Overcoming Contamination in Surface Plasmon Resonance

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Overcoming Contamination in Surface Plasmon Resonance Spectroscopy Diptabhas Sarkar and P. Somasundaran* NSF IUCR Center for Studies in Novel Surfactants, Langmuir Center for Colloids and Interfaces, Columbia University, 911 Mudd Building, 500 West 120th Street, New York, New York 10027 Received February 7, 2002. In Final Form: August 8, 2002 Surface plasmon resonance spectroscopy is a technique used for detection of subtle changes in the optical properties of materials and finds wide application in biosensors and chemical tranducers. Due to the extreme sensitivity, contamination of the sensor surface is a major problem in experiments involving surface plasmon resonance measurements. We report here that aluminum oxide (alumina) can be used as a perfect protective coating for the metal in surface plasmon resonance experiments. The alumina is electron beam evaporated to create a relatively thick layer on the sensor (gold) surface, immediately after the gold deposition process without exposing the latter to environmental conditions. During experiment, this protective coating was removed by dissolving the alumina in an alkaline solution, thus exposing the uncontaminated gold surface for further experiments. By the study of the shift in the waveguide modes supported in the dielectric alumina layer, the dissolution of alumina was monitored. Theoretical simulation studies using Fresnel’s equations were done to explain the dissolution plots obtained. Atomic force microscopy of the alumina surface provided information on the variation of the roughness parameter, with the progress in dissolution. It was found that the roughness of the surface at all points during the dissolution process remained much below the wavelength of the incident radiation, thus justifying the modeling of the system as stacked Fresnel’s layers.

Introduction The interest in the use of surface plasmon oscillations as a transduction mechanism to measure refractive index began with the landmark paper by Kretschmann where he used the surface plasmon oscillations to measure the dielectric constant of metals.1 Kretschmann used a prism to allow for light of sufficient momentum to couple into the fundamental resonance mode of a thin metal layer on the face of a prism. In 1983 Liedberg first demonstrated the exploitation of surface plasmon oscillations for chemical sensing.2 Since then surface plasmon resonance (SPR) has grown into a versatile technique used in a variety of applications. These include absorbance studies of selfassembled monolayers,3,4 biokinetics5,6 and biosensing measurements,7-9 bulk liquid refractive index measurements, gas detection,10-13 immunosensing, binding of proteins on surfaces,14 light modulation, and SPR microscopy.15 Owing to the recent surge in studies involving * Corresponding author: e-mail, [email protected]; tel, 212854 2926; fax, 212-854 8362. (1) Kretchmann, E. Z. Phys. 1971, 241, 313. (2) Liedberg, B.; Nylander, C.; Lundstrom, I. Sens. Actuators, B 1983, 4, 299. (3) Peterlinz, K. A.; Georgiadis, R. Langmuir 1996, 12, 4731. (4) Ehler, T. E.; Malmberg, N.; Noe, L. J. J. Phys. Chem. B 1997, 101, 1268. (5) Bunjes, N.; Schmidt, E. K.; Jonczyk, A.; Rippmann, F.; Beyer, D.; Ringsdorf, H.; Graber, P.; Knoll, W.; Naumann, R. Langmuir 1997, 13, 6188. (6) Stenberg, E.; Persson, B.; Roos, H.; Urbaniczky, C. J. Colloid Interface Sci. 1991, 143, 2. (7) Dubs, M.-C.; Altschuh, D.; Van Regenmortel, M. H. V. Immunol. Lett. 1991, 31, 59. (8) Morgan, H.; Taylor, D. Biosens. Bioelectron. 1992, 7, 405. (9) Severs, A. H.; Schassfoort, R. B. M.; Salden, M. H. L. Biosens. Bioelectron. 1993, 8, 185. (10) Miwa, S.; Arakawa, T. Thin Solid Films 1996, 281-282, 466. (11) Abdelghani, A.; Chovelon, J. M.; Jaffrezic-Renault, N.; RonotTrioli, C.; Veillas, C.; Gagnaire, H. Sens. Actuators, B 1997, 38-39, 407. (12) Zhu, D. G.; Petty, M. C.; Harris, M. Sens. Actuators, B 1990, 2, 265. (13) Vukusic, P. S.; Sambles, J. R. Thin Solid Films 1992, 221, 311.

nanosized materials, surface plasmon excitations in metallic nanoparticles,16 the effect of adsorbates17 on the plasmon properties, and the binding kinetics18 of adsorption are being studied. Recent studies report the improvements in the sensing capabilities of traditional SPR instruments by binding metallic nanoparticles on flat metal surfaces.19,20 Surface plasmons are electromagnetic waves trapped at an interface between a metal and a dielectric, which have fields decaying exponentially in both media.21 This surface excitation is tied to the oscillating surface charge density and propagates in a direction parallel to the interface. The presence of exponentially decaying fields makes it sensitive to changes in refractive indices next to the interface, a property that is exploited for optical monitoring of changes in the local environment. Any experiment involving surface plasmon resonance spectroscopy involves the interaction of analyte molecules with a thin metal surface, normally gold, which acts as the sensor surface. The gold layer, which is normally of the order of 50 nm, is coated either directly on to the base of a prism or on a glass coverslip made of the same material as the prism and then attached to the base of the prism by an index matching material. The coating process involves the vapor phase deposition of gold in an ultrahigh vacuum chambersa setup that is expensive and not (14) Curtiss, L. K.; Bonnet, J. D.; Rye, K. Biochemistry 2000, 39, 5712. (15) Nelson, B. P.; Grimsrud, T. E.; Liles, M. R.; Goodman, R. M.; Corn, R. M. Anal. Chem. 2001, 73, 1. (16) Klar, T.; Perner, M.; Grosse, S.; von Plessen, G.; Spirkl, W.; Feldmann, J. Phys. Rev. Lett. 1998, 80, 4249. (17) Malinsky, M. D.; Kelly, K. L.; Schatz, G. C.; Van Duyne, R. P. J. Am. Chem. Soc. 2001, 123, 1471. (18) Wagner, N. J.; Vaynberg, K. A. Langmuir 2001, 17, 957. (19) Hutter, E.; Cha, S.; Liu, J.-F.; Park, J.; Fendler, J. H.; Roy, D. J. Phys. Chem. B 2001, 105, 8. (20) Hutter, E.; Fendler, J. H.; Roy, D. J. Phys. Chem. B 2001, 105, 11159. (21) Raether, H. Excitation of Plasmons and Interband Transitions by Electrons; Springer: Berlin, Heidelberg, New York, 1980; Vol. 88.

10.1021/la020130g CCC: $22.00 © 2002 American Chemical Society Published on Web 09/26/2002

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Figure 1. Schematic representation of the surface plasmon resonance setup.

readily available in all chemical laboratories. Furthermore the calibration and deposition process to get the optimum thickness of metal (proper control of which is the most important criterion for the success of SPR experiments) is an area of science that requires a different focus from those practiced by the analytical laboratories. It is a complicated, time-consuming process and requires a different focus for these institutions. One option is to use glass coverslips coated with the gold from an external source, but owing to the high surface energy of the pure metal, this procedure leads to poor results due to the contamination of the metal surface when kept exposed to the environment over extended periods of time. This problem of contamination makes the storage of precoated slides difficult, and obtaining a freshly coated sample every time an experiment is performed is time-consuming. One alternative is to use the technique of optical waveguide spectroscopy (OWS).22-24 OWS has the advantage of offering a solid surface of the user’s choice, but even that is subject to contamination from external sources. This paper discusses a method that minimizes the problem of contamination of the gold sensor surface encountered in SPR experiments. It makes the storage of precoated coverslips easy, thus removing the necessity of any special protective environment. This technique involves the use of alumina as a protective layer on top of the gold. The alumina is electron beam evaporated over the gold surface immediately after the metal deposition, in the same vacuum chamber, without exposing the metal surface to external environmental conditions. This prevents any kind of foreign molecules from reaching the metal surface thus keeping it clean. In the beginning of a desired experiment the alumina layer is dissolved away using a concentrated sodium hydroxide solution, and the gold surface, which is free of any kind of contamination, is thus exposed. The presence of alumina allows for the possibility of supporting optical waveguide modes in the dielectric layer,25 which makes monitoring the alumina dissolution process easy. (22) Guemouri, L.; Ogier, J.; Ramsden, J. J. J. Chem. Phys. 1998, 109, 3265. (23) Lavers, C. R.; Wilkinson, J. S. Sens. Actuators, B 1994, 22, 475. (24) Homola, J.; Ctyroky, J.; Skalsky, M.; Hradilova, J.; Kolarova, P. Sens. Actuators, B 1997, 38-39, 286. (25) Tien, P. K. Rev. Mod. Phys. 1977, 49, 361.

Instrument The schematic representation of the in-house built surface plasmon resonance spectroscope is shown in Figure 1. A diode laser, operating at 635 nm, is used as the driving beam. To obtain a more uniform and coherent photon source, the beam is made to pass through a spatial filter, which removes the spurious bands and the Bessel rings that are associated with these photon sources. Two crossed polarizers are placed in the beam path which serve to attenuate the beam intensity to a level commensurate with the CCD’s dynamic range and also provide a beam polarized in a plane, parallel to the plane of incidence. Presence of a half-wave plate after the crossed polarizers allows for the change of polarization of the driving beam from “p” to “s” and vice versa, if required. A beam expander is subsequently used to increase the beam diameter 10fold, which provides the necessary angle spread and thus serves to remove any mechanical moving parts in the instrument, which are present in the more conventional SPR setups utilizing the θ-2θ goniometers. A cylindrical lens, whose line coincides with the axis of rotation of the prism/flowcell assembly is subsequently used to focus the beam at the prism/metal interface. The laser beam that is reflected from the base of the prism is collimated using a cylindrical lens, and the collimated beam is captured using a CCD camera. The image from the CCD camera is stored in a computer in real time with the help of a national instrument image grabber card. The data acquisition and analysis processes were programmed using the NI-IMAQ and LabVIEW software, respectively, from National Instruments. The current time resolution of our instrument is 30 ms and can be improved upon availability of faster data transfer protocols from the CCD to the computer. The raw reflectance signal, obtained from the CCD, is Fourier transformed26 to obtain the frequency components. An inverse Fourier transformation using the lowfrequency components yields the purified signal. The angle of minimum reflection is obtained by fitting a parabola to the data points around the region of minimum reflection. The light from the driving beam is reflected from the prism metal interface. The prism (SF-11; refractive index (n) ) 1.78)/metal interface is produced by vacuum sput(26) Mayo, C. S.; Hallock, R. B. Rev. Sci. Instrum. 1989, 60, 739.

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tering the metal layer on a coverslip (SF-11; n ) 1.78) and attaching this coverslip to the base of the prism with a melt-mount (Cargille) with n ) 1.69. This material was chosen instead of an index-matching fluid for the following two reasons: (a) the index matching fluid with n ) 1.78 has dissolved elemental sulfur as one of its components, the fumes of which reacted with the pure gold surface on the coverslip; (b) the index matching fluid was flowing out quite rapidly, when the prism-coverslip setup was placed vertically in the flow-cell assembly. No interference patterns arising from the index mismatch were detectable, as the thickness of the index-matching layer is of the order of a fraction of a micrometer. The prism-coverslip assembly is clamped on the flow cell, with silicone O-rings in the middle, to form a fluidtight seal. Chemical solutions are circulated under laminar flow conditions through chemical-resistant Tygon tubing and pumped using a Perkin-Elmer peristaltic pump. All solutions are thermostated using a RMS Lauda waterbath. Preparation of the metal sensor surface was done at Universal Thin Films Laboratory, in a custom-built electron beam equipped vacuum chamber. The deposition process was done under a pressure of ca. 1 × 10-5 Torr, with the electron gun operating under an applied voltage of 7 kV and100 mA current. The substrates were at a temperature of 150 °C. Prior to the coating of the metal, the coverslips were cleaned thoroughly with chromic acid to remove any organic contaminants. Before the gold was coated, a ca. 5 Å thick chromium layer is placed on the glass surface, which works as an adherent layer for the gold. The gold layer is then evaporated over the chromium. The rate of deposition for the gold was strictly controlled at ∼1 Å/s, so that the deposition proceeded evenly and the final coating had minimum roughness. The progress of the deposition process is followed using a quartz crystal microbalance, which is present inside the vacuum chamber. An approximately 500 nm thick aluminum oxide layer is then electron beam evaporated on top of the gold, without exposing the slides to the laboratory environment. This was done at ca. 5 Å/s, as the roughness of the alumina surface was not a critical parameter in the actual SPR experiments. It is known that metal oxides on electron beam evaporation produce molecularly dissociated species, and the coating thus produced is also not stoichiometrically balanced. To minimize the presence of dissociated species on the surface, pure oxygen is “bled” into the chamber after alumina deposition until the vacuum is reduced from 1 × 10-5 to 5 × 10-5 Torr. The substrates are exposed to the environment on attainment of room temperature. Experiment Surface plasmon resonance experiments were performed with the setup described above. A glass slide was precoated with ∼50 nm gold and ∼500 nm of alumina on top as a protective layer and then stored for over 6 months as is. The slide was attached to the glass prism using Cargille meltmount (n ) 1.69). Once the slide-prism block was attached to the flow cell and the entire assembly mounted on to the sample holder, a central incident angle was set from previous knowledge of the possible position of the resonance minimum, thus choosing the desired angle window and care was taken to ensure that the system assembly was not disturbed for extended periods of time. The dissolution of the protective alumina layer was affected using 1 N NaOH solution. As the alumina dissolved, the shift in the minimum reflectance position of the waveguide modes was recorded in real time. Atomic force microscopy experiments were performed to study the progress in dissolution of alumina in terms of change in surface roughness. A commercial atomic force microscope (Nanoscope IIIa, Digital Instruments, Santa Barbara, CA), equipped

Figure 2. Kinetics of dissolution of alumina using NaOH solution (1 N, 25 °C).

Figure 3. Experimentally obtained reflectance spectrum. with a 10.0 µm piezoscanner and a fluid cell was used to study the topology of the surfaces. All images were recorded using the “tapping mode” to minimize the damage to the sample surface and under filtered fluids to remove the possibility of any dust entering the system. The images were subjected to second-order flattening as the only postrecording image processing. All the images were taken at the same point of the sample surface so as to obtain a systematic and uniform reference scale for comparison purposes. The sample surface was a glass slide coated in the same batch as the ones used in SPR experiments, to provide a better comparison. NaOH (1 N) was flowed through the fluid cell and was replaced with water at regular intervals, during which the topography of the surface was monitored. It is to be noted that due to the inherent scanning process in atomic force microscopy, there is always a time lag between two regions in an image. Thus an alumina surface imaged under sodium hydroxide would result in an image with significantly different exposure times to different regions and would possibly show a slope in the topography. To minimize this effect, the surfaces were imaged under water, which has a much lower rate of alumina dissolution.

Results and Discussion Figure 2 shows the kinetics of dissolution of alumina from the surface, using 1 N sodium hydroxide solution. There are two sections in the plot, with a region in the middle where no reflectance dip was observed. Figure 3 shows the change in reflectance spectrum as a function of time. It can be seen that at one point the reflectance dip disappears. The absence of reflectance minimum is manifested as a break in the kinetic plot. Note, in Figure 3 the time/thickness of alumina axis is not divided into

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Figure 4. (a) Three-dimensional plot of waveguide mode resonances as a function of the angle of incidence, with changing thickness of the alumina layer. (b) Density plot of Figure 4a.

equal intervals of time/thickness. The plot has been compressed in time and certain plots have been left out for clarity. To better understand the kinetic plot and the change in reflectance and to explain the disappearance of the reflectance, simulation studies on the expected reflectance from a four-layer system (glass/gold/alumina/water) were performed employing the method of multiplication of transfer matrixes.27,28 The layers were assumed to be flat. Figure 4a shows the theoretically calculated reflectance (27) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized Light; North-Holland Personal Library: Amsterdam, 1987. (28) Sprokel, G. J. Mol. Cryst. Liq. Cryst. 1981, 68, 39.

for transverse magnetic radiation, obtained from the glass/ gold/alumina/water multilayer structure as a function of the angle of incidence, and as a function of the thickness of the alumina layer. The density plot (Figure 4b) shows clearly that with increase in the thickness of the waveguide (alumina layer), more waveguide modes are launched and supported. The first mode is called zeroth order mode (m ) 0) and it starts when the alumina thickness is minimum. The higher order modes are first, second, third, and so on. A thickness of 400-500 nm was assumed for the alumina layer, from prior knowledge of the evaporation data. In this thickness range, there is only the first-order mode in the 49-55° angle window (the present setup

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Figure 5. AFM pictures of alumina surface with progress in dissolution: (a) ethanol; (b) 1 N NaOH solution, 0 min; (c) 1 min; (d) 6 min, (e) 11 min; (f) 16 min; (g) 31 min; (h) higher resolution image of gold surface at z ) 5 nm/division. Panels a-g have a resolution of x ) y ) 0.2 mm/division, and z ) 50 nm/division.

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allows monitoring in a 14° spread around the central angle, of which only 6° are shown here for better resolution). As the alumina layer is dissolved away by the flowing sodium hydroxide solution, the first-order mode resonance shifts to lower angles and finally disappears. This process is represented in the first segment of the kinetic plot (Figure 2). At the same time the zeroth order mode shifts too but is not visible as it is still outside the angle window (on the higher side). It takes some time for the alumina to dissolve and shift the zeroth order mode such that it comes within the angle window. This explains the time lag between the first and the second segments of the plot. The second segment represents the shifting of the zeroth order mode to lower angles until all the alumina has dissolved and the reflectance spectrum of the gold/sodium hydroxide solution interface is obtained. On following the shift in mode positions with decreasing thickness of the waveguide, one can see that the zeroth-order mode is most sensitive to thickness changes (relatively greater shift in angle with change in thickness) and furthermore the sensitivity is more in the lower thickness range. Tiefenthaler29 had theoretically proven the fact that lower order modes are more sensitive to changes in refractive index and thickness than modes of higher order. Atomic force microscopic studies were performed to study the topological changes on the stacked structure with progress in dissolution of alumina. Roughness was defined as the vertical distance between the highest and lowest points in the section analysis of a representative portion on the surface. Figure 5a shows the topography of the initial alumina-coated slide under ethanol, with a roughness of 5.25 nm. Ethanol was chosen to prevent any kind of dissolution of the alumina. Parts b and c of Figure 5 shows the change in topography of the surface, when ethanol was first replaced with water and subsequently water replaced by 1 N NaOH solution. When ethanol was replaced with water, the roughness of the surface increased to 15.6 nm, which is possibly due to the dissolution of some of the more soluble defect points on the surface. A 1 N NaOH solution initially causes the dissolution of the sharper peaks on the surface left from the water treatment, with a consequent decrease in the roughness to 10.4 nm, which is in agreement with the fact that the regions of high surface area (sharp peaks) have a higher surface energy and hence are more reactive. On further exposure as is shown in Figure 5d, the roughness increased significantly and the surface showed rounded humps. With progress in time, the alumina gradually dissolves and the rounded features, which were seen in Figure 5d, first became more jagged (Figure 5, parts e and f) and then the roughness decreased, depicting the complete dissolution of alumina and the appearance of the bare gold surface (Figure 5g). Figure 5h shows the surface at an increased resolution. The roughness (Figure 6a shows an illustrative section analysis) of the surfaces, as shown in Figure 6b, increases from 36.9 to 78.8 nm and then decreases to 31.3 nm and finally reaches 3.6 nm when the dissolution process is complete. The atomic force microscopy (AFM) pictures of topological changes during the dissolution of alumina show that while the roughness parameter increases, at no point does it increase beyond the wavelength of the incident radiation. Furthermore the bare gold surface also show a roughness, which is 2 orders of magnitude smaller than the wavelength of the incident radiation (635 nm). This implies that scattering is negligible from the waveguide modes and the surface plasmons, which (29) Tiefenthaler, K.; Lukosz, W. J. Opt. Soc. Am. B 1989, 6, 209.

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Figure 6. (a) Illustrative section analysis showing the roughness parameter, (vertical distance between the two arrowheads). (b) Progression in the roughness of the surface as a function of time.

Figure 7. Reflectance spectrum of gold/water interface: (a) unprotected slide; (b) alumina-protected slide. The dotted line in the case of (b) shows the theoretical fit.

justifies our assumption of the layers in the stacked multilayer structure as flat Fresnel’s layers. Figure 7b shows the reflectance spectrum obtained for the gold/water interface. The spectrum obtained for a similar gold-coated coverslip stored for 6 months, but without any alumina protective layer, is presented in Figure 7a for comparison. The broadening of the SPR reflectance spectrum is a clear indication of the presence of contamination on the metal surface. The dotted line in Figure 7b shows the experimental spectrum. The experimental data were visually fitted with the predicted spectrum using a refractive index of 1.330 for water. The theoretical results, shown in solid line, match the experimental data very well. The thickness obtained was 57 nm, while the dielectric constant obtained for gold was -10.150 + 0.950i. These results match prior knowledge on the thickness (measured during evaporation) and the literature dielectric constant for bulk gold (-11.0 + 1.7i). It is to be noted that the dielectric constant of metallic thin films can differ from that of the bulk dielectric constant by as much as 15%. Conclusions Alumina was shown to be an excellent protective coating material for experiments using surface plasmon resonance spectroscopy. Following the shift in the “angle of minimum

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reflectance” of the waveguide modes, which requires no extra instrumentation over the standard SPR setup, the dissolution process of alumina can be monitored. AFM studies showed that while the roughness of the surface increased during the dissolution process, it never went beyond the wavelength of the incident radiation, which means that the dissolution proceeded relatively uniformly over the entire surface and that the layers could be modeled as Fresnel’s layers. Thus by using this procedure, fabrication of contamination-free surface plasmon resonance

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based sensor surfaces can become a less expensive and less time-consuming process. Acknowledgment. The authors gratefully acknowledge the support for this work from the National Science Foundation (NSF/EEC-98-13309) and the I/UCRC for Advanced Studies in Novel Surfactants (NSF/EEC-98-04618). LA020130G