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Surface Plasmon Resonance Spectroscopy: A Versatile Technique in a


Biochemists 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 uorescence signal. During
the last two decades, SPR has been broadly applied to study of noncovalent interactions of proteinDNA, proteincell, RNADNA,
DNADNA, proteinprotein, proteincarbohydrate, small molecule
macromolecule (e.g., receptorinhibitor complex), proteinpeptide,
and self-assembled monolayers. In addition, SPR has been successfully
applied to drug discovery ligand-shing and clinical immunogenicity
studies (i.e., to monitor an immune response against a therapeutic
agent). SPR spectroscopy can address questions such as specicity of
an interaction, kinetics, anity, 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

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 reect as opposed to refract. In refraction, the
light ray changes direction and bends as it passes through two
media. In reection, the light beam rebounds after impinging
on a surface with angles of incident and reection being the
same. When the light beam seizes to cross the boundary and
is entirely reected, a total internal reection (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 ber optics where light travels along the optical
ber, reecting o 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 reected light beam leaks
some electrical eld intensity into the medium that has the
lower refractive index. The leaked electrical eld is referred to
as the evanescent eld. The evanescent eld waves amplitude
decays with distance from the interface in an exponential fashion. In
SPR, the evanescent wave excites electrons within the metal layer of
a metaldielectric (the two nonabsorbing media with dierent
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: specicity of an
interaction; binding anity; 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 antibodyantigen interactions,3 carbohydrateRNA interactions,4 protein conformational changes,5 self-assembly monolayers,6 proteincarbohydrate 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.1012 The objective of this manuscript is to briey
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.1316
2012 American Chemical Society and
Division of Chemical Education, Inc.

Published: November 20, 2012


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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
ows (e.g., 1100 L/min) over the chip surface, is referred to as
the analyte. The analyte is introduced by injection of a sample
(e.g., 2080 L depending on the application) into a continuous
ow of a buer solution or the running buer. The resonance
angle change is proportional to changes in the mass on the sensor
surface (i.e., owing to analyteligand 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 buer. The continuous ow of
the running buer delivers the analyte molecules near the
sensors gold surface and to the close proximity of immobilized
ligand molecules. Once an analyte molecule engages in specic
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 nonspecic 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 25100 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 rst 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
nonspecic bindings, allow sucient 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 nonspecic 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 anity capture molecule. The
anity ligand must demonstrate sucient binding to ensure little
or no ligand dissociation. Examples of capture molecules are
monoclonal antibodies (mAbs), avidinbiotin via biotinylation,
or a puried tag commonly used in protein purication, 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
reected angle of polarized light is recorded. The reected
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

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 conguration). The circles and inverted
Y entities in the ow 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 heliumneon
(HeNe) 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-eciency light-emitting diode


(LED). The second main component is a sensor chip with a
thin layer of gold (4050 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 buers, and can be
coated chemically to enhance surface immobilization. The
goldglass 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
ow 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 105 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 conguration. Theoretical details
of SPR spectroscopy have been elegantly described elsewhere.11

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
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capture method oers the alternative of using less puried or


crude ligand samples because the anity capture can also serve
as a purication step. Another added benet 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 anity-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 uid
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 lipidbilayer 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
buer, 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 dened 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 buer. The cycle number of the sensor chips
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 ne-tuning.

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. Briey, 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 les). However, if the analyte molecule in
the running buer 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 t (a pseudo-rst-order approximation) between two
dierent 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 dierent 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 dierent 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, dierent
dilutions (typically at least 45 concentrations, plus a baseline run) of
the Ag were used to determine KD values (3240 nM). A simple 1:1
interaction model (i.e., mAb + Ag mAbAg) with binding kinetics
of pseud-orst-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 M1 s1, dissociation rate constant (kd) or o-rate
(koff) in s1, equilibrium association constant (KA; KA = ka/kd)
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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

1100 L/min, through flow cell;


steps of 1 L

20200 L

From 1 to 100 L/min

Required Sample Volume

injected volume + 2080 L,


depending on application

95 L (25 L minimum injected volume +


35 L system dead volume + 25 L vial
dead volume + 8 L number of
bubbles used as separators)

Injection volume plus 2050 L (application


dependent)

Refractive Index Range

1.331.40 RIU

1.331.37 RIU

1.331.40 RIU

Analysis Temperature

440 C (max 20 C below


ambient)

1540 C

445 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

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

100120 V; 220240 V

100240 V

Processing Unit: Autorange 100240 VAC


(10%), 5060 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 SPRMALDIMS (matrixassisted laser desorption/
ionization mass spectrometry)
interface

This protein interaction array (6 6)


system is applicable to antibody
screening, proteinprotein interactions,
protein interface mapping,
proteinnucleic 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 Scientic
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 M1, and equilibrium dissociation constant (KD; KD = kd/ka)


in M. By denition, anity 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-rstorder binding kinetics or a universal tting 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 tting 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 vant Ho 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
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temperature range studied).23 A host of experimental designs


can be applied to SPR protocols in a wide range of scientic
disciplines. For example, experiments could involve sequencespecic DNAprotein 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

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 buers 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 simplied representative workow for a three-part laboratory session on SPR spectroscopy.
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the scope of this technology to scientic 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.

AUTHOR INFORMATION

Corresponding Author

*E-mail: rbakhtiar@msn.com.
Notes

The authors declare no competing nancial interest.

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 oer 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 (SPRMS),31 ow-through electrochemical detection (ECSPR),32 or high-performance liquid
chromatography (HPLCSPR).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 aects the biological functionality of the analyte precludes the
use of SPR spectroscopy. Small molecules (e.g., <150180 Da)
are not easily amenable to SPR experiments and dicult to detect.
In most cases, association and dissociation rate constants outside
of 103108 M1s1 and 1051.0 s1 windows, respectively, will
be relatively more challenging to study.24 In addition, nonspecic binding, sample viscosity, mass-transport artifacts,
quality of reagents used, cleanness of instrument, and baseline
stability are worth consideration during analysis.19 Generally,
high anity interactions (e.g., KD < 1 nM) tend to have a
slower o-rate or dissociation rate constant and pose diculties
for SPR kinetic studies. Conversely, weak interactions (e.g., KD
in M range) are well suited to SPR spectroscopy applications.
Figure 5 depicts a theoretical workow for a three-session
laboratory exercise involving SPR label-free measurements of
proteinprotein interactions. As shown in Figure 5 and prior
demonstrations,1315 much of the preparations can be performed in advance, in order to devote more time to biosensor
optimization experiments. By the same token, data modeling
and analysis can be performed in classroom, in conjunction
with follow-up lectures. A good starting point is to examine
well-established and understood models such as those captured
in vendors application notes.
Lastly, the cost of an SPR instrument and its follow-on
consumables (e.g., biosensor chip) remains relatively high and
could play a limiting factor in a wider implementation of this
technology at the academic teaching laboratory level. Alternatively, clever studies have recently been reported using a
patented portable homemade device, constructed for less than
$2000, and implemented at various undergraduate and high
school laboratories.34 The homemade unit (8 4 2 in.) was
used in a three-part undergraduate laboratory session involving
multiple experiments. One experiment focused on monitoring
the oxidation of poly(vinyl ferrocene) during a conductive polymer deposition process. The second experiment centered on
protein A binding with rabbit IgG and rabbit IgG binding with
anti-rabbit IgG Fab. This oers an exciting prospect to broaden

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