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Novel Silicon Dioxide Sol-Gel Films for Potential Sensor Applications: A Surface Plasmon Resonance Study Dev K. Kambhampati,*,† Thomas A. M. Jakob,*,† Joseph W. Robertson,‡ Mei Cai,‡ Jeanne E. Pemberton,‡ and Wolfgang Knoll*,† Max Planck Institut fuer Polymer Forschung, Ackermannweg 10, D-55128 Mainz, Germany, and Department of Chemistry, University of Arizona, Tucson, Arizona 85721 Received August 30, 2000 Stable silicon dioxide (silica, SiO2) films on noble metals were synthesized using a novel sol-gel technique. Surface plasmon resonance (SPR) spectroscopy was used to compare the stability of silica films generated by this sol-gel technique with those deposited by using thermal evaporation, in the presence of PBS buffer. These films were later chemically functionalized with various reactive groups as a test for their versatility and usefulness in sensoric applications. A surface grating enclosed in a high-pressure and elevated temperature cell was used in the investigation of polymer brushes while SPR coupled with fluorescence detection schemes was used in the Kretschmann prism configuration to monitor DNA hybridization interactions.
Introduction The use of analytical sensing instruments based on the principle of surface plasmon resonance is finding widespread applications in the area of molecular diagnostics and drug discovery assays and in characterizing surfaces in real time.1-7 Although these instruments are fast and reliable, the sensing technique is based upon the utilization of a noble metal (typically gold or silver) to generate the surface plasmon electromagnetic field which is then used to probe changes of the optical (film) properties, e.g., by binding reactions, occurring within the vicinity of the surface. Thiol-based self-assembly techniques8-12 work well with noble metals and in most cases are sufficient to generate supramolecular architectures which can be functionalized for potential biosensing applications. However, in certain cases, such an immobilization strategy is not suitable and as a result requires the use of other complex coupling chemistries. * Corresponding authors. Prof. Dr.Wolfgang Knoll: e-mail knoll@ mpip-mainz.mpg.de; phone 49-6131379160; Fax 49-6131379360. Dev Kambhampati: e-mail
[email protected]; phone 49-6131379264; Fax 49-6131379100. Thomas Jakob: e-mail jakob@ mpip-mainz.mpg.de; phone 49-6131379157; Fax 49-6131379100. † Max Planck Institut fuer Polymer Forschung. ‡ University of Arizona. (1) Karlsson, R.; Roos, H.; Fagerstam, L.; Persson, B. Methods: A Companion to Methods in Enzymology 1994, 6, 99-110. (2) Jordan, C. E.; Frutos, A. G.; Thiel; A. J.; Corn, R. Anal. Chem. 1997, 69, 4939-4947. (3) Jung, L. S.; Campbell, C. T.; Chinowsky, T. M.; Mar, M. N.; Yee, S. S. Langmuir 1998, 14, 5636-5648. (4) Knoll, W.; Liley, M.; Piscevic, D.; Spinke, J.; Tarlov, M. J. Adv. Biophys. 1997, 34, 231-251. (5) Piscevic, D.; Lawall, R.; Veith, M.; Liley, M.; Okahata; Y.; Knoll, W. Appl. Surf. Sci. 1995, 90, 425-436. (6) Schuck, P. Annu. Rev. Biophys. Biomol. Struct. 1997, 26, 541566. (7) Zizlsperger, M.; Knoll, W. Prog. Colloid Polym. Sci. 1998, 109, 244-253. (8) Chen, X.; Zhang, X. E.; Chai, Y. Q.; Hu, W.; Zhang, Z.; Zhang, X.; Cass, A. E. S. Biosens. Bioelectron. 1998, 13, 451-458. (9) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 89168920. (10) Allara, D. L. Cambridge University Press: New York, 1996; Chapter 11; pp 180-200. (11) Whitesides, G. M.; Gorman, C. B. CRC Press: Boca Raton, FL, 1995; Chapter 52, pp 713-731. (12) Porter, M. D.; Walczak, M. M. CRC Press: Boca Raton, FL, 1995; Chapter 53, pp 733-740.
Supramolecular architectures of biomolecules based on silane coupling chemistries13 on silicon dioxide substrates are commonly used in many applications, e.g., in genomic research where these substrates are employed to immobilize nucleic acids or proteins and their interaction behavior with complementary molecules is detected by using fluorescence techniques.14-16 Another example for the use of these substrates is in immobilizing polymer brushes17 which serve as a three-dimensional matrix for studying host-guest interactions and also in fundamental studies of polyelectrolytes.18 One way to combine the use of the surface plasmon spectroscopy optical method for monitoring surface reactions and silicon dioxide (SiO2) substrates for the coupling chemistry of one of the reactants is to thermally evaporate a thin layer of SiO2 in a vacuum chamber onto the metal layer used for surface plasmon excitation. However, we observe that in most cases the interface SiO2/aqueous medium is not stable, and usually the thin layer of SiO2 comes off the surface within a few minutes and as a result is not suitable for the above-mentioned applications. In this paper, we report the use of sol-gel chemistry in generating stable silicon oxide films on noble metals which can then be easily functionalized for sensing purposes. The stability of these films was tested by using a stringent aqueous solution containing PBS buffer, which is commonly used in many biological studies. To display the versatility of these films, we applied them in investigations of DNA hybridization reactions using surface plasmon resonance-enhanced fluorescence spectroscopy in the Kretschmann19 format using a coupling prism. A second example concerns the investigation of polymer brushes under high-pressure and elevated temperature conditions employing a specially designed high-pressure/temperature cell and using gratings for surface plasmon excitation. (13) Plueddemann, P., 2nd ed.; Plenum Press: New York, 1991. (14) Chee, M.; Yang, R.; Hubbell, E.; Berno, A.; Huang, X. C.; Stern, D.; Winkler, J.; Lockhart, D. J.; Morris, M. S.; Fodor, S. P. A. Science 1996, 271, 610-614. (15) Fodor, S. P. A. Science 1997, 277, 393-395. (16) Sapolsky, R. J.; Hsie, L.; Berno, A.; Ghandour, G.; Mittmann, M.; Fan, J. B. Genet. Anal.: Biomol. Eng. 1999, 14, 187-192. (17) Prucker, O.; Ruehe, J. Macromolecules 1998, 31, 592-601. (18) Biesalski, M.; Ruehe, J. Macromolecules 1999, 32, 2309-2316. (19) Kretschmann, E.; Raether, H. Z. Naturforsch. 1968, A23, 2135.
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Figure 1. Schematic of the experimental setup used for the recording of scans and kinetics of the reflectivity in surface plasmon spectroscopy. Onto the goniometer G can be mounted (a) a Kretschmann configuration type of cell which allows for monitoring the fluorescence of chromophores excited by the surface plasmon electromagnetic field at the metal/dielectric interface or (b) a highpressure surface-plasmon grating cell in which hydrostatic pressure and temperature can be tuned separately (T, p) and therefore a complete set of thermodynamical parameters can be evaluated. In synopsis, the laser beam is confined by the iris I, and its intensity and polarization are adjusted with the two polarizers P; then it is reflected off the mirror M onto the sample, which is mounted on top of the θ/2θ goniometer G, and from there onto the detector D. Furthermore, the lens L collimates the laser beam on the detector, and the Fresnel rhombus FR is used to easily adjust the polarization. A chopper C is used to generate a certain reference frequency of the laser beam and is used in combination with a lock-in amplifier, which then reads out the signal detected by the detector. The lock-in amplifier is interfaced to a computer for data collection purposes. In our fluorescence measurements, an interference filter F is used in front of the photomultiplier tube PM to allow light of a certain wavelength (in our case λ ) 670 nm) to pass while restricting other wavelengths, including the incident laser beam. A flow cell FC was also designed for fluid handling purposes.
Experimental Materials and Methods Materials. Sol-Gel. All materials employed were of research grade and were used as received from commercial sources without any additional purification with the exception of ethanol (Riedel Haeen), which was dried using a distillation column, and 3MPT (Fluka), which was distilled prior to usage. Drying of ethanol and distillation of 3MPT were carried out to minimize the presence of moisture in these reagents. Tetramethoxysilane (TMOS) and hydrochloric acid (HCl) were purchased from Fluka while methanol was obtained from Riedel de Haen. Highly pure Millipore water (18.2 MΩ cm) was used for preparing the HCl and TMOS solutions. DNA Architectures. Streptavidin and the biotinylated DNA catchers and fluorescent targets were obtained from Boehringer Mannheim. Biotin silane was synthesized in-house while the amino spacer silane was purchased from ABCR Chemicals. Potassium hydroxide, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate were purchased from Fluka. A 0.1 M PBS buffer (Sigma) solution was used in our stability analysis tests and in the actual DNA hybridization experiments. PMMA Brush. Toluene and triethylamine were both obtained from Fisher Chemicals and dried while methyl methacrylate, purchased from Fluka, was purified by distillation. Azodichlorinesilane was kindly provided by O. Prucker and J. Ru¨he, Freiburg, Germany. Methods. Thermal Evaporation of SiOx Films. Thin films of SiOx on metal layers were deposited by thermal evaporation of silicon oxide (SiO) in the presence of oxygen within an evaporating chamber (Baltzers Instruments) maintained at low pressure (∼2 × 10-4 mbar). We call the thin film of silica deposited on the metal layer as SiOx because the SiO vapor can react with oxygen with different stoichiometries. SPR Instrumentation. For the surface plasmon field-enhanced fluorescence spectrocopy (SPFS)20 measurements, the Kretschmann prism coupling concept was used for generating surface plasmons while in the case of the high-temperature and -pressure measurements21 conducted in a pressure cell, a surface grating22,23 was used for surface plasmon excitation. The schematic of the experimental setup used for SPFS and for high-temperature and (20) Liebermann, T.; Knoll, W. Colloids Surf. A 2000, 171, 115-130.
-pressure measurements is shown in Figure 1. Except for the additional fluorescence and high-pressure cell extensions, the equipment that was used in our measurements was a regular SPR unit. In a typical surface plasmon experiment, a p-polarized (transverse magnetic, TM) He-Ne laser beam (632.8 nm) is reflected off the sample which is mounted on top of a θ/2θ goniometer with the detector D measuring the reflectivity (R) changes as a function of the angle of incidence of the in-coming laser beam. A lock-in amplifier coupled to both the detector and chopper then reads out the actual reflectivity signal. Modes of SPR Measurement. There are two modes of monitoring interfacial interactions in the normal prism coupling version. In the scan mode, the variation of reflected light as a function of the angle of incidence of the laser beam is monitored. The critical angle and the angle of resonance are characteristics of the system under study, and any change in the refractive index of the dielectric medium, e.g., by formation of a monolayer, will cause the resonance angle to shift to a higher angle. To illustrate this point, Figure 2a shows the shift in the resonance angle after a monolayer of 3MPT was formed by a self-ssembly process on gold. Note that there is no change in the position of the critical angle as the same solvent (in this case, ethanol) was used before and after the self-assembly process. The formation of this monolayer (Figure 2b) can also be monitored in real time by recording the changes in the reflectivity at a fixed angle of excitation/observation. This is called the kinetic mode and is useful in real-time monitoring of interfacial reactions. SPFS Measurements. Although SPR is a highly sensitive technique, it suffers from low signal-to-noise ratio problems in ultrathin film systems where the refractive index changes are very small. To overcome such problems, one can use fluorescently tagged molecules which generate a substantially higher signal, which can then be readily detected by using the recently developed SPFS technique (cf. Figure 1a). In this technique, the fluorescent markers are excited by the strong plasmon electromagnetic field at resonance, and the wavelength shifted fluorescence light can then be detected by using a photomultiplier containing a band(21) Kleideiter, G.; Sekat, Z.; Kreiter, M.; Lechner, M. D.; Knoll, W. J. Mol. Struct. 2000, 521, 167-178. (22) Raether, H. Springer-Verlag: Berlin, 1988. (23) Knoll, W. Annu. Rev. Phys. Chem. 1998, 49, 569-638.
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Figure 2. SPR measurement modes. (a) Angular scans of reflectivity indicating the shift in the resonance angle due to the formation of a thin film of 3MPT on gold by self-assembly. (b) Kinetic measurement of the self-assembly process of the 3MPT layer in real time. pass filter to block elastically scattered light. A glass flow cell (volume 150 µL) was fabricated in-house and was used for the purpose of exchanging biomolecules and buffer solutions. Glass was used as our material of choice as we detect the fluorescent signal from the rear end of the flow cell. The flow cell was conveniently coupled to the gold-prism assembly by the use of elastic O-rings. A 50 nm gold film coated with the SiO2 sol-gel film (see below) was used in all our experiments High-Pressure Measurements. For systems involving confined geometries and rigorous experimental conditions, e.g., highpressure environments, a Kretschmann19 prism coupling mode for exciting surface plasmons is possible but difficult to implement. In such cases, a surface grating serves as a much more convenient tool in fulfilling the experimental requirements.21 For our high-pressure measurements (Figure 1b), a syringe pump controlled by a pressure detector was responsible for the buildup of the pressure in the cell. Temperature of the high-pressure cell was varied by including a heating mantle and was monitored by using a Peltier type thermal detector. The grating coupler consisted of a glass substrate with a surface relief grating prepared by reactive ion etching and subsequently coated with 150 nm of gold by thermal evaporation. Synthesis of Silica Sol-Gel Films. Ultrathin silica films (Figure 3a) were prepared on our metallic surfaces immediately after the Au or Ag deposition, using a combination of self-assembly and sol-gel techniques. During the initial self-assembly step (cf. Figure 2), 3-(mercaptopropyl)trimethoxysilane (3MPT) layers were formed by immersing the substrates in ∼20 mM solution of 3MPT (previously vacuum-distilled) in dry ethanol or hexane for 2 h (Ag) or 3 h (Au). These films were then rinsed with copious amounts of dry ethanol and Millipore Q-UV-purified water. Further hydrolysis and condensation of the 3MPT were induced immediately following these rinsing steps by immersing the samples in aqueous 0.1 M HCl for 1-12 h.24,25 These 3MPTmodified surfaces were then rinsed again with copious amounts
Figure 3. (a) A schematic of the supramolecular architecture of silica sol-gel films on noble metals. The chemically anchored 3MPT layer on Ag or Au is subjected to hydrolysis by a 0.1 M HCl solution, generating silanol groups which are then used in a condensation reaction with the spin-coated TMOS solution to generate stable silicon dioxide films. The thickness of the films can be varied by varying the concentration of the TMOS solution and also by the rotation speed used during spin-coating. (b) Ellipsometric measurements of the sol-gel film thickness. This figure illustrates the easy tunability of the thickness of the gel films by varying the H2O/Si ratio in the deposited sol. of water and immediately placed on a sample rotator. Silica films were formed by delivering small aliquots of premixed solutions (24) Thompson, W. R.; Cai, M.; et al. Langmuir 1997, 13, 22912302.
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of tetramethoxysilane (TMOS), methanol, water, and 0.1 M HCl. Typical compositions were 163 µL of H2O, 55 µL of CH3OH, 81 µL of 0.1 M HCl, and 2-40 µL of TMOS, covering H2O:Si molar ratios of 50-1000. Solutions were shaken for ∼30 min to facilitate TMOS hydrolysis prior to deposition. Small aliquots of ∼45 µL/ cm2 of these solutions were delivered to the 3MPT-modified metal surface, and the sample spun at ∼3400 rpm for 1 min. The metal/ 3MPT/SiO2 surfaces so formed were stored in a desiccator at room temperature for a minimum of 2 days to complete the condensation process and to remove any residual solvent. Ellipsometric measurements (cf. Figure 3b) were made on films formed on Ag substrates with a Rudolph Research model 43603-200E ellipsometer using a He-Ne laser at 632.8 nm at an incident angle of 70°. In the determination of silica film thickness, a refractive index of 1.457, equal to that of dense silica,26 was used. Procedure for the Immobilization of a PMMA Brush. The synthesis of the polymer brush followed the “grafting from” technique17,27 and consisted of two steps: The formation of a self-assembled monolayer of the initiator molecules for free radial polymerization in a toluene-triethylamine-azodichlorinesilane solution represents the first step. Subsequently, the polymerization was realized in a 4/1 v/v solution of MMA in toluene at 50 °C for 16 h. After polymerization, the samples were rinsed with copious amounts of toluene and exposed to a Soxhlet extraction procedure for at least 24 h. Coupling Biotin Silanes to Silicon Dioxide Surfaces. Immobilization of biotin on silicon oxide surfaces28,29 is normally preceded by an activation step in order to generate surface silanol groups. The presence of these groups is essential for biotin and spacer silane binding, thus functionalizing the SiO2 surface. In our experimental protocol, gold wafers containing 15 nm thick silicon dioxide sol-gel films were immersed in an activating solution of 0.1 M potassium dihydrogen phosphate (10% v/v) and 0.1 M dipotassium hydrogen phosphate (90% v/v) for half an hour. The above solutions were prepared in distilled water, and the pH of the activating solution was adjusted to 9.4 by adding a few drops of 1 M potassium hydroxide solution. After the activation step, the wafers were thoroughly rinsed in distilled water and thereafter dried in a stream of nitrogen. The dried wafers were then heat-treated at 50 °C under vacuum for a period of 2-3 h. After this step, the wafers were ready for the biotin immobilization step. The structures of biotin and amino diluent silanes that were used in the immobilization procedure are shown in Figure 4a. Diluent silanes are required to optimize the interaction between biotin-binding proteins and also to prevent any unspecific binding on the SiO2 surface. Here, we prepared a 0.2 mM solution containing 10 mol % biotin silane and 90 mol % amino silane using methanol as a solvent. This ratio was selected as it produced optimal protein binding results.30,31 The wafers after the activation step were subsequently immersed in the silane solution and maintained under nitrogen for a period of 24 h. Thereafter, they were thoroughly rinsed in methanol and dried in nitrogen. Covalent linking of the silane compounds to the silicon dioxide surface was carried out by heat-treating the dry wafers at 120 °C under vacuum for 2 h. They were now ready for biomolecular interaction studies. The supramolecular architecture that was used in studying DNA hybridization interactions is shown in Figure 4b.
Results and Discussion Silicon Dioxide Layers on Noble Metals at Ambient Conditions. Vapor-deposited silicon oxide (SiOx) films on noble metals are plagued by stability problems when (25) Thompson, W. R.; Pemberton, J. E. Chem. Mater. 1995, 7, 130136. (26) Brinker, C. J.; Scherer, G. W. Academic Press: San Diego, 1990; Chapter 3, p 97. (27) Ruehe, J. Nachr. Chem Rech. Lab. 1994, 42, 1237. (28) Busse, S. Ph.D. Thesis, Universitaet Mainz, 2000. (29) Tovar, G.; Paul, S.; Knoll, W.; Prucker, O.; Ruehe, J. Supramol. Sci. 1995, 2, 89. (30) Spinke, J.; Liley, M.; Schmitt, F. J.; Guder, H. J.; Angermaier, L.; Knoll, W. J. Chem. Phys. 1993, 99, 7012-7019. (31) Spinke, J.; Liley, M.; Guder, H. J.; Angermaier, L.; Knoll, W. Langmuir 1993, 9, 1821-1825.
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Figure 4. (a) Structural formula of the biotin and aminoterminated silanes. A 1:9 biotin/amino molar ratio is used to generate a stable self-assembled monolayer capable of binding a layer of streptavidin (see below). The amino diluent silanes are required to prevent steric hindrance and also to minimize unspecific binding. (b) A detailed schematic of the supramolecular architecture used in investigating DNA hybridization interactions. The mixed biotin/amino silane layer was used for immobilizing a monolayer of streptavidin followed by the immobilization of probe DNA molecules containing a biotin molecule at their 5′ end. DNA target molecules in solution, containing a fluorescent label at their 5′ end, were used for monitoring the hybridization interactions with the surfaceimmobilized DNA probes.
tested in PBS buffer solution. PBS, however, is one of the most commonly used buffers in biomolecular interaction studies and serves as a useful test reference for the stability of the SiOx films on noble metals. One of the inherent problems of this system is the interface between the noble metal and the SiOx layer which is quite unstable and can be easily eroded by the presence of solvent molecules which are in contact with the SiOx surface. The reactivity of the noble metal also has an important part to play in the overall stability of these layers. Thin films of gold are normally quite stable (cf. Figure 5a) and resist oxidation in aqueous environments, unlike those made from silver which are easily oxidized under the influence of salt present in the buffer solution (Figure 5g). Although thin films made on gold are typically sufficiently stable for biomolecular interaction studies, we will also test for the robustness of deposited silicon dioxide films on Ag substrates. Since silver shows a very narrow surface plasmon resonance and also generates a high field enhancement, stable films of SiOx obtained on that substrate will be very useful for many fluorescence studies and sensoric applications.
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Figure 5. Stability analysis of sol-gel films in PBS buffer. Shown are the reflectivities measured at a fixed angle of observation for (a) bare gold, (b) 15 nm sol-gel film on gold, (c) 30 nm sol-gel film on gold, (d) thermally evaporated 15 nm SiOx film on gold, (e) 30 nm sol-gel film on silver, (f) thermally evaporated 15 nm SiOx film on silver, and (g) bare silver.
SPR kinetic plots of silver and evaporated SiOx films on silver and gold are shown in parts g, f, and d of Figure 5, respectively. The decrease in reflectivity with time indicates that the SiOx film is eroding off the interface, which is a qualitative measure of the instability of the interface. This instability limits the usefulness of these substrates for various SiOx coupling schemes. The presented scheme to chemically synthesize silicon dioxide films circumvents this problem. Sol-gel processing allows for exquisite control of the micro- and nanostructure and thickness of these ultrathin coatings. The sol-gel preparations used here utilize extreme dilution of the Sicontaining reactants to ensure complete hydrolysis of the starting alkoxide and to inhibit solution condensation reactions of hydrolyzed monomers and oligomers. Thus, the sizes of the resulting sol particles are limited leading to their greater packing density and to the exclusion of most solvent (H2O/EtOH) in the final film and hence leads to a very low porosity. The thickness of the SiO2 could be varied by spin-coating TMOS on to the hydrolyzed 3MPT monolayer at different speeds and concentrations. The stability of the 15 and 30 nm silica films on gold (cf. Figure 5b,c) and 30 nm film on silver (cf. Figure 5e) is quite remarkable. We attribute this behavior mainly to two reasons: first we have effectively anchored the SiO2 film on to the noble metal by forming covalent bonds between some of the Si atoms of the SiO2 film and the Si atoms of the 3MPT molecule, which in turn is strongly coordinated via its S-terminal to the noble metal surface, and second we have a uniformly distributed sol-gel film which forms a protective layer and thereby prevents the buffer from undermining the coating. All the above stability experiments were performed at 20 °C and under atmospheric pressure. Next, we will show that these films are also stable under high-temperature and high-pressure conditions. Stability of Sol-Gel Films at Elevated Temperatures and Pressures. In addition to presenting a platform to which further functional layers can be assembled utilizing different processing steps, the sol-gel films in some cases also have to withstand severe thermal and chemical conditions. One example is the binding of biotin onto the sol-gel film at 120 °C, as explained above. Here, we focus on the variation of the temperature and pressure to investigate the stability of the sol-gel films. The measurements were carried out using the high-
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Figure 6. Comparison of the minimum resonance angle of gold (dashed) and gold containing a sol-gel film on top (solid) monitored as a function of time. While recording the minimum angle the temperature is increased in 10 °C steps from 50 to 140 °C and decreased continuously back to 50 °C again. The data of part a was measured at 10 MPa while part b was recorded at 80 MPa.
pressure cell with the pressure medium being a mixture of acetone and ethanol (47.7%/52.3%). This solvent was chosen with regard to measurements on PMMA brushes because this mixture, at 25 °C, is a Θ solvent for that particular polymer32 in solution. Figure 6 summarizes the results obtained at 10 and 80 MPa. The minimum resonance angle is monitored as a function of time while the temperature was simultaneously increased stepwise from 50 to 140 °C in 10 °C steps and at each temperature was kept constant for 2 h. At the end of the measurement cycle, the starting temperature was adjusted again in order to verify whether the high temperatures had damaged the film. As can be seen, the resonance minimum stays constant at the different temperatures over the 2 h period, which implies that also the optical thickness of the layer is constant and that the film is stable. Also, after the entire temperature cycle is completed by cooling again to room temperature, the minimum angle matches the one measured at the beginning of the cycle, which is another evidence of the stability of the layer. The decreasing resonance angle with increasing temperature can be quantitatively attributed to the change of refractive index of the pressure medium with the optical thickness of the sol-gel layer staying virtually constant. This can be also seen by comparing the curves of plain gold with those having a sol-gel film. When increasing the pressure, the shape of the curves remains the same whereas the respective resonance minima are shifted to somewhat larger angles. It appears that pressure has effectively no influence on the stability of sol-gel films. PMMA Brush Immobilization on Silicon Oxide Substrates. The demonstrated stability of the sol-gel layers is only a prerequisite for further functionalization of the surface. To test the surface activity of the silanol groups, we prepared a PMMA brush on the SiO2 in two steps: The azodichlorinesilane adsorbed to the surface by self-assembly was used as a platform for radical polymerization of the MMA monomer. Figure 7 summarizes the various steps of the sample preparation in terms of SPR scans on gratings at 24 °C and 10 MPa in an acetone/ethanol (47.7%/52.3%) environment. The dotted curve represents the gold reference with the reflectivity minimum at 15°. A sol-gel film of 23.5 nm thickness then (32) Brandrup, J., Immergut, E. H., et al., Eds.; Polymer Handbook, 4th ed.; John Wiley & Sons: New York, 1999; Section VII, p 309.
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Figure 7. High-pressure studies of PMMA brushes immmobilized on sol-gel films using both s- and p-polarized light.
shifts the minimum to approximately 17° whereas the angle of the total internal reflection stays the same (dashed curve). One also notes that the plasmon is somewhat broader, indicating that the film is not perfectly homogeneous in (optical) thickness. The deposition of the brush, again, moves the resonance minimum to larger angles (solid curve). However, the homogeneity of the surface decreases again as can be concluded from both the further broadening of the resonance and the drop in reflectivity at small angles. Besides the minimum due to the surface plasmon, another minimum at very small angles occurs as a result of the excitation of a waveguide mode. Thus, a scan at s-polarized light (dash dotted curve) completes the measurements at this set of parameters, and the thickness and refractive index of the polymer brush can readily be calculated by means of the Fresnels formalism. In this case the thickness was calculated to be 770 nm at a refractive index of the polymer of n| ) 1.383 parallel and n⊥ ) 1.387 perpendicular to the surface by assuming n0 ) 1.362 as the refractive index of the pressure medium, which was measured separately. The brush must therefore be in a swollen state since the refractive index of the polymer brush is only slightly larger than that of the surrounding medium. DNA Hybridization. The second example for a surface functionalization that we discuss in order to demonstrate the applicability of the sol-gel films for (bio)sensing studies concerns a DNA hybridization experiment. All kinetic analysis experiments (cf. Figure 4b) were performed in PBS buffer, and once the interaction had reached equilibrium, the fluid cell was copiously flushed with PBS buffer to remove any unspecificly bound molecules and also to investigate the desorption behavior of these systems. The kinetics of the interaction of streptavidin with biotin is shown in Figure 8a. As can be seen from the figure, the binding is quite rapid and results in a stable monolayer formation. This monolayer was then used for immobilizing probe DNA molecules containing a biotin unit at their 5′ end. The kinetics of the interaction of the DNA probe strands with the streptavidin layer is shown in Figure 8b. These probe strands were then applied for studies of hybridization interactions with their complementary DNA target strands associating from solution. All the above steps were monitored by using normal SPR. For the DNA hybridization step, however, surface plasmon field-enhanced fluorescence spectroscopy was used as a detection method because SPR alone was not able to monitor these interactions due to poor signal-to-noise ratio (cf. Figure 8c). The DNA target strands were labeled with a fluorescent dye (MR121, Boehringer Mannheim) at their 5′ end
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Figure 8. Kinetic plots of the buildup of the supramolecular architecture which was used in investigating DNA hybridization interactions. (a) Interaction of streptavidin with the amino and biotinylated silane mixed monolayer. Vertical arrows at the end of the adsorption/binding indicate the start of PBS buffer rinsing, which was carried out to remove any unspecifically bound adsorbates and also to monitor desorption behavior. (b) Interaction of the biotinylated probe DNA molecules with the streptavidin monolayer. (c) Plain SPR response of the DNA hybridization process. This plot illustrates the poor signal-tonoise ratio obtained from normal SPR measurements. (d) Time dependence of the fluorescence emission, indicating the interaction of fluorescently labeled DNA targets in solution, with the surface-immobilized probe DNA molecules. SPR coupled with fluorescence results in such a remarkable signal-to-noise ratio where we can readily monitor the various stages of the hybridization interaction.
which could be excited by using a He-Ne laser (λ ) 632.8 nm) and emitted fluorescence light at 670 nm. We utilize the surface plasmon electromagnetic field for enhancing the fluorescent signal while monitoring these interactions in real time. The interaction of target DNA strands at a concentration of 1.0 µM present in solution, binding to their surface bound complementary DNA probe (5.0 µM) strands, is shown in Figure 8d. This interaction is quite rapid, and even after substantial rinsing with buffer, the fluorescent signal still remains at a high level. Fluorescence quenching losses occurring by Foerster energy tranfer can be neglected in our system as we have a 15 nm silicon dioxide layer between the gold and the fluorescently tagged DNA target strands. In addition to the silicon oxide layer, the biotin, streptavidin, and the probe DNA layers also cummulatively add up to approximately 8 nm, which also increases the distance of the target strands from the gold layer even farther. Conclusions We have demonstrated that a stable silica film on noble metals can be generated by applying sol-gel chemistry. These films are resistant to buffer solutions containing various salts and can be readily used in a liquid cell format for investigating the buildup of supramolecular architectures in real time. The stability of these films under high-pressure and -temperature conditions was found to be quite remarkable, which enhances their applicability for experiments even at harsh conditions. We have also shown that these films could be chemically treated to introduce various reactive groups which were then used for various surface studies. We demonstrated this by giving experimental data on polymer brushes and DNA hybridization interactions. The flexibility of introducing various silane coupling agents on these stable silica sol-gel films on noble metals will greatly enhance their applicability
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in studying problems related to fluorescence quenching, biomolecular interactions, or polymers at interfaces. Acknowledgment. The authors thank Gretl Dworschak for her skillful laboratory assistance during the synthesis of the silicon oxide sol-gel films. We also thank
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Boehringer Mannheim for providing us with the biotin silane supplies and Prof. Juergen Ruehe and Dr. Oswald Prucker for helpful discussions regarding the synthesis and characterization of polymer brushes. LA001250W
