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Interference Enhanced Raman Spectroscopy as a Promising Tool for the Detection of Biomolecules on Raman-compatible Surfaces Susanne Pahlow, Thomas Mayerhöfer, Marie van der Loh, Uwe Hübner, Jan Dellith, Karina Weber, and Jürgen Popp Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01234 • Publication Date (Web): 11 Jul 2018 Downloaded from http://pubs.acs.org on July 14, 2018

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Analytical Chemistry

Interference Enhanced Raman Spectroscopy as a Promising Tool for the Detection of Biomolecules on Raman-compatible Surfaces Susanne Pahlow1,2, Thomas Mayerhöfer1,3, Marie van der Loh3, Uwe Hübner3, Jan Dellith3, Karina Weber*1,2,3 and Jürgen Popp1,2,3 1) Friedrich Schiller University Jena, Institute of Physical Chemistry and Abbe Center of Photonics, Helmholtzweg 4, 07743 Jena, Germany 2) InfectoGnostics Research Campus Jena, Centre for Applied Research, Philosophenweg 7, 07743 Jena, Germany 3) Leibniz Institute of Photonic Technology - Member of the research alliance “Leibniz Health Technologies“, AlbertEinstein-Straße 9, 07745 Jena, Germany ABSTRACT: Raman spectroscopy in combination with appropriate sample preparation strategies, for example enrichment of bacteria on metal surfaces, has been proven to be a promising approach for rapidly diagnosing infectious diseases. Unfortunately, the fabrication of the required chip substrates is usually very challenging due to the lack of feasible instruments that can be used for quality control in the surface modification process. The intrinsically weak Raman signal of the biomolecules, employed for the enrichment of the microorganisms on the chip surface, does not allow monitoring the successful immobilization by means of a Raman spectroscopic approach. Within this contribution we demonstrate how a simple modification of a plain aluminum surface enables enhancing (or decreasing, if desired) the Raman signal of molecules deposited on that surface. The manipulation of the Raman signal strength is achieved via exploiting interference effects that occur, if the highly reflective aluminum surface is modified with thin layers of transparent dielectrics like aluminum oxide. The thicknesses of these layers were determined by theoretical considerations and calculations. For the first time it is shown that the interference effects can be used for the detection of biomolecules as well by investigating the siderophore ferrioxamine B. The observed degree of enhancement was approximately one order of magnitude. Moreover, the employed aluminum/aluminum oxide layers have been thoroughly characterized using atomic force and scanning electron microscopy as well as X-ray reflectometry and UV-Vis measurements.

Raman spectroscopy including all its variants is a very powerful spectroscopic technique with a very broad application area ranging from the investigation from inorganic solid-state materials 1 to biochemical applications like tissue imaging and cell and bacteria identification 2 due to its fingerprint specificity based on the large variety of vibrational modes in matter. The intrinsically weak effect of Raman scattering is a disadvantage of its usage. Accordingly, a number of different ways have been found to enhance this comparably weak signal. The most well-known enhancement effect is called surface enhanced Raman spectroscopy (SERS).3 This term is, at least partly, a misnomer, as it is not a typical surface effect. Instead, the enhancement is according to literature, intimately connected to the existence of a in most cases, structured gold or silver surface. The structure allows the excitation of surface plasmon-polaritons which lead to locally high electric field strengths. The Raman scattering is, to a first approximation, proportional to the fourth power of these electric field strengths.3 Additionally, a chemical effect seems to contribute, the magnitude of which is hard to estimate.4,5 SERS has undeniable its merits for a large number of different applications.6-8 However, employing SERS for thin film analysis can be difficult, in particular if SERS labels cannot be incorporated into the layer and structured surfaces have to be used instead.3 As a consequence, the inhomogeneity of the electric field enhancement on this surface, the low range of the enhancement, which

is usually on the order of a few nm and the roughness of the surface, which is a necessity for SERS do not render SERS the method of choice. Therefore, for thin films another enhancement technique is favored. This technique is called interference-enhanced Raman scattering (IERS)9 and is based, as the name suggests, on the interference effect that occurs on the surface of coated metallic substrates or Bragg reflectors.10 While the enhancement is compared to SERS relatively modest (enhancement factors or on the order of 101-102),9 the substrates are more easy to fabricate, long-term stable, have a homogenous enhancement over the whole surface and can be atomically smooth if fabricated by atomic layer deposition.3 As in case of SERS, however, the presence of sample changes the wavenumber of highest enhancement and the magnitude of this enhancement. While this was never an obstacle for the application of SERS, it might have prevented IERS from routine use. So far, IERS has only been used to enhance the Raman signals for well-defined layers and mostly inorganic or metalorganic materials like tellurium11, graphene10,12-14, MoS215 and F16CuPc9. In this contribution, IERS is applied for the first time for investigating a biomolecule relevant within the context of the infectious diseases.16,17 As previously indicated, Raman microspectroscopy is a highly efficient tool for the rapid and precise identification of bacterial pathogens.18,19 Due to the specific

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chemical information, concealed in the Raman spectra of the investigated cells, a reliable assignment on species level is enabled. However, in order to exploit the full benefits of this approach, elaborate sample preparation strategies have to be applied before real world samples, such as body fluids20,21 or soil samples22, can be successfully investigated. Most samples matrices contain components which will interfere with the Raman spectra of the bacterial cells and consequently hamper a correct identification.23 In this context chip based approaches are very attractive as they allow performing the enrichment of the bacterial cells and subsequent Raman measurements on the same platform.2,24 An important requirement for investigating the isolated bacteria directly on the chip is the utilization of Raman compatible materials, which do not interfere with the spectra of the bacterial cells. Metals, such as Ni and Al, have been proven to be suitable choices.2,24-27 A major challenge in the development of such chip substrates is the control of the surface modification with specific capture elements for the bacterial cells. The detection of these capture molecules on the chip surface in order to evaluate the actual success of the immobilization method is often quite complicated or not possible at all. If antibodies are employed as capture probes, an immunoassay with an enzyme24 or dye labelled secondary antibody can be performed in order to proof a successful immobilization. The implementation of less common capture probes, for example siderophores2,17,28,29, makes applying a comparable antibody based detection scheme significantly more difficult. Also, it has to be considered that the prerequisite for this approach is the availability of proper primary antibodies for the detection of the immobilized biomolecules. The production of these can be both difficult and expensive. These deliberations illustrate that a simple and straightforward approach for the detection of biomolecules on Ramancompatible chip surfaces would be highly desirable. Therefore, it is the main objective of this work to evaluate the potential applicability of IERS as an instrument of quality control in the development of chip-based isolation strategies. We decided to establish the proof of concept for using IERS for the detection of biomolecules with the siderophore ferrioxamine B as a model substance. To that end, we modified Raman-compatible substrates featuring an aluminum surface with defined layers of aluminum oxide for providing either enhancement or decrease of the Raman signal. The characteristics of the substrates were carefully studied employing both theoretical calculations and Raman spectroscopic measurements. In order to experimentally confirm the calculated interference effects of the aluminum oxide layers, we investigated films of Poly(methyl methacrylate) (PMMA) as model analyte first, before we studied the interference effects for the siderophore ferrioxamine B.

EXPERIMENTAL SECTION Chip substrates. The fabrication of the chips started on wafer scale by using silicon-wafers (100 mm, Si). First the wafers are prepared with photoresist (AZ 5214, thickness 1.8 µm, spin-process) and tempered 20 min at 80 °C in an oven. Then the photolithography is performed using a mask aligner tool (EVG 620TP) and chromium-masks. After the exposure the resist is tempered 2 min at 120 °C on a hotplate and floodexposed in UV-light to make an image reversal. The image reversal is needed to get good lift-off-results in the next fabrication step, the metal-evaporation. The development of the resist is done for 40 s in AZ MIF-726 developer. Then the

wafer is placed in a vacuum evaporation tool (Roth&Rau “Microsys 600”) and a 200 nm thick metal stack is deposited. After the evaporation the wafer is soaked in an acetone bath to perform the lift-off-process. The result is the formation of metallic pad-like patterns on the wafer-surface. These then get covered with a (60, 75, 112, 135 or) 150 nm thick alumina film by using a 120 °C atomic layer deposition process (plasma enhanced ALD, Oxford “Opal”). This special deposition technique ensures a closed and conformal coating in combination with a precise thickness control of the alumina film. The wafer is separated into chips by using wafer dicing. To avoid contaminations during the dicing process the wafer was covered with photoresist beforehand. After dicing the separated chips are cleaned in an ultrasonic acetone bath, stopped in isopropanol and spin-dried. Modification of the substrates with ferrioxamine B. Desferrioxamine B and anhydrous FeCl3 were purchased from sigma aldrich. A ferrioxamine B stock solution of 25 mM was prepared by combining equal volumina of 50 mM FeCl3 and desferrioxamine solutions in purified water. This stock solution was diluted with purified water to achieve the desired concentrations. We tried three approaches for depositing the analyte solution on the chip substrates: spin coating, dip coating and spotting of defined volumes as drops. Both spin and dip coating did not succeed. In both cases no uniform layers of analyte were formed, but the liquid would gather in irregular shaped droplets on random areas of the chip, so that we would have not been able to determine how much analyte was deposited on the chips and a comparison of different chip substrate would not be possible. Drop coating enabled us to specifically modify each chip with the same amount of analyte, so we were able to compare the Raman spectra recorded on different chips. Accordingly, the chip substrates were modified with ferrioxamine B by applying a droplet of 1 µL of each concentration per measuring field. Prior to the modification with ferrioxamine B, the substrates were silanized as previously described.2 The substrates were dried for three hours. After the drying step the Raman spectra were immediately recorded. Coating of the substrates with PMMA. For further experiments a number of chips were additionally coated with a 30 nm thin layer of PMMA. The e-beam-resist ARP679.04 (diluted 1:2 with thinner AR 600-09) was spun with 6000/min and subsequently dried 10 min at 180 °C on a hotplate (ARP and AR are trademarks and products from Allresist GmbH Berlin). Raman measurements. The Raman measurements of the drop dried ferrioxamine B were conducted under ambient conditions in backscattering geometry using a Raman microscope setup (BioParticle Explorer, rap.ID Particle Systems GmbH, Berlin, Germany) with 532 nm excitation wavelength and a 100x magnification objective (MPLFLN 100xBD, NA = 0.9, Olympus Corporation, Tokyo, Japan). Further information about the system can be found elsewhere.18,25,26 The integration time per spectrum was 5 s, while the laser power was adjusted to approximately 12 mW. The spectra shown within this publication were acquired in the middle of the spots only, as a coffee ring effect occurs via drop drying. The layer thickness in the outer parts of the dried spots is too thick for observing the interference enhancements. For each concentration 25 spectra were recorded. The Raman measurements of the PMMA were performed as well in backscattering geometry using a Raman microscope (WITec GmbH, Ulm, Germany) with 514 nm excitation wavelength using a 100x magnifica-


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Analytical Chemistry tion objective (Zeiss Plan-Neofluar, 100× , NA = 0.9, Oberkochen, Germany). Detailed information on the setup can be

found elsewhere.30 The integration time per spectrum was 15 s

Figure 1: a) and b) Electric field maps in the system air/Al2O3-layer/Al. The layer thickness amounts to 60 nm on the left side and 135 nm on the right side (indicated by green lines). c) and d) electric field strength on the surface of the layer (y = 0 µm) in dependence of the wavelength while the laser power was 15 mW. For each layer thickness 400 spectra were recorded. Regarding polarization or orientation related effects, note that, molecular alignment might play a role if monolayers are investigated but since field components parallel to the surface are not suppressed in contrast to metallic surfaces we do not expect any restriction in this regard. Furthermore, for biological samples which are heterogeneous any incident polarized light will be depolarized due to scattering effects so that even in the case that molecular alignment occurs it will be hard to detect it. UV-Vis measurements. The reflection spectra of the surfaces were measured with a custom-designed microspectroscopy setup, which is realized by combining an optical microscope (AxioImager Z1.m, Zeiss, Göttingen, Germany) with a grating (150 gr/mm) based spectrometer (SpectraPro 2300i, Princeton Instruments, Trenton, NJ) by an optical fiber (FT400EMT, ThorLabs, Newton, NJ). The reflectance spectrum of the substrate and the corresponding background spectrum in the vicinity of the substrate were measured with an exposure time of 750 ms. AFM measurements. The AFM measurements were carried out on a Dimension Edge system by Bruker. The system was operated in the contact mode and silicon nitride tips (MSCT) with a specified tip radius of ≤ 10 nm were used. SEM measurements. The SEM images were performed on a FEI Helios Nanolab G3 UC system which enables ultra-high

resolution power even at lowest electron energies (E0). In this manner great detail visibility and best surface sensitivity can be ensured. The images shown in Figure S2 were taken at E0= 1 keV and a probe current of 13 pA. For the detection of secondary electrons an in-lens detector (TLD) was used and the final lens of the system was operated in the immersion mode (field mode). In order to enhance the surface topography impression the specimens were tilted by 45°. All samples were coated in previous with approximately 10 nm carbon in order to avoid charging effects.

RESULTS AND DISCUSSION Theoretical background. Interference in thin layers is a well-known phenomenon and can be quantitatively understood based on the interaction of coherent waves, in this particular case of a wave and its reflected counterpart.31 Formalisms have been developed which allow to calculate the electric field strength due in the presence of interference for arbitrary layer stacks, angle of incidence and polarization32, even in the presence of thick layers which may lead to the loss of coherence within them33. Ultimately, these calculations are based on a 2×2 matrix formalism that was devised by Abelès34. Here, we do not want to recite the corresponding formulas, as nowadays commercial program packages are available (e.g. FDTD Solutions from Lumerical), which can be used to illustrate the electric fields in an intuitively accessible way. In its most simple concept, an IERS substrate consists of a metal substrate (or a film of a highly-reflecting metal) and a non-absorbing



dielectric thin film (a quarter wave layer) on top of it. A typical example of the field map of such a substrate consisting of an Al2O3 layer on top of aluminum for normal light incidence is illustrated in Figure 1. The left side of Figure 1 shows the electric field map for a 60 nm thick overlayer of Al2O3 on top of Al. For this thickness, the first order maximum of the electric field strength on the interface vacuum/layer appears at 518 nm and the relative electric field strength |E|/|E0| amounts to 1.88 (2 would be the maximum for a totally reflecting substrate). The use of the first order maximum is of particular advantage, as the half maximum full width of the second order maximum is much lower as can be seen in Fig. 1d) for a 135 nm thick Al2O3 overlayer. For this overlayer, the relative electric field strength features a minimum at 526 nm. Roughly, the Raman signal is proportional to |E|4/|E0|4, also in interference-enhanced Raman scattering9, therefore in the first case the maximum enhancement would be 1.884 ≈ 12.5, while in the second case the signal would be decreased by 0.0394 ≈ 2.4×10-6. In practice, it has to be taken into account that the electric field enhancement consists of two parts. The first part is the enhancement of the electric field of the incoming radiation, e.g. a laser with a wavelength of 514 nm, which is proportional to |E|2/|E0|2 at this wavelength (about 3.53 for the substrate with the 60 nm). The second part is the enhancement of the Raman scattered radiation. A Raman shift of e.g. 3000 cm-1 would be equivalent to photons having a wavelength of 607 nm. At this wavelength |E|2/|E0|2 would amount to about 3.03 (cf. Figure 2). The overall enhancement taking into account both parts contributing to the enhancement would be with about 10.7 still an order of magnitude over the whole surface of the substrate. This also shows that the enhancement does not vary much over the Raman spectral range (12.5 at 0 cm-1 vs. 10.7 at 3000 cm-1). From Figure 1, it is also obvious that the spatial dependence of the electric field intensity is comparably weak normal to the surface. Under the precondition that the biological sample layer should experience a more or less uniform enhancement the sample layer thickness should not exceed up to 50 nm with the additional assumption that biological materials with an index of refraction of about n ≈ 1.5 should not disturb the system strongly, in particular as there is no very large index of refraction mismatch with Al2O3 (n ≈ 1.77 in this spectral range 35 ). Above that the gain in signal becomes weaker since the increase in sample volume is compensated by a decrease in enhancement and finally even a weakening of the signal once the sample thickness reaches regions where the signal is suppressed (always provided that the sample thickness is at least locally homogenous). For sample thicknesses larger than 250 nm the gain in signal should be mostly provided from the gain in volume. Figure 1 has been generated assuming normal incidence. The non-collimated beam of a microscope leads to a distribution of angles of incidences so that strictly speaking the field intensity would have to be calculated by averaging over the angle of incidence and the polarization. Based on the fact that reflectance from a metal is already high, therefore only slowly varying with the angle of incidence and in the opposite way for perpendicular and parallel to the incidence plane polarized light, the simplifying assumption of normal incidence for numerical apertures up to 0.9 should be justified, which is also fully confirmed by the results of our experiments.

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Overall, we investigated substrates with five different Al2O3 thicknesses: 60, 75, 112.5, 135 and 150 nm. The comparison of the different electric field enhancement factors in dependence of wavelengths at the surface of the substrates is depicted in Figure 2.

Figure 2: Electric field strength on the surface of the layer (y = 0 µm) in dependence of the wavelength for the different thicknesses of the Al2O3 layer used in this work. Characterization of the chip substrates. In order to characterize the surface of the aluminum oxide layer on top of the chip substrates the two complementary methods atomic force microscopy (AFM) and scanning electron microscopy (SEM) have been applied for all layer thicknesses. The root-meansquared roughness for the Al2O3 coated substrates ranges between 9.1 and 20 Å. There is no significant difference to the roughness of the uncoated aluminum surface, which was determined to be 9.9 Å. As expected, there is no increase of roughness for an increasing thickness of the Al2O3 layer. However, on the Al2O3 coated substrates the growth of some larger particles or tips with a diameter of 100 to 200 nm was observed, as can be seen in AFM images of Figure 3b. In Figure S1 of the supporting information the corresponding SEM images can be found. Furthermore, the desired layer thickness was verified exemplarily using X-Ray reflectometry (XRR). Layers of transparent materials on highly reflecting surfaces were investigated already in the 60ies and 70ies of the last century due to their potential application for high-temperature solar absorber surfaces. 36,37 For this particular application the absorption should be at maximum in the visible spectral region. The absorption for these types of composite materials is actually induced by interference, since highly reflecting materials usually show only very weak absorption as the radiation is not able to penetrate more than a few ten nanometers into the metal. However, since the layer on top of the metal is nonabsorbing, absorption has to take place inside the metal. Interference is connected with an electric field standing wave at certain wavelengths λm which are given by:

4n2 d = λm . 2m + 1

(1)

Here, n2 is the index of refraction (function) of the transparent layer, d its thickness and m the order of the minimum in the reflectance. Being strict, eqn. (1) only holds for an ideal-


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Analytical Chemistry ized perfect reflector like a perfect electric conductor, but for good conductors like Au, Ag and Al eqn. (1) is a good approximation 38. The stronger electric fields at these wavelengths induce absorption which scales with E

2

E0

2 38

. For a

thickness of d = 60 nm the corresponding minimum of zeroth

Verification of the interference effects with PMMA as a model analyte. Before applying the fabricated IER substrates for the investigation of biomolecules, we experimentally verified their functionality by utilizing PMMA as a model analyte first. The advantage of this polymer is that homogeneous layers with defined thicknesses can be easily deposited on the

Figure 4: Experimental (solid line) and calculated (dashed line) UV-Vis spectra of aluminum substrates with layers of Al2O3 with different thicknesses.

Figure 3: AFM 3D images of a) the plain aluminum surface and b) aluminum oxide layer of 112 nm thickness. order is at about 530 nm as can be seen both in the corresponding calculation using the optical constants of Al as well as in the experimental spectrum in Figure 4 (for the perfect electric conductor eqn. (1) predicts a thickness of 75 nm, the spectrum of this sample is also depicted in Figure 4). Using the zeroth order is in particular of advantage as the

E

2

2

E 0 curves show a broad peak and the Raman en-

hancement depends in a second approximation on E at the exciting wavelength and on E

2

E0

2

2

E0

2

at the wave-

9

length of the inelastically scattered photon. For the substrates with larger thickness the corresponding zeroth order minimum in the spectra is redshifted. In particular the samples with d = 135 nm and d = 150 nm show a maximum in the reflectance spectrum around 532 nm. Accordingly E

2

E0

2

should be

minimum in this spectral range, and, Raman scattering should effectively be suppressed if an excitation wavelength is chosen in this range. In the experimental spectra the spectra of the samples with d = 135 nm and d = 150 nm are interchanged in comparison with the calculated spectra. A probable reason for this swap could be errors in the optical constants of Al which were taken over from 39 or of those of Al2O3 35. Note that the changes in the reflectance for highly reflecting substrate materials like metals are too small to have a substantial effect on the visual appearance, while corresponding changes manifest itself by a different color for silicon substrates (cf. Figure S2 in the supporting information).

chip surface so that the interference effects can be studied unambiguously. To that end the substrates were modified with 30 nm PMMA via spin coating and Raman spectra were subsequently recorded. Figure 5 shows the Raman spectra of the PMMA films acquired on substrates with five different layer thicknesses of Al2O3. While the Raman signature of the PMMA coating on plain aluminum is barely visible (and allows a fair assessment of signal increase due to interference enhancement as in this case the exciting laser beam also passes twice through the layer and the forward scattered Raman signal is back reflected and collected by the microscope objective as well), distinct spectral features of the polymer are clearly observed for the thicknesses 60 and 75 nm Al2O3. In good agreement with the previously discussed theory (Figure 1 and Figure 2), the signal enhancement for the 60 nm layer of Al2O3 even exceeds the one for the 75 nm substrate as predicted by the calculations of the electric field enhancement taking into account by their optical constants that real metals are no perfect reflectors. The Raman spectrum acquired on the substrate featuring a 112 nm layer of Al2O3 only shows a weak signal in the C-H stretching region between 3100 - 2800 cm-1, roughly comparable to that on the bare Al. Overall we find a signal enhancement of about 10 as predicted. Compared to a transparent substrate the enhancement might even be higher due to the higher interaction length of the beam and the reflection of forward Raman-scattered photons (in practice, however, the signal enhancement from strongly reflecting substrates might be depending on the spectral region, since the situation is more complex as just depicted.40 The peak visible in all spectra at 1550 cm-1 arises from ambient O2. Furthermore, using Al2O3 layer thicknesses of 135 and 150 nm the Raman signal can be effectively suppressed. Such substrates offer the interesting prospect to selectively eliminate any unwanted background signal from e.g. capture molecules, even if they show strong Raman signatures.



Proof of concept for ferrioxamine B. Regarding the chip based isolation of microorganisms the choice of capture probes for the proposed application is of great importance. Antibodies are a well-established and frequently applied option. However, when the sample is supposed to be screened for several pathogens at once, a whole array of numerous different

with the one recorded on the plain aluminum a slight enhancement in the signal intensity can be observed. For the 150 nm Al2O3 layer the Raman intensity is weakened in relation to the unmodified surface (see Figure 6).

Figure 6: Raman spectra of 100 µM ferrioxamine B recorded on aluminum substrates with different layer thicknesses of Al2O3. Figure 5: Raman spectra of PMMA recorded on aluminum substrates with different layer thicknesses of Al2O3.

species specific antibodies would be required to do so. As the identification of the species can be easily achieved using Raman microspectroscopy after the chip based isolation, applying species specific capture probes is unnecessary. Recently, the potential of siderophores as capture probes for bacteria has been investigated.2,17,28,29 Ferrioxamine B as well as pyoverdine were found to be promising candidates for the enrichment of bacterial cells on surfaces. For investigating the interference effects on the Raman spectrum of ferrioxamine B, the chip substrates with Al2O3 layers of 75, 112 and 150 nm were modified with a 100 µM solution of the siderophore iron complex and subsequently Raman spectra using an excitation wavelength of 532 nm were recorded. For comparison also a chip substrate without Al2O3 layer was included. As it can be seen in Figure 6 the Raman intensity clearly depends on the thickness of the Al2O3 layer. While on the plain aluminum chip surface only two distinct Raman bands at 579 and 1569 cm-1 can be observed, the fingerprint region of the ferrioxamine B measured on the 75 nm Al2O3 coated substrate is significantly more pronounced. The strong band at 579 cm-1 can be assigned to the Fe-O stretching vibration. This band is absent of course for the desferrioxamine B compound. In Figure S3 of the supporting information a comparison of the Raman spectra as well as the structural formulas of both ferrioxamine B and desferrioxamine B are depicted. The Raman bands at 993 and 1052 cm-1 can be assigned to the symmetric C-N-C and the C-O stretching vibrations. The prominent band at 1569 cm-1 arises from C=N stretching vibrations.41 Comparing the ferrioxamine B Raman spectrum measured on the 112 nm thick Al2O3 layer

Further spectra of lower analyte concentrations in the range between 0 and 100 µM were recorded on the different Al2O3 layer thicknesses in order to compare the signal intensities for each type of substrate. The observed interference effects (see Figure 7), namely strong signal enhancement for the 75 nm layer, slight increase for the 112 nm substrate and a decrease in intensity for the 150 nm layer are consistent with the expectations as well as the previously discussed results from Figure 6. For better visualization the spectra acquired on the different substrates are displayed in Figure S4 of the supporting information ordered by layer thickness. For the aluminum substrate without Al2O3 layer a minimum concentration of 10 µM ferrioxamine B is necessary in order to recognize weak spectral features of the analyte. On the substrates with 75 nm Al2O3 at a concentration of 1 µM ferrioxamine B the typical fingerprint region is still visible. This result is in good agreement with the predicted electromagnetic enhancement of the signal of about one order of magnitude. Additionally, we plotted the areas of the peak with its maximum at 1579 cm-1 against the analyte concentration for the different substrate types (see Figure S5). While the peak areas generally increase with higher concentrations, quantification is difficult, because no linear correlation can be observed. Conducting interference enhanced Raman measurements for analytes such as ferrioxamine B under well-defined conditions is not easy to achieve. In order to actually observe the interference effects the layer of the analyte has to be very thin (approximately 100 nm or thinner). During drop drying typically a coffee ring effect (see Figure S6) occurs, so that the outer border of the spot is much thicker than the layer in the center of the spot. The previously discussed results (Figure 6 and


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Analytical Chemistry Figure 7) have been achieved by measuring in the center of the spot exclusively. Spectra of the outer region of the spots have

been recorded as well. The corresponding results can be found in Figure S7 of the supporting information. Accordingly,

Figure 7: Raman spectra of ferrioxamine B recorded on aluminum substrates with different layer thicknesses of Al2O3: a) 0 nm, b) 150 nm c) 112.5 nm and d) 75 nm. strategies have to be found to generate thin layers with homogenous thicknesses, if those are not automatically formed by chemically binding of the analytes to the substrate surface.

ASSOCIATED CONTENT

CONCLUSIONS

Supporting Information

To the best of our knowledge, we have demonstrated for the first time how IERS can be applied for the detection of biomolecules. The results indicate that IERS might be a highly promising tool for quality control in the development of chip based Raman compatible sample preparation strategies. Using the iron loaded siderophore ferrioxamine B and the polymer PMMA as model analytes, we could proof the enhancement as well as the decrease of the Raman signal due to interference effects occurring on aluminum substrates coated with aluminum oxide layers of specific thicknesses. Overall, our experimental results of Raman signal enhancement of about one order of magnitude agree well with the theoretically predicted electromagnetic enhancement. While the constructive interference effects obviously can be employed for verifying a successful surface modification with biomolecules, the destructive interference effects are of interest as well in the context of Raman microspectroscopic investigations of bacteria. For example, they enable using previously not applicable surface modifications of the chip substrates, the signals of which would have interfered with the spectra of the investigated bacterial cells. Now it has been demonstrated that a simple layer of aluminum oxide can suppress the Raman signal of polymers, which so far were not a suitable option as surface modification in Raman compatible chip based sample preparation strategies. We foresee that this will allow a new degree of freedom in surface modification towards full biocompatibility of Raman substrates for numerous applications.

The Supporting Information is available free of charge on the ACS Publications website. The following information is included: SEM characterization of the aluminium surface and Al2O3 layers, photographic images of the chip substrates, Raman spectra of ferrioxamine B and desferrioxamine B, additional Raman spectra of ferrioxamine B recorded on different substrate types, peak areas plotted against the analyte concentration, microscopic image of drop dried ferrioxamine B. (PDF)

AUTHOR INFORMATION Corresponding Author *corresponding author: [email protected], phone: +49 3641 948390

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT We would like to thank Christa Schmidt for performing FE-SEM, AFM and XRR measurements. Furthermore we thank David Zopf for conducting the UV-Vis measurements. Funding of the research projects “Intersept” (13N13852), “EXASENS” (13N13856) and “InfectoGnostics” (13GW0096F)



by the Federal Ministry of Education and Research, Germany is gratefully acknowledged.

REFERENCES (1) Kranert, C.; Sturm, C.; Schmidt-Grund, R.; Grundmann, M. Phys. Rev. Lett. 2016, 116, 127401. (2) Pahlow, S.; Stöckel, S.; Pollok, S.; Cialla-May, D.; Rösch, P.; Weber, K.; Popp, J. Anal. Chem. 2016, 88, 1570-1577. (3) Jahn, M.; Patze, S.; Hidi, I. J.; Knipper, R.; Radu, A. I.; Muhlig, A.; Yuksel, S.; Peksa, V.; Weber, K.; Mayerhofer, T.; Cialla-May, D.; Popp, J. Analyst 2016, 141, 756-793. (4) Moskovits, M. Physical chemistry chemical physics : PCCP 2013, 15, 5301-5311. (5) Jensen, L.; Aikens, C. M.; Schatz, G. C. Chemical Society reviews 2008, 37, 1061-1073. (6) Cialla-May, D.; Zheng, X. S.; Weber, K.; Popp, J. Chemical Society reviews 2017, 46, 3945-3961. (7) Jahn, I. J.; Zukovskaja, O.; Zheng, X. S.; Weber, K.; Bocklitz, T. W.; Cialla-May, D.; Popp, J. The Analyst 2017, 142, 1022-1047. (8) Zheng, X. S.; Jahn, I. J.; Weber, K.; Cialla-May, D.; Popp, J. Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy 2018, 197, 56-77. (9) Solonenko, D.; Gordan, O. D.; Milekhin, A.; Panholzer, M.; Hingerl, K.; Zahn, D. R. T. Journal of Physics D: Applied Physics 2016, 49, 115502. (10) Lee, Y.-C.; Wang, E.-Y.; Liu, Y.-L.; Chen, H.-L. Chem. Mater. 2015, 27, 876-884. (11) Connell, G. A. N.; Nemanich, R. J.; Tsai, C. C. Applied Physics Letters 1980, 36, 31-33. (12) Wang, Y. Y.; Ni, Z. H.; Shen, Z. X.; Wang, H. M.; Wu, Y. H. Applied Physics Letters 2008, 92, 043121. (13) Yoon, D.; Moon, H.; Son, Y.-W.; Choi, J. S.; Park, B. H.; Cha, Y. H.; Kim, Y. D.; Cheong, H. Physical Review B 2009, 80, 125422. (14) Dyakov, S. A.; Perova, T. S.; Miao, C. Q.; Xie, Y. H.; Cherevkov, S. A.; Baranov, A. V. J. Raman Spectrosc. 2013, 44, 803-809. (15) Zhang, H.; Wan, Y.; Ma, Y.; Wang, W.; Wang, Y.; Dai, L. Applied Physics Letters 2015, 107, 101904. (16) Chu, B. C.; Garcia-Herrero, A.; Johanson, T. H.; Krewulak, K. D.; Lau, C. K.; Peacock, R. S.; Slavinskaya, Z.; Vogel, H. J. Biometals 2010, 23, 601-611. (17) Kim, Y.; Lyvers, D. P.; Wei, A.; Reifenberger, R. G.; Low, P. S. Lab on a Chip 2012, 12, 971-976. (18) Silge, A.; Schumacher, W.; Rösch, P.; Da Costa Filho, P. A.; Gérard, C.; Popp, J. Syst. Appl. Microbiol. 2014, 37, 360-367. (19) Lorenz, B.; Wichmann, C.; Stöckel, S.; Rösch, P.; Popp, J. Trends Microbiol. 2017, 25, 413-424. (20) Kloß, S.; Kampe, B.; Sachse, S.; Rösch, P.; Straube, E.; Pfister, W.; Kiehntopf, M.; Popp, J. Anal. Chem. 2013, 85, 9610-9616. (21) Kloß, S.; Rösch, P.; Pfister, W.; Kiehntopf, M.; Popp, J. r. Anal. Chem. 2014, 87, 937-943. (22) Schwarz, M.; Kloß, S.; Stöckel, S.; Pollok, S.; Holländer, A.; CiallaMay, D.; Weber, K.; Popp, J. Anal. Bioanal. Chem. 2017, 409, 3779-3788. (23) Pahlow, S.; Meisel, S.; Cialla-May, D.; Weber, K.; Rösch, P.; Popp, J. Advanced Drug Delivery Reviews 2015, doi:10.1016/j.addr.2015.1004.1006. (24) Pahlow, S.; Kloss, S.; Blättel, V.; Kirsch, K.; Hübner, U.; Cialla, D.; Rösch, P.; Weber, K.; Popp, J. Chemphyschem 2013, 14, 3600-3605. (25) Stöckel, S.; Meisel, S.; Elschner, M.; Melzer, F.; Rösch, P.; Popp, J. Anal. Bioanal. Chem. 2015, 407, 787-794. (26) Meisel, S.; Stöckel, S.; Rösch, P.; Popp, J. Food Microbiol. 2014, 38, 36-43. (27) Stöckel, S.; Stanca, A. S.; Helbig, J.; Rösch, P.; Popp, J. Anal. Bioanal. Chem. 2015, 407, 8919-8923. (28) Doorneweerd, D. D.; Henne, W. A.; Reifenberger, R. G.; Low, P. S. Langmuir 2010, 26, 15424-15429. (29) Wolfenden, M. L.; Sakamuri, R. M.; Anderson, A. S.; Prasad, L.; Schmidt, J. G.; Mukundan, H. Advances in Biological Chemistry 2012, 2, 396-402. (30) Mühlig, A.; Bocklitz, T.; Labugger, I.; Dees, S.; Henk, S.; Richter, E.; Andres, S.; Merker, M.; Stöckel, S.; Weber, K.; Cialla-May, D.; Popp, J. Anal. Chem. 2016, 88, 7998-8004. (31) Yeh, P. Optical Waves in Layered Media; Wiley, 2005.

(32) Ohta, K.; Ishida, H. Applied Optics 1990, 29, 1952-1959. (33) Ohta, K.; Ishida, H. Applied Optics 1990, 29, 2466-2473. (34) Abelès, F. Ann. Phys. 1950, 12, 596-640. (35) Palik, E. D. Handbook of Optical Constants of Solids, Five-Volume Set: Handbook of Thermo-Optic Coefficients of Optical Materials with Applications; Elsevier Science, 1997. (36) Park, K. C. Appl. Opt. 1964, 3, 877-881. (37) Yoshida, S. Thin Solid Films 1979, 56, 321-329. (38) Mayerhöfer, T. G.; Popp, J. 2017. (39) Weber, M. J. Handbook of Optical Materials; Taylor & Francis, 2002. (40) Mikoliunaite, L.; Rodriguez, R. D.; Sheremet, E.; Kolchuzhin, V.; Mehner, J.; Ramanavicius, A.; Zahn, D. R. T. Sci. Rep. 2015, 5, 13150. (41) Cozar, O.; Leopold, N.; Jelic, C.; Chiş, V.; David, L.; Mocanu, A.; Tomoaia-Cotişel, M. J. Mol. Struct. 2006, 788, 1-6.


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