Surface plasmon resonance (SPR) — a label-free technique for measuring biomolecular interactions in real time — benefits a wide variety of applications, from basic science to drug discovery. The technique monitors the refractive index of materials adsorbed on a surface by recording the SPR phenomenon that occurs when light is reflected off the surface while a solution containing an interactant washes over it. In this way, researchers can measure DNA-DNA, DNA-protein and lipid-protein interactions, as well as interactions between biomolecules and nonbiological surfaces.By coupling localized surface plasmon resonance with interferometry, researchers in Japan have devised a label-free DNA biosensorthat addresses some of the shortcomings of the individual techniques. Furthermore, the instrument requires only a single optical fiber and, thus, paves the way for a portable, handheld diagnostic device based on surface plasmon resonance. Reprinted with permission of Analytical Chemistry.There is an increasing demand for portable SPR devices for clinical use and for use in the field with environmental applications. At the same time, researchers are expanding the scope of the technique by measuring additional parameters. With such developments, users can take the already robust method to places where it has never been before to obtain important new information about biomolecular interactions.Toward handheld devicesSeveral groups are working to realize handheld devices based on surface plasmon resonance. In the March 1 issue of Analytical Chemistry, researchers with Japan Advanced Institute of Science & Technology in Ishikawa, Tokyo Institute of Technology in Yokohama, Japan, and Dong-A University in Busan, South Korea, reported a study in which they showed that a combination of methods — specifically, localized surface plasmon resonance and interferometry — could enable development of such a device.Measuring changes in absorbance is difficult with many conventional interferometric devices because the porous anodic alumina structure often used is insensitive to these changes as a result of its transparency. The researchers therefore capped the structures with a gold coating, facilitating measurement of absorbance (through localized surface plasmon resonance) as well as wavelength shift (through interferometry). Shown here are atomic force microscopy analyses of the chip surface before (A) and after (B) deposition of the gold. Panels (C) and (D) show the top view of the surface before and after deposition, respectively. Profiles of the chip surface corresponding to the overlaid lines, before and after deposition, are shown in panel (E). Reprinted with permission of Analytical Chemistry.As early as 2002, investigators at Duke University described a label-free biosensor based on localized surface plasmon resonance that takes advantage of changes in the refractive index induced by the biomolecular recognition events confined to the interface. They measured biomolecular interactions using a self-assembled monolayer of colloidal gold nanoparticles on glass. A number of similar reports have appeared since then. However, the utility of this approach is limited by the complicated chemistry required to form a self-assembled monolayer of gold nanoparticles — even the slightest defects during mass fabrication of chips could lead to significant reproducibility and reliability errors.Researchers have developed a multimode fiber biosensor based on surface plasmon resonance. The biosensor avoids the relatively bulky components and complicated signal processing often associated with conventional approaches to surface plasmon resonance and, thus, could serve as the basis of a portable system for biochemical sensing and environmental monitoring. The top panel shows a side view of the optical fiber biosensor, with the 0.5-mm polished surface used for surface plasmon resonance. The bottom panel shows the experimental setup used to test the efficacy of the new biosensor, with a halogen light source, sensing and reference fibers, and an optical spectrum analyzer . Eight degrees indicates a method used to reduce reflective light from the end of the fiber. Reprinted with permission of Applied Optics.Much earlier, investigators had sought to measure biomolecular recognition events with porous, silicon-based optical interferometric biosensors. One of the initial challenges associated with this technique — the difficulty in fabricating nanopore arrays with uniform diameter —was addressed by using porous anodic alumina chips. These offer easy control of electrochemical anodization conditions, which determine the uniformity of porosity, pore diameter and thickness of nanopore arrays. However, the resulting interferometric chip enabled measurement of biomolecular interactions based almost solely on wavelength shift. Because of the transparency of the porous anodic alumina structure, the change in absorbance of the biomolecular interaction appeared to be negligible.In the Analytical Chemistry study, the researchers coated the surface of a porous anodic alumina structure with gold, thus facilitating measurement of localized SPR coupled with interfometry. “Using our gold-capped oxide nanostructure,” said researcher Do-Kyun Kim, “one can measure both the change in the refractive index — which depends on the absorbance intensity change — and the wavelength shift in a single event using a single optical fiber.” He added that the need for only a single optical fiber makes the technique both user-friendly and suitable for the development of handheld diagnostic devices.The researchers demonstrated the technique by detecting picomolar quantities of untagged oligonucleotides as well as hybridization with synthetic and PCR-amplified DNA samples. The experimental setup for localized SPR spectroscopy microscopy consisted of a tungsten halogen light source with a wavelength range of 360 to 2000 nm, a UV-visible spectrophotometer and an optical fiber probe bundle with a fiber core diameter of 200 μm and a wavelength range of 450 to 850 nm, all from Ocean Optics Inc. of Dunedin, Fla. Light emerging from the probe bundle was vertically incident onto the porous anodic alumina layer chip. The reflected light was coupled into a detection fiber probe in the same bundle and sent to the spectrophotometer.Eiichi Tamiya, principal investigator of the study, noted that the technique could contribute to a variety of diagnostic applications, including those involving DNA hybridization, DNA-protein interaction and protein antibody-antigen interaction in real blood samples. To this end, he said, the researchers have begun experiments with human blood samples. He added that, to develop the technique for use in handheld diagnostic devices, they must understand the influence of molecular shape, structure and size on biomolecular interactions, as well as the mechanisms of the interactions and more.Miniaturizing devicesDevelopment and implementation of portable SPR devices can be hindered by the size, instability and temperature sensitivity associated with the prism coupling technique traditionally used for observation of surface plasmon resonance. In the Feb. 10 issue of Applied Optics, investigators with National Defense and Tatung universities, both in Taiwan, described a multimode optical fiber biosensor based on surface plasmon resonance that could help to address these potential obstacles.Researchers have described a surface plasmon resonance technique that enables measurement of scattering as well as reflection. Measuring multiple parameters yields important additional information and could open up a variety of applications.An optical fiber biosensor could offer many advantages. “With properties such as small size, low power, low weight, good performance, environmental ruggedness and immunity to electromagnetic interference, [such biosensors] are of interest in both life sciences and biomedical applications,” said Yu-Chia Tsao, a researcher with Tatung University and one of the authors of the study. Tsao also noted that optical fiber biosensors are ideally suited for portable SPR devices.The first optical fiber biosensor based on SPR was reported in 1993. Since then, a number of additional biosensors have been described, focusing especially on miniaturization of SPR devices. Examples include a plastic-clad silica fiber with partly removed cladding, a side-polished optical fiber, a tapered single-mode fiber and a tip-polished fiber. In the recent Applied Optics study, the investigators reported a side-polished multimode optical fiber biosensor based on SPR with a halogen light source. They tested some of the other types of fiber, Tsao noted, but failed to obtain satisfactory results with them.They produced the biosensor by side-polishing the fiber until half of the core was closed, creating a polished surface 5 mm long and 62.5 μm deep. Then they coated the surface with a thin gold layer using a sputtering method. To maximize the SPR response, they performed a series of calibration experiments to optimize the thickness of the gold layer — finding that the optimal thickness was approximately 37 nm.The biosensor works on the following principle: The linearly polarized light propagating through the fiber generates surface plasmon waves on the gold surface. The transmitted light then tracks the SPR response, which is sensitive to the refractive index on the surface. By modifying the surface, researchers can make it hybridize the probe DNA — which will then hybridize the target DNA — and create a sensor suitable for biological studies.The researchers demonstrated the optical fiber biosensor by using it to detect DNA hybridization. The experimental setup consisted of a halogen light source and an optical spectrum analyzer, both made by Ando Electric Co. Ltd. of Taiwan, as well as a 2 × 2 coupler, a side-polished sensing fiber and a side-polished reference fiber. Both of the fibers were graded-index multimode fibers made by Prime Optical Fiber Corp., also of Taiwan, with a 62.5-μm core diameter and a 125-μm cladding diameter.The experiments showed that the performance of the optical fiber biosensor is comparable to that of SPR devices that use prism coupling techniques. In fact, Tsao said, the researchers have been able to detect the concentration of the bacterium Legionella pneumophila with a detection limit of 102 to 105 CFU/ml —though they have not been able to achieve the quantitative reproducibility needed to perform statistical research. They are currently working to address this issue.Tsao noted a number of advantages of the optical fiber biosensor, including its low cost and relatively high stability. In addition, molecules are easily immobilized on the polished surface for hybridization. Thus, it offers potential for a variety of applications, including medical diagnosis, preventive medicine, environmental monitoring and biochemical analysis.The researchers continue to work with the optical fiber biosensor. Besides working toward statistical research with Legionella pneumophila, they are tackling detection of Pseudomonas aeruginosa, Escherichia coli O157:H7, ochratoxin A, aflatoxin and more. Furthermore, they are continuing to develop a portable SPR system based on the optical fiber biosensor. To this end, Tsao said, they must reduce the size of the system while maintaining the sensitivity of the biosensor.The drive toward miniaturization —and subsequent development of portable devices — is only one of the current trends in surface plasmon resonance measurements. Recent research also has focused on use of surface plasmon resonance for multiparametric monitoring. Investigators at National Academy of Sciences in Kiev, Ukraine, and at Karolinska Institute in Stockholm, Sweden, have developed a technique in which scattered light is measured under surface plasmon resonance conditions. As reported in an Analytical Chemistry paper published online on Jan. 18, the technique maintains the functional advantages of traditional SPR measurements while increasing the dynamic range of analysis and incorporating information from surface plasmon field-enhanced fluorescence spectroscopy.The use of surface plasmon resonance as a multiparametric sensor could benefit a variety of applications, said researcher Boris Snopok. Measuring parameters other than simple reflection may yield more information on interfacial structures because measurements of evanescent wave light scattering depend strongly on variation of permittivity within interfacial structures, for example. In the current study, the researchers looked only at reflection — the measure used with conventional SPR techniques — and total integrated scattering, but they hope to expand the technique to measure other parameters.To measure scattered light under surface plasmon resonance conditions, they used a scanning imaging SPR spectrometer developed at the National Academy of Sciences and based on a single-channel scanning SPR spectrometer. The instrument used a simple gold-covered glass slide and an optical system, including a 650-nm semiconductor laser and collimating optics, and a concave cylindrical lens to keep the beam from focusing on the convex surface of the main lens. A DIN (“Deutsche Industrie Normen” or “German Industry Norm”) standard 20× microscope objective focused the light scattered from the sample onto the surface of a CCD camera made by Motorola. The instrument offered roughly 4-μm resolution when measuring scattered light.By varying the angle of incidence, the researchers measured both the integral intensity of reflected light and the two-dimensional matrix of scattered light intensity as functions of angles. Thus, they performed measurements in a microarray format to test the technique’s ability to measure parallel protein-protein interactions, using protein A and rabbit antibodies. They registered immobilization of the antibodies in the protein A spots as well as at the free gold surface of the substrate, thus demonstrating the potential of the technique for measurements in the microarray format. In addition, they detected protein-protein interactions in human serum, validating the measurements with traditional immunochemistry.Shown here are protein microarrays before and after reaction with human serum, as measured by the multiparametric technique described by researchers. The spot positions in the microarray correspond to protein A (A3, B3, C3), polyclonal rabbit antibodies (A1, A2, B1) and unmodified gold (B2, C1). The spot intensity corresponds to the angular position for the scattered light peak for a given point.The technique offers a variety of advantages over conventional approaches to surface plasmon resonance. Snopok noted that the ability to measure both scattered light and fluorescence intensity is especially promising, as it allows detection of multiprotein complexes in real time. Also, analysis of angle-dependent scattered light could allow monitoring of in-plane processes on the nanometer scale — in contrast to the classical approach to surface plasmon resonance, in which relying on reflectivity leads to surface plasmons with propagation lengths of a few microns.The researchers are working to develop a multiparametric SPR sensor based on surface plasmon resonance and gold nanoparticles. “The present article is the first step in this direction,” Snopok explained, “since we must initially show that we have similar results for uniform systems with ‘normal’ optical properties (with respect to nanosystems). Our next step is to use gold nanoparticles as immobilization support for proteins and DNA (as interfacial layers or embedded in polymer matrix) for testing the integrated light scattering for such systems.” He added that they also are interested in monitoring the interactions of interfacial ensembles of nanoparticles with low-molecular-weight analytes, through measurement of both reflection and scattering.