Raman and Surface Enhanced Raman Microscopy of Microstructured

Jan 7, 2006 - The Raman signature of PEI has not been compensated for and contributes positive difference peaks. Polyethylenimine/DNA Multilayers...
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Langmuir 2006, 22, 1735-1741

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Raman and Surface Enhanced Raman Microscopy of Microstructured Polyethylenimine/DNA Multilayers Rolf Dootz,†,‡ Jingjing Nie,†,§ Binyang Du,†,§ Stephan Herminghaus,†,‡ and Thomas Pfohl*,†,‡ Applied Physics Department, UniVersity of Ulm, Albert-Einstein-Allee 11, 89069 Ulm, Germany, and Max-Planck-Institut for Dynamics and Self-Organization, Bunsenstr. 10, 37073 Go¨ttingen, Germany ReceiVed October 10, 2005. In Final Form: NoVember 17, 2005 We analyze microstructured multilayer films of poly(ethyleneimine) (PEI) and DNA by employing Raman and surface enhanced Raman spectroscopy (SERS). The microstructuring of the samples allows a simultaneous measurement of signal and reference in a single analytic process. Silver nanoparticles are implemented in the microstructured multilayers for SERS measurements. The recorded SERS spectra of PEI/DNA are dominated by the Raman bands of the DNA bases which show a larger mean enhancement than bands belonging to DNA backbone vibrations. Our results show that the combination of SERS and microstructured multilayer films provides an adapted way to characterize the polyelectrolytes as well as to measure the enhancement factor and the distance dependence for the SERS active silver nanoparticles. Furthermore, microstructured polyelectrolyte films containing SERS active nanoparticles are used for sensing molecules.

1. Introduction Aggregates of DNA and polyvalent cations play an important role for cellular mechanisms.1 Cellular DNA is situated in an environment consisting of macromolecules such as proteins and cationic molecules, e.g., histones and polyamines. In the cell nucleus, the DNA is very highly concentrated (ca. 200-400 mg/mL)2 and associated with polyamines.3 These polyamine/ DNA interactions are speculated to be essential for the organization of the genetic material taking part in relevant cellular processes such as protein translation and DNA double-strand stabilization.4,5 Wide interest has been attracted to polyaminemediated DNA condensation. For example, polyamine/DNA aggregates are currently being investigated for drug delivery, gene detection, and gene therapy using not only cellular polyamines such as spermine and spermidine but applying also molecules such as poly(ethyleneimine) (PEI) which cannot be found in cells.4,6,7 Thus, due to the increasing use of artificial polyamines for drug delivery, more detailed information on the interactions between DNA and artificial polyamines such as PEI is required. The overall negative charge of DNA allows for the formation of DNA-containing polyelectrolyte multilayers (PEM) by the layer-by-layer technique8 enabling the use of multilayer films for analyzing the interaction between DNA and PEI. Using alternate adsorption of positively and negatively charged poly* Corresponding author. Phone: +49 551 5176 240. Fax: +49 551 5176 202. E-mail: [email protected]. † University of Ulm. ‡ Max-Planck-Institut for Dynamics and Self-Organization. § Present address: Institute of Physical Chemistry, Technical University of Clausthal, Arnold-Sommerfeld Str. 4, 38678 Clausthal-Zellerfeld, Germany. (1) Cohen, S. S. A Guide to the Polyamines; Oxford University Press: New York, 1998. (2) Torbet, J.; DiCapua, E. EMBO J. 1989, 8, 4351. (3) Ruiz-Chica, J.; Medina, M. A.; Sa´nchez-Jime´nez, F.; Ramı´rez, F. J. Biophys. J. 2001, 80, 443. (4) McMurry, L. M.; Algranati, I. D. Eur. J. Biochem. 1986, 155, 383. (5) Thomas, T.; Thomas, T. J. Cell. Mol. Life Sci. 2001, 58, 244. (6) Schu¨ler, C.; Caruso, F. Biomacromolecules 2001, 2, 921. (7) Dudnik, V.; Sukhorukov, G. B.; Radtchenko, I. L.; Mo¨hwald, H. Macromolecules 2001, 34, 2329. (8) Decher, G.; Hong, J. D.; Schmitt, J. Thin Solid Films 1992, 210, 831.

electrolyte molecules, PEMs can be built on various templates such as silicon wafers9 and colloid particles.10 Surface analysis techniques such as ellipsometry, X-ray,11 and neutron scattering12 can then be performed on the layers. The primary advantage of analyzing PEMs lies in their high reproducibility with respect to thickness and internal properties. A broad scope of materials can be incorporated into the multilayers with precision including synthetic polymers,13 fluorescent dyes, charged nanoparticles, and biological macromolecules, e.g., proteins.14-16 This inclusion of molecular moieties offers potential industrial and bio-chemical application including the fabrication of films engineered to either promote or inhibit the attachment of cells or proteins to surfaces17,18 and chemical or biological sensors.19 In the context of controlled release, the layer-by-layer fabrication offers potential advantages by providing the possibility of tuning the concentrations of incorporated materials by varying the number of polymer layers.20 An additional breadth of possible applications is achieved by combining the nanostructure of the multilayer films perpendicular to the substrate with a tailored microstructuring along the surface.21-25 (9) Pardo-Yissar, V.; Katz, E.; Lioubashevski, O.; Willner, I. Langmuir 2001, 17, 1110. (10) Caruso, F.; Mo¨hwald, H. J. Am. Chem. Soc. 1999, 121, 6039. (11) Ibn-Elhaj, M.; Riegler, H.; Mo¨hwald, H.; Schwendler, M.; Helm, C. A. Phys. ReV. E 1997, 56, 1844. (12) Loesche, M.; Schmitt, J.; Decher, G.; Bouwman, W. G.; Kjaer, K Macromolecules 1998, 31, 8893. (13) Bertrand, P.; Jonas, A.; Laschewsky, A.; Legras, R. Macromol. Rapid Commun. 2000, 21, 319. (14) Schwinte, P.; Voegel, J. C.; Picart, C.; Haikel, Y.; Schaaf, P.; Szalontai, B. J. Phys. Chem. B 2001, 105, 111906. (15) Jin, W.; Shi, X. Y.; Caruso, F. J. Am. Chem. Soc. 2001, 123, 8121. (16) Tiourina, O. P.; Antipov, A. A., Sukhorukov, G. B., Larionova, N. L.; Lvov, Y.; Mo¨hwald, H. Macromol. Biosci. 2001, 1, 209. (17) Richert, L.; Lavalle, P.; Vautier, D.; Senger, B.; Stoltz, J. F.; Schaaf, P.; Voegel, J. C.; Picart, C. Biomacromolecules 2002, 3, 1170. (18) Mrksich, M.; Dike, L. E.; Tien, J.; Ingber, D. E.; Whitesides, G. M. Exp. Cell Res. 1997, 235, 305. (19) Wang, B. Q.; Rusling, J. F. Anal. Chem. 2003, 75, 4229. (20) Zhang, J.; Chua, L. S.; Lynn, D. M. Langmuir 2004, 20, 8015. (21) Clark, S. L.; Montague, M.; Hammond, P. T. Macromolecules 1997, 30, 7237. (22) Jang, H.; Kim, S.; Char, K. Langmuir 2003, 19, 3094. (23) Yang, S. Y.; Rubner, M. F. J. Am. Chem. Soc. 2002, 124, 2100. (24) Delamarche, E.; Bernard, A.; Schmid, H.; Michel, B.; Biebuyck, H. Science 1997, 276, 779.

10.1021/la052739y CCC: $33.50 © 2006 American Chemical Society Published on Web 01/07/2006

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The interaction of DNA and PEI is monitored here using Raman microscopy. Raman microscopy is a rapid, nondestructive, chemically sensitive method of analysis which provides a high lateral resolution and enables quantitative analysis of molecular interactions and in situ detection. Due to the fact that all molecules in the analyzed volume take part in the scattering process, multiple components can be detected at one time. The use of microstructured samples enables us to measure simultaneously signal and reference in one measurement. Since Raman spectroscopy provides weak signal intensities only species which are present in a sufficient high concentration can be detected. To correct for this problem surface enhanced Raman scattering (SERS) is used. Metallic nanostructures can strongly enhance the Raman signal of molecules adsorbed on them. Therefore, the SERS effect enables the study of very low concentrations of biomolecules, close to those at which they are in physiological environment. In this paper, we describe the layer-by-layer assembly of microstructured PEI/DNA films by using poly(dimethyl siloxane) (PDMS) microchannels produced by soft lithography methods.26,27 Raman microscopy is used for sensing molecular interactions of PEI and DNA. SERS measurements of PEI/DNA multilayer films implementing silver nanoparticles are described. We show that the combination of SERS and microstructured multilayer films enables both the characterization of the polyelectrolytes and an estimation of the properties of the SERS active particles by using the well-defined multilayer structure. Finally, we show an application of microstructured multilayer films containing SERS active particles for molecule detection which can be used for combinatory investigations. 2. Materials and Methods Preparation of Polyelectrolyte Complexes and Multilayers. The used polyelectrolytes are the following: branched PEI (Mw ∼ 750 000), sodium poly(styrene-sulfonate) (PSS, Mw ∼ 70 000), polydisperse deoxyribonucleic acid sodium salt (DNA, highly polymerized from calf thymus, 10-15 million daltons), and poly(allylamine hydrochloride) (PAH, Mw ∼ 70 000). All polyelectrolytes were purchased from Sigma Aldrich and used as received except for PSS, which is dialyzed before use to remove molecules with molecular weight less than Mw ∼ 20 000. Aqueous polyelectrolyte solutions are produced with concentrations ranging from 1 to 2 mg/mL. The ultrapure water used in all experiments is prepared in a Milli-Q synthesis A10 purification system and has a resistivity higher than 18.2 MΩ cm. Aggregates of PEI and DNA are produced by adding 1 mL of a DNA solution with a concentration of 10 mg/mL to 1 mL of a PEI solution with a concentration of 2 mg/mL. The relative charge ratio of the formed aggregates is N/P ) 0.6. Here, N denominates the number of positive amine charges of the dendrimers (assuming full protonation), whereas P is the number of the negative phosphate charges of the DNA backbone. The bundle like shape of the formed aggregates can be seen visually. Polyelectrolyte multilayer films are formed by adsorption on silicon wafers. The silicon wafers are cleaned by immersing them in 5:1:1 (vol %) H2O/H2O2/NH3 at 80 °C for 15 min, rinsed with ultrapure water, dried with a stream of dry nitrogen, and used as templates for the formation of PEMs. First, a precursor PEI layer is coated onto the silicon wafers by dipping into PEI solution for 9 min, followed by rinsing three times (1 min per step) with water, and drying with nitrogen afterwards. The multilayer films are produced by alternated absorption of negatively and positively charged polyelectrolytes from aqueous solutions using the same procedure as for PEI until the desired number of layers is achieved. The thickness (25) Schmitt J.; Ma¨chtle, P.; Eck, D.; Mo¨hwald, H. and Helm, C. A. Langmuir 1999, 15, 3256. (26) Xia, Y.; Mrksich, M.: Kim, E.; Whitesides, G. M. J. Am. Chem. Soc. 1995, 117, 9576. (27) Kumar, A.; Whitesides, G. M. Appl. Phys. Lett. 1993, 63, 2002.

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Figure 1. Pumping system used consists of a PC controlled stepping motor linked to a microliter syringe which is connected to the PDMS stamp containing the microchannels sealed with a silicon wafer. of the polyelectrolyte films is determined by using a null ellipsometer operated at 632.8 nm at an incidence angle of 70° (Multiskop Ellipsometer, Optrel GBR, Berlin). Microstructuring of the Polyelectrolyte Multilayers. For producing samples which allow simultaneous measurement of signal and reference in a single analytic process, we use parallel microchannel systems for microstructuring the multilayer films. These microfluidic systems consist of 500 or 250 channels with widths of 7 and 17 µm, respectively. The depth of the channels is 50 µm, and the length is 15 mm. The microchannels are produced by standard soft lithography technique.26,27 This method involves the casting of liquid Sylgard 184 poly(dimethylsiloxane) (PDMS, purchased from Dow Corning) against a master with a relief structure. This relief structure consists of a SU 8-50 resist (MicroChem) and is fabricated by using photolithography. The ratio of PDMS to cross-linker is 10:1, and the baking procedure takes 90-120 min at 70-80 °C. The elastomeric properties of the cross-linked PDMS stamp enable either reversible or irreversible sealing of the microchannels with a substrate forming a microfluidic device. For an irreversible sealing, the PDMS stamp is exposed for 2-10 s to air plasma using a PDC-32G Basic Plasma Cleaner (Harrick) at 100 W, whereas for a reversible sealing, this manufacturing step is skipped. For microstructured patterning of PEMs this microfluidic device is connected to a microliter syringe system by Teflon tubes with an inner diameter of 0.7 mm. The used setup is sketched in Figure 1. The pumping system allows flow speeds ranging from nm/s to mm/s (corresponding to pL/s and µL/s). For the buildup of PEMs inside the microfluidic devices, a precursor layer is adsorbed by pumping a 1-2 mg/mL aqueous solution of PEI through the microchannels for 30 min. Additional layers are produced by alternated pumping of 1-2 mg/mL aqueous solutions of negatively and positively charged polyelectrolytes, respectively. To remove excessive polyelectrolytes from the channel system, each adsorption step is followed by pumping ultrapure water through the system for 30 min. Fabrication of the Nanoparticles and the SERS Active PEMs. AgNO3 (99.9%) and NaBH4 (99.5%) were purchased from SigmaAldrich and used as aqueous solutions. For the fabrication of the silver nanoparticles, 10 mL of a 2.5 mM aqueous solution of AgNO3 is added to 90 mL of a fresh, ice cold, 4 mM aqueous solution of NaBH4 under agitation.28,29 The reaction follows a series of welldefined stages which can be observed visually. For a more detailed description of the ongoing reaction see refs 28 and 29. All solutions are stored under exclusion of light and discarded after one week. The radius, R, of the negatively charged silver particles is R ) 12.5 ( 2.5 nm measured on adsorbed particles by AFM in tapping mode using a Nanoscope IIIa-AFM (DI, Santa Barbara, CA). For all experiments, the nanoparticles are adsorbed on silicon wafers with a supporting PEI layer. The particles form a monolayer with a two-dimensional filling factor usually in the range of 0.40.5. Every adsorption step of nanoparticles is followed by rinsing with water. After the adsorption of a particle layer, a further film buildup can be achieved by consecutively adsorbing cationic and anionic polyelectrolytes on the substrate. For a microstructured (28) Van Hyning, D. L.; Zukoski, C. F. Langmuir 1998, 14, 7034-7046. (29) Van Hyning, D. L.; Klemperer, W. G.; Zukoski, C. F. Langmuir 2001, 17, 3120.

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Figure 2. Dependence of the thickness of PEI/DNA multilayer films on the number of adsorbed layers. Error bars in (b) correspond to the standard deviation. The arrow indicates the film thickness of the microstructured multilayer film used in Figure 4. pattern, the silver particle solution is pumped through the microchannel system for 30 min. Again, to remove excessive nanoparticles from the channel system, each adsorption step is followed by a 30 min pumping of ultrapure water. Confocal Raman Microscope. A confocal Raman microscope CRM200 manufactured by Witec (Ulm, Germany) is used for roomtemperature Raman microscopy experiments of the adsorbed PEMs. The microscope is equipped with a piezo scanning table which can be moved 200 ( 0.004 µm in the x and y directions and 20 ( 0.001 µm in z direction. The laser beam (SGL-2200 laser, 532 nm, controlled by SUWTech LGD-2500, Shanghai Uniwave Technology) is focused on the sample using a diffraction limited spot size by an Olympus LMPlanFl 100x/0.80 objective. The measured signal is transmitted to the spectrograph via an optical fiber with a diameter of 50 µm. The end of this fiber is placed at the image plane of the microscope and used as a pinhole. The microscope provides the recording of spectra with an air-cooled CCD-chip (1340 × 1000 pixel) behind a grating spectrograph with a 600 lines per mm grating and a resolution of 6 cm-1. Additionally, high resolution 2D-Raman images of the sample at fixed wavenumbers can be recorded by laterally (x-y) and vertically (x-z, y-z) rasterizing the sample through the excitation spot using the scanning table and detecting the Raman signal with an avalanche photodiode detector (APD). The presented Raman spectra are accumulated averages of 50-60 exposures of 150 s each. For SERS spectra, exposure times of 5 s are used.

3. Results and Discussion Buildup of PEI/DNA Multilayer Films. The thickness of PEI/DNA multilayers as a function of the number of adsorbed polyelectrolyte layers is shown in Figure 2. The generated films offer a high reproducibility with respect to their thickness and internal properties. Contrary to other salt-free polyelectrolyte systems, which show a linear increase in film thickness with increasing layer number, for PEI/DNA multilayers, a superlinear growth in film thickness is found for the first layers.30-35 The growth mechanism of multilayer films is not yet fully understood. Generally, it is believed that the formation of multilayers is mainly driven by electrostatic interactions.36,37 (30) Pei, R. J.; Cui, X. Q.; Yang, X. R.; Wang, E. K. Biomacromolecules 2001, 2, 463. (31) Decher, G.; Hong, J. D.; Schmitt, J. Thin Solid Films 1994, 244, 772. (32) Ramsden, J. J.; Lvov, Y. M.; Decher, G. Thin Solid Films 1995, 254, 246. (33) Lavalle, P.; Picart, C.; Mutterer, J.; Gergely, C.; Reiss, H.; Voegel, J.-C.; Senger, B.; Schaaf, P. J. Phys. Chem. B 2004, 108, 635. (34) Elbert, D. L.; Herbert, C. B.; Hubbell, J. A. Langmuir 1999, 15, 5355. (35) Picart, C.; Mutterer, J.; Richert, L.; Luo, Y.; Prestwich, G. D.; Schaaf, P.; Voegel, J.-C.; Lavalle, P. Proc. Natl. Acad. Sci. 2002, 20, 12531. (36) Grosberg, A. Y.; Nguyen, T. T.; Shklovskii, B. I. ReV. Mod. Phys. 2002, 74, 329. (37) Netz. R. R.; Joanny, J.-F. Macromolecules 1999, 32, 9013.

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Figure 3. Raman spectra of PEI/DNA obtained from analyzing complexes. The difference spectrum achieved by subtracting the spectrum of DNA (obtained from 20 mg/mL aqueous solution) from that of PEI/DNA in the region of 670-1160 cm-1 (inlet picture) shows the prominent reduction of the two Raman bands at 782 and 1087 cm-1 belonging to phosphate group vibrations of the DNA backbone. The Raman signature of PEI has not been compensated for and contributes positive difference peaks.

Each adsorption step overcompensates and reverses the surface charge of the supporting substrate, leading to a subsequent buildup of multilayer films. For higher salt concentrations, the change from a linear to a super-linear increase in thickness has been attributed to roughness effects.13,33 Additionally, a diffusion mechanism is assumed to explain the exponential growth of multilayers formed in lower salt concentrations.38 Raman Spectroscopy of PEI/DNA Films and Complexes. Raman spectroscopy has been shown to be a suitable tool for analyzing polyamine/DNA films and aggregates.39,40 The characteristic Raman bands of DNA are situated in the wavenumber region of 600-1700 cm-1 and can be divided into two categories according to their origin: bands originated from vibrations of the bases and bands from vibrations of the DNA backbone, i.e., the desoxyribose and the phosphate groups. A detailed description of the Raman spectrum of DNA is given elsewhere.39 Due to the fact that the phosphate groups are the carrier of the negative charge of DNA, Raman bands assigned to them are of special interest. The most prominent among these bands are observed at 782 and 1087 cm-1. The Raman band at 782 cm-1 is due to a vibration of the 5′C-O-P-O-C3′ network of B-form DNA, the form of DNA which can be found in almost all living cells, and is overlapped by bands belonging to vibrations assigned to the bases thymine (785 cm-1) and cytosine (775 cm-1), whereas the Raman band at 1087 cm-1 is due to a symmetric stretching vibration of the PO2- moiety and is easily distinguished from other Raman bands of B-form DNA.39-41 Raman spectroscopy is used for sensing molecular interactions of PEI and DNA. The Raman spectrum of PEI/DNA is largely consistent with the sum of the intensities of the corresponding Raman bands in the PEI and DNA spectra. However, salient negative difference features are revealed at 782 and 1087 cm-1 indicating that the negatively charged groups along the DNA backbone are the main targets of PEI/DNA interaction. Figure 3 shows the Raman spectrum of PEI/DNA complexes. The inlet (38) Boulmedais, F.; Ball V.; Schwinte, P.; Frisch, B.; Schaaf, P.; Voegel, J. C. Langmuir 2003, 19, 440. (39) Deng, H.; Bloomfield, V. A.; Benevides, J. M.; Thomas, G. J. Jr. Nucleic Acids Res. 2000, 28, 17, 3379. (40) Deng, H.; Bloomfield, V. A.; Benevides, J. M.; Thomas, G. J., Jr. Biopolymers 1999, 50, 656. (41) Guan, Y.; Choy, G. S.-C.; Glaser, R.; Thomas, G. J., Jr. J. Phys. Chem. 1995, 99, 12054.

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Figure 4. Raman scans of microstructured PEI/DNA films consisting of 5 layers at a fixed wavenumber of 1576 and 1087 cm-1, respectively. Due to the microstructuring of the sample, reference and signal can be recorded in a single measurement. Using the averaged intensity ratio 〈I1087/I1576〉 a reduction in intensity of the interaction band at 1087 cm-1 of 25% is measured for both PEI/ DNA complexes and multilayers in comparison to DNA.

picture shows the difference spectrum obtained by subtracting the spectrum of DNA (recorded from a 20 mg/mL aqueous solution) from that of PEI/DNA in the region of 670-1160 cm-1. This reflects the fact that electrostatic attraction between both molecules is the driving force for the DNA condensation.39 It is known that the complexation of DNA by polyamines preserves and stabilizes the B duplex structure of DNA without significantly perturbing base pairing or base stacking interactions of DNA, thus leaving Raman bands belonging to base vibrations unaffected by complexation.39 Therefore, the intensity of the Raman band at 1576 cm-1, which arises from vibrations of the bases guanine and adenine, remains unchanged during the complexation and can be used as reference. The reduction in intensity of the Raman bands belonging to phosphate group vibrations of DNA relative to the reference intensity of the band at 1576 cm-1 can be used as benchmarks for the complexation reaction. Although the Raman band at 782 cm-1 shows a more prominent change in intensity during complexation, the Raman band at 1087 cm-1 is preferred as the benchmark due to the fact that it is, contrary to the one at 782 cm-1, not overlapped by other Raman bands in its vicinity. To estimate the reduction in intensity of the interaction Raman band at 1087 cm-1 during complexation, the intensity ratio 〈I1087/ I1576〉 is calculated for the DNA and the PEI/DNA spectra. The results show that due to the complexation with PEI a reduction in intensity of this DNA Raman band of 25% can be observed (see also Figure 4). The intensity of the Raman signal is strongly dependent on the amount of material taking part in the scattering process. Due to their micrometer scale, structure analysis of PEI/DNA complexes with the CCD camera can be accomplished. However, the camera is not sensitive enough for recording spectra of thin PEI/DNA multilayer films. Therefore, we use the knowledge of

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the Raman spectrum obtained from PEI/DNA complexes to systematically scan the multilayer films on specific wavenumbers using the more sensitive avalanche photodiode (Figure 4a,b). Lateral scans of the microstructured polyelectrolyte films are accomplished at 1576 cm-1 (reference band) and 1087 cm-1 (interaction band) and the intensity of the signal areas averaged. From the intensity ratio, 〈I1087/I1576〉 the resulting intensity reduction of the benchmark band at 1087 cm-1 due to the interaction of PEI and DNA forming multilayer films is calculated accounting for 25%. Therefore, it is equivalent to the one observed in PEI/DNA complexes (Figure 4). Surface Enhanced Raman Scattering (SERS) of PEI/DNA Multilayers. Due to the fact that only species present in sufficiently high amounts can be detected, conventional Raman spectroscopy is not suitable for analyzing or sensing thin PEM films. Therefore, Raman measurements of thin PEI/DNA films are accomplished by using the signal enhancement in the vicinity of silver nanoparticles. Implementation of SERS active nanoparticles in the polyelectrolyte films is not only suitable for sensing thin polyelectrolyte films but also provides an elegant way to measure the properties of the SERS active particles by using the well-defined characteristics of PEMs.42 (A) Determination of the SERS Enhancement Factor. The enormous enhancement of the Raman signal arises from two different types of mechanism contributing to the SERS effect. The first is an electromagnetic effect which is associated with large local fields due to electromagnetic resonances occurring near metal surface structures. This causes the largest part of the enhancement.43-45 The second, minor contribution is due to a chemical effect involving chemical interactions between the adsorbed molecules and the metal surface.46,47 Whereas the chemical enhancement depends on the used analytes, the electromagnetic one is strongly influenced by the local metal surface geometry which determines the local electromagnetic field. Therefore, large differences in local enhancement can be observed.44-48 To determine the mean enhancement factor a SERS inactive and a SERS active sample are compared (Figure 5). The SERS inactive sample consists of a multilayer film of 410 nm thickness, whereas for the SERS active sample a PEI/DNA film of 4 nm thickness is adsorbed on SERS active nanoparticles. Particular with respect to surface roughness and inhomogeneities due to the adsorbed nanoparticles, it cannot be ruled out, if a homogeneous covering of the surface is achieved for the SERS active sample. Assuming that the intensity of the Raman signal is proportional to the layer thickness d of the adsorbed polyelectrolyte film, the enhancement factor may be measured by comparing the total mean intensities of the Raman signals from both samples weighted with the respective thickness of the PEI/DNA film. A vertical scan at a fixed wavenumber of 1576 cm-1 is recorded for both samples in order to measure the mean intensity of the Raman signal. The scanning data are shown in Figure 5. The intensity of both samples averaged over all x positions is plotted in dependence of the z axis. The total mean intensities are estimated by integrating over the data points. By comparing the total mean intensities weighted with the layer (42) Dong, W.-F.; Sukhorukov, G. B.; Mo¨hwald, H. Phys. Chem. Chem. Phys. 2003, 5, 3003. (43) Wang, D.-S.; Kerker, M. Phys. ReV. B 1981, 24, 1777. (44) (a) Wang, D.-S.; Chew, H.; Kerker, M. Appl. Opt. 1980, 19, 2256. (b) Wang, D.-S.; Chew, H.; Kerker, M. Appl. Opt. 1980, 19, 4159. (45) Weaver, G. C.; Zou, S.; Chan, H. Y. H. Anal. Chem. 2000, 72, 38A. (46) Campion, A.; Kambhampati, P. Chem. Soc. ReV. 1998, 27, 241. (47) Jiang, J. D.; Burstein, E.; Kobayashi, H. Phys. ReV. Lett. 1986, 57, 1793. (48) Kneipp, K.; Kneipp, H.; Itzkan, I.; Dasri, R. R.; Feld, M. S. J. Phys. Condens. Matter 2002, 18, R597.

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Figure 5. Determining the SERS enhancement factor by comparing the mean intensities of the Raman signal of a SERS inactive (13 layers PEI/DNA) and a SERS active sample (1 doublelayer PEI/DNA) weighted with the respective thickness of the multilayer films. To determine the mean intensities, vertical scans of the samples (sketched on the left) are made.

Figure 6. Dependence of the Raman intensity of the SERS active sample on the position along the x axis (white dots). The image obtained by vertically scanning the SERS active sample (1 doublelayer PEI/DNA) is underlayed.

thicknesses of every sample, the total mean enhancement factor accounts for A ˆ ) 2.1 × 105. This is in good agreement with theoretical estimations (A ˆ ) 104-106).43 In Figure 6, the dependence of the SERS intensity on the position along the x axis is displayed. Significant variations in total intensity and therefore in the SERS enhancement factor are observed resulting from local differences in the relative arrangement of the nanoparticles and the adsorbed analyte molecules. (B) Distance Dependence of the SERS Effect in PEM. Contrary to the chemical contribution to the enhancement, the electromagnetic contribution is not restricted to directly on the metal nanoparticle surface adsorbed molecules but depending on the distance between the SERS active substrate and the analyte molecules. Again, one can use the characteristic increase of film thickness of PEM with every polyelectrolyte layer to determine the distance dependence. Figure 7a shows a sketch of the experiment demonstrating this principle. Silver nanoparticles are absorbed on a silicon wafer coated with a supporting PEI layer. Analyte molecules are deposited in the microchannels. The deposition is carried out by drying up an aqueous solution of the analyte molecules in the microchannels ensuring that approximately the same amount of material remains on the sample.

The typical stripe pattern of the channels is observed when the sample is scanned with the Raman microscope at the fixed wavenumber of one of the Raman bands of the analyte molecules (Figure 7b). The distance d between these analyte molecules and the SERS active nanoparticles is tuned by an intermediate polyelectrolyte multilayer film consisting of PAH and PSS which is adsorbed prior to the use of the microchannels. This enables measurements of the distance dependence of the SERS signal. PAH and PSS are used due to the linear and reproducible increase in film thickness d with each layer. This is verified by measuring on a silicon wafer by using ellipsometry (data not shown). Lactose was chosen as analyte due to the fact that these molecules have prominent Raman bands in a region of wavenumbers (300-500 cm-1) where none of the used polyelectrolytes provides significant contributions to the Raman signal (Figure 7a).32 The mean intensity of the SERS signal is given by subtracting the averaged intensity of the reference regions between the lactose stripes from the averaged intensity of the Raman signal from the lactose molecules at 349 cm-1. Five samples with different distances d between the SERS active nanoparticles and the lactose stripes are produced. To ensure comparability of the five samples the adsorption of the silver nanoparticles is carried out simultaneously from the same silver nanoparticle solution. Figure 7b shows the dependence of the mean intensity of the SERS signal of lactose on the distance d. The following function is used to fit the distance dependence of the SERS signal in Figure 7b44,45

R12 I(d) ) B (R + d)12 R and B are fitting parameters. The distance dependence of the SERS signal shows that for distances d g 9 nm no SERS enhancement is detectable corresponding to the theoretical results.46,48 The parameter R, the mean radius of the used silver particles, is determined to be R ) 12.2 nm which is in good agreement with the AFM measured radius of R ) 12.5 ( 2.5 nm. Theses results show that embedding SERS active nanoparticles in microstructured PEM provides simple and effective means to characterize them.

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Figure 7. Control of the distance between the SERS active nanoparticles and the analyte lactose molecules by varying the thickness of an intermediate PEM. Lateral Raman scans recorded at 349 cm-1 and the dependence of the mean intensity of the lactose SERS signal on the distance between the SERS active nanoparticles and the lactose molecules are shown. Error bars in (b) correspond to the standard deviation.

Due to the increased substrate roughness caused by the adsorbed nanoparticles, a changed increase in polyelectrolyte film thickness d compared to smooth substrates may occur. This could affect especially the first polyelectrolyte layers.49 However, from the measured distance dependence of the SERS effect, which is in good agreement with theory, it is possible to conclude the thickness of the intermediate polyelectrolyte film. Comparing this thickness with the reproducible thickness of PAH/PSS multilayers on smooth substrates, which has been assumed for the calculations, shows that this assumption is adequate since a similar increase in film thickness of the PAH/PSS multilayer film on the used nanoparticles and on a silicon wafer is found. (C) Sensing Molecular Groups in Thin Multilayer Films by Using Microstructured SERS Active Domains. Molecules accumulate unselectively on the surface of the silver nanoparticles showing a nonspecific and irreversible adsorption behavior. This can be used for trace analysis. In Figure 8a, setup for sensing molecules in multilayer films using microstructured SERS active (49) Lvov, Y. M. Thin Film Nanofabrication by Alternate Adsorption of Polyions, Nanoparticles and Proteins. In Handbook for Surfaces and Interfaces; Nalwa, H., Ed.; Academic Press: New York, 2002.

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domains is sketched. The SERS active domains consist of adsorbed silver nanoparticles applied by using microchannels in a PDMS stamp which is reversible sealed to the silicon wafer. After stripe patterning the wafer surface with SERS active nanoparticles, the used microfluidic device can be removed and followed by another microstructured PDMS stamp in order to adsorb a 4 nm thick PEI/DNA film in stripes perpendicular to the first patterning. Remarkably, the second stamp can be used for accurately adsorbing microstructured layers (Figure 8), although the substrate is already partially coated leading to an adhesion between substrate and stamp which can locally be very weak. To demonstrate the possibility of using different PDMS stamps, two different microchannel systems are used with 7 and 17 µm channel width, respectively. Figure 8 shows a lateral scan of the sample at a fixed wavenumber of 757 cm-1. At this wavenumber, the DNA base thymine gives rise to a prominent SERS enhanced Raman band and is therefore used for DNA detection. The resulting pattern is composed of the overlapping regions of the two perpendicular stripe systems showing the effectiveness of SERS for tracing molecular groups. Having determined the overlapping regions, SERS spectra are obtained at these points. Depending on the local surface geometry as well as on the distance of intramolecular groups relative to the SERS active particles, changes in relative intensity and position of the Raman bands in the SERS spectra can occur. Compared to the DNA signal, the SERS signal of PEI is weak when exiting at the used wavelength.50 Therefore, the obtained SERS spectra are dominated by Raman bands of DNA, which can be divided into two categories according to their origin vibrations: Raman bands belonging to DNA backbone vibrations and bands assigned to DNA base vibrations (Figure 9). However, it is significant that Raman bands whose origin is in base vibrations show a higher mean enhancement than Raman bands belonging to backbone vibrations. In a qualitative sense, the different enhancement of backbone and base vibrations may be explained by the different orientation of the appropriate polarizability tensor relative to the surface of the metallic nanoparticles. Since the electric field components are largest perpendicular to the surface of the conductive sphere, modes involving large changes in the perpendicular polarizability are most strongly enhanced.51,52 For base vibrations, the direction of maximum change in polarizability is oriented in average rather perpendicular to the conductive surface, and therefore, they experience a higher mean enhancement than the parallel oriented backbone vibrations. Due to the distance dependent decrease of the SERS enhancement, it cannot be ruled out on which length scale this preferential orientation of the DNA molecules persists. Despite this, the SERS spectrum shown in Figure 9 provides an accurate determination of the adsorbed PEI/DNA aggregates (compare with the PEI/DNARaman spectrum in Figure 3). The distance dependence of the SERS enhancement can be used by coating the SERS active surface of the nanoparticles depending on the experimental needs. Being an ideal platform for a simple and reproducible build-in of various functional moieties, e.g., proteins, PEMs can be used as supporting templates to functionalize the surface of the SERS active particles. Therefore, a tunable and specific adsorption of analyte molecules is possible. Diverse functionalizing of different surface areas (50) Sanchez-Cortes, S.; Marsal Berenguel, R.; Madejo´n, A.; Pe´rez-Me´ndez, M. Biomacromolecules 2002, 3, 655. (51) Moskovits, M.; Suh, J. S. J. Phys. Chem. 1984, 88, 1293. (52) Schatz, G. C.; van Duyne, R. P. Handbook of Vibrational Spectroscopy Chalmers, J. M., Griffiths, P. R., Eds.; John Wiley & Sons Ltd.: Chichester, U.K., 2002.

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Langmuir, Vol. 22, No. 4, 2006 1741

Figure 8. Setup for sensing molecules using microstructured SERS active domains demonstrated for a PEI/DNA doublelayer. Lateral Raman scans of the sample are carried out at a fixed wavenumber of 757 cm-1.

Figure 9. Typical SERS spectrum of PEI/DNA multilayer films obtained from one of the overlap regions of Figure 8. Wavenumber regions containing Raman bands belonging to backbone vibrations of DNA are marked.

can be achieved by use of microchannel systems which allow an individual filling of single channels. Using such devices for a deposition of molecules and particles in stripes oriented perpendicular to one another enables the detection of various molecules during one SERS measuring process and can be applied for combined investigations. Furthermore, such a microstructured arrangement is suitable for monitoring spreading behavior of analyte molecules along the surface.

4. Conclusion In this paper, we have studied microstructured PEI/DNA multilayer films by employing Raman and surface enhanced

Raman spectroscopy (SERS). Our results show the importance of using microstructured PEM films which allow for simultaneous measurement of signal and reference in a single analytic process. For the SERS measurements, silver nanoparticles have been implemented in the microstructured PEI/DNA multilayers. Results in the present work show that the implementation of SERS active nanoparticles in microstructured multilayer films provides an adapted way to determine the SERS enhancement factor and the distance dependence of the used SERS active particles. Furthermore, we have shown that this combination of SERS active nanoparticles and PEM can be used for sensing molecular groups in thin polyelectrolyte films. Presumably due to the differences in the mean orientation of the base and backbone vibrations relative to the conductive, SERS active surface, the more strongly enhanced Raman bands of the DNA bases dominate the SERS spectra of PEI/DNA multilayer films. Using the distance dependence of the SERS effect, the SERS active surface can be modified depending on the experimental needs, e.g. by polyelectrolyte layers with incorporated functional groups. Such a setup has a large potential for a specific detection of analyte molecules. Acknowledgment. We thank Udo Krafft, Jutta Wolf, Alexander Otten, and Heather Evans for their helpful assistance. This project has been supported by the DFG (Pf 375/2) in the framework of the Emmy-Noether program. LA052739Y