product review
New choices for SPR Emerging products are filling niches in the SPR market. by Elizabeth Zubritsky
T
ARTWORK BY GH MULTIMEDIA
o date, surface plasmon resonance (SPR) has appealed primarily to biological scientists, who have used it to study a wide range of molecular interactions, including receptor binding, competitive binding, and pharmacokinetic assays. Because SPR instruments have few components—typically, a light source, a detector, and a prism coated with a thin film of metal, usually gold— many of them are home-built. However, commercial instruments are also available. The market has been dominated by the Uppsala, Sweden-based company Biacore since 1990, but some new products, aimed at specific segments of the market, are being introduced. Table 1 provides a sampling of those currently offered. Part of the reason that the market has been rather confined in the past is because users’ needs are so varied, says Sinclair Yee at the University of Washington, whose group developed a fiber-optic probe that was acquired by Biacore and has also worked with Texas Instruments on its SPR product. The variety of needs is the same problem faced by vendors of other biological sensor technologies, he says. “Different sensor configurations are required for different applications,” he explains. “This is a huge challenge in almost all sensor indus-
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Table 1. Summary of selected commercial SPR products1.
1 2
Model
Biacore 3000
FT-SPR 100 2
Spreeta
Manufacturer
Biacore, Inc. Suite 100 200 Centennial Ave. Piscataway, NJ 08854 800-242-2599
GWC Instruments 505 South Rosa Rd. Madison, WI 53719 608-441-2720
Texas Instruments 13536 N. Central Expressway P.O. Box 655012 Mail Station 945 Dallas, TX 75265 214-480-7800
URL
www.biacore.com
www.gwcinstruments.com
www.ti.com/spreeta
Dimensions
760 3 350 3 610 mm
Design
Angle shift with fixed source
Wavelength shift, with measurements at a variety of fixed angles possible
Angle shift with fixed geometry and divergent beam
Incident angle range (degrees)
65–72
40–70
57–81
Resolution (degrees)
1.0 3 10 –4
Depends on the host spectrometer
3.3 3 10 –4
Flow cells
4
1
1
Light source
760-nm LED
Depends on the host spectrometer; spectral range is 850–1750 nm
LED centered at ~830 nm and 20- to 30-nm wide
Price (USD)
~$200,000
~$20,000
~$3000 for evaluation kit (includes 50 sensors)
Features
Microfluidics with subnanoliter dead volume and robotics for sample delivery; automated, unattended sample analysis possible; software for data analysis, curve fitting, and modeling; wide variety of sensor surfaces provided
Submonolayer sensitivity; in situ and ex situ capability; broad wavelength range out to 2 µm
Small, inexpensive instrument; designed for both bulk refractive index applications
Reader service number
401
402
403
127 3 101 3 25 mm
Some manufacturers offer instruments other than those shown. Contact the individual vendors for descriptions of their full product lines. This is a kit for use with a commercial FT-IR spectrometer, not a stand-alone instrument.
tries.” Another reason, noted by several sources, is that SPR is a relatively unknown technology, and vendors must first educate potential users. Finally, SPR must compete with rival sensor technologies, including other optical sensor technologies. Nevertheless, more companies are willing to try their luck. Interestingly, none of them are tackling Biacore and its open-platform instrument headon. Instead, they appear to be going after particular market segments. Some are catering exclusively to med-
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ical diagnostics. Other targets are users who want to add an SPR accessory to a spectrometer rather than buy a separate instrument. Still others are banking on users wanting smaller, less expensive devices. Not to be outdone, Biacore has continued to develop new products of its own.
Sensitive detection SPR has several advantages in biological studies. First, it is sensitive and can be made specific. Second, it provides kinetics data and can be performed on
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samples in solution, unlike some mass spectrometry techniques. Finally, unlike enzyme-linked immunoselective assays, SPR does not require the labeling of analytes, nor does it rely on fluorescence, luminescence, or colorimetry for detection. Instead, SPR is an optical technique that measures the intensity of light reflected inside a prism or optical fiber, which has been coated with a thin metal film (see Anal. Chem. 1998, 70, 449 A–455 A). The light enters the prism at an angle guaranteed to produce total internal reflection. The minimum angle at which this occurs is the critical angle, and it depends on the indexes of refraction of the prism and the external medium. Thus, the system is very sensitive to anything on the prism’s surface. Specifically, the metal film supports the propagation of charge density waves—or plasmons—along the surface. A prism or optical fiber can couple light into the surface plasmon. At certain wavelengths and incident angles, an evanescent wave propagates through the thin metal film to induce a resonance in the plasmon on the surface. This registers on the detector as a loss of light or a dip in the plot of the intensity of reflected light. If the experimental conditions remain constant, the dip will occur in the same place each time. SPR becomes interesting when particular molecules are attached to the metal film. If the film is coated with a specific antigen, for example, the binding of antibodies to the antigens will register as a shift in the location of the dip, and this change can be related to the density of the bound antibody. Not only can the binding affinity of this antigen–antibody pair be assessed, but the binding kinetics can be observed in real time. Thus, detailed information such as the on- and offrates of the antibody can be obtained. SPR is also seen as a possible replacement for ellipsometry when measuring the thickness of organic films or certain metal films, says
product review
Robert Corn at the University of Wisconsin–Madison and a co-founder of GWC Instruments. “It has a little bit higher sensitivity than ellipsometry on metal surfaces,” he says, “so it is potentially useful in those applications.” However, he adds that SPR is unlikely to replace all ellipsometry, because it can’t be used on silicon and several other metal films.
terms of the wavelength and not the angle. This approach is preferred by some researchers, and it is particularly useful for fiber-optic probes, in which white light is sent down the fiber. None of the sources for this article could specify an overall sensitivity advantage for either angle- or wavelength-shift measurements. However, Corn and Yee say that better sensitivi-
pany GWC Instruments is introducing an SPR imager. The sensitivity improvement can be obtained by using incoherent near-IR excitation wavelengths, he explains. In return, SPR imaging provides location-specific information about binding on surfaces such as DNA or protein microarrays. Preliminary work also has been done using SPR with commercial
Angle versus wavelength shift Because the location of the absorption loss is a function of both wavelength and incident angle, changes in its position can be detected in several ways. The most common approach is the angle-shift method (also called angle scanning), in which the wavelength is held constant, and the angle of incidence is varied. This can be done either by literally scanning the beam through a range of angles or by using a divergent beam that covers a range of angles at once. In either case, the setup is simple. A light-emitting diode (LED) or lowpower laser is used—high-intensity sources are not needed—with an offthe-shelf detector. For basic angleshift setup, a single detector registers the intensity during scanning. For a divergent beam, a diode array detects the intensity for the various angles simultaneously. Although most instruments move only the light beam, it is also possible to scan by moving the
Either angle- or wavelength-shift measurements are possible.
ty is possible at longer wavelengths. With the wavelength-shift technique, theoretical studies suggest longer wavelengths can enhance the sensitivity by ~10 times, Yee says. Using both angle-shift methods and SPR imaging, Corn’s group experimentally demonstrated better sensitivity in the near IR (Anal. Chem. 1999, 71, 3928–3934; 3935–3940).
SPR imaging Another way to perform SPR measurements is to fix both the angle and the wavelength and measure the change in reflectivity at various positions across a surface. Researchers at the Max Planck Institute for Polymer
SPR gets its specificity from coatings applied to the thin gold film.
detector. In any case, the location of the dip is specified in terms of the angle at which it occurs. On the other hand, the wavelength-shift method (or wavelength scanning) uses a light beam that includes a range of wavelengths, and the location of the dip is specified in
microscopes. A small probe, such as a modified atomic force microscope tip, is placed within the evanescent field region and scanned across a surface. The tip acts as a scatterer, resulting in conical radiation, which has an intensity proportional to the original surface plasmon field intensity. Researchers at Northwestern University currently are using this technique to create high-resolution maps of surfaces.
Research in Mainz (Germany), who have done some of the early work, call this approach “SPR microscopy”. Others call it “SPR imaging” because it can analyze an entire surface. SPR imaging requires better sensitivity than the angle- or wavelength-shift methods, according to Corn, whose com-
Surface coatings For bulk refractive index measurements—which can be useful for process monitoring, for example— SPR can be performed with the metal-coated prism. However, for biosensing, the metal film must be coated with another film that permits the attachment of molecules. This secondary coating can be either a selfassembled monolayer or a thicker (~100 nm) hydrogel polymer film. According to Mike Robinson of Biacore, a hydrogel allows molecules to move freely on the surface, thus potentially reducing some problems with binding interference. A hydrogel also provides more attachment points than a monolayer and acts as a buffer to reduce nonspecific binding to the surface, he says. On the other hand, Corn says, some researchers think it is harder for molecules to diffuse
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product review
through a hydrogel, which can potentially alter the measured absorption kinetics. Monolayer films, which have lower sensitivity, do not have these diffusion problems, he adds. However, Robinson points to another researcher’s paper, in which there were no differences in binding kinetics between monolayers and a dextran hydrogel (Anal. Chem. 1999, 71, 777–790). Whether the secondary coating is a monolayer or a hydrogel, the surface
trolled. In addition, Yee’s group is exploring optical compensation techniques based on critical angle measurements.
Flow cells In most SPR instruments, the sample is introduced to the coated surface of the prism via a flow cell. Depending on the complexity of the instrument, there may be microfluidics and robotics to deliver a variety of samples automatically. The exception to this
Some instruments have multiple flow cells for simultaneous measurements.
recognition properties can be defined either directly or indirectly, Robinson says. One possibility for direct recognition is modifying the carboxyl groups in the coating to form reactive esters. Other options include thiol couplings, hydrazine, and self-assembled monolayers. Indirect recognition, on the other hand, includes biotin–streptavidin binding for DNA, RNA, or antibody capture. All companies sell sensors that users can modify themselves, and some also offer sensors outfitted with various modified surfaces.
rule is the fiber-optic probe-based instruments for remote sensing, which, loosely speaking, bring the detection surface to the sample. Most SPR instruments have only one flow cell, but some have several cells, which permit the analysis of multiple samples simultaneously. Even in this case, however, there is still only one buffer stream. Thus, different molecules can be attached to the surface of each flow cell. However, because the same buffer stream will pass through all the cells, they will all be exposed to the same analytes.
Temperature control Because SPR results depend on the index of refraction, knowing the temperature precisely can be important. “People worry most about the temperature of the liquid being measured,” says Jerry Elkind of Texas Instruments, “because the refractive index of water changes by ~10–4 per °C.” In some instruments, a rapidly responding thermistor within the flow cell compensates for changes in the temperature of the liquid being analyzed. In others, both the temperature within the flow cell and the temperature of the initial sample are con-
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The future SPR instrumentation is still undergoing quite a lot of development, and continuing efforts to miniaturize it figure prominently. Among them are attempts by Yee and others to put SPR into capillaries for high-throughput applications. Yee says his group has demonstrated that capillary-based SPR is possible, but several challenges remain, including depositing gold in the capillaries and optimizing the sensor. Biacore is also targeting the highthroughput market with plans to introduce systems based on high-den-
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sity arrays. Other research is aimed at increasing SPR’s sensitivity and resolution. In addition to the aforementioned work by Corn and Yee, the latter’s group is working on a new class of sensors based on the “long-range” plasmon wave. Another approach is simply to use a different detection method. For example, N. J. Tao and colleagues at Florida International University improved sensitivity by 1–2 orders of magnitude with a bicell detector, which “works like a balance,” Tao says. Initially, the dip is centered between the two photocells, and there is equal intensity on both. But if the dip shifts, one photocell detects more intensity. This differential signal provides better sensitivity, and the common noise can be stripped out, so it can be used in ambient light, he adds. It may also be possible to get new kinds of information from SPR. For example, some researchers, including a group at Boston University, are performing wavelength-shift measurements at a variety of fixed angles to get “colored SPR” data. Tao’s group recently measured SPR angular shifts at various wavelengths in a fashion similar to absorption spectrometry to study conformational changes of proteins (Anal. Chem. 2000, 72, 222–226). The researchers collected spectroscopic information by choosing wavelengths where there were chromophores, which allowed them to determine if particular groups on the protein were being changed. Researchers hope that developments such as these will create more SPR applications in the future. And, no doubt, current and potential vendors will be waiting to see if that means more markets. Elizabeth Zubritsky is an assistant editor at Analytical Chemistry.
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