product review
Surface plasmon resonance instruments diversify An increasing number of vendors have entered the SPR spectroscopy and imaging market. Rajendrani Mukhopadhyay
T
here was a time when the commercial space for surface plasmon resonance (SPR) instruments could be summed up in one name: Biacore. Now, that space holds more players. Biacore is arguably still the most prominent vendor of SPR instruments, but since Analytical Chemistry last reviewed these instruments (Anal. Chem. 2000, 72, 289A–292A), a number of companies have sprung up. “There are more and more companies looking at this type of technology,” says Helena Nilshans of Biacore. “That’s a measurement of market development!” Some of the companies have joined Biacore in offering conventional SPR spectroscopic instruments. Others, such as GWC Technologies, have introduced instruments for SPR imaging. “The market is growing rapidly, and as more people become familiar with the technique and also its limitations, there will be better data published, which will attract more people,” says Thomas Ryan of Reichert Analytical Instruments (formerly a part of Leica Microsystems). SPR instruments are mainly used in biological applications— the classic example is the characterization of antibody–antigen interactions. “People who use SPR for their research basically screen to see if anything binds to the molecule of interest,” says Quan “Jason” Cheng at the University of California, Riverside. SPR measurements are used to study how quickly and how strongly an analyte binds to the molecule of interest. The instruments also have applications in materials science and food analysis. Table 1 lists some commercially available SPR spectroscopy and imaging instruments. The table is meant to be representative, not comprehensive. Some vendors may offer similar products not listed here.
Overview of SPR spectroscopy and imaging Two types of SPR instruments are commercially available: those for spectroscopy and those for imaging. They all have a laser, a detector, and a glass prism covered with a thin metal © 2005 AMERICAN CHEMICAL SOCIETY
layer (gold is commonly used). SPR spectroscopy instruments can be further subdivided into systems based on angle-shift or wavelength-shift measurements. In angle-shift instruments, total internal reflection is observed above the critical angle of incidence at the interface of the gold-covered glass and the medium. The electromagnetic field component of the light penetrates the metal, and energy is transferred to the metal’s electrons. The energy transfer produces charged density waves called surface plasmons at the metal–medium interface. Alternatively, in wavelength-shift instruments, resonance conditions are created by keeping the incident angle constant while changing the wavelength of the light source. The resonance conditions are influenced by molecules, usually called probes, immobilized on the metal layer. When an analyte, also known as the target, binds to the surface-immobilized probes, the change in mass can be detected as a shift in resonant angle (or wavelength). The change in mass can be monitored to measure the amount of bound target, the affinity of the target for the probe, and the association and dissociation kinetics between the target and the probe. In SPR spectroscopy, the size of the laser spot determines the size of the area studied—usually only one analysis point A U G U S T 1 , 2 0 0 5 / A N A LY T I C A L C H E M I S T R Y
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product review
Table 1. Selected SPR instruments.1 Product
Biosuplar-2 and Biosuplar-3
Biacore T100
Autolab ESPRIT
SPRimager II
Company
Analytical µ-Systems Dept. of Mivitec GmbH Eichenstr. 24 93161 Sinzing Germany +49-941-461-28-33 www.biosuplar.com
Biacore AB Rapsgatan 7 SE-754 50 Uppsala Sweden +46-18-675700 www.biacore.com
Eco Chemie BV Kanaalweg 29 G 3526 KM Utrecht The Netherlands +31-30-2893154 www.ecochemie.nl
GWC Technologies 505 S. Rosa Rd., Ste. 120 Madison, WI 53719 608-441-2720 www.gwctechnologies.com
Cost (U.S.D.)
>$9000
$320,000
$97,500
$47,000
Wavelength- or angle-shift design
Both (angle dependence or reflection at fixed angle)
Angle
Angle
Fixed angle, fixed wavelength
Dimensions (cm)
22 13 10
60 61.5 69
60 51 45
61 45.7 22.9
Light source
Low-power GaAs LED, = 630– 670 nm (depends on model)
LED
670 nm
800 nm
Incident angle range (degrees)
17 (standard), 18.5 (on demand); any starting point between 0° and 90° can be selected
63–67
62–78, dynamic range 4000
40–70
Resolution (degrees)
$8990
$55,000
$29,000
$45,000 (excluding FTIR spectrometer)
Wavelength- or angle-shift design
Angle
Angle
Angle
Wavelength shift based on FT multiplexing
Dimensions (cm)
21.5 13 10
5 5 4.5
39.4 32 14
40 69 33
Light source
GaAs semiconductor laser, = 650 nm
HeNe laser or laser diode
780-nm laser LED with 780-nm bandpass filter
Near-IR tungsten–halogen broad–1 band source: 5800–12,000 cm
Incident angle range (degrees)
17
20 –140
46–67
40–70 (uses a fixed angle for each measurement)
Resolution (degrees)
0.0028
Tracking accuracy 0.0001
3.25 10–5
Degree resolution does not apply to FT-SPR; wavenumber shift of 4 cm–1 in FT-SPR corresponds to ~1 Å film thickness change; wavenumber resolution of as high as 0.09 cm–1 is available.
Other features
Measurements in gas and ambient liquid; 2 optical channels; additional channel for parallel electric measurements (±5 V); electrochemical function; opencell configuration; closed cell for measurements in flow regime; recording of full SPR curve; maximum time resolution of measurements is 0.2 s (slope mode).
Modular, open-platform setup with options for ellipsometry, Brewster-angle microscopy, surface plasmon and waveguide spectroscopy, SPR microscopy, imaging ellipsometry, waveguide modes, and contact angle module; time resolution in surface plasmon kinetic measurements is 50 µs.
HPLC fluidics can be modified to suit experimental needs; optional autosampler; 10–90 °C temperature range; provides response vs time and reflectivity data; open architecture; National Instruments Labview data acquisition and handling.
The FT-SPR module offers SPR capabilities with FT advantages: high throughput, multiplexing, and wavelength accuracy. OMNIC software supports FT-SPR data collection over a wide range of kinetic measurements. The center-of-gravity algorithm in Thermo’s TQ analysis software allows rapid determination of SPR minimum shifting. The FT-SPR system has high sensitivity because of the FT component. The SPR 100 module is compatible with Thermo’s Magna-IR, Nexus, and Nicolet X700 FTIR spectrometers, provided the system has a near-IR configuration.
LED: light-emitting diode. 1 Some companies may offer similar products not listed here. Contact the vendors for their full product lines.
The second advantage is obtaining real-time data. It “makes a huge difference,” says Burland. With fluorescencebased methods, irreversible photobleaching of the fluorescence signal is a concern. “Now, you don’t have to worry about that. You can set things up so you can simply watch on the screen what’s happening. That’s very handy for methods development. . . . [If] something goes wrong early, you can see it,” he says. Commercial SPR spectroscopy and imaging instruments come in two general formats—open-platform and closed-box. The two instrument formats appeal to different types of customers. For example, biochemists often prefer to work with
closed-box systems. They want instruments that work “according to the recipe provided by the vendor,” says Raymond Dessy of Virginia Polytechnic Institute and State University. They don’t want to modify the instrument or understand how it works; rather, they just want to get numbers from it, he says. Open-platform instruments are designed for researchers who are comfortable tinkering with optical systems. Dessy says that the users of these instruments “don’t mind modifying [the instrument], want to know how it’s working, and are interested in the basic physics of the unit.” Because of easy access to the innards, open-platform systems can also be useful for teaching purposes. A U G U S T 1 , 2 0 0 5 / A N A LY T I C A L C H E M I S T R Y
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Problems
some conditions that will drive it off without denaturing what is immobilized on the surface.” Apart from sample preparation, other factors can thwart SPR measurements. For one, the refractive index is dependent upon temperature. If the room temperature fluctuates, or samples are introduced into the instrument at different temperatures, then a change in the baseline SPR spectroscopic signal is recorded. “A lot of the instruments leave something to be desired in temperature control,” says Dessy. Researchers “may not even be aware that [the measurement] is primarily based on refractive index and thickness. They forget [that] changes in temperature are going to affect the solutions.” Dessy says that many people don’t take into account temperature drift when they are looking at small changes in the signal. Jennifer Shumaker-Parry of the University of Utah also says that changes in the refractive index of the bulk solution can occur as different samples are introduced. Once again, the baseline SPR spectroscopic signal is sensitive to the change in bulk refractive index even though the measurements occur at the surface. “You either have to have a way to account for the bulk changes by having multiple flow channels,” she says. “Or, you have to control it very well so that, from sample to sample, the bulk reResearchers try to combat nonspecific interactions fractive index is essentially the same.” by manipulating the surface and making sure In some SPR spectroscopy instruments, multiple flow samples are as pure as possible. Despite these channels can incorporate controls to account for changes efforts, miscellaneous materials do stick to the in bulk refractive index. However, it can be difficult to sisurface and errant molecules in samples multaneously run the experiment and the control in the adjacent channels with high compete with the target to bind to the probe. precision and correct for changes in background. Background changes aren’t as much of a problem in SPR dodecyl sulfate, which makes hybridization work better for imaging because the microarray format allows controls to be DNA, is used, “You’ll find it sticks all over the surface. If you easily included on the same chip as the experiment. Shumakerdo fluorescence, you never see it, so you don’t care about it. Parry explains, “You’re using one solution, you’re doing exBut in SPR, you see it,” says Corn. periments and controls all at the same time, and you’re using Researchers try to combat nonspecific interactions by manipulating the surface and making sure that the samples are as one surface.” But in the end, the instrument itself is a lesser cause for pure as possible. Despite these efforts, miscellaneous materials concern than the purity of the samples and the surface on do stick to the surface and errant molecules in samples comwhich the experiment is being conducted. “I think it’s surface pete with the target to bind to the probe. Surface problems aren’t just limited to nonspecific binding. chemistry that limits you,” says Corn. “That’s true for fluorescence, too. It’s not a question of signal. It’s a question of Ryan says getting probes “to stick down and stay active” on background. You’re hardly ever limited by your ability to dethe metal surface is a problem. Often probes will denature on tect photons—you’re limited by the other stuff that gets in the surface and will not yield any useful information. the way.” But even if the probes are active and interacting properly, sometimes the very nature of the interaction with their binding partner confounds an experiment. If the interaction has a The future very high affinity or has a very slow rate of unbinding, Ryan Several experts say that the combination of SPR spectroscopy says, “You can’t wait till it all comes off. . . . You have to find and imaging with other analytical techniques is a new direcExperts agree that most of the difficulties with SPR measurements lie in sample preparation. “The challenge is more with your sample. The optics is relatively straightforward,” says Wolfgang Knoll of the Max Planck Institute for Polymer Research (Germany). Attaching a probe to a gold surface is usually easier said than done. A myriad of surface-chemistry techniques, ranging from hydrogels to antibodies, is often explored to find the best immobilization method for the probe of interest. For this purpose, vendors such as Biacore offer a variety of ready-made chips with different surface chemistries. Dessy points out that if an immobilization technique ends up holding the probe well above the surface, problems in measurement can arise. “One of the things we look for are tethering systems that keep us as close to the surface as possible. One can improve the performance of the instrument that way,” he says. Nonspecific interactions are the bane of SPR spectroscopy and imaging. Robert Corn of the University of California, Irvine, says, “The good news is everything has an SPR signal. The bad news is everything has an SPR signal.” If sodium
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product review
tion for the field. “With SPR imaging, you can get the specifics of biomolecule interactions,” says Shumaker-Parry. “But, because you don’t get identification, due to a lack of labeling, combining it with MS gives identification information, especially for proteomics.” Knoll suggests a combination of SPR spectroscopy with electrochemistry because “with the gold or the noble metal layer you need to excite the plasmon; you already have the working electrode in place.” He also suggests incorporating the quartz crystal microbalance with SPR spectroscopy. “One [method] is measuring the optical thickness, the other is measuring the mass load or the viscoelastic mechanical properties,” he says. Some companies manufacture instruments that offer a combination of techniques. Optrel produces a modular instrument called Multiskop that carries out measurements on the basis of optical methods such as SPR spectroscopy, ellipsometry, and Brewster-angle microscopy. Thermo Electron will be launching an FT-SPR system at the end of July 2005 that consists of an SPR module in conjunction with a Thermo FTIR spectrometer. “The SPR accessory is driven by the FTIR spectrometer,” explains Eric Jiang of Thermo Electron. “This system isn’t only giving you the high-sensitivity SPR capabilities. It also
does both routine and advanced FTIR analysis.” The basic SPR instrument setup is not expected to dramatically change in the future; however, vendors say that current challenges exist. “There’s a huge potential market out there for an instrument that’s affordable. What we’re trying to do is come up with an instrument that a standard university researcher with an average NIH grant can afford,” says Ryan. SPR companies expect new types of customers to enter the market as the applications expand and mature. Some vendors note that the availability of more ready-made applications will make it easier to sell the instruments to industry. Experts are optimistic about the future of SPR technology but recognize what lies ahead. “I’m pretty confident SPR will play an important role in the future of bioanalytical chemistry,” says Cheng. “But we need to see this technique get wide acceptance, and there is still a lot of work to be done.” Rajendrani Mukhopadhyay is an associate editor of Analytical Chemistry.
Upcoming product reviews October 1: 2-D gel accessories December 1: Atomic force microscopes
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