Anal. Chem. 2007, 79, 1349-1355
Imaging Technique for the Screening of Protein-Protein Interactions Using Scattered Light under Surface Plasmon Resonance Conditions Andrej Savchenko,†,‡ Elena Kashuba,‡,§,| Vladimir Kashuba,§ and Boris Snopok*,†
V. Lashkaryov Institute of Semiconductor Physics, National Academy of Sciences, 41 Prospect Nauki, 03028 Kyiv, Ukraine, Department of Microbiology, Tumor and Cellular Biology (MTC), Karolinska Institute, S17177 Stockholm, Sweden, and IRIS (Center for Integrative Recognition in the Immune System), Karolinska Institute, S17177 Stockholm, Sweden
We propose a novel technique to detect protein-protein interactions in microarray format. The technique involves measuring scattered light under surface plasmon resonance (SPR) conditions. We have shown that the maximum scattering angle correlates with the traditionally employed reflection minimum. Panoramic scanning of scattered light under SPR conditions has all the functional advantages of the SPR technique. In addition, the proposed technique simplifies device design, increases the dynamic range of analysis, and integrates data with those from surface-plasmon field-enhanced fluorescence spectroscopy. We demonstrate the technique by showing direct protein-protein interaction between protein A and either rabbit antibodies or human serum. Sensor systems based on surface plasmon resonance (SPR) are widely used in the basic and applied sciences.1-3 SPR is valuable for studying biofilm formation processes, the interaction of proteins with small molecules or viruses,4-7 and protein-protein interactions.8,9 Many available commercial single-channel SPR systems have low throughput capacity and require large amounts of material for analysis.10,11 Users would value a microarray SPR spectrometer detecting hundreds of concurrent interactions. Such * To whom correspondence should be addressed. Phone +380445255626. Fax: +380445258342. E-mail:
[email protected],
[email protected]. † National Academy of Sciences. ‡ Contributed equally. § Department of Microbiology, Tumor and Cellular Biology (MTC), Karolinska Institute. | IRIS (Center for Integrative Recognition in the Immune System), Karolinska Institute. (1) Rich, R. L.; Myszka, D. G. Drug Discovery Today: Technologies 2004, 1, 301-308. (2) Ramsden, J. J. Optical biosensors. J. Mol. Recognit. 1997, 10, 109-120. (3) Homola, J. Anal. Bioanal. Chem. 2003, 377, 528-539. (4) Kim, S. J.; Gobi, K. V.; Harada, R.; Shankaran, D. R.; Miura, N. Sens. Actuators B: Chem. 2006, 115, 349-356. (5) Snopok, B. A.; Boltovets, P. N.; Rowell, F. J. Theor. Exp. Chem. 2006, 42, 106-112. (6) Oh, B.-K.; Kim, Y.-K.; Park, K. W.; Lee, W. H.; Choi, J.-W. Biosens. Bioelectron. 2004, 19, 1497-1504. (7) Boltovets, P. M.; Snopok, B. A.; Boyko, V. R.; Dyachenko, N. S.; Shirshov, Yu. M. J. Virol. Methods 2004, 121, 101-106. (8) Lee, H. J.; Marriott, G.; Corn, R. M. J. Physiol. 2005, 563, 61-71. (9) Snopok, B. A.; Kostukevich, E. V. Anal. Biochem. 2006, 348, 222-231. (10) Chiena, F.-C.; Chen, S.-J. Biosens. Bioelectron 2004, 20, 633-642. (11) Mukhopadhyay, R. Anal. Chem. 2005, 313A-317A. 10.1021/ac061456n CCC: $37.00 Published on Web 01/18/2007
© 2007 American Chemical Society
techniques form one of the most promising approaches to functional protein studies.12-15 Traditional optical SPR instruments are simple, compact, and sufficiently sensitive to measure functional dependence of the angle/wavelength-resolved reflection coefficient.1-12 One measurable quantity is a shift in the minimum of angle or wavelength functional dependence on reflection. This quantity (by SPR theory) is proportional to the mass at the interface when the thickness of the nonabsorbing coating layer is lower than the wavelength.16-18 In general, an optoelectronic technique measuring parameters other than simple reflection may yield more information on interfacial structure as measurements (intensity, polarization, and Stokes factors) of evanescent wave light scattering depend strongly on variation of permittivity within interfacial architectures and, consequently, on the presence of foreign molecules.19-22 Despite the high sensitivity of light scattering to small variations in thin film optical parameters, scattering is still not used in SPR sensor devices. Here, we describe a novel technique in sensing based on the monitoring of scattered light under SPR conditions. The aim of the work was to first design a system and then use it to experimentally show that measurement of peak angle dependence of scattered light might yield valuable information on surface processes. The data are derived in real time from microarrays. The utility of the approach is exemplified by the detection of direct protein-protein interactions between protein A and either rabbit antibodies or human serum. (12) Rella, R.; Spadavecchia, J.; Manera, M. G.; Siciliano, P.; Santino, A.; Mita, G. Biosens. Bioelectron. 2004, 20, 1140-1148. (13) Otsuki, S.; Tamada, K.; Wakida, S. Appl. Opt. 2005, 44, 3468-3472. (14) Smith, E.; Thomas, W. D.; Kiessling, L. L.; Corn, R. M. J. Am. Chem. Soc. 2003, 125, 6140-6148. (15) Jung, J. M.; Shin, Y. B.; Kim, M. G.; Ro, H. S.; Jung, H. T.; Chung, B. H. Anal. Biochem. 2004, 330, 251-256. (16) De Feijter, J. A.; Benjamins, J.; Veer, F. A. Biopolymers 1978, 17, 17591772. (17) Ball, V.; Ramsden, J. J. Biopolymers 1998, 46, 489-492. (18) Snopok, B. A.; Kostyukevych, K. V.; Rengevych, O. V.; Shirshov, Yu. M.; Venger, E. F.; Kolesnikova, I. N.; Lugovskoi, E. V. Semiconductor Physics, Quantum Electron. & Optoelectronics 1998, 1, 121-134. (19) Barnes, W. L.; Sambles, J. R. Solid State Commun. 1985, 55, 921-923. (20) Barnes, W. J Opt. A: Pure Appl. Opt. 2006, 8, S87-S93. (21) Sterligov, V. A.; Cheyssac, P.; Lysenko, S. I.; Fidali, Y. E; Kofman, R.; Stella, A. Eur. Phys. J. 1999, D 9, 581-584. (22) Lysenko, S. I.; Kaganovich, E. B.; Kizyak, I. M.; Snopok, B. A. Sensor Lett. 2005, 3, 117-125.
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Figure 1. Attenuated total reflection method, in the Kretschmann configuration, for excitation of surface plasmons. Schematic sketch of the plasmon propagation picture on the rough surface (a): onestep scattering (I) and stochastic surface interference (II) mechanisms. kSP, k*SP, krf are wave vectors of the generated plasmon, the in-planescattered nonradiative plasmon, and the surface roughness, respectively. (b) Measured reflectivity (R) and scattering (S) of a gold film in water as a function of the angle of incidence (the refractive index of glass is 1.61).
II. BACKGROUND: RADIATIVE SCATTERING OF SURFACE PLASMON STATES AT A ROUGH SURFACE The presence of the interface between the crystal and the surrounding medium causes the appearance of the collective excitations that are localized on the surface. These excitations are related to features of the electron and photon spectra of the system as a whole. In metals (Au, Ag, etc.23) coherent superposition of electron-hole pairs creates a wavelike excitation of the surface charge density (a coupled state between the plasma oscillations and photons, i.e., the “plasmon surface polaritons” or the surface plasmon4); the plasmon is a quasiparticle describing a coupled normal longitudinal mode of the surface (Figure 1).20,24,25 It is known that surface normal modes do not interact directly (23) de Bruijn, H. E.; Kooyman, R. P. H.; Greve, J. Appl. Opt. 1992, 31, 440442. (24) Abeles, F. In Modern Problems in Condensed Matter Sciences; Agranovitch, V. M., Loudon, R., Eds.; North-Holland: Amsterdam, 1982; Vol 9 (Surface Excitations). (25) Knoll, W. In Handbook of Optical Properties; Hummel, R. E., Wiesmann, P., Eds.; CRC Press, Inc.: Boca Raton, FL, 1997; Vol II (Optics of Small Particles, Interfaces, and Surfaces), pp 373-400.
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with electromagnetic waves because their dispersion regions do not correspond to electromagnetic frequency ranges. In line with this fact, an incident electromagnetic wave cannot directly excite surface plasmons at any angle and vice versa. The most convenient way to generate surface plasmons uses Kretschmann’s configuration2,25,26 to measure reflected light in total internal reflection condition. This technique is widely used as it is experimentally simple and gives reliable results. A photon with a large impulse in the prism interacts with the plasmon state through a thin metal layer deposited directly on the base of the glass prism (Figure 1). The surface plasmon resonance creates a sharp minimum (position ΘSPR) on the curve of the reflection coefficient for the p-polarized light (Figure 1b). The characteristic length of plasmon propagation along the surface is tens of micrometers. This restricts the use of evanescent wave excitations (i.e., SPR) in conventional microscopy techniques.25 Simultaneously, surface plasmon is a nonradiative mode with field amplitude that is maximal on the surface. The penetration depth is several hundreds of nanometers (above wavelength λ of used light) in a dielectric medium. These facts explain the high selectivity and sensitivity of SPR to surface processes, characterized by changes in the thicknesses and optical properties of interfacial architectures.1-3 Coupling between external radiation and plasmon states is also possible through diffraction of the external radiation on the rough or artificially structured surfaces.27 This diffraction causes changes in the longitudinal impulse of the light (or surface wave) dependent on the surface profile.26 In recent years, surface gratings (created by microlithography) have become available and studies of surface plasmon excitation using these gratings have become most useful.28,29 Moreover, surface waves decay by spreading along a rough surfacesthe surface relief is the most important factor affecting changes in power of surface plasmons.19,26,30 The decay of the surface state may be caused by different mechanisms, in particular, such as single or multiple radiative scatterings.27 It should be noted that damping caused by light radiation is absent on a smooth surface.26 The traditional description of radiation under SPR conditions is as a single elastic scattering of surface plasmons that propagates on the surface with a small value of mean-square roughness.27,30,31 In this model the metal surface is considered as flat with a stochastic distribution of relief height, so that the modulations in the relief height are considered to be perpendicular to the surface (process I, Figure 1a). According to this model, the intensity of radiative scattering in the plane of incidence is caused not only by the amplification of the electric field on the surface but also by dipole radiation W (∆k) and the power of spectral density PSD (∆k) of the surface. Thus, if the surface has relatively low roughness, the angle distribution of the scattering will depend mostly on dipole radiation W (∆k). In the case of increased roughness the influence of W (∆k) would diminish and PSD (∆k) would determine the angle distribution. However, the tight correlation between the scattering maximum and the reflection (26) Raether, H. Surface plasmons on smooth and rough surfaces and on gratings; Springer-Verlag: Berlin, 1985. (27) Kretschmann, E.; Raether, H. Z Naturforschung. 1968, 23a, 2135-2136. (28) Michel, T. R. J. Opt. Soc. Am. A 1994, 11, 1874-1885. (29) Worthing, P. T.; Barnes, W. L. J. Mod. Opt. 2002, 49, 1453-1462. (30) Depine, R. A.; Gigli, M. L. Opt. Commun. 1996, 129, 318-322. (31) O’Donnell, K. A.; Mendez, E. R. J. Opt. Soc. Am. A 2003, 20, 2338-2346.
minimum would not change because both are caused by amplification of the surface field and metal film characteristics. We will mention here that analysis of scattered light indicatrix yields much additional information about the processes taking place in such systems.32-35 Recently, great progress has been made in the description of processes on stochastic relief. This allows consideration of processes not previously included in classical theory. This process includes repetitive nonradiative elastic scattering of plasmon waves at a rough surface with changes in propagation directions (process II, Figure 1a).36-38 Thus, elastic repetitive nonradiative scattering on the surface plane and consequent single radiative scattering may be a principal decay mechanism of surface electromagnetic excitations. By “elastic nonradiative scattering” we mean that the interaction of plasmon quasiparticles with nonuniform surface features changes the direction of propagation without loss of energy. We have here a good example of strong interference of surface waves.39 This leads to a decrease of wave propagation in media and creation of surface states detectable by near-field microscopy.40 As discussed earlier,41 formation of localized states of the surface wave is followed by radiative decay and irradiation of the electromagnetic waves into surrounding media. We emphasize, however, that detailed measurements of such scattering as commonly used for biosensor applications polycrystalline gold films are not yet available. Moreover, no single model yet suffices to describe radiative decay of coupled surface waves on a stochastic surface. III. EXPERIMENTAL PROCEDURES The scanning imaging SPR spectrometer “BioSketch”, registering scattered light under SPR conditions, was developed at the V. Lashkaryov Institute of Semiconductor Physics of the National Academy of Sciences of the Ukraine and is derived from the singlechannel scanning SPR spectrometer “BioHelper”.42 The SPR spectrometer uses open measurement architecture and employs a simple gold-covered glass slide for microarray scanning (Figure 2a). The optical part of the instrument includes a semicylindrical lens (1) and a replaceable glass plate (chip) with a layer of gold (3) that is in optical contact with the lens through an immersion liquid (polyphenyl ether, refractive index above 1.61). A cell (4) is made of transparent material (glassed plexiglass). The cell supplies material to the transducer in the flow-through mode. A laser beam formation unit (dashed line) is placed on the (32) Bennett, J. M.; Mattsson, L. Introduction to Surface Roughness and Scattering; Optical Society of America: Washington, D.C., 1989. (33) Lysenko, S. I.; Snopok, B. A.; Sterligov, V. A.; Kostyukevich, E. V.; Shirshov, Yu. M. Opt. Spectrosc. 2001, 90, 606-616. (34) Sterligov, V. A.; Cheyssac, P.; Lysenko, S. I.; Kofman, R. Opt. Commun. 2000, 177, 1-8. (35) Stover, C. Optical Scattering: Measurement and Analysis; SPIE Optical Engineering Press: Bellingham, 1995. (36) Bozhevolnyi, S. I.; Volkov, V. S.; Boltasseva, A.; Leosson, K. Opt. Commun. 2003, 223, 25-29. (37) O’Donnell, K. A. Opt. Soc. Am. A 2001, 18, 1507-1518. (38) Evlyukhin, A. B.; Bozhevolnyi, S. I. Surf. Sci. 2005, 590, 173-180. (39) Ditlbacher, H.; Krenn, J. R.; Schider, G.; Leitner, A.; Aussenegg, F. R. Appl. Phys. Lett. 2002, 81, 1762-1764. (40) Bozhevolnyi, S. I.; Markel, V. A.; Coello, V.; Kim, W.; Shalaev, V. M. Phys. Rev. 1998, B58, 11441-11448. (41) In Optical Properties of Random Nanostructures; Shalaev, V. M., Ed.; Topics in Applied Physics; Springer-Verlag: Berlin, Heidelberg, 2002. (42) Snopok, B. A.; Yurchenko, M.; Szekely, L.; Klein, G.; Kasuba, E. Anal. Bioanal. Chem., 2006, 386, 2063-2073.
Figure 2. Experimental setup of the scanning imaging SPR spectrometer “BioSketch” with registration of scattered light under SPR conditions (a) and microarray configuration (b) with the printed spots of protein A (“A”) and polyclonal rabbit antibodies (“IgG(r)”) against caspase-3. The SPR spectrometer includes a semicylindrical lens (1), a semiconductor laser (λ ) 650 nm) and collimating optics (2), a replaceable glass plate (chip) with a layer of gold (3), a cell (4) from glassed plexiglass, the DIN standard 20× microscope objective (5), a charged coupled device (CCD) camera (6), and a photodiode (7).
angle scanning system. The unit consists of a semiconductor laser (λ ) 650 nm) and collimating optics (2). A concave cylindrical lens (which prevents the collimated laser beam from focusing on the convex surface of the main lens (1)) also is placed in the scanning system. A stepper motor maintains the scanning angle φ at 23° (from a starting angle of 52°). The angular resolution is 0.035°. Analytical Chemistry, Vol. 79, No. 4, February 15, 2007
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This system makes it possible to detect both the integral intensity of reflected light (the classical version of the SPR spectrometer, Figure 1b, beam II) and the two-dimensional matrix of scattered light intensity (Figure 1b, beam III), as functions of angles, by varying the angle of incidence (Figure 1b, beam I). The DIN standard 20× microscope objective (5) focuses scattered light onto the surface of a charge-coupled device (CCD) camera (a 10-bit digital image sensor; Motorola MCM20027B) (6). The resolution of the system when used to measure scattered light is about 4 µm (smaller than the surface plasmon propagation length; some tens of micrometers). The intensity of the reflected beam is registered with a photodiode (7). A microcontroller-based system (using an Atmel RISC processor) coordinates operation of all units. Materials and Methods. Chemicals. Chemical reagents were purchased from Sigma (St. Louis, MO) and used without further purification. The reagents were as follows: a 30% (w/w) solution of hydrogen peroxide, a 37% (v/v) solution of hydrochloric acid, guanidine thiocyanate, glucose, glycerin, protein A of Staphylococcus aureus, and polyclonal rabbit antibodies against caspase-3. Total human serum was obtained from peripheral blood of a healthy donor (EK). The secondary rabbit anti-human IgG, FITCconjugated antibody, was from DAKO (Glostrup, Denmark). Chip Manufacture. A glass chip (n ) 1.61) with a 45 nm gold layer sputtered on a chromium adhesion layer (5 nm) served as physical transducer. We removed various contaminants from the gold surface of the chip just before array printing. Such materials have unfavorable effects on formation of the sensitive surface layer and the optical parameters of the sensitive interface. The etchant solution contained 2 vol % of 37% (v/v) hydrochloric acid and 2 vol % of 30% (w/w) hydrogen peroxide in distilled water. The chip was held in this etchant for 15 min at room temperature. To prevent protein denaturation at the metal surface, we performed (immediately after cleaning) metal passivation with a self-assembling layer of guanidine thiocyanate (0.25 mmol in water, 30 min at room temperature). As shown earlier,5,42 this treatment makes it possible to retain biological activity of proteins at the surface. Microarray Production. The microarrays were printed (Figure 2b) on the surface of modified (see above) SPR chips using a spotter (QArrayMini; Genetix, New Milton, England) according to the manufacturer’s protocol (humidity of 80% and room temperature).43 The single spot size was about 200 µm, and the distance between spot centers was 300 µm. Aqueous solutions of concentrations 150 µg/mL and 1 mg/mL were used to deposit antibodies and protein A, respectively. After printing, the microarrays were dried for 1 h at a humidity of 80%, then washed in phosphate buffered saline (PBS), and kept in the same buffer at 4 °C until use. Experimental Procedure and Data Processing. Experiments were performed in the flow-through mode. The peristaltic pump (Alitea, U1-M) provided constant flow (200 µL/min) through the experimental cell. To investigate protein-protein interactions, a typical adsorption experiment was carried out in the following manner. The running buffer (PBS) was allowed to flow through the cell with the printed chip and then rapidly substituted by the first protein solution (with rabbit antibodies at a concentration higher (43) http://www.genetix.com/instr/qarraymini.asp.
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than 1 µg/mL, IgG(r), or human serum, HS). The protein solution was replaced by buffer after 10 min. When HS was used, after baseline stabilization, the second protein solution (with FITCconjugated rabbit anti-human antibody, IgGFITC) was introduced and buffer washing recommenced after 10 min. The intensities of the reflected and scattered light were measured by a total integrated regime over a wide range of angles. The positions of extremes were determined by approximating the parts of the curves near the extremes (a 17% curve amplitude was used for approximation) with a third-degree polynomial using the least-squares method. For microarrays, the intensity of the scattered light was averaged over a square of constant side inscribed in the spot area. Optical Microscopy. Fabricated microarrays were visually checked with transmitted light. For routine documentation, the images were captured with a Hamamatsu dual-mode-cooled CCD camera C4880 as described elsewhere44 using a DAS microscope (Leitz DM RB). A 10-fold magnification was used. IV. VALIDATION OF SCATTERING MEASUREMENTS Typical experimental functional dependencies of reflected and scattered light intensities on the angle of excitation are shown (Figure 1b). The scattering curve is slightly asymmetric and, at large angles, falls off more smoothly (as does the SPR reflection curve), in full agreement with theoretical considerations. The position of the peak of angular dependence is determined mainly by the conditions for the maximum of the squared amplitude of electric field of the plasmon state at the interface.3,45,46 At the same time, the reflection curve is determined by superposition of the field of plasmon excitation (on the metal side of the metaldielectric interface) and that of the penetrating wave that appears under total internal reflection (at the prism-metal interface, on the metal side) (Figure 1). Therefore, both geometrical and morphological features of the metal film as well as the degree of prism-SPR chip optical matching (when replaceable chips with optical contacts on the immersion layer are used) may lead to additional variations in the curve form due to changes in the parameters of wave damping in the metal.3,45,46 Concurrently, scattered light (the radiation losses in this system) parameters will be determined mainly by the intensity of plasmon excitation and power spectral density of surface roughness. Finally, this difference between the physical mechanisms of reflection and scattering results in distinct maxima and minima positions of the angular dependencies of scattering and reflection, respectively (Figure 1b). The shape of the reflection curve is thus determined to a great extent by the effective macroscopic morphological parameters of the metal film, but the scattering curve depends on local topographic features. Similar conclusions are valid also for fluorescence spectroscopy, Raman scattering, or surface plasmon-coupled emission with excitation by the evanescent wave field.47-49 (44) Kashuba, E.; Mattsson, K.; Klein, G.; Szekely, L. Mol. Cancer 2003, 2, 1-9. (45) Ekgasit, S.; Thammacharoen, C.; Knoll, W. Anal. Chem. 2004, 76, 561568. (46) Ekgasit, S.; Thammacharoen, C.; Yu, F.; Knoll, W. Anal. Chem. 2004, 76, 2210-2219. (47) Ekgasit, S.; Stengel, G.; Knoll, W. Anal. Chem. 2004, 76, 4747-4755. (48) Ekgasit, S; Tangcharoenbumrungsuk, A.; Yu, F.; Baba, A; Knoll, W. Sens. Actuators B 2005, 105, 532-541.
Figure 3. Relation between angle position shifts (relative to pure water) of integrated scattered light intensity maximum and reflection minimum for water solutions of glucose (0), glycerol (O), and a protein A-rabbit IgG(r) layer (4).
Measurements of Bulk Refractive Index Changes. To check linear dependency of the shifts in extremes of the angular dependences of scattering and reflection under SPR, as predicted from the theoretical description outlined above, we carried out two sets of experiments. These tests enabled us to obtain the dependencies for two limiting cases. We investigated variation in bulk refractive index of the volume phase over the bare physical transducer and after formation of a thin homogeneous film on the transducer surface. To determine the effect of bulk refractive index variation on the shifts of both the peaks of angular dependence of scattering (∆S) and minimum of reflection (∆R) under SPR (∆R ) f(∆S)) we used water solutions of glucose and glycerin in the concentration range from 1.25 to 10.00 wt % (in this range the solutes do not form adsorbed films on a gold surface). We showed (Figure 3) that variation of the refractive index of the solution with variation of concentration leads to coordinated shifting of the extreme of angle dependencies. A linear correlation (>99%) between these quantities is observed as the conditions for SPR excitation change due to variation of the refractive index of the solution. This result indicates that sensitivity of the new technique (scattering) is the same as that of the conventional practice (reflection). Moreover, the lower detection limit of the instrumentation is determined by water temperature variations for both systems. The main difference is that the upper detection limit of scattering is higher than that of SPR. Correspondingly, the dynamic range of the new method is greater than that of SPR. This is an essential difference. Indeed, determination of SPR angle position using the angular dependence of scattered light is more universal because it enables one to make an analysis even when the reflection curve is weak and the position of the minimum cannot be determined.50 Study of Protein Film Formation. The practical value of SPR lies in an ability to register effective variations of refractive index (49) Matveeva, E.; Gryczynski, Z.; Gryczynski, I.; Lakowicz, J. R. J. Immunol. Methods 2004, 286, 133-40. (50) Sterligov, V. A.; Kretschmann, M. Opt. Express 2005, 13, 4134-4140.
at distances of several (or several tens) nanometers from the interface rather than the possibility of SPR use in the direct refractometric measurements described above. Indeed, since the penetration depth for an evanescent wave is on the order of the wavelength, the SPR excitation angle depends on the optical parameters of the medium in the immediate vicinity of the metal surface (as a wave decays exponentially from the surface). The refractive indices of different biological substances (proteins, DNA, etc.) are usually 1.45-1.50 and thus higher than those of solutions prepared for experiments (1.33-1.37). Therefore, such molecules (either immobilized or bound at a surface) contribute significantly to the effective refractive index of a near-surface layer, thus changing considerably the SPR excitation angle. To confirm linearity of the dependence ∆R ) f(∆S) in our work, we studied both immobilization of proteins on the transducer surface and protein interactions with a selective partner. We examined the reaction between protein A of Staphylococcus aureus and the Fc fragments of rabbit immunoglobulin IgG. It should be noted that such architecture serves as the basis for the antibody-based microarrays intended for functional investigation of proteins.42 It has already been shown6,7,42 that protein A forms stable continuous films on a gold surface modified with a self-assembled guanidine thiocyanate layer and that protein A thus bound retains the capacity to bind with the Fc fragment of IgG. The data of Figure 3 show a complete cycle of the standard experiment analyzing protein-protein interaction. After adsorption of protein A, the cell was washed with working buffer and an antibody layer was then formed. Figure 3 shows that the shifts of extremes at immobilization and interaction between proteins at the surface are mutually proportional to each other with a high degree of accuracy (the correlation coefficient is >0.99). Thus, the data allow us to contend that the measurement of scattered light under SPR conditions may be efficiently used to obtain information on processes occurring at the interface. V. MICROARRAYS Measurement of Model Objects. To test the value of panoramic scanning for investigating parallel protein-protein interactions, we performed measurements in the microarray format using protein A of Staphylococcus aureus and rabbit IgG. The following areas were introduced into an (800 × 800 µm) nineelement fragment of a microarray. First, we made three spots with protein A (positive control; we expected a considerable shift in the scattering peak due to binding of IgG). Second, we included an area of unmodified gold surface in the middle of the microarray (to estimate nonspecific immobilization of antibodies). Third, we introduced five spots coated with the rabbit antibodies against caspase-3 (negative control; these antibodies should not react with rabbit IgG). The data on interaction processes are shown (Figure 4). After the response level was initially stabilized (absolute value of the initial SPR position depends on the quantity of deposited substance and can vary from spot to spot), the rabbit antibody solution was injected into the working cell. This resulted in considerable signal increase (the informative parameter is the difference between new (after interaction process and running buffer wash) and initial SPR positions) in the areas with protein A. The signals sometimes returned to the initial level after washing with working buffer due Analytical Chemistry, Vol. 79, No. 4, February 15, 2007
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Figure 4. Kinetic behavior of the SPR angle shift ΘSPR for a ninespot array under IgG(r) exposure.
to removal of the weakly bound nonspecific component. Indeed, in those areas where anti-caspase rabbit antibody was introduced during microarray fabrication, the increase in signal was insignificant (3 ( 2 angle minutes) in comparison with one in areas with preimmobilized protein A (19 ( 3 angle minutes). In the free gold surface area a medium response was observed due to nonspecific binding (10 angle minutes)sit indicates the necessity to modify the gold surface for sensing application. Washing had only a slight effect in areas with protein A, however, indicating formation of strong bonds between the given proteins. The response (19 ( 3 angle minutes) with rabbit antibodies for different spots is comparable to that obtained in the single-channel experiment discussed earlier (15.2 ( 0.2 angle minutes). This confirms that our procedure quantitatively registers proteinprotein interaction in the microarray format. In other words, IgG rabbit antibody immobilization occurred in the protein A spots and at the free gold surface, as expected. Analysis of Biological Subjects. Experiments with single protein solutions, as described above, are valuable for drug screening and analysis of protein-protein interactions in microarray format. Another experimental design might use a mixture of proteins in a protein microarray. As an example, we used human serum (a mixture of proteins) to bind to a printed layer of protein A. Determination of HS immunoglobulin capture by protein A was performed using secondary rabbit FITC-conjugated antibodies against human IgGs. A standard array (Figure 2b) involved the following areas: protein A spots that could bind human immunoglobulins; a free gold surface at which antibody immobilization might also occur; spots of anti-caspase rabbit antibodies that do not interact with human immunoglobulins; and an area with a strike of an empty spotter needle (this area is marked “empty”). We included the “empty” spots because we found, during microarray fabrication, that the spotter needle cripples the gold coating when depositing microdrops of solution. The needle leads to the appearance of a scratch about 10 × 4 µm (see Figure 7a, left upper spot). Thus, the “empty” area serves to check the effect of scratches on detected signals because scattering intensities were determined by signal integration over the whole area of a protein spot. The results of measurements for nine areas are given in Figure 5. Increases in signal occurred predominantly in spots with protein 1354 Analytical Chemistry, Vol. 79, No. 4, February 15, 2007
Figure 5. Kinetic behavior of the SPR angle shift ΘSPR for a ninespot array (Figure 2b) under human serum (HS) exposure and following interaction with FITC-conjugated secondary rabbit antihuman immunoglobulin antibody (IgGFITC).
A due to interaction with human immunoglobulins. A slight signal increase due to nonspecific adsorption onto metal is also observed in the unmodified gold surface areas. No signal increases were observed in areas where rabbit antibodies were applied. Washing with buffer resulted in insignificant signal decreases in protein A spots due to removal of nonspecifically bound proteins from the transducer surface. It should be noted that no additional specific/unspecific interactions were registered in the microarray area where a strike of the spotter needle without a reagent had been made. A response from this area was identical to that from the free gold surface. This indicates that spotter needle artifacts do not contribute to the signal that carries information. The same microarray panel was then treated with secondary rabbit anti-human immunoglobulin FITC-conjugated antibodies. As expected, we detected the binding of rabbit FITC-conjugated antibodies to human immunoglobulin/protein A complexes (Figure 5). A slight increase in response to rabbit FITC-conjugated antibodies was observed also in rabbit anti-caspase spots due to weak cross-reactivity of the antibodies applied. Moreover, essential increasing of the response (artifact) was observed for one of the IgG(r) spots contrary to others; this observation indicates an advantage of the microarray technology with repeated reaction spots in comparison with single measurements. Figure 6 illustrates the space of scattering peaks for a protein microarray at different stages of the analysis procedure. The axes correspond to the coordinate axes of a microarray (as for the optical images in Figures 7 and 2a), while the intensities correspond to the angular positions (ΘSPR) of the scattering peaks for a given point. It is possible to conclude that the panoramic scanning procedure based on an analysis of scattered light allows high-quality images of microarray functional elements, in the space of scattering peaks, to be obtained with signal-to-noise ratios no less than 4.5. This allows adequate detection of most protein-protein interactions in an actual biological mixture. Comparative Measurements Using Fluorescence Microscopy. In order to confirm the results described above, we studied microarray images by fluorescence microscopy. Figure 7a shows
Figure 6. Space of scattered light peaks for a protein microarray at different stages of the analysis procedure. (Left) Initially printed array. (Right) After reaction with human serum. The spot positions are indicated in a microarray outline in Figure 2a, while spot intensity corresponds to the angular position (ΘSPR) of the scattered light peak for a given point.
Figure 7. Array images obtained after reactions with human serum and rabbit anti-human FITC-conjugated antibody using transmitted light (phase contrast) (left), and the same microarray after illumination with light to detect fluorescence at 400 nm (right). Note intense fluorescence in those spots where protein A-human serum-rabbit anti-human IgG structures had been formed.
an image of a microarray obtained by transmitted light (phase contrast). It shows clearly all microarray features corresponding to areas in the scattering peak space (Figure 6a). An image of the same microarray under UV light (λ ) 400 nm) for detection of fluorescence is also shown (Figure 7b). Intense fluorescence was observed in those spots where protein A/human immunoglobulins/rabbit anti-human FITC-conjugated antibody complexes were formed. Weak fluorescence signal was detected in spots where rabbit anti-caspase antibodies were deposited. That can be due to the slight cross-reactivity of antibodies. This is in full agreement with data obtained by the novel technique. VI. CONCLUSIONS Here we show, for the first time, that measurements on scattered light yield valuable information under SPR conditions in sensor applications. A panoramic scanning spectrometer records variations in both the volume refractive index and protein layer
formation on a metal surface and has all the functional advantages of the SPR technique. Using the proposed technique we detected protein-protein interactions in microarrays using protein A, commercial antibodies, and human serum. The information obtained with the scattered light technique was confirmed by traditional immunochemistry. The novel technique to measure scattered light intensity seems to be very promising. Considering that the design is simple, the technological and measurement reliability of the device optical system is very good. Images of micrometer size are formed on replacement chips without device adjustment. The instrument is based on well-developed elements of optical microscopy, thus allowing attainment of high performance at low cost. The proposed technique simplifies device design, increases the dynamic range of analysis, and integrates data with those from surface-plasmon field-enhanced fluorescence spectroscopy.45-48 All this will result in increased universality, reliability, and cheapness of analytical equipment, thus permitting development of a new generation of efficient and scalable instrumentation using SPR phenomena. ACKNOWLEDGMENT The Swedish Cancer Society, a matching grant from the Concern Foundation, Los Angeles, the Cancer Research Institute, New York, the Swedish Institute, the Swedish Foundation for Strategic Research, and an INTAS grant 05-109-5077 supported this work. Received for review August 6, 2006. Accepted November 20, 2006. AC061456N
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