Surface plasmon resonance (SPR) is a physical phenomenon that was first investigated in the 1950s. Today, SPR is the basis of a measurement technique that can be used for biosensors. Commercial SPR instruments allow researchers to measure binding events occurring a few nanometers above a gold surface. The resolution of such instruments can detect molecular changes that are as minute as 20 picograms binding over a square millimeter. Such measurements are used for a range of applications, including research in proteomics, drug discovery, antibody isolation, DNA hybridization, and pollution monitoring. This article will discuss how SPR works, examples of commercial systems, the variants of these systems, and potential applications of SPR as the technology miniaturizes.
How Does SPR Work?
Surface plasmons are oscillations of free electrons in a thin conductive film that are excited by incident light. At a critical angle of incidence, a maximum coupling of energy from the incident light with the plasmons occurs, which makes them resonate. Under these conditions, the energy absorbed by the resonating electrons makes a shadow at a particular angle in the light reflected off the metal. This shadow is measured as the SPR effect.
Figure 1. (click to enlarge) Commonly used Kretschmann configuration of glass prism with gold as the metal coating (a). The dip in intensity of the light due to the surface plasmon resonance phenomenon as a function of angle of incident light (b). The change in phase through resonance (c).
All of this happens at the surface because the surface itself is a glass-metal interface where light energy couples with the plasmons in the metal. However, the wave of energy created by the plasmons can penetrate the glass-metal interface by only a fraction of a micron. By making the metal layer 30–50 nanometers thick, the wave can emerge through the other side of the metal and interact with whatever material is present.
SPR is a powerful tool for biosensors because the angle of the shadow is affected by minuscule changes in the index of refraction of material above the metal layer (e.g., proteins sticking to the metal coating, antibodies binding to protein layers).
The index of refraction is the property of a transparent material to change the angle of an obliquely incident ray of light. For example, the index of refraction for air is approximately 1 (air causes negligible refraction of light); for water, it is approximately 1.33. This difference may be compared to why a straight stick in water appears to be kinked. The SPR effect is exploited by putting a liquid over a metal coating, and allowing the analytes to bind to either clean metal or a surface precoated with reagents. The binding event is then measured as a tiny change in the angle of the shadow, which is related to the binding chemistry taking place on the metal surface.
In biosensors using SPR, the metal layer is usually made of gold, with a thickness of 30–50 nanometers. Gold is preferred because it shows the SPR effect at convenient wavelengths and is chemically stable. The prism of glass onto which the gold is placed can be as simple as the triangular prism known as the Kretschmann configuration (see Figure 1a). The measurement taken is based on a detection of a slight change of intensity of the reflected shadow of light. Figure 1b shows a sharp dip in intensity near the shadow. The steepest part of the curve is where the highest sensitivity of measurement can be made, corresponding to as small as one part in 1 million refractive index units (1e-6 RIU).
Since light beams carry phase information and amplitude information, the second configuration of SPR systems is based on measuring phase change with an interferometer. Interferometry measures the relative phase of two light beams.
If two light beams have a phase difference, their highs and lows do not match. The interferogram that results from this interference consists of a sinusoidal intensity pattern called a fringe field. In a fringe field, the minimum intensity occurs when the beams interfere destructively; the maximum occurs when the beams interfere constructively.
In an SPR system, a fringe image can be made by combining the reflected light from the sample with reference light that does not see the sample. The phase difference due to a protein binding event can be measured by small changes in the fringe field. The key to using phase difference in SPR is that the phase change varies linearly with respect to refractive index through SPR over a linear range of three radians (see Figure 1c). The steep phase change in Figure 1c is similar to a phase plot for a mechanical mass spring-damper or an electrical LRC circuit exhibiting resonance. The variation of phase with respect to refractive index is a function of the gold thickness. Under certain conditions, it may be tuned to give higher sensitivity than the corresponding intensity domain measurement.
Whether an SPR system uses intensity or phase for making measurements, the outcome is a label-free measurement of the binding event. Compared with SPR techniques, fluorescent detection methods require analytes to be labeled with a fluorophore molecule that will emit light to pinpoint the analyte’s location. The necessary labeling reaction complicates the experimental protocol and introduces additional sources of uncertainty. Fluorophore portions may also interfere with biomolecular interactions; the labeling itself may not be homogeneous across the target molecule population, rendering quantitative interpretation more difficult. Such challenges become more pronounced when analyzing biomolecular interactions in raw or minimally processed complex biological samples (e.g., blood, saliva, serum, milk). Another advantage of SPR in the case of such complex samples is that monitoring the interaction on a surface reduces the cross-sensitivity of the method to higher-concentration bulk interferents.
SPR systems can detect kinetic information, such as the rate of complex formation and disintegration of biological species. One of the leading commercial SPR systems is the Biacore 3000 by Biacore International AB (Uppsala, Sweden). The system is a lab-based instrument capable of automatically analyzing 192 samples per run, giving real-time point measurements from the flow cells. Using SPR intensity measurement, the system’s applications include protein identification, protein function analysis, quality control of recombinant proteins, and detection of impurities in protein therapeutics. Biacore reports that more than 4000 scientific publications have used results from its system.
Another commercial SPR system is Spreeta by Sensata Technologies (Attleboro, MA). This system is a low-cost SPR-based biosensing platform that enables real-time quantitative concentration, affinity, and kinetic analysis of biomolecular interactions. Available as either a benchtop or handheld instrument, the system also uses intensity for real-time point measurements. The system’s applications include a competitive assay format for the determination of biotin, avidin-biotin ligand immobilization, and affinity analysis of antibody binding.
Lumera Corp. (Bothell, WA) develops proprietary polymer materials to improve the design, performance, and functionality of devices used primarily in bioscience, communications, and computing. One of its products is the Proteomic Processor, which uses SPR to interrogate high-density microarrays in a flow cell. This instrument also uses a microelectromechanical system (MEMS) mirror to redirect the light beam and scan the microarray rapidly, allowing it to obtain acquisition rates of more than 10,000 interactions per hour.
Figure 2. (click to enlarge) Example of kinetic measurement enabled by spatial imaging SPR. A moving front of betamercaptoethanol binding to gold (a). The top image is taken a few seconds after the bottom one (b).
These three companies have successfully introduced laboratories to instruments utilizing the SPR technique, and they have pushed the envelope of what can be measured. The SPR effect is a passing mention when describing these instruments, which indicates how mainstream the technique has become, and how the technical complexities are handled by efficient instrument design. In order to make further advancements in instrument development, the next generation of SPR systems will need to build on these successes and enable more information by taking the point measurements and creating area imaging. Lumera’s Proteomic Processor achieves near-simultaneous measurement of microarrays by rapidly scanning spots with light from the MEMS mirror. However, true simultaneous measurement of an area requires an image-based system.
Spatial imaging can measure an area of binding locations, which could be populated with a microarray to be monitored simultaneously. In March 2005, Biacore acquired the Flexchip from HTS Biosystems (Hopkinton, MA). The Flexchip is an array-based SPR system that can simultaneously measure up to 400 kinetic interactions in a single flow condition. GWC Technologies (Madison, WI) has also developed kinetic spatial-array imaging with its SPRimager II. This instrument uses prism-coupled SPR to generate an image of a 16–25-spot array that is referenced against a control region to monitor the differences in signal from the array. The instrument’s applications include DNA:RNA, peptide:antibody, and protein:protein interactions.
Figure 3. (click to enlarge) Novel monolithic construction of an interferometer by Cambridge Consultants (Cambridge, UK) for phase-imaging SPR.
Cambridge Consultants Ltd. (Cambridge, UK) has developed an SPR system that provides improvements in SPR measurements. Based on phase differences, the system generates spatial imaging of the sample area over time. As with the Flexchip and SPRimager II systems, binding kinetics can be observed over an area. As an example of the kinetics involved in surface interactions, Figure 2 shows the moving front of betamercaptoethanol (BME) binding to a horizontal gold surface at the bottom of an oval well.
Figure 4. (click to enlarge) Example fringe field for double-well phase imaging SPR (a). Example of processed image showing different binding density of antibodies (b).
The green area is the background where there is no fluid, and the left image shows the sample well accumulating surface-bound material. The sample was injected in the well below the center, and the binding front advances toward the top of the image. The right image shows the final coverage of BME throughout the well. The spatial resolution is down to 50 µm, and the reflectivity resolution of the system is down to 1e-6 RIU.
The Cambridge Consultants system uses a monolithic construction of an interferometer that limits vibrational interference (see Figure 3). The gold surface has two illumination regions: the left region is the optical reference area and the right is the sample area. The reference area is flooded with the same buffer solution as the sample area, but without the relevant chemistry. Both areas are maintained at the same temperature to minimize thermal drift throughout the experiment.
The interferometric fringe fields are captured at chosen time intervals and processed with Fourier methods in the frequency domain, yielding slight changes in the sample area’s index of refraction relative to the reference area. Figure 4 shows an example fringe field and a resulting spatial image of the sample area for specific and nonspecific antibody binding. The sample area is divided into two oval wells instead of one in order to interrogate the two antibodies simultaneously. The aspect ratio of the wells is actually exaggerated by the angle of incidence in the same way that viewing a circle obliquely makes it appear elliptical. Similarly, the circular collimated illumination forms an elliptical sample area, which for these experiments was approximately 3.5 × 6 mm.
Figure 5. (click to enlarge) The average DC-corrected signal from double-well SPR measurement of antibody binding to engineered proteins, one of which contains the epitope for antibody binding.
The system has demonstrated differential binding by using two antibodies challenging the same protein in each well. The antibody in the left well, Anti-HA, binds specifically to the epitope on the exposed top of the protein. Conversely, a nonmatching antibody, cMyc, does not bind to the epitope and shows little differential signal.
Figure 6. (click to enlarge) Hapten:protein interaction using biotin array patterned on imaging surface. Processed image showing triplets of spots with binding, where the blue areas are binding spots and the green background does not have specific binding.
The image area contains two oval wells and a control region in between, which is achieved by a polydimethylsiloxane mask. The control region is monitored to provide a DC correction that accounts for thermal drift. The oval wells where binding occurs show a reduction in the index of refraction. This corresponds to a local increase in thickness at the gold surface due to binding of the antibody with the protein in each well.
The magnitude of this change denotes the amount of binding. The wells are washed repeatedly with a buffer solution to remove any nonspecifically bound molecules, which are monitored to yield a final level of specifically bound material. Figure 5 shows the average of the DC-corrected signal over three suitable areas of the image: the left well, the reference area in between, and the right well. The magnitude of the final binding level for the antibody with the matching protein is noticeably different from the level for the antibody with a nonmatching protein. The result is the ability to discern the protein that contains the antibody-binding site.
Another example of the system’s spatial feature is demonstrated by hapten:protein interactions using a biotin array patterned on the right half of the sample area. With the entire area covered with liquid, the binding occurs to the triplets in the array with affinity (see Figure 6). The two illustrations in Figure 7 are the association-dissociation curves that show the difference in binding affinity between the array elements.
Figure 7. (click to enlarge) Two association-dissociation curves for a small rectangular region encompassing a blue spot.
Richard F. Day, PhD,
is a senior consultant in the medical technology division at Cambridge Consultants Ltd. (Cambridge, UK).
He can be reached at
The phase-imaging feature of this SPR arrangement could be used to detect binding events in arrays of spots on an exposed area over which sample is flowed. This feature could also be used to monitor binding in separate channels arranged in arrays of microchannels, each flowing with different analytes. Many exciting and commercially promising applications for SPR are emerging from various research fields every year. Some examples include the following: protein detection for clinical diagnostics, biowarfare pathogen detection, protein-protein interaction studies, protein expression analysis, protein-drug interaction analysis, vaccine development, food safety for allergens, antibiotic residue, or pathogens, and detection of chemical pollutants.
The challenge ahead is repackaging an SPR system from a lab-based instrument into a low-cost, hand-held, and potentially disposable system, while exploiting the image-based methods. Two university groups developing image-based methods using phase differences are the Shun Hing Institute of Advanced Engineering (Hong Kong) and the National Cheng Kung University (Tainan City, Taiwan). Researchers at the University of Washington (Seattle) are also developing an SPR-based measurement of analytes in saliva. A competitive immunoassay for phenytoin was developed using SPR to measure variously treated preconditioned saliva samples.
The development and use of SPR systems will continue to contribute to the emergence of new IVDs with high sensitivities. The search for IVDs accessible to global healthcare providers may turn to using SPR. In developed-world laboratories, SPR systems are being used to search for new drugs and to better understand protein interactions. As numerous SPR systems gain commercial maturity and lead to significant medical and scientific discoveries, the elusive plasmon will have a greater influence beyond the few nanometers above a thin gold layer.