd or ka in order to estimate the transition state energies for a given complex.24 The dissociation or association rate constants can also be used to calculate the activation 206
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temperature range studied).23 A host of experimental designs can be applied to SPR protocols in a wide range of scientific disciplines. For example, experiments could involve sequencespecific DNA−protein complex formation, assay for interleukin-8 in saliva, kinetic analysis of bovine serum albumin (BSA) and an anti-BSA antibody, or screening of several inhibitors against an enzyme. Readers are encouraged to consult the references provided herein to explore such applications.
energy of association or dissociation from an Arrhenius plot (assuming that the activation energy is unchanged over the
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COMMERCIAL EQUIPMENT A number of vendors manufacture commercial SPR instruments (Tables 1 and 2),30 with estimated costs varying between $35,000 to $300,000, depending on the application, choice of accessories, and model. We envision that in some instances, an academic discount could apply; used demo-units could perhaps be obtained at a reduced price. The utility of an SPR unit in upper-level undergraduate as well as graduate laboratories is immense and cannot be overstated. GE Healthcare (which acquired Biacore in 2006) and Bio-Rad Laboratories are among the SPR manufacturers (Table 1 and 2). For example, Figure 4 shows two examples of SPR biosensor chips that are available commercially. The biosensor chips are costly (e.g., ranging from $55 to $500 each) yet in most cases can be regenerated and reused. In addition, running buffers and immobilization
Figure 4. Representative biosensor chips used in SPR instruments. The gold surface areas are indicated using arrows. Ligands can be covalently coupled to the sensor surface via amine, thiol, aldehyde, or carboxyl groups. In some cases, the chip surface contains active carboxylic groups for covalent immobilization using primary amine groups.
Figure 5. A simplified representative workflow for a three-part laboratory session on SPR spectroscopy. 207
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the scope of this technology to scientific teaching institutions in countries with underdeveloped education infrastructures.
solutions are needed for routine use. In general, the SPR instruments are robust and seldom break down under proper care and clean up. In larger academic institutions, the SPR spectroscopy facility is used for teaching and research and often is self-funded by fees generated for sample analysis (e.g., $35/h to $125/h). A quick search of the National Science Foundation (NSF) Award database revealed a number of seed grants (∼$100,000 and up) in support of acquisition of a SPR instrument. The sources of the SPR instrument funding were programs such as Major Research Instrumentation, as well as others.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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INSTRUCTIONAL OPPORTUNITIES AND CONCLUSIONS With the advent of technological advancements, SPR spectroscopy applications have been underrepresented in teaching biochemistry, biophysics, and physical chemistry laboratories. The current software packages offer a user-friendly platform for general practitioners, in conjunction with robust hardware that can be used as a stand alone or hyphenated to other detectors such as mass spectrometry (SPR−MS),31 flow-through electrochemical detection (EC−SPR),32 or high-performance liquid chromatography (HPLC−SPR).33 While the SPR technology has been popular in analytical laboratories, it has several limitations. Although a variety of immobilization chemistries has been introduced, any approach that affects the biological functionality of the analyte precludes the use of SPR spectroscopy. Small molecules (e.g.,
