Label-Free in Situ SERS Imaging of Biofilms - The Journal of Physical

Jul 21, 2010 - Telephone: +49 89 2180 78238 (N.P.L); +49 89 2180 78242 (C.H.). ... In this paper, we report for the first time on the application of S...
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Label-Free in Situ SERS Imaging of Biofilms Natalia P. Ivleva,*,† Michael Wagner,‡ Agathe Szkola,† Harald Horn,‡ Reinhard Niessner,† and Christoph Haisch*,† Chair for Analytical Chemistry, Institute of Hydrochemistry, Technische UniVersita¨t Mu¨nchen, Marchioninistrasse 17, D-81377, Munich Germany, and Institute of Water Quality Control, Technische UniVersita¨t Mu¨nchen, Am Coulombwall, D-85478, Garching, Germany ReceiVed: March 18, 2010; ReVised Manuscript ReceiVed: June 22, 2010

Surface-enhanced Raman scattering (SERS) is a promising technique for the chemical characterization of biological systems. It yields highly informative spectra, can be applied directly in aqueous environment, and has high sensitivity in comparison with normal Raman spectroscopy. Moreover, SERS imaging can provide chemical information with spatial resolution in the micrometer range (chemical imaging). In this paper, we report for the first time on the application of SERS for in situ, label-free imaging of biofilms and demonstrate the suitability of this technique for the characterization of the complex biomatrix. Biofilms, being communities of microorganisms embedded in a matrix of extracellular polymeric substances (EPS), represent the predominant mode of microbial life. Knowledge of the chemical composition and the structure of the biofilm matrix is important in different fields, e.g., medicine, biology, and industrial processes. We used colloidal silver nanoparticles for the in situ SERS analysis. Good SERS measurement reproducibility, along with a significant enhancement of Raman signals by SERS (>104) and highly informative SERS signature, enables rapid SERS imaging (1 s for a single spectrum) of the biofilm matrix. Altogether, this work illustrates the potential of SERS for biofilm analysis, including the detection of different constituents and the determination of their distribution in a biofilm even at low biomass concentration. Introduction Biofilms are interface-associated communities of microorganisms enclosed in a matrix of extracellular polymeric substances (EPS). Biofilm formation represents a protected mode of microbial growth (over 99% of microorganisms on Earth are organized within biofilms) that allows cells to survive in diverse environments.1,2 Depending on the type of biofilm and microorganisms involved, up to 90% of the particulate fraction of the biofilm can be EPS (biopolymers such as polysaccharides, proteins, nucleic acids, lipids, and humic-like substances).3 EPS play a major role in the formation and maintenance of the biofilm structure. The EPS matrix mediates the cell/cell communications (quorum sensing), protects microorganisms against environmental stress, caused, e.g., by antibiotics, and prevents biofilm dehydration (water can make up to 98% of the biofilm mass).4 Detailed information on the chemical composition and the structure of the biofilm matrix is important in medicine, biology, and technological processes. For instance, the development of new biocides, optimization of antifouling strategies, and biological wastewater treatment depend on the quality of information about the EPS matrix. However, the actual chemical composition and structure of EPS matrixes varies significantly, depending on several factors: the microbial cells present and their metabolic activity, the nutrients available, the prevailing physicochemical conditions, and the biofilm age.5,6 That results in contradictory data on the physico-chemical properties of EPS. In order to achieve comprehensive information on the biofilm * To whom correspondence should be addressed. Telephone: +49 89 2180 78238 (N.P.L); +49 89 2180 78242 (C.H.). Fax: +49 89 2180 78255 (N.P.L); +49 89 2180 99 78242 (C.H.). E-mail: [email protected] (N.P.L); [email protected] (C.H.). † Institute of Hydrochemistry. ‡ Institute of Water Quality Control.

matrix and to understand the influence of different factors on biofilm development, especially on the composition and the distribution of EPS, it is highly desirable to establish a rapid analytical tool for in situ biofilm analysis. This technique should provide valuable chemical information along with high spatial resolution, sensitivity, and reproducibility. For decades, fluorescence-induced confocal laser scanning microscopy (CLSM) has been the standard technique used for three-dimensional in situ visualization of biofilm structure.3,7 In combination with different staining protocols, CLSM allows for quantitative assessment of the biofilm constituents (e.g., DNA and EPS). For example, the introduction of a lectin binding analysis enables the detection of EPS glycoconjugates.8,9 However, staining of the total EPS is complicated, since EPS are complex mixtures of different biopolymers with a large number of potential binding sites that limit the specificity of CLSM. Raman microscopy (RM), which does not require staining, and provides chemical information about complex biofilm matrixes, has been successfully tested for nondestructive biofilm analysis including microbial constituents and EPS.10-15 Recently, it has been shown that the combination of CLSM and RM can provide deeper insights into the composition and the structure of the biofilm matrix.9 Raman microscopy is based on the effect of an inelastic light scattering by molecules. This nondestructive analytical technique combines spectroscopic and optical methods and provides whole-organism Raman vibrational fingerprint spectra of the biological samples (all biologically relevant molecules, such as proteins, nucleic acids, carbohydrates, and lipids, exhibit distinct spectroscopic signatures) with spatial resolution in the micrometer range. Thus, RM enables correlations between optical and chemical images. Unlike IR spectroscopy, RM is characterized by a low water background which is beneficial for in situ

10.1021/jp102466c  2010 American Chemical Society Published on Web 07/21/2010

SERS Imaging of Biofilms analysis of biomatrixes. RM has already been acknowledged as a powerful technique for the characterization and identification of a wide range of biological systems (e.g., microorganisms, yeasts, plants, and pollen).16-22 Due to the low quantum efficiency of the Raman effect (typically 10-6-10-8), RM suffers from limited sensitivity. That usually results in long collection times for biofilm analysis even when a high excitation laser power is applied. However, Raman scattering can be significantly enhanced if a molecule is attached, or in immediate proximity, to nanometer-roughened metal (Ag, Au, or Cu) surfaces. This effect, known as surface-enhanced Raman scattering (SERS), leads to Raman signal enhancements in the range of 103-106. Under certain conditions (at “hot spots”sclosely spaced particles or rough nanostructures), enhancement factors up to 1014-1015 (sufficient for singlemolecule sensitivity) can be achieved.23-25 At least two effects contribute to the observed total enhancement.26 The electromagnetic enhancement effect is based on “localized surface plasmon resonance”, which takes place on the nanometer scale of SERS substrates, while chemical enhancement or “charge transfer” is assumed to involve an electronic coupling between the adsorbed analyte and metallic substrate. Beyond signal enhancement, a potential fluorescence background can be reduced by SERS. This effect is important for the analysis of biological samples which are often prone to fluorescence when excited in the visible region. SERS offers many advantages in bioanalysis due to the highly specific fingerprint spectra with very narrow and highly resolved bands that are suitable for the identification of multicomponent samples or for the simultaneous measurement of multiple analytes in a nondestructive and rapid manner. In recent times, SERS has been applied, among others, for the identification of bacterial cells27-39 (for recent reviews, see refs 33 and 40) and viruses.41 We have demonstrated the applicability of SERS for in situ characterization of multispecies biofilms. We have employed colloidal silver nanoparticles for the measurements and obtained reproducible SERS spectra from different constituents of the complex biofilm matrix with enhancement factors of up to several orders of magnitude.42 In this paper, we report on chemical imaging of biofilms by SERS. To the best of our knowledge, this is the first application of SERS imaging for an in situ analysis of biomatrixes as complex as multispecies heterotrophic biofilms. In particular, good SERS measurement reproducibility, in combination with the achieved enhancement factors of several orders of magnitude (>104), enables rapid (1 s for a single spectrum) SERS imaging of biofilms. Thus, the potential of SERS for sensitive chemical analysis, including the detection of different components and the determination of their distribution in the biofilm matrixes, has been illustrated. Materials and Methods Reference Samples. The following biofilm-specific polysaccharides were analyzed by SERS: cellulose (Sigma-Aldrich, Germany), dextran (100 kDa, Fluka/Sigma-Aldrich, Germany), xanthan (Fluka/Sigma-Aldrich, Germany), gellan (Fluka/SigmaAldrich, Germany), and alginic acid (Fluka/Sigma-Aldrich, Germany). As reference materials for proteins, bovine serum albumin (BSA, Sigma-Aldrich, Germany) and L-phenylalanine (Phe, Sigma-Aldrich, Germany) were chosen. Adenine (SigmaAldrich, Germany) was applied as an example for DNA respectively RNA base molecules. Biofilm Cultivation. Multispecies biofilms were grown in a homemade funnel reactor, manufactured from PVC, at a

J. Phys. Chem. B, Vol. 114, No. 31, 2010 10185 Reynolds number Re ) 4000, i.e., under turbulent flow conditions.9 Inoculation was performed with activated sludge supernatant from the municipal wastewater treatment plant of Garching (Germany). Heterotrophic biofilms were cultivated on marked glass slides (epoxy resin marking, Paul Marienfeld GmbH & Co. KG, Lauda-Ko¨nigsfeld, Germany) with 3 mg L-1 Phe, 8 mg L-1 (NH4)2SO4, 2.8 mg L-1 CaCl2, 7 mg L-1 MgSO4 · 7H2O, 6 mg L-1 NaNO3, 5 mg L-1 FeSO4 · 7H2O, and 0.2 mg L-1 K2HPO4. Preparation of Colloidal Silver Nanoparticles. The silver colloids were produced by reduction of silver nitrate with hydroxylamine hydrochloride at alkaline pH and at room temperature, following the Leopold and Lendl43 method with slight modifications.44 In short, 10 mL of AgNO3 (99.8%, Merck KGaA, Germany) solution (10-2 M) was rapidly added to 90 mL of NH2OH · HCl (>99.0%, Fluka/Sigma-Aldrich, Germany) solution (1.67 × 10-3 M) containing 3.33 × 10-3 M NaOH (10-1 M solution, Merck KGaA, Germany). By dividing the original reactant volumes into 10 batches, a more uniform development of silver colloids was achieved. The reaction was completed within a few seconds, resulting in a stable greenishgray, relatively monodisperse colloid with silver particles of 20-30 nm diameter.44 Stored in the dark and cool (4 °C), the colloids remained stable for at least 3 weeks. Raman and SERS Measurements. The Raman microscope system used here was a Renishaw 2000 (Renishaw, U.K.). All measurements were carried out employing a He-Ne laser (633 nm, 7 mW at the sample). For the SERS studies of the reference compounds (Phe, BSA, adenine), the samples were mixed with the silver colloidal solution to final concentrations in the range 10-8-1 M. Subsequently, they were analyzed without drying in microtiter plates (96 wells, PP, Greiner Bio-One International AG, Austria) with an exposure time of 10 s, using a 50× lens (NA ) 0.75). The polysaccharides were mixed with 1 mL of silver colloid and the measurements were performed with a 50 s exposure time; CaF2 plates were used as substrata. The multispecies biofilm samples were examined directly on the marked glass slides in a homemade transparent PMMA box filled with cultivation medium, using a 63× water immersion lens (NA ) 0.9, lateral and axial resolution ca. 1 and 3 µm, respectively). Normal Raman spectra from 2000 to 300 cm-1 were obtained within 100 s. For SERS measurements, the glass slides with biofilm were immersed in the silver colloid suspension and spectra from 2000 to 300 cm-1 were collected with 10 s exposure time. We did not observe any fluctuating carbon bands or other signals indicating sample decomposition, which is probably due to the fact that the SERS measurements were performed in situ in aqueous environment. For distinction of the SERS spectra from the Raman measurements, the second are labeled as normal Raman (NR) spectra throughout this paper. No noticeable bands of the cultivation medium were observed in the NR and SERS spectra. For better comparison, neither baseline correction nor normalization of the NR or SERS spectra collected from 2000 to 300 cm-1 was performed. NR and SERS maps were created by raster scans of the biofilm with 3 µm steps, employing a computer-controlled xyz motorized stage. The spectra were recorded in the so-called static acquisition mode, i.e., without movement of the spectrometer grating, with 10 s (NR) respectively 1 s (SERS) integration time for each single spectrum. This acquisition mode restricts the spectral range (from ca. 900 to 300 cm-1 for NR and from ca. 1500 to 950 cm-1 for SERS spectra); the short measuring time for a single spot makes the collections of large maps in reasonable

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time possible (e.g., 30 or 5 min for NR or SERS maps of 30 µm × 30 µm area with 121 points, respectively). Subsequent band analysis for the collected spectra was applied (MATLAB 7.0.4, The Mathworks Inc., Natick, MA) to create the intensity-correlated maps for relevant NR and SERS bands. Thus, chemical images corresponding to optical images of the biofilm were generated based on the area beneath the baselinecorrected NR band at 377 cm-1 (400-340 cm-1) or the area under baseline-corrected SERS bands, e.g., at 1383 cm-1 (1400-1350 cm-1), 1280 cm-1 (1300-1255 cm-1), 1125 cm-1 (1160-1090 cm-1), and 1000 cm-1 (1010-980 cm-1). CLSM Measurements. In addition to Raman microscopy, confocal laser scanning microscopy (CLSM) was performed to visualize the three-dimensional structure of the biofilm samples. Lectin binding analysis was used to mark EPS glycoconjugates with fluorescently labeled lectin isolated from Aleuria aurantia (LINARIS Biologische Produkte GmbH, Germany), combined with SYTO60 (Invitrogen/Molecular Probes, Carlsbad, CA) to stain nucleic acids.3,45 A Zeiss LSM510 META confocal microscope system (Zeiss MicroImaging GmbH, Germany), controlled by the AIM software (version 3.2), was employed for the monitoring of the fluorescence-marked biofilm constituents. The biofilm samples were analyzed in situ by use of a 40× water immersion lens (NA ) 0.8). Three-dimensional reconstruction of CLSM image stacks was created with the Avizo 5.0 (VSG Visualization Sciences Group, Burlington, MA) software. Results and Discussion SERS Analysis of Reference Samples. Although SERS has been established as a powerful analytical tool to study different biomolecules (proteins, nucleic acids) and biological samples (microorganisms, mammal cells, tissues),26,40,46-51 no comprehensive database of the SERS spectra for microbiological samples has been available up to now. Thus, band assignment for SERS spectra of biofilms was a challenge and required an extended analysis of already published data. Additionally, for a better understanding of the SERS signature of complex biofilm matrixes, SERS studies of a large variety of compounds that could be found in biofilms were performed. Information on the SERS analysis of carbohydrates is particularly sparse and limited to studies of glucose, sodium and calcium alginates, and alditol bearing polysaccharides.52-54 However, polysaccharides are one of the major components of the EPS matrix.6 Some polysaccharides can be present in the form of neutral macromolecules (cellulose, dextran), but the majority of polysaccharides that are found in biofilms are polyanionic due to the presence of either uronic acids (alginate, xanthan), acetyl group (xanthan, gellan), or ketal-linked pyruvate (xanthan). We found significant differences in the SERS signature of the biofilm-specific neutral and polyanionic carbohydrates (Figure 1). Only low enhancement (not shown) is characteristic for cellulose and dextran (neutral macromolecules). For these analytes, the SERS bands are in good agreement with the Raman bands of the corresponding polysaccharides and appear in three major regions (CH/CH2 deformation at 1500-1200 cm-1; C-C stretching, C-O-C glucosidic link, and ring breathing modes at 1200-950 cm-1; and side group deformation at 950-700 cm-1).15 Polyanionic polysaccharides such as xanthan, gellan, and alginic acid, in contrast, exhibit high signal enhancement (not shown) in the SERS spectra. Additionally, significant differences between the SERS and Raman peak numbers, positions, intensities, and widths were observed. Besides the

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Figure 1. SERS spectra of biofilm-specific polysaccharides (normalized and shifted vertically for better comparison).

SERS bands in the region, characteristic for glucosidic ring mode vibrations (1200-950 cm-1), SERS peaks in the region 1450-1270 cm-1 are very strong here. Similar SERS signatures were found for sodium and calcium alginates.52 These prominent bands in the SERS spectra of the polyanionic polysaccharides are considered characteristic for carboxylic acids adsorbed on silver, and correspond to symmetric carboxylate ion (COO-) stretching vibrations.52,53 The band near 730 cm-1 can be attributed to COO- deformation55 and CH2 rocking vibrations.56-58 Moreover, comparison of the spectra of the polysaccharides with various structures reveals significant differences in the SERS band positions, shapes, and intensities. These specific SERS signatures can be helpful for characterization/distinction of various polysaccharides in EPS matrixes. Besides polysaccharides, proteins are another major component in biofilm matrixes.6 The spectra collected from protein (BSA) with silver colloids (see Supporting Information, Figure S1a) exhibit characteristic amide I and III vibrations as well as a typical band of Phe (aromatic ring breathing). These SERS lines are in good agreement with the literature.59 A SERS spectrum of Phe is shown in the Supporting Information, Figure S1b. As an example for SERS spectra of DNA, respectively RNA, base molecules, adenine mixed with silver colloids was analyzed (Supporting Information, Figure S1c). This compound is characterized by two well-known strong sharp bands at 1333 and 733 cm-1.24,26,60-62 These peaks of adenine are often taken as markers for the analysis of biological samples and are typical for the DNA in SERS spectra of bacteria.40 Raman (NR) and SERS Spectra of Biofilm. For the NR and subsequent SERS analyses, a mature multispecies biofilm was chosen. The 82-day-old biofilm with a thickness of approximately 200 µm was present in the form of clusters (see three-dimensional reconstruction of a CLSM image stack in Figure 2). As mentioned above, these clusters are microorganisms embedded in a matrix formed by extracellular polymeric substances. Parts a and b of Figure 3 show the optical microscope image and some typical normal Raman (NR) spectra of multispecies biofilm (82 days old, upper layer of ca. 200 µm thick matrix), obtained with 633 nm excitation wavelength and a 100 s integration time. The NR spectra are characterized by two broad bands around 1630 and 1320 cm-1. Additionally, peaks from carotenoids at 1505 and 1152 cm-1, typical for colored bacteria,63,64 are present. This NR vibration signature of biofilms grown with Phe as substrate is in good agreement with NR spectra of biofilms cultivated with glucose42 or methanol.65 The bands around 1630 and 1320 cm-1 could be assigned to humiclike substances, which are characterized by two broad and overlapping bands with intensity maxima near 1608-1580 and

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Figure 2. Three-dimensional isosurface view of CLSM image stack. The squared base has the area 230 µm × 230 µm. Color allocation: green ) EPS glycoconjugates (Aleuria aurantia lectin marked with AlexaFluor488); red ) nucleic acids (SYTO60).

1350-1290 cm-1, respectively.66,67 The contribution of antisymmetric and symmetric carboxylate stretching vibrations (generally found at 1650-1540 and 1450-1360 cm-1, respectively68) cannot be excluded. The strong band at 377 cm-1 can be attributed to largely hydrated iron oxide, most likely to FeO(OH), which is often found in corrosion products.69,70 This compound causes a light orange color of the analyzed biofilm and could be the oxidation product of Fe2+ from the nutrients used during biofilm cultivation (see Materials and Methods). This band has already been found in NR spectra of biofilm cultivated with glucose42 or methanol65 as substrate instead of Phe, and under similar nutrient conditions. Thus, RM provides in situ chemical information about biofilm components. However, this technique suffers from limited sensitivity. At least 100 s accumulation time is required in order to obtain the NR spectra with a reasonable signal-to-noise ratio even at the maximal available laser power. Moreover, the obtained NR spectra typically contained only a few bands and therefore chemical information is limited. Recently, we have shown that improving the sensitivity of RM by surface-enhanced Raman scattering (SERS) allows for much faster biofilm analysis.42 For in situ SERS measurements we employed hydroxylamine hydrochloride reduced silver colloids that can be reproducibly synthesized at room temperature, resulting in a relatively monodisperse hydrogel with silver particles of 20-30 nm diameter.43,44,71 This SERS substrate can be used for biofilm analysis without adding an aggregation agent. In consequence, uniform distributions of metal nanoparticles within a biofilm matrix are to be expected. Thus, reproducible SERS spectra with a considerable enhancement factor can be achieved.42 Figure 3c illustrates representative SERS spectra measured at the biofilm spots already analyzed by NR (Figure 3b). The spectra were collected with a 633 nm laser and 10 s signal accumulation time. As a general rule, neither baseline correction nor normalization was applied to the presented spectra, thus facilitating comparison of the NR and SERS vibration signatures.

J. Phys. Chem. B, Vol. 114, No. 31, 2010 10187 Apparently, the replicated SERS spectra from the biofilm matrix are characterized not only by the reproducible band positions and relative intensities, but also by the absolute enhancement of the signal. It is important to underline that the Raman cross section can be enhanced only for those molecular components that are sufficiently close (within ∼10 nm) to the SERS active surface, since the electromagnetic enhancement scales with the 12th power of the distance (d) between the analyte and SERS substrate (∝d-12).26 Additionally, molecular components should have a certain spatial orientation to the SERS surface.34,72 Therefore, NR and SERS signatures of the biofilm are not supposed to coincide. Moreover, spectral differences may be explained by the chemical enhancement effect, induced by direct interactions between the analyte and metal surface. That could result in significant band shifts in SERS spectra. Thus, the number and position of vibrational lines and their relative intensities can be expected to be different for NR and SERS spectra of biofilms. That was confirmed in our previous work.42 Comparison of the NR and SERS spectra of a biofilm (Figure 3b,c) reveals that, besides the significant enhancement of the Raman signal, the SERS spectra are characterized by a higher number of peaks. Thus, the chemical information content is larger. Although it is very difficult to estimate the enhancement factor for the biofilm SERS spectra, from the intensities of the strongest bands and the used acquisition conditions, we assume a minimum enhancement of 104 in this case. The SERS spectra of the biofilm clusters are characterized by multiple bands with the intensity maxima near 1555, 1380, and 1280 cm-1. This SERS signature is in agreement with the recently published data on the SERS study of the multispecies biofilm.42 A tentative band assignment was performed, referring to already published Raman and SERS studies of biological samples (see Table 1) and our own database of the SERS fingerprint spectra from the reference compounds (see SERS Analysis of Reference Samples). Typical vibrational lines of polysaccharides, proteins, nucleic acids, and carotenoids are anticipated to contribute to the SERS spectra of the biofilm (for details, see Table 1). The strong bands near 1555 and 1380 cm-1 in the SERS spectra of the biofilm (Figure 3c) are characteristic for COOantisymmetric and symmetric stretching vibrations,33,41,58,73 respectively. In the SERS spectra of viruses, Bao et al.41 identified the signals at 1582 and 1396 cm-1 as COOantisymmetric and symmetric stretching, respectively. The strong vibration near 1400 cm-1 is often assigned in the literature to the carbohylate stretching mode of R-amino acids and has been associated with aspartic and glutamic acids for SERS spectra of E. coli where silver colloids have been produced inside the cells.40 Based on our SERS analysis of the polysaccharides, we believe that the strongest band observed in the SERS spectra of the biofilm matrix near 1380 cm-1 is predominantly due to the symmetric carbohylate stretching mode of the polyanionic polysaccharides (which are common for EPS matrix). Hence, this prominent band can be applied as a marker for polyanionic carbohydrates in SERS spectra of biofilm matrixes. Polysaccharides, especially polyanionic macromolecules, are supposed to predominantly account for the multiple bands in the biofilm SERS spectra due to direct interactions between carboxyl groups and the silver surface that results in the peaks at 1555 and 1380 cm-1. The SERS bands at 1280 cm-1 (COH, HCO, and HCC deformation,58,74,75 COO- stretching symmetric, C-O stretching52), 1145 cm-1 (C-C74,75 and C-O75 antisymmetric ring breathing), 1125 cm-1 (C-C stretching, C-O-C

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Figure 3. Optical image of the biofilm (82 days old, upper layer of ca. 200 µm thick matrix) with the marked mapping area (a). Corresponding NR (b) and SERS (c) spectra. NR (d) and SERS (e) maps for 377 and 1383 cm-1 band areas, respectively. Spectra for maps were collected during 10 s (NR) or 1 s (SERS) from 30 µm × 30 µm biofilm area scanned with 3 µm step.

glycosidic link, and ring breathing58,74,75), and below 580 cm-1 should also be assigned to carbohydrates (Table 1). Proteins can contribute to the peaks near 1635 cm-1 (amide I76-78), 1280 cm-1 (amide III56-59,72,76,78), 1145 cm-1 (NH2 twist58), and 1000 cm-1 (Phe ring breathing57-59,76). The amide I region is relatively weak, which is typical for SERS of bacteria.34,38,56 Moreover, the amide I and III modes are most likely overlapped with antisymmetric and symmetric COO- stretching vibrations from polysaccharides, respectively. However, the Phe band near 1000 cm-1 can be used for the detection of proteins in the biofilm matrixes. A contribution of nucleic acids to the weak bands near 1335 cm-1 and near 730 cm-1 can be also assumed. Although based on our analysis of reference samples, these lines can also be associated with polysaccharides; the peaks at 1335 and 735 cm-1 are often used as markers of adenine and DNA by SERS analysis of biological samples.40 Thus, the significant enhancement of Raman signals by SERS along with the higher number of sharp peaks in the SERS spectra suggests the suitability of SERS for sensitive in situ chemical

analysis of various components within biofilm matrixes with the spatial resolution of an optical microscope. NR and SERS Maps of Biofilm. Information on the distribution of different components in complex biofilm matrixes can be obtained by Raman mapping. The spectra from a distinct biofilm area are obtained by raster scans. By subsequent processing of the collected spectra, maps of the relevant band intensities can be calculated, corresponding to the optical images of the biofilms. Figure 3d shows the NR map of the band intensity at 377 cm-1, obtained from an area of 30 µm × 30 µm (see square in Figure 3a; the scale remains the same) with 3 µm steps. Acquisition times of 10 s for a single spectrum (spectral region from 900 to 300 cm-1) were used. The NR map illustrates the distribution of iron oxide hydroxide (FeO(OH)) within the biofilm matrix. However, NR mapping is very time-consuming because at least 10 s are required for each single spectrum, resulting in ca. 30 min acquisition time for a relatively small map of 121 grid points.

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TABLE 1: Tentative Band Assignment for SERS Spectra Acquired from Biofilm Matrix with Silver Colloidal Nanoparticlesa assignment peak position/cm 1680-1612 1640-1540 1550-1500 1450-1440 1450-1360 1383, 1353 1345-1315 1335-1330 1300-1270 1350-1200 1290-1230 1280, 1270 1170-1150 1160-1145 1155 1145-1135 1127-1122 1125 1095-1090 1085-1080 1060 1060-1025 1020-1000 1000 899-855 852 828 800-780 780 760 735-720 580-530 480-305 a

-1

carbohydrates

proteins amide I

COO- str antisym42,52,68,72

DNA/RNA

carotenoids

lipids

76-78

CdC str63,64,81 CH2 def57,58,76 COO- str sym42,52,68 COO- str sym42,52,68 CH def17,57,58

CH2 def58,76

CH2 def58,76,82

CH def17,53,57,58,72 A24,26,60-62

COO- str sym, C-O str52 amide III56-59,72,76,78 42,75

HCC, HCO, COH def, CH2 def COO- str sym, C-O str,52 HCC, HCO, COH def, CH2 def42,75

amide III56-59,72,76,78 C-C str63,64,81

C-C, C-O ring breath, antisym74,75,82 C-C, C-O ring breath, antisym74,75,82 C-C str, C-O-C glycosidic link; ring breath, sym57,58,74-76,82,83 C-C str, C-O-C glycosidic link; ring breath, sym57,58,74-76,82,83 C-C str, C-O-C glycosidic link; ring breath, sym57,58,74-76,82,83 C-O-H bend, CH bend74

NH2 twist58 C-N, C-C str57,76

C-C str76

C-N, C-C str57,76 PO2- str, sym58

PO2- str, sym58

C-C; C-N76 C-O, C-C str

74,76

C-CH3 def63,64,81 ring breath Phe57-59,76 C-C str, C-O-C 1,4-glycosidic link57,58,76 ring breath Tyr76,84 ring breath Tyr76,84

COO- def;55 CH2 rock56,57 C-O-C glycosidic ring def76,85 skeletal modes CC, CCC ring def74,85

O-P-O str84 C, U26,57,58,76 T26

ring breath Trp84 A24,26,60-62,76

bend, bending; breath, breathing; def, deformation; rock, rocking; scis, scissoring; str, stretching.

The enhancement factor of several orders of magnitude and the good reproducibility of the SERS measurements offer significant improvement in this regard. Owing to the signal enhancement, the accumulation time for each grid point can be reduced to 1 s, allowing for relatively fast SERS mapping (5 min for a 121-point SERS map instead of 30 min for a similar NR map). Figure 3e illustrates the SERS map for the strongest band at 1383 cm-1 collected from the biofilm area of 30 µm × 30 µm (already analyzed by NR mapping, Figure 3d) with 1 s spectra (1500-950 cm-1). The SERS map of polyanionic polysaccharides (COO- symmetric stretching) represents the complete biofilm cluster. These data suggest the relatively uniform distribution of the signal enhancement through the entire biofilm matrix by the SERS measurements and illustrate the potential of SERS imaging for sensitive and rapid chemical analysis of biofilm matrixes. By increasing the signal enhancement, allowing for shorter exposure times, extended biofilm areas can be imaged in reasonable measurement times. The application of a 633 nm excitation laser for NR and SERS analysis enables direct comparison of NR spectra and maps vs SERS data. In our future work we plan to use shorter excitation wavelengths (514 and 532 nm) for SERS studies. However, significantly higher fluorescence in NR spectra in this case can complicate the comparison of NR and SERS data.

SERS Imaging of Biofilm. For analyzing the distribution of different components (e.g., polysaccharides, proteins) in the biofilm matrix, it is important to choose characteristic frequency regions and/or marker bands for these substances. In the case of polysaccharides, symmetric carboxylate ion stretching near 1380 cm-1 seems to be suitable for visualization of polyanionic macromolecules within the biofilm matrix (Figure 3e). Additionally, glucosidic ring mode vibrations near 1125 cm-1 can be used for the imaging of carbohydrates (predominantly neutral macromolecules, see SERS Analysis of Reference Samples and Figure 1). For proteins, the SERS band at ca. 1280 cm-1 can also have a contribution of the amide III mode. On the other hand, the line of Phe at ca. 1000 cm-1 should not be interfered by polysaccharide vibrations and can be applied for the detection of proteins within the biofilm matrix. Figure 4a shows again the optical microscope image of the upper layer of ca. 200 µm biofilm (age 82 days), where the measurement points for the exemplary fingerprint SERS spectra (Figure 4b) are marked. An area of 60 µm × 60 µm was chosen for SERS imaging (Figure 4c-f; the scale remains the same). The maps of the SERS bands at 1383, 1125, 1280, and 1000 cm-1, presented in Figure 4c-f, visualize the spatial distribution of polysaccharides and proteins in the upper biofilm layer. As illustrated in Figure 4c,e, the SERS lines at 1383 cm-1 (polyanionic polysaccharide) and 1280 cm-1 (complex band

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Figure 4. Optical image of the biofilm (82 days old, upper layer of ca. 200 µm thick matrix) with the marked mapping area (a). Exemplary fingerprint SERS spectra (b). SERS maps for bands at 1383 (c), 1125 (d), 1280 (e), and 1000 cm-1 (f). Spectra for maps were collected during 1 s from 60 µm × 60 µm biofilm area scanned with 3 µm step.

comprising different carbohydrate vibrations and amide III mode of proteins) are characteristic for the entire clusters. At the same time, the bands at 1125 and 1000 cm1 (Figure 4d,f), which can be attributed to glucosidic link and Phe vibrations, are obviously expressed only in restricted areas of the biofilm matrix. However, the differences in the distribution of the polysaccharide bands at 1125 cm-1 (glucosidic link) and 1383 cm-1 (COOsymmetric stretching) could be associated with the differences in their relative intensities. As shown in Figure 3b, the bands at 1125 cm-1 (as well as at 1000 cm-1) are characterized by

significantly lower intensity compared to the prominent band at 1383 cm-1 and therefore could be partially hidden in the noise by 1 s SERS spectra used for mapping. The reproducibility of these results is confirmed by multiple analyses of different clusters from the upper layer of 82-day-old biofilm from the same as well as of adjacent sample carriers. The clusters in these mature biofilms attached directly to the glass surface (Figure 5a) exhibit a slightly different SERS signature (Figure 5b) compared to the biofilm fraction located on the top (Figure 4b). Although the position of the prominent

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Figure 5. Optical image of the biofilm (82 days old, on glass) with the marked mapping area (a). Exemplary fingerprint SERS spectra (b). SERS maps for bands at 1383 (c), 1125 (d), 1280 (e), and 1000 cm-1 (f). Spectra for maps were collected during 1 s from 60 µm × 60 µm biofilm area scanned with 3 µm step.

bands remains invariable, the relative intensities of these lines show changes in the spectra of the different axial sections of the biofilm samples. For example, the intensity of the complex vibration mode at 1280 cm-1 seems to be comparable to or higher than the intensity of the SERS band at 1383 cm-1 (Figure 5b). Nevertheless, the maps of bands at 1383, 1125, 1280, and 1000 cm-1 of this biofilm type (Figure 5c-f) reveal information on the relative abundance of polysaccharides and proteins within the biomatrix. To get more information about the relative intensities of the bands used for SERS imaging, their ratios were calculated based

on the absolute band areas estimated from the SERS spectra. Analysis of marker bands at 1383 and 1280 cm-1 of 82-dayold biofilm reveals significant differences in their ratio (I1383/ I1280) for the different axial sections of the sample, e.g., for biofilm on the top of clusters (4.1 ( 1.3) and for biofilm located near the substratum (0.8 ( 0.4). That indicates the dominance of COO- stretching vibrations characteristic for polyanionic polysaccharides (see Table 1) in the upper biofilm layer. We suppose that the ratio of two marker bands could be used in order to derive information about the metabolic activity of biofilm. It must be stressed that additional experiments/

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Figure 6. Optical image of the biofilm (22 days old) with the marked mapping area (a). Exemplary fingerprint SERS spectra (b). SERS maps for bands at 1353 (c), 1155 (d), 1270 (e), and 1003 cm-1 (f). Spectra for maps were collected during 1 s from 60 µm × 60 µm biofilm area scanned with 3 µm step.

experiences are needed to prove the applicability of band ratios in terms of space (z-dimension) and/or time dependencies. The analysis of a biofilm cultivated for 22 days instead of 82 days (for an optical microscope image, see Figure 6a) reveals that the younger biofilm is characterized by slightly shifted frequencies of the prominent bands with the strongest peak at 1270 cm-1, instead of at 1280 cm-1 for 82-day-biofilm (compare Figure 6b with Figures 4b and 5b). Symmetric carboxylate stretching mode of polyanionic polysaccharides for a 22-day-old biofilm is present in the form of the broad SERS signature with a maximum at 1353 cm-1. Figure 6c-e illustrates the maps of the SERS lines at 1353,

1155 (glucosidic mode, see Table 1), 1270, and 1003 cm-1 for this biofilm and allow for the visualization of polysaccharides and proteins in the biofilm matrix. Apparently, the differences in the SERS spectra and hence in the chemical composition of the biofilms are expressed more clearly for samples cultivated for different times. Indeed, the study of samples cultivated only for 8 days (initial phase of biofilm growth) shows significant variations in the SERS spectra of the biofilm concerning band positions and their relative intensities. Nevertheless, even for these clusters of early stage biofilm, the bands at 1353, 1155, 1270, and 1004 cm-1 seem to be suitable for the analysis of the relative abundance of

SERS Imaging of Biofilms polysaccharides and proteins in biomatrix (for details see the Supporting Information, Figure S2). It should be underlined that we tested here for the first time the applicability of SERS imaging for in situ analysis of biofilms. In further studies we plan to focus on the characteristic differences in SERS spectra (band positions and their relative intensities) of biofilms cultivated at various environmental conditions, and on the influence of a biofilm type (thickness and/or its metabolic activity) and cultivation stage on their SERS signature. Especially SERS analysis of initial growth is important. We believe that the application of SERS imaging in biofilm research can enable a deeper insight into the chemical composition and structure of complex biofilm matrixes. For the reported SERS measurements of multispecies biofilms, we employed hydroxylamine hydrochloride reduced silver colloids that can be reproducibly synthesized at room temperature, resulting in a stable, relatively monodisperse hydrogel. Although reproducible SERS spectra with an enhancement factor of several orders of magnitude can be achieved with this SERS substrate, we plan to study the application of gold colloids for in situ analysis of biofilm. This should help in eliminating possible artifacts due to microbial toxicity of silver nanoparticles79 and silver ions79,80 formed by dissolution/oxidation of the colloidal silver nanoparticles. Additionally, the use of gold SERS substrates could enable online monitoring of the biofilm formation, development, and aging. Conclusions In this paper, we presented the first application of SERS imaging for in situ chemical analysis of biofilms. For better understanding of the SERS signature of complex biofilm matrixes, a large variety of compounds that can be found in biofilm were analyzed. We achieved substantial enhancement of the Raman signals by SERS (>104), highly informative SERS signatures, and good SERS measurement reproducibility. Altogether, this indicates significant potential of SERS imaging for sensitive in situ chemical analysis of different components in biofilm matrixes even at low concentrations, with the spatial resolution in the micrometer range. We believe that SERS can help reveal more detailed information on the chemical composition of EPS matrixes compared to normal Raman spectroscopy, since the concentration of biomolecules can be below the sensitivity of NR, especially when the initial growth phase of biofilms is investigated. Moreover, compared to CLSM analysis, which often suffers from limited specificity, SERS enables labelfree sensitive analysis of biomatrixes. Our future research will focus on the interpretation of SERS signatures from different biofilms, e.g., position and relative intensities of the relevant bands, to characterize growth, aging, and storage of biofilms. Acknowledgment. Financial support by the German Research Foundation (DFG, Project HA3507/2-2&HO1910/7-2) is gratefully acknowledged. The authors thank Maria Knauer for her help preparing the SERS substrates and for many useful discussions. Supporting Information Available: Additional data about the SERS analysis of biofilm are reported. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Hall-Stoodley, L.; Costerton, J. W.; Stoodley, P. Nat. ReV. Microbiol. 2004, 2, 95–108. (2) Vu, B.; Chen, M.; Crawford, R. J.; Ivanova, E. P. Molecules 2009, 14, 2535–2554.

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