Article pubs.acs.org/ac
Preaggregated Ag Nanoparticles in Dry Swellable Gel Films for Offthe-Shelf Surface-Enhanced Raman Spectroscopy Wendy W. Y. Lee,† Victoria A. D. Silverson,† Colin P. McCoy,‡ Ryan F. Donnelly,‡ and Steven E. J. Bell*,† †
Innovative Molecular Materials Group, School of Chemistry and Chemical Engineering, Queen’s University Belfast, David Keir Building, Stranmillis Road, Belfast BT9 5AG, United Kingdom ‡ School of Pharmacy, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, United Kingdom S Supporting Information *
ABSTRACT: Large, thin (50 μm) dry polymer sheets containing numerous surface-enhanced Raman spectroscopy (SERS) active Ag nanoparticle aggregates have been prepared by drying aqueous mixtures of hydroxyethylcelloulose (HEC) and preaggregated Ag colloid in 10 × 10 cm molds. In these dry films, the particle aggregates are protected from the environment during storage and are easy to handle; for example, they can be cut to size with scissors. When in use, the highly swellable HEC polymer allowed the films to rapidly absorb aqueous analyte solutions while simultaneously releasing the Ag nanoparticle aggregates to interact with the analyte and generate large SERS signals. Either the films could be immersed in the analyte solution or 5 μL droplets were applied to the surface; in the latter method, the local swelling caused the active area to dome upward, but the swollen film remained physically robust and could be handled as required. Importantly, encapsulation and release did not significantly compromise the SERS performance of the colloid; the signals given by the swollen films were similar to the very high signals obtained from the parent citrate-reduced colloid and were an order of magnitude larger than a commercially available nanoparticle substrate. These “PolySERS” films retained 70% of their SERS activity after being stored for 1 year in air. The films were sufficiently homogeneous to give a standard deviation of 3.2% in the absolute signal levels obtained from a test analyte, primarily due to the films’ ability to suppress “coffee ring” drying marks, which meant that quantitative analysis without an internal standard was possible. The majority of the work used aqueous thiophenol as the test analyte; however, preliminary studies showed that the Poly-SERS films could also be used with nonaqueous solvents and for a range of other analytes including theophylline, a therapeutic drug, at a concentration as low as 1.0 × 10−5 mol dm−3 (1.8 mg/dm3), well below the sensitivity required for theophylline monitoring where the target range is 10−20 mg/dm3.
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purchased in advance and then stored until required.7−9 Indeed, the high unit cost of the substrates means that the approach is really only economically viable for users who require reasonably small numbers of measurements. In contrast, the colloids, which are commonly used in SERS analysis, are inexpensive to produce and also give the highest enhancements; however, they are inherently unstable systems which eventually aggregate and precipitate. One approach to this problem is to avoid storage of particles by preparing them shortly before use and to avoid the waste and effort that would be associated with making fresh batches of bulk colloid by preparing small amounts of enhancing materials either in microfluidic systems or by in situ reduction. For example, photoreduction of matrix-stabilized silver halides has been reported10 while chemical reduction using borohydride has been used to prepare agarose gels loaded
ince its discovery in the 1970s, surface-enhanced Raman spectroscopy (SERS) has developed into a powerful and accurate analytical tool capable of providing quantitative as well as qualitative data.1−3 However, SERS has still not been widely adopted within the mainstream analytical community; this is not due to the instrumental requirements that previously set a high barrier to adoption since conventional Raman measurements have genuinely now become routine and the same instruments are capable of carrying out SERS measurements, but instead, it can clearly be traced to the lack of appropriate enhancing media. Despite the numerous reports of novel enhancing media which continue to appear in the literature, the vast majority of researchers either prepare their own enhancing media or purchase solid substrates from one of the small number of commercial vendors.4−6 These solid substrates do not give the high signal levels associated with colloidal systems, but they do have the distinct advantage, particularly for users with an intermittent need for SERS measurements, that they can be © 2014 American Chemical Society
Received: March 21, 2014 Accepted: July 21, 2014 Published: July 21, 2014 8106
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hydroxyethylcellulose (HEC) 250 pharm HX was purchased from Ashland Inc. Tris(bipyridine)ruthenium(II) nitrate was the generous gift of Dr. N. C. Fletcher. The colloidal Ag nanoparticles were prepared by citrate reduction according to the Lee and Meisel method15 and had a λmax = 405 nm. Sufficient gel for a single 10 × 10 cm sheet could be prepared by mixing 54.2 mL of colloid with 10.8 mL of 0.1 mol dm−3 MgSO4 in a beaker for a few seconds before gradually adding 0.8 g of HEC powder (i.e., sufficient to give 1.2% w/v polymer) over 10 min to the preaggregated colloid as it was stirred at 400 rpm using a mechanical stirrer. The stirring was continued until a smooth, glossy, and thickened gel-like consistency was obtained, which typically took approximately 30 min. The resulting gel was then immediately poured into a 10 × 10 × 0.5 cm casting mold made with a Perspex bottom and removable sides held in place with wing nuts. The bottom surface of the mold was covered with silicone release sheet which was held in place using vacuum grease. The gel was then left to dry in an open space at room temperature (relative humidity ≤50%) for approximately 48 h or until fully dry, at which point the resulting film could be easily peeled away from the surface and cut into smaller sections, typically 5 × 5 mm, using normal scissors. The extinction spectrum of the films showed a broad extinction from 300 to 1000 nm (see Supporting Information, Figure S1), which was due to a combination of light scattering by the semiopaque film and absorption by the aggregated particles. When in use, the films were either rehydrated by applying a droplet of aqueous analyte (normally 5 μL) or fully immersed into bulk analyte solution. In both cases, the film would be placed onto a glass slide covered in aluminum foil before the probe laser from a PerkinElmer RamanMicro 200 Raman microscope using a 4× objective lens at 55% laser power was directed onto the film surface and a SERS spectrum was collected. The microscope uses a 785 nm cavity diode laser (80 mW) with a Czerny-Turner spectrometer with a spectral range of 95−3200 cm−1. It is composed of an Olympus microscope (BX51 reflected illumination frame) which is connected to a spectrometer through fiber-optic cables. The spot diameter is 230 μm. Thiophenol was initially dissolved in ethanol, and serial dilutions were carried out using 25% ethanol/water. Adenine, crystal violet dye, (s)-(−)-nicotine, melamine, potassium thiocyanate, and theophylline were dissolved and diluted with water only. For experiments with benzylmercaptan, the analyte was diluted in the required solvent to 1 × 10−3 mol dm−3 and was applied directly onto the films as a 10 μL droplet. Alternatively, in experiments where the films were preswelled, 20 μL of water was spread over a ca. 5 × 5 mm area of the film and allowed to partly dry before a 10 μL analyte solution droplet was applied.
with silver nanoparticles by a process in which the gel was formed, treated with silver nitrate, and subsequently reduced.11 However, these in situ methods generally tend to be complex and more importantly offer limited opportunities for controlling the size and shape of the particles formed. An alternative to generating the particles at point of use is to improve colloid stability. A promising approach is to prepare the enhancing particles and then incorporate them in a polymeric support. For example, gold-silica nanoshells have been incorporated within highly porous nitrocellulose membranes,12 but the disadvantage of this approach is that it leaves the particles exposed to the environment. Alternatively, we have previously trapped the active particles in swellable polycarbophil (PC) polymers, where they were fully protected from the environment. One format was to prepare thin polycarbophil (PC) coatings containing unaggregated colloid13 which could be stored dry but upon rehydration with aqueous analyte and aggregating agent would become SERS active. A significant disadvantage of these PC coatings was that they had to be prepared with unaggregated colloid since the performance decreased significantly when the colloids were aggregated before being isolated in the polymer. This was believed to be due to the polymers not fully releasing the colloid upon rehydration so that the original enhancement level was not restored when the aqueous analyte was applied to preaggregated coatings. Similarly, even when the coatings were aggregated during the swelling step, the enhancement was reduced compared to simple aqueous conditions. A partial solution to this problem was found in the observation that the nanoparticles within PC remained more accessible to analytes if the gels formed by mixing colloids with PC polymer were stored in the gel form, and indeed in this case, the particles could be preaggregated and still remain active.14 However, although the wet SERS gels were useful for routine laboratory analysis and had the advantage that they prolonged colloid storage lifetime up to one year, the fact that they were liquid meant that they were not much better than conventional colloids for on-site use because they had the same handling problems. The examples above are just a small sample drawn from the vast body of published research involving the stabilization of SERS nanoparticles within various organic and inorganic hosts. However, within this large body of research, apart from the examples above, there has been surprisingly little work on the use of swellable polymers for stabilizing colloids, possibly because it seems more obvious to use a porous host to allow analytes to penetrate than to depend on swelling to release the particles. However, here we describe a new method for preparing free-standing films which allows Ag nanoparticles to be stored and stabilized in the dry state but to become active when required by swelling with aqueous analyte. Importantly, these films overcome previous difficulties in that the particles are fully released on swelling and can even be preaggregated so that no postswelling aggregation step is necessary and simple aqueous (or nonaqueous) solutions can be applied directly to the films.
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RESULTS AND DISCUSSION The preparation method produces large flexible polymer film sheets (10 × 10 cm) which we term “Poly-SERS” films. These are the same color as aggregated silver colloid solution but have mechanical properties similar to common 80 gsm printer paper. They are sufficiently rigid to be free-standing, but sections can be cut to the desired size with scissors. Figure 1 shows a SEM image of a Poly-SERS film showing that its surface is smooth and flat16 and that it has a uniform thickness of approximately 50 μm, which can of course be adjusted by altering the amount of polymer used. This thickness was chosen because it gives
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EXPERIMENTAL SECTION Silver nitrate (99.9999%), magnesium sulfate, thiophenol (≥99%), adenine (99%), crystal violet dye (anhydrous, ≥90.0%), (s)-(−) nicotine (98%), melamine (99%), potassium thiocyanate (99%), benzylmercaptan (99%), and theophylline (≥99%) were purchased from Sigma-Aldrich. Natrosol 8107
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structure of the films is therefore very similar to that of a conventional aggregated aqueous colloid with aggregates of various sizes randomly distributed throughout the sample volume, except that in the dry film they are fixed in place by the rigid polymer host rather than floating free in solution. The average diameter of the aggregates was ca. 800 nm, but a broad range of diameters was observed; a plot showing the distribution of aggregate sizes is shown in Figure S2, Supporting Information. Manual counting showed this average diameter corresponded to approximately 13 Ag particles, fewer than would be in a close packed structure due to the loose arrangement of the particles in the aggregates. The main challenge with encapsulated particle films is the requirement that the host polymer does not interfere with the measurements; this means that it should allow the particles to be accessed when required and it should not itself give large interfering SERS signals. In this case, the blank spectra of the films are completely dominated by a strong Ag−Cl band at 241 cm−1 which arises from binding of chloride ions from the polymer to the surface of the citrate-reduced colloids.17 The HEC itself is such a poor scatterer that the SERS bands of the polymer are not detectable even in the blank spectra (Supporting Information, Figure S3). The Poly-SERS films have been tested using two different methods to bring the analyte solution into contact with the film. The first was simply immersing a section of film directly into the bulk analyte solutions; the second was by placing a droplet of aqueous analyte onto the surface of the film. The Raman spectra were then measured under both sets of experimental conditions in order to determine the rate at which the application of analyte solution caused swelling of the film and the associated contact between the analyte and particles which gives the SERS signal. In the first instance, individual sections of Poly-SERS film (5 × 5 mm) were fully immersed into 11 separate sample vials containing 5 mL, 1 × 10−4 mol dm−3 thiophenol solutions for times ranging from 2 to 240 min. The films were then removed from their corresponding sample vials and placed flat onto aluminum foil covered glass slides, and their SERS spectra were recorded. The absolute signal intensities are shown in the Supporting Information (Figure S4), but the result was not surprising; essentially, the signal was large right from the first accumulations (105 counts in 30 s), and although there was a gradual increase in SERS signal intensity with increasing time spent in the thiophenol solutions, the difference was less than a factor of 2. This suggests that, even though there was some further diffusion of the analyte molecules from the bulk solution into the SERS films after they had been initially swollen in the first 2 min, this postswelling diffusion process was not the main mechanism by which the analyte adsorbed onto the particles. The next experiments involved applying sample droplets onto the surface of the Poly-SERS films. Figure 3b shows a section of Poly-SERS film which was rehydrated with a 5 μL water droplet applied to the upper surface and allowed to swell for approximately 10 min before the sample was sectioned and photographed using an optical stereomicroscope. In this case, the droplet retained its initial diameter during the swelling process with the result that the area of the film under the droplet swelled and domed upward toward the droplet. The swelled region was notably more elastic than the dry film but still retained its physical integrity. On standing in the open air, the aqueous solvent evaporated, reversing the effect, until the
Figure 1. SEM image showing a face/edge view of a Poly-SERS film at 470× magnification. The inset is a photograph showing a ca. 1 cm2 section of a Poly-SERS film cut from a standard 10 × 10 cm sheet.
sufficient mechanical strength to allow easy handling but is still thin enough to swell rapidly. Critically, using an appropriate concentration of salt in the preaggregation step before adding polymer gives clusters of particles which are retained in the dried film, as shown in the SEM image Figure 2a. The range of salt concentrations which
Figure 2. SEM image showing a face on view of the individual Ag aggregates held within a Poly-SERS film. Image (a) was recorded at 12 000× magnification. Image (b) shows an exposed Ag nanoparticle cluster held within a film at 52 000×.
give acceptable results is quite large; thus, this does not cause any problems during preparation, but outside the range, either the particles show insufficient aggregation at low salt concentration (≤1.25 × 10−3 mol dm−3) or the films break up during drying at high salt concentrations (≥2.5 × 10−2 mol dm−3). The SEM image shows that the aggregates are distributed approximately evenly across the film, and those which lie deeper within the polymer are increasingly out of focus. This is not due to limited depth of focus of the SEM, although it gives a result which is strikingly similar to what would be expected in that case, but instead, it is due to scattering of the electron beam by the polymer host, with deeper clusters suffering more scattering. Figure 2b demonstrates that this is the case because it shows an image of an adventitious crack in a Poly-SERS film which has exposed a section of a nanoparticle cluster which sits deep within the film. In the image, the exposed particles are in sharp focus while those of the same cluster which were imaged through the polymer at each side of the crack are quite blurred. The overall 8108
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Figure 3. SERS spectra of 15 μL droplets of 1 × 10−4 mol dm−3 thiophenol obtained from 2 to 240 min after the initial deposition of the analyte droplet onto the surface of the SERS film. The spectra are on the same vertical axis, apart from being offset for clarity. Inset (a) is a graph comparing the changes in the absolute SERS signal intensities against the change in mass of an evaporating droplet of the same composition over time. Inset (b) shows a side view of a section of a Poly-SERS film which was rehydrated with a 5 μL water droplet for 10 min before being cut and photographed.
film returned to its original shape and thickness. The effect of this swelling on the SERS signals from the films was tested by depositing 15 μL droplets of 1 × 10−4 mol dm−3 aqueous thiophenol onto their surfaces and recording their SERS spectra as a function of time. The data, plotted in Figure 3, show that strong thiophenol bands were already present in the first spectrum recorded (accumulation finished 2 min after droplet application). The intensity of these bands then increased over time, eventually reaching a plateau at approximately 60 min, after which the signal remained stable. The initial signal was clearly due to the primary swelling process, but the subsequent slow increase in signal could arise from either diffusion of any unadsorbed analyte into the film and onto the surface of the aggregates or increased concentration due to evaporation of the aqueous solvent. The experiments with submerged films showed that diffusion into the films following the initial swelling had only a small effect on the signal while in this case the signal level at the plateau was approximately 5× of that found after the films were first swollen (Supporting Information, Figure S4) which seems too large for secondary diffusion to be the main cause. In contrast, the plot in Figure 3, which compares the absolute SERS intensities of thiophenol and the change in mass of the evaporating droplet with time, shows that the rate of signal increase and mass decrease were very similar and that the maximum SERS signals were obtained when the solvent had fully evaporated. This suggests that as the analyte molecules became increasingly concentrated with evaporation of the droplet solvent they were forced into the films to interact with the nanoparticles. In this case, the films acted more like other conventional solid substrates where a
small droplet is typically applied and allowed to evaporate before the signal is recorded. With droplet drying, the possibility of coffee ring effects, in which solute is preferential deposited in a ring at the edge of the droplet, needs to be considered. The effect occurs due to pinning of the droplet combined with capillary flow which causes the solution to move to the edge of the drop, increasing the local concentration. While the coffee ring effect can be useful in some circumstances, such as separating components within complex biological samples, in this context it is undesirable because if a coffee ring forms the concentration detected during SERS analysis depends on where the laser beam is directed on the dried droplet. However, with the PolySERS films, the coffee ring effect is suppressed compared to simple supports. Figure 4 shows an experiment where 10 μL droplets of 1 × 10−3 mol dm−3 Rose Bengal dye were deposited onto either a simple glass slide or a HEC only film and allowed to dry. Not surprisingly, a significant coffee ring effect occurred with the glass slide; however, with the HEC film, the dyed area was much more uniform. This may be due to HEC disrupting the normal capillary flow of drying droplets and giving something more comparable to Marangoni circulation.18 This type of circulation is able to homogenize the drying of analyte droplets so that their deposition onto the surface is more uniform. The coffee ring suppression was studied further by depositing a 5 μL, 1 × 10−4 mol dm−3 thiophenol droplet onto a PolySERS film and recording data at various points across the diameter of the dried analyte droplet. As shown in Figure 4, the intensities show no increased intensity at the edges and, across 8109
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whole field of view in Figure 2 and therefore that the SERS signal arises from more than 100 clusters of various sizes. It is therefore more likely that the observed point-to-point variation is due to differences in the average particle concentration occurring over length scales >50 μm rather than the local variations seen in the SEM images. To determine the extent to which the absolute signal levels from the enhancing films can be used for quantitative analysis, the SERS signals from 5 μL thiophenol droplets at various concentrations were recorded, as shown in Figure 5a. The data show the expected rise in signal with increasing concentration up to a plateau region. Figure 5a includes a Langmuir type fit of the data which shows reasonable agreement with the experiment but plateaus early and does not follow the gradual rise of the data points even at high concentration, which we have found empirically to fit to a simple logarithmic curve, as shown in Figure 5a and as the semilog plot in Figure 5b. The data show a strong correlation between the concentration of the analyte and the absolute SERS signals and suggest that the reproducibility of the method is sufficient for at least semiquantitative analysis using absolute signal intensities. Of course, this calibration could be improved using an internal standard if required and by averaging over a larger number of points (as described above), but for current purposes, the intention is to demonstrate the level of quantification that can be achieved using the simplest possible approach of measuring absolute signal intensity at a single point. The absolute signal levels provided by enhancing materials with different physical characteristics are difficult to compare in absolute terms due to difficulties in finding meaningful figures of merit. For this reason, we have carried out simple side-byside experiments to compare the Poly-SERS films with conventional silver colloids, whose performance most researchers will be familiar with, and a Q-SERS substrate, which is a commercially available material based on layers of 15 and 60 nm Au particles on a solid support.5 In this test, 5 μL portions of 1 × 10−4 mol dm−3 thiophenol solution (aqueous with a small concentration of ethanol to aid solvation) were used. In one case, the droplet was preconcentrated onto the Poly-SERS film and its spectrum was recorded using a 4× objective lens (this objective was used because some sample damage was observed with the 20× objective). This was compared to a droplet dried onto a Q-SERS surface whose spectra were
Figure 4. Absolute SERS signal intensities of a 5 μL, 1 × 10−4 mol dm−3 thiophenol droplet preconcentrated on a Poly-SERS film obtained at various horizontal sample stage positions (0.3 mm increments). The inset shows a photograph of 15 μL, 1 × 10−3 mol dm−3 Rose Bengal preconcentrated onto (a) a glass slide and (b) HEC film.
the spot, the intensities show random fluctuations which are due to point-to-point variation in the film. The point-to-point variation in Figure 4 suggested that the homogeneity of the Poly-SERS film should be tested. Five μL droplets of 1 × 10−4 mol dm−3 thiophenol were deposited at 15 random points over one film, and the absolute SERS signal intensities of thiophenol within each spot after drying was measured. Using an average of three spectra for each point gave a relative standard deviation in the absolute SERS signal intensities of 3.2%. The origin of this variation was presumably that some parts of the film may have been more highly doped with the Ag nanoparticles than others, and averaging the signal from 3 points, although it reduced the variation, was not sufficient to completely eliminate it. The SEM image in Figure 2 shows that the aggregates are well spread throughout the films, but they are not tightly packed together so there are still areas up to 5 μm across which have no aggregates present. This could be a difficulty if the film was probed using a microscope with a diffraction limited spot since there would be a significant chance of either completely missing a cluster or alternatively focusing on a large cluster and obtaining an anomalously high signal. However, this local variation should not be the origin of the variations observed in Figure 4 since the laser spot diameter used in these measurements was ca. 230 μm, which means that the area sampled in the SERS experiments is larger than the
Figure 5. (a) A calibration curve showing the absolute SERS signal intensities of the strong 1572 cm−1 band of thiophenol at various concentrations using a Poly-SERS film. The data have been fitted to a Langmuir isotherm (dashed line) and a logarithmic increase (solid line). (b) A semilog plot of the same data. 8110
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at an extremely low cost (≪ $1) since the films are cast in 10 × 10 cm molds, and therefore, even a single sheet can provide 400 individual 5 × 5 mm sections. Finally, an important parameter is the long-term stability of the substrates. The SERS activity of a representative film was tested using 5 μL of 1 × 10−4 mol dm−3 thiophenol over a 12 month period, taking an average of three spectra from different parts of the film for each time point. It was found that the films retained 78% of the original absolute signal intensity after storing for 6 months and 70% of the original value after storing for 12 months. The most likely cause of aging of the Poly-SERS films is oxidation of the nanoparticles since the films were not stored under inert conditions; they were simply kept in the dark, under atmospheric air, and at laboratory temperature. It would therefore be expected that storage under sealed anaerobic conditions (similarly to those used with commercially available solid substrates) would dramatically increase the lifetime. All the above measurements used thiophenol as the analyte which is widely used as a test material because it gives large and therefore easy to measure SERS signals and binds strongly to Ag and Au surfaces. However, with any new substrate, it is important to test it using a more challenging analyte; in this case, theophylline was chosen, and in addition, the measurements were carried out using films that had been stored for >6 months before use. Theophylline is a therapeutic drug which is used in the treatment of asthma, chronic obstructive pulmonary disease, bronchitis, and heart failure.19 Rapid monitoring of theophylline is important because it has a narrow therapeutic window which sets the target range of detection at 10−20 mg/ dm3 (5.6 × 10−5−1.1 × 10−4 mol dm−3).20 15 μL drops of 1 × 10−3, 1 × 10−4, and 1 × 10−5 mol dm−3 theophylline were tested with the Poly-SERS films and, as shown in Figure 7, the theophylline could easily be detected at 1 × 10−5 mol dm−3 (1.8 mg/dm3), well below the limit required for therapeutic drug monitoring. Of course, this result was obtained in aqueous
recorded using a 20× objective lens; this lens gives a higher signal than the 4× objective used for the Poly-SERS measurements so both systems were compared under their best conditions. The signal from conventional aqueous colloid was recorded by mixing 100 μL of 1 × 10−4 mol dm−3 thiophenol with 100 μL of CRSC and aggregating with 50 μL of 0.1 mol dm−3 MgSO4. The results are shown in Figure 6.
Figure 6. SERS spectra of 5 μL of 1 × 10−4 mol dm−3 thiophenol obtained with (a) a Poly-SERS film, (b) Q-SERS, and (c) normal aggregated citrate-reduced silver colloid solution. The spectra are on the same vertical axis, apart from being offset for clarity.
For 30 s accumulations, the intensity of the thiophenol 1572 cm−1 band was ca. 110 000 counts with the Poly-SERS films, ca. 8000 counts with Q-SERS, and ca. 85 000 counts using aqueous CRSC and MgSO4 aggregating agent. These results therefore demonstrate that the Poly-SERS films are a considerably more sensitive SERS substrate than Q-SERS. Indeed, the signals from the Poly-SERS films were also slightly larger than those obtained with aqueous silver colloids, which are known to be among the best of all SERS enhancing materials, despite numerous attempts to better them.1 This was presumably due to a combination of the much larger number of SERS active nanoparticles in the probed volume, and the preconcentration was due to solvent evaporation. The Poly-SERS films were only 50−250 μm thick, depending on the stage of swelling; however, they contained the same number of particles as the 5 mm depth of gel which was initially used to prepare them, and in the swollen films, all these particles lay within the depth of focus of the low confocality Raman spectrometer used in this work. In comparison, with the aqueous colloid, a 5 mm deep sample will contain the same number of particles as the film, however, many of these particles will lie outside the focal depth of the system and therefore not contribute to the signal. This ability of the films to keep more particles in the focal volume combined with the preconcentration effect clearly more than compensates for any reduction in accessability of the analyte to the surface that polymer encapsulation causes. Indeed, even without any solvent evaporation, the films give signals that are comparable to those of aqueous colloids, at ca. 1/3 the intensity, which is not an important difference in this context. We note that 5 × 5 mm squares of Poly-SERS film are easily large enough for a single measurement so they can be prepared
Figure 7. SERS spectra of 15 μL of (a) 1 × 10−3, (b) 1 × 10−4, and (c) 1 × 10−5 mol dm−3 theophylline and (d) water only obtained with a Poly-SERS film. Inset is the molecular structure of theophylline. All spectra are shown on the same vertical axis, apart from being offset for clarity. 8111
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Figure 8. SERS spectra obtained with a Poly-SERS film of (a) 1 × 10−4 mol dm−3 adenine, (b) 1 × 10−5 mol dm−3 crystal violet, (c) 5 × 10−3 mol dm−3 potassium thiocyanate, (d) 1 × 10−3 mol dm−3 melamine, (e) 1 × 10−4 mol dm−3 nicotine, and (f) 1 × 10−6 mol dm−3 tris(bipyridine)ruthenium(II) nitrate. The concentrations of the analytes were adjusted to give approximately the same absolute signals, and in this figure, all spectra are shown on the same vertical axis, apart from being offset for clarity.
solution rather than body fluid, but nonetheless, it clearly demonstrates that the films are effective for more challenging analytes than alkyl/aryl thiols which are commonly used to demonstrate SERS enhancements in novel substrates. Another important feature is that the films should be broadly applicable with a range of analytes. Figure 8 shows the high signal-to-noise SERS spectra which were obtained when 5 μL of various aqueous analytes, including dye, drug, and nucleobase, were tested with Poly-SERS films (which had again been stored in air for six months) by directly depositing droplets onto the surface. The samples were chosen to span a broad range of organic/inorganic/organometallic analytes with different sizes and charges. The ability of the Poly-SERS films to detect such
analytes and to do this after prolonged storage clearly demonstrates that the films have considerable potential for application in multiple fields. Finally, the films are not confined to aqueous media, a range of solvents has been tested, initially by simply depositing droplets of analyte on the surface in the usual way. Benzylmercaptan was chosen as the target compound due to its solubility in a wide range of solvents. Figure 9a shows unscaled spectra obtained using solvents ranging from acetone to hexane where it is clear that the solvents that would be expected to swell the films, such as acetone and methanol, give good spectra while hexane, which would not be expected to swell the film, gives no SERS signal from the analyte. More 8112
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Fax: +44 2890 976534. Tel: +44 2890 974470. E-mail: s.bell@ qub.ac.uk. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Support for this work by the EPRSC (grant number EP/ H021647/1) is acknowledged. Figure 9. SERS spectra obtained for 1 × 10 −3 mol dm −3 benzylmercaptan dissolved in the solvents marked using a PolySERS film. (a) Sample applied directly to the film. (b) Film preswelled with 20 μL of water before addition of solution. All spectra in (a) or (b) are shown on the same vertical axis, apart from being offset for clarity.
REFERENCES
(1) Bell, S. E. J.; Sirimuthu, N. M. S. Chem. Soc. Rev. 2008, 37, 1012− 1024. (2) Schlücker, S. In Surface Enhanced Raman Spectroscopy: Analytical, Biophysical and Life Science Applications; John Wiley & Sons: Hoboken, NJ, 2013. (3) Kneipp, K.; Moskovits, M.; Kneipp, H. In Surface-Enhanced Raman Scattering: Physics and Applications; Springer: New York, 2006. (4) http://www.renishawdiagnostics.com/en/klarite-sers-substrates-17076 (accessed March 20, 2014). (5) http://www.q-sers.com/ (accessed March 20, 2014). (6) http://www.rta.biz/Content/SERS_Vials.asp (accessed March 20, 2014). (7) Hankus, M. E.; Holthoff, E. L.; Stratis-Cullum, D. N.; Pellegrino, P. M. Proc. SPIE 8018, Chemical, Biological, Radiological, Nuclear, and Explosives (CBRNE) Sensing XII, 80180P, 2011, DOI:10.1117/ 12.886779. (8) Almaviva, S.; Botti, S.; Cantarini, L.; Fantoni, R.; Lecci, S.; Palucci, A.; Puiu, A.; Rufoloni, A. J. Raman Spectrosc. 2014, 45, 41−46. (9) Lee, V.; Farquharson, S. In Photonic Detection and Intervention Technologies for Safe Food (SPIE Proceedings); SPIE: Bellingham, WA, 2001; Vol. 4206, pp 140−146. (10) Sagmuller, B.; Schwarze, B.; Brehm, G.; Schneider, S. Analyst 2001, 126, 2066−2071. (11) Keating, M.; Chen, Y.; Larmour, I. A.; Faulds, K.; Graham, D. Meas. Sci. Technol. 2012, 23, 1−9. (12) Bishnoi, S. W.; Swarup, V.; Lin, Y.; Tibudan, M.; Huang, Y.; Nakaema, M.; Keiderling, T. A. Anal. Chem. 2011, 83, 4053−4060. (13) Bell, S. E. J.; Spence, S. J. Analyst 2001, 126, 1−3. (14) Bell, S. E. J.; Sirimuthu, N. M. S. Analyst 2004, 129, 1032−1036. (15) Lee, P. C.; Meisel, D. J. Phys. Chem. 1982, 86, 3391−3395. (16) The reverse face (upper side during drying) has some cosmetic defects which are left by residual air bubbles that rise to the surface during drying and leave patches with a cracked appearance when they burst. However, these are surface features which do not affect the distribution of the particles and which disappear when the films are swollen in any case. (17) Bell, S. E. J.; Sirimuthu, N. M. S. J. Phys. Chem. A 2005, 109, 7405−7410. (18) Hu, H.; Larson, R. G. J. Phys. Chem. B 2006, 110, 7090−7094. (19) http://www.nhs.uk/Conditions/Asthma/Pages/ MedicineOverview.aspx?condition=Bronchitis&medicine= Theophylline (accessed March 20, 2014). (20) http://www.nlm.nih.gov/medlineplus/ency/article/003430.htm (accessed March 20, 2014).
interestingly, it was found that, if the films were initially swollen by applying water and, subsequently (2 min), a droplet of the test solution was added, the SERS signal of the analyte could be recorded. Indeed, the intensities of signals recorded using this method for both polar and nonpolar solvents were all within a factor of 2× of each other.
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CONCLUSION The Poly-SERS films developed in this work are highly sensitive and have the potential to transform SERS into a convenient, inexpensive, and reproducible routine analytical technique for a wide range of analytes. The use of a swellable polymer allows the particles to be protected in storage but released when required. Numerous polymers, swellable or nonswellable, could be used for encapsulation; the difficulty is in finding a polymer which when treated with analyte solution releases the particles so they are free to interact with the target compounds. Here, HEC was found to have all the required characteristics of a host material, in that it could be formed into thin films which contained high concentrations of Ag nanoparticle aggregates of the type known to be highly SERS active in solution. These films were easy to cut to the appropriate size and either could be dipped into test solutions or have the solutions applied as small droplets to their surface which caused the films to swell, drawing the solution into the films and allowing the analyte to come into contact with the released particles. The swollen films could be either probed immediately or allowed to dry, which gave a further increase in signal intensity. The drying did not give a noticeable coffee ring effect after evaporation. The absolute signal heights given by the Poly-SERS films were comparable to those given by the parent colloid in normal solution, which are known to be extremely high and were more than an order of magnitude larger than those given by a commercial SERS substrate based on Au particles. These films gave signals 70% of their original value even after being stored in air for 12 months, so they have the potential for genuine offthe-shelf use either for occasional users or for routine analysis, where the low cost and scalability of the preparation method is just as important as the storage lifetime. 8113
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Preaggregated Ag Nanoparticles in Dry Swellable Gel Films for Offthe-Shelf Surface-Enhanced Raman Spectroscopy Wendy W. Y. Lee,† Victoria A. D. Silverson,† Colin P. McCoy,‡ Ryan F. Donnelly,‡ and Steven E. J. Bell*,† †
Innovative Molecular Materials Group, School of Chemistry and Chemical Engineering, Queen’s University Belfast, David Keir Building, Stranmillis Road, Belfast BT9 5AG, United Kingdom ‡ School of Pharmacy, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, United Kingdom S Supporting Information *
ABSTRACT: Large, thin (50 μm) dry polymer sheets containing numerous surface-enhanced Raman spectroscopy (SERS) active Ag nanoparticle aggregates have been prepared by drying aqueous mixtures of hydroxyethylcelloulose (HEC) and preaggregated Ag colloid in 10 × 10 cm molds. In these dry films, the particle aggregates are protected from the environment during storage and are easy to handle; for example, they can be cut to size with scissors. When in use, the highly swellable HEC polymer allowed the films to rapidly absorb aqueous analyte solutions while simultaneously releasing the Ag nanoparticle aggregates to interact with the analyte and generate large SERS signals. Either the films could be immersed in the analyte solution or 5 μL droplets were applied to the surface; in the latter method, the local swelling caused the active area to dome upward, but the swollen film remained physically robust and could be handled as required. Importantly, encapsulation and release did not significantly compromise the SERS performance of the colloid; the signals given by the swollen films were similar to the very high signals obtained from the parent citrate-reduced colloid and were an order of magnitude larger than a commercially available nanoparticle substrate. These “PolySERS” films retained 70% of their SERS activity after being stored for 1 year in air. The films were sufficiently homogeneous to give a standard deviation of 3.2% in the absolute signal levels obtained from a test analyte, primarily due to the films’ ability to suppress “coffee ring” drying marks, which meant that quantitative analysis without an internal standard was possible. The majority of the work used aqueous thiophenol as the test analyte; however, preliminary studies showed that the Poly-SERS films could also be used with nonaqueous solvents and for a range of other analytes including theophylline, a therapeutic drug, at a concentration as low as 1.0 × 10−5 mol dm−3 (1.8 mg/dm3), well below the sensitivity required for theophylline monitoring where the target range is 10−20 mg/dm3.
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purchased in advance and then stored until required.7−9 Indeed, the high unit cost of the substrates means that the approach is really only economically viable for users who require reasonably small numbers of measurements. In contrast, the colloids, which are commonly used in SERS analysis, are inexpensive to produce and also give the highest enhancements; however, they are inherently unstable systems which eventually aggregate and precipitate. One approach to this problem is to avoid storage of particles by preparing them shortly before use and to avoid the waste and effort that would be associated with making fresh batches of bulk colloid by preparing small amounts of enhancing materials either in microfluidic systems or by in situ reduction. For example, photoreduction of matrix-stabilized silver halides has been reported10 while chemical reduction using borohydride has been used to prepare agarose gels loaded
ince its discovery in the 1970s, surface-enhanced Raman spectroscopy (SERS) has developed into a powerful and accurate analytical tool capable of providing quantitative as well as qualitative data.1−3 However, SERS has still not been widely adopted within the mainstream analytical community; this is not due to the instrumental requirements that previously set a high barrier to adoption since conventional Raman measurements have genuinely now become routine and the same instruments are capable of carrying out SERS measurements, but instead, it can clearly be traced to the lack of appropriate enhancing media. Despite the numerous reports of novel enhancing media which continue to appear in the literature, the vast majority of researchers either prepare their own enhancing media or purchase solid substrates from one of the small number of commercial vendors.4−6 These solid substrates do not give the high signal levels associated with colloidal systems, but they do have the distinct advantage, particularly for users with an intermittent need for SERS measurements, that they can be © 2014 American Chemical Society
Received: March 21, 2014 Accepted: July 21, 2014 Published: July 21, 2014 8106
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hydroxyethylcellulose (HEC) 250 pharm HX was purchased from Ashland Inc. Tris(bipyridine)ruthenium(II) nitrate was the generous gift of Dr. N. C. Fletcher. The colloidal Ag nanoparticles were prepared by citrate reduction according to the Lee and Meisel method15 and had a λmax = 405 nm. Sufficient gel for a single 10 × 10 cm sheet could be prepared by mixing 54.2 mL of colloid with 10.8 mL of 0.1 mol dm−3 MgSO4 in a beaker for a few seconds before gradually adding 0.8 g of HEC powder (i.e., sufficient to give 1.2% w/v polymer) over 10 min to the preaggregated colloid as it was stirred at 400 rpm using a mechanical stirrer. The stirring was continued until a smooth, glossy, and thickened gel-like consistency was obtained, which typically took approximately 30 min. The resulting gel was then immediately poured into a 10 × 10 × 0.5 cm casting mold made with a Perspex bottom and removable sides held in place with wing nuts. The bottom surface of the mold was covered with silicone release sheet which was held in place using vacuum grease. The gel was then left to dry in an open space at room temperature (relative humidity ≤50%) for approximately 48 h or until fully dry, at which point the resulting film could be easily peeled away from the surface and cut into smaller sections, typically 5 × 5 mm, using normal scissors. The extinction spectrum of the films showed a broad extinction from 300 to 1000 nm (see Supporting Information, Figure S1), which was due to a combination of light scattering by the semiopaque film and absorption by the aggregated particles. When in use, the films were either rehydrated by applying a droplet of aqueous analyte (normally 5 μL) or fully immersed into bulk analyte solution. In both cases, the film would be placed onto a glass slide covered in aluminum foil before the probe laser from a PerkinElmer RamanMicro 200 Raman microscope using a 4× objective lens at 55% laser power was directed onto the film surface and a SERS spectrum was collected. The microscope uses a 785 nm cavity diode laser (80 mW) with a Czerny-Turner spectrometer with a spectral range of 95−3200 cm−1. It is composed of an Olympus microscope (BX51 reflected illumination frame) which is connected to a spectrometer through fiber-optic cables. The spot diameter is 230 μm. Thiophenol was initially dissolved in ethanol, and serial dilutions were carried out using 25% ethanol/water. Adenine, crystal violet dye, (s)-(−)-nicotine, melamine, potassium thiocyanate, and theophylline were dissolved and diluted with water only. For experiments with benzylmercaptan, the analyte was diluted in the required solvent to 1 × 10−3 mol dm−3 and was applied directly onto the films as a 10 μL droplet. Alternatively, in experiments where the films were preswelled, 20 μL of water was spread over a ca. 5 × 5 mm area of the film and allowed to partly dry before a 10 μL analyte solution droplet was applied.
with silver nanoparticles by a process in which the gel was formed, treated with silver nitrate, and subsequently reduced.11 However, these in situ methods generally tend to be complex and more importantly offer limited opportunities for controlling the size and shape of the particles formed. An alternative to generating the particles at point of use is to improve colloid stability. A promising approach is to prepare the enhancing particles and then incorporate them in a polymeric support. For example, gold-silica nanoshells have been incorporated within highly porous nitrocellulose membranes,12 but the disadvantage of this approach is that it leaves the particles exposed to the environment. Alternatively, we have previously trapped the active particles in swellable polycarbophil (PC) polymers, where they were fully protected from the environment. One format was to prepare thin polycarbophil (PC) coatings containing unaggregated colloid13 which could be stored dry but upon rehydration with aqueous analyte and aggregating agent would become SERS active. A significant disadvantage of these PC coatings was that they had to be prepared with unaggregated colloid since the performance decreased significantly when the colloids were aggregated before being isolated in the polymer. This was believed to be due to the polymers not fully releasing the colloid upon rehydration so that the original enhancement level was not restored when the aqueous analyte was applied to preaggregated coatings. Similarly, even when the coatings were aggregated during the swelling step, the enhancement was reduced compared to simple aqueous conditions. A partial solution to this problem was found in the observation that the nanoparticles within PC remained more accessible to analytes if the gels formed by mixing colloids with PC polymer were stored in the gel form, and indeed in this case, the particles could be preaggregated and still remain active.14 However, although the wet SERS gels were useful for routine laboratory analysis and had the advantage that they prolonged colloid storage lifetime up to one year, the fact that they were liquid meant that they were not much better than conventional colloids for on-site use because they had the same handling problems. The examples above are just a small sample drawn from the vast body of published research involving the stabilization of SERS nanoparticles within various organic and inorganic hosts. However, within this large body of research, apart from the examples above, there has been surprisingly little work on the use of swellable polymers for stabilizing colloids, possibly because it seems more obvious to use a porous host to allow analytes to penetrate than to depend on swelling to release the particles. However, here we describe a new method for preparing free-standing films which allows Ag nanoparticles to be stored and stabilized in the dry state but to become active when required by swelling with aqueous analyte. Importantly, these films overcome previous difficulties in that the particles are fully released on swelling and can even be preaggregated so that no postswelling aggregation step is necessary and simple aqueous (or nonaqueous) solutions can be applied directly to the films.
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RESULTS AND DISCUSSION The preparation method produces large flexible polymer film sheets (10 × 10 cm) which we term “Poly-SERS” films. These are the same color as aggregated silver colloid solution but have mechanical properties similar to common 80 gsm printer paper. They are sufficiently rigid to be free-standing, but sections can be cut to the desired size with scissors. Figure 1 shows a SEM image of a Poly-SERS film showing that its surface is smooth and flat16 and that it has a uniform thickness of approximately 50 μm, which can of course be adjusted by altering the amount of polymer used. This thickness was chosen because it gives
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EXPERIMENTAL SECTION Silver nitrate (99.9999%), magnesium sulfate, thiophenol (≥99%), adenine (99%), crystal violet dye (anhydrous, ≥90.0%), (s)-(−) nicotine (98%), melamine (99%), potassium thiocyanate (99%), benzylmercaptan (99%), and theophylline (≥99%) were purchased from Sigma-Aldrich. Natrosol 8107
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structure of the films is therefore very similar to that of a conventional aggregated aqueous colloid with aggregates of various sizes randomly distributed throughout the sample volume, except that in the dry film they are fixed in place by the rigid polymer host rather than floating free in solution. The average diameter of the aggregates was ca. 800 nm, but a broad range of diameters was observed; a plot showing the distribution of aggregate sizes is shown in Figure S2, Supporting Information. Manual counting showed this average diameter corresponded to approximately 13 Ag particles, fewer than would be in a close packed structure due to the loose arrangement of the particles in the aggregates. The main challenge with encapsulated particle films is the requirement that the host polymer does not interfere with the measurements; this means that it should allow the particles to be accessed when required and it should not itself give large interfering SERS signals. In this case, the blank spectra of the films are completely dominated by a strong Ag−Cl band at 241 cm−1 which arises from binding of chloride ions from the polymer to the surface of the citrate-reduced colloids.17 The HEC itself is such a poor scatterer that the SERS bands of the polymer are not detectable even in the blank spectra (Supporting Information, Figure S3). The Poly-SERS films have been tested using two different methods to bring the analyte solution into contact with the film. The first was simply immersing a section of film directly into the bulk analyte solutions; the second was by placing a droplet of aqueous analyte onto the surface of the film. The Raman spectra were then measured under both sets of experimental conditions in order to determine the rate at which the application of analyte solution caused swelling of the film and the associated contact between the analyte and particles which gives the SERS signal. In the first instance, individual sections of Poly-SERS film (5 × 5 mm) were fully immersed into 11 separate sample vials containing 5 mL, 1 × 10−4 mol dm−3 thiophenol solutions for times ranging from 2 to 240 min. The films were then removed from their corresponding sample vials and placed flat onto aluminum foil covered glass slides, and their SERS spectra were recorded. The absolute signal intensities are shown in the Supporting Information (Figure S4), but the result was not surprising; essentially, the signal was large right from the first accumulations (105 counts in 30 s), and although there was a gradual increase in SERS signal intensity with increasing time spent in the thiophenol solutions, the difference was less than a factor of 2. This suggests that, even though there was some further diffusion of the analyte molecules from the bulk solution into the SERS films after they had been initially swollen in the first 2 min, this postswelling diffusion process was not the main mechanism by which the analyte adsorbed onto the particles. The next experiments involved applying sample droplets onto the surface of the Poly-SERS films. Figure 3b shows a section of Poly-SERS film which was rehydrated with a 5 μL water droplet applied to the upper surface and allowed to swell for approximately 10 min before the sample was sectioned and photographed using an optical stereomicroscope. In this case, the droplet retained its initial diameter during the swelling process with the result that the area of the film under the droplet swelled and domed upward toward the droplet. The swelled region was notably more elastic than the dry film but still retained its physical integrity. On standing in the open air, the aqueous solvent evaporated, reversing the effect, until the
Figure 1. SEM image showing a face/edge view of a Poly-SERS film at 470× magnification. The inset is a photograph showing a ca. 1 cm2 section of a Poly-SERS film cut from a standard 10 × 10 cm sheet.
sufficient mechanical strength to allow easy handling but is still thin enough to swell rapidly. Critically, using an appropriate concentration of salt in the preaggregation step before adding polymer gives clusters of particles which are retained in the dried film, as shown in the SEM image Figure 2a. The range of salt concentrations which
Figure 2. SEM image showing a face on view of the individual Ag aggregates held within a Poly-SERS film. Image (a) was recorded at 12 000× magnification. Image (b) shows an exposed Ag nanoparticle cluster held within a film at 52 000×.
give acceptable results is quite large; thus, this does not cause any problems during preparation, but outside the range, either the particles show insufficient aggregation at low salt concentration (≤1.25 × 10−3 mol dm−3) or the films break up during drying at high salt concentrations (≥2.5 × 10−2 mol dm−3). The SEM image shows that the aggregates are distributed approximately evenly across the film, and those which lie deeper within the polymer are increasingly out of focus. This is not due to limited depth of focus of the SEM, although it gives a result which is strikingly similar to what would be expected in that case, but instead, it is due to scattering of the electron beam by the polymer host, with deeper clusters suffering more scattering. Figure 2b demonstrates that this is the case because it shows an image of an adventitious crack in a Poly-SERS film which has exposed a section of a nanoparticle cluster which sits deep within the film. In the image, the exposed particles are in sharp focus while those of the same cluster which were imaged through the polymer at each side of the crack are quite blurred. The overall 8108
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Figure 3. SERS spectra of 15 μL droplets of 1 × 10−4 mol dm−3 thiophenol obtained from 2 to 240 min after the initial deposition of the analyte droplet onto the surface of the SERS film. The spectra are on the same vertical axis, apart from being offset for clarity. Inset (a) is a graph comparing the changes in the absolute SERS signal intensities against the change in mass of an evaporating droplet of the same composition over time. Inset (b) shows a side view of a section of a Poly-SERS film which was rehydrated with a 5 μL water droplet for 10 min before being cut and photographed.
film returned to its original shape and thickness. The effect of this swelling on the SERS signals from the films was tested by depositing 15 μL droplets of 1 × 10−4 mol dm−3 aqueous thiophenol onto their surfaces and recording their SERS spectra as a function of time. The data, plotted in Figure 3, show that strong thiophenol bands were already present in the first spectrum recorded (accumulation finished 2 min after droplet application). The intensity of these bands then increased over time, eventually reaching a plateau at approximately 60 min, after which the signal remained stable. The initial signal was clearly due to the primary swelling process, but the subsequent slow increase in signal could arise from either diffusion of any unadsorbed analyte into the film and onto the surface of the aggregates or increased concentration due to evaporation of the aqueous solvent. The experiments with submerged films showed that diffusion into the films following the initial swelling had only a small effect on the signal while in this case the signal level at the plateau was approximately 5× of that found after the films were first swollen (Supporting Information, Figure S4) which seems too large for secondary diffusion to be the main cause. In contrast, the plot in Figure 3, which compares the absolute SERS intensities of thiophenol and the change in mass of the evaporating droplet with time, shows that the rate of signal increase and mass decrease were very similar and that the maximum SERS signals were obtained when the solvent had fully evaporated. This suggests that as the analyte molecules became increasingly concentrated with evaporation of the droplet solvent they were forced into the films to interact with the nanoparticles. In this case, the films acted more like other conventional solid substrates where a
small droplet is typically applied and allowed to evaporate before the signal is recorded. With droplet drying, the possibility of coffee ring effects, in which solute is preferential deposited in a ring at the edge of the droplet, needs to be considered. The effect occurs due to pinning of the droplet combined with capillary flow which causes the solution to move to the edge of the drop, increasing the local concentration. While the coffee ring effect can be useful in some circumstances, such as separating components within complex biological samples, in this context it is undesirable because if a coffee ring forms the concentration detected during SERS analysis depends on where the laser beam is directed on the dried droplet. However, with the PolySERS films, the coffee ring effect is suppressed compared to simple supports. Figure 4 shows an experiment where 10 μL droplets of 1 × 10−3 mol dm−3 Rose Bengal dye were deposited onto either a simple glass slide or a HEC only film and allowed to dry. Not surprisingly, a significant coffee ring effect occurred with the glass slide; however, with the HEC film, the dyed area was much more uniform. This may be due to HEC disrupting the normal capillary flow of drying droplets and giving something more comparable to Marangoni circulation.18 This type of circulation is able to homogenize the drying of analyte droplets so that their deposition onto the surface is more uniform. The coffee ring suppression was studied further by depositing a 5 μL, 1 × 10−4 mol dm−3 thiophenol droplet onto a PolySERS film and recording data at various points across the diameter of the dried analyte droplet. As shown in Figure 4, the intensities show no increased intensity at the edges and, across 8109
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whole field of view in Figure 2 and therefore that the SERS signal arises from more than 100 clusters of various sizes. It is therefore more likely that the observed point-to-point variation is due to differences in the average particle concentration occurring over length scales >50 μm rather than the local variations seen in the SEM images. To determine the extent to which the absolute signal levels from the enhancing films can be used for quantitative analysis, the SERS signals from 5 μL thiophenol droplets at various concentrations were recorded, as shown in Figure 5a. The data show the expected rise in signal with increasing concentration up to a plateau region. Figure 5a includes a Langmuir type fit of the data which shows reasonable agreement with the experiment but plateaus early and does not follow the gradual rise of the data points even at high concentration, which we have found empirically to fit to a simple logarithmic curve, as shown in Figure 5a and as the semilog plot in Figure 5b. The data show a strong correlation between the concentration of the analyte and the absolute SERS signals and suggest that the reproducibility of the method is sufficient for at least semiquantitative analysis using absolute signal intensities. Of course, this calibration could be improved using an internal standard if required and by averaging over a larger number of points (as described above), but for current purposes, the intention is to demonstrate the level of quantification that can be achieved using the simplest possible approach of measuring absolute signal intensity at a single point. The absolute signal levels provided by enhancing materials with different physical characteristics are difficult to compare in absolute terms due to difficulties in finding meaningful figures of merit. For this reason, we have carried out simple side-byside experiments to compare the Poly-SERS films with conventional silver colloids, whose performance most researchers will be familiar with, and a Q-SERS substrate, which is a commercially available material based on layers of 15 and 60 nm Au particles on a solid support.5 In this test, 5 μL portions of 1 × 10−4 mol dm−3 thiophenol solution (aqueous with a small concentration of ethanol to aid solvation) were used. In one case, the droplet was preconcentrated onto the Poly-SERS film and its spectrum was recorded using a 4× objective lens (this objective was used because some sample damage was observed with the 20× objective). This was compared to a droplet dried onto a Q-SERS surface whose spectra were
Figure 4. Absolute SERS signal intensities of a 5 μL, 1 × 10−4 mol dm−3 thiophenol droplet preconcentrated on a Poly-SERS film obtained at various horizontal sample stage positions (0.3 mm increments). The inset shows a photograph of 15 μL, 1 × 10−3 mol dm−3 Rose Bengal preconcentrated onto (a) a glass slide and (b) HEC film.
the spot, the intensities show random fluctuations which are due to point-to-point variation in the film. The point-to-point variation in Figure 4 suggested that the homogeneity of the Poly-SERS film should be tested. Five μL droplets of 1 × 10−4 mol dm−3 thiophenol were deposited at 15 random points over one film, and the absolute SERS signal intensities of thiophenol within each spot after drying was measured. Using an average of three spectra for each point gave a relative standard deviation in the absolute SERS signal intensities of 3.2%. The origin of this variation was presumably that some parts of the film may have been more highly doped with the Ag nanoparticles than others, and averaging the signal from 3 points, although it reduced the variation, was not sufficient to completely eliminate it. The SEM image in Figure 2 shows that the aggregates are well spread throughout the films, but they are not tightly packed together so there are still areas up to 5 μm across which have no aggregates present. This could be a difficulty if the film was probed using a microscope with a diffraction limited spot since there would be a significant chance of either completely missing a cluster or alternatively focusing on a large cluster and obtaining an anomalously high signal. However, this local variation should not be the origin of the variations observed in Figure 4 since the laser spot diameter used in these measurements was ca. 230 μm, which means that the area sampled in the SERS experiments is larger than the
Figure 5. (a) A calibration curve showing the absolute SERS signal intensities of the strong 1572 cm−1 band of thiophenol at various concentrations using a Poly-SERS film. The data have been fitted to a Langmuir isotherm (dashed line) and a logarithmic increase (solid line). (b) A semilog plot of the same data. 8110
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at an extremely low cost (≪ $1) since the films are cast in 10 × 10 cm molds, and therefore, even a single sheet can provide 400 individual 5 × 5 mm sections. Finally, an important parameter is the long-term stability of the substrates. The SERS activity of a representative film was tested using 5 μL of 1 × 10−4 mol dm−3 thiophenol over a 12 month period, taking an average of three spectra from different parts of the film for each time point. It was found that the films retained 78% of the original absolute signal intensity after storing for 6 months and 70% of the original value after storing for 12 months. The most likely cause of aging of the Poly-SERS films is oxidation of the nanoparticles since the films were not stored under inert conditions; they were simply kept in the dark, under atmospheric air, and at laboratory temperature. It would therefore be expected that storage under sealed anaerobic conditions (similarly to those used with commercially available solid substrates) would dramatically increase the lifetime. All the above measurements used thiophenol as the analyte which is widely used as a test material because it gives large and therefore easy to measure SERS signals and binds strongly to Ag and Au surfaces. However, with any new substrate, it is important to test it using a more challenging analyte; in this case, theophylline was chosen, and in addition, the measurements were carried out using films that had been stored for >6 months before use. Theophylline is a therapeutic drug which is used in the treatment of asthma, chronic obstructive pulmonary disease, bronchitis, and heart failure.19 Rapid monitoring of theophylline is important because it has a narrow therapeutic window which sets the target range of detection at 10−20 mg/ dm3 (5.6 × 10−5−1.1 × 10−4 mol dm−3).20 15 μL drops of 1 × 10−3, 1 × 10−4, and 1 × 10−5 mol dm−3 theophylline were tested with the Poly-SERS films and, as shown in Figure 7, the theophylline could easily be detected at 1 × 10−5 mol dm−3 (1.8 mg/dm3), well below the limit required for therapeutic drug monitoring. Of course, this result was obtained in aqueous
recorded using a 20× objective lens; this lens gives a higher signal than the 4× objective used for the Poly-SERS measurements so both systems were compared under their best conditions. The signal from conventional aqueous colloid was recorded by mixing 100 μL of 1 × 10−4 mol dm−3 thiophenol with 100 μL of CRSC and aggregating with 50 μL of 0.1 mol dm−3 MgSO4. The results are shown in Figure 6.
Figure 6. SERS spectra of 5 μL of 1 × 10−4 mol dm−3 thiophenol obtained with (a) a Poly-SERS film, (b) Q-SERS, and (c) normal aggregated citrate-reduced silver colloid solution. The spectra are on the same vertical axis, apart from being offset for clarity.
For 30 s accumulations, the intensity of the thiophenol 1572 cm−1 band was ca. 110 000 counts with the Poly-SERS films, ca. 8000 counts with Q-SERS, and ca. 85 000 counts using aqueous CRSC and MgSO4 aggregating agent. These results therefore demonstrate that the Poly-SERS films are a considerably more sensitive SERS substrate than Q-SERS. Indeed, the signals from the Poly-SERS films were also slightly larger than those obtained with aqueous silver colloids, which are known to be among the best of all SERS enhancing materials, despite numerous attempts to better them.1 This was presumably due to a combination of the much larger number of SERS active nanoparticles in the probed volume, and the preconcentration was due to solvent evaporation. The Poly-SERS films were only 50−250 μm thick, depending on the stage of swelling; however, they contained the same number of particles as the 5 mm depth of gel which was initially used to prepare them, and in the swollen films, all these particles lay within the depth of focus of the low confocality Raman spectrometer used in this work. In comparison, with the aqueous colloid, a 5 mm deep sample will contain the same number of particles as the film, however, many of these particles will lie outside the focal depth of the system and therefore not contribute to the signal. This ability of the films to keep more particles in the focal volume combined with the preconcentration effect clearly more than compensates for any reduction in accessability of the analyte to the surface that polymer encapsulation causes. Indeed, even without any solvent evaporation, the films give signals that are comparable to those of aqueous colloids, at ca. 1/3 the intensity, which is not an important difference in this context. We note that 5 × 5 mm squares of Poly-SERS film are easily large enough for a single measurement so they can be prepared
Figure 7. SERS spectra of 15 μL of (a) 1 × 10−3, (b) 1 × 10−4, and (c) 1 × 10−5 mol dm−3 theophylline and (d) water only obtained with a Poly-SERS film. Inset is the molecular structure of theophylline. All spectra are shown on the same vertical axis, apart from being offset for clarity. 8111
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Figure 8. SERS spectra obtained with a Poly-SERS film of (a) 1 × 10−4 mol dm−3 adenine, (b) 1 × 10−5 mol dm−3 crystal violet, (c) 5 × 10−3 mol dm−3 potassium thiocyanate, (d) 1 × 10−3 mol dm−3 melamine, (e) 1 × 10−4 mol dm−3 nicotine, and (f) 1 × 10−6 mol dm−3 tris(bipyridine)ruthenium(II) nitrate. The concentrations of the analytes were adjusted to give approximately the same absolute signals, and in this figure, all spectra are shown on the same vertical axis, apart from being offset for clarity.
solution rather than body fluid, but nonetheless, it clearly demonstrates that the films are effective for more challenging analytes than alkyl/aryl thiols which are commonly used to demonstrate SERS enhancements in novel substrates. Another important feature is that the films should be broadly applicable with a range of analytes. Figure 8 shows the high signal-to-noise SERS spectra which were obtained when 5 μL of various aqueous analytes, including dye, drug, and nucleobase, were tested with Poly-SERS films (which had again been stored in air for six months) by directly depositing droplets onto the surface. The samples were chosen to span a broad range of organic/inorganic/organometallic analytes with different sizes and charges. The ability of the Poly-SERS films to detect such
analytes and to do this after prolonged storage clearly demonstrates that the films have considerable potential for application in multiple fields. Finally, the films are not confined to aqueous media, a range of solvents has been tested, initially by simply depositing droplets of analyte on the surface in the usual way. Benzylmercaptan was chosen as the target compound due to its solubility in a wide range of solvents. Figure 9a shows unscaled spectra obtained using solvents ranging from acetone to hexane where it is clear that the solvents that would be expected to swell the films, such as acetone and methanol, give good spectra while hexane, which would not be expected to swell the film, gives no SERS signal from the analyte. More 8112
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Fax: +44 2890 976534. Tel: +44 2890 974470. E-mail: s.bell@ qub.ac.uk. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Support for this work by the EPRSC (grant number EP/ H021647/1) is acknowledged. Figure 9. SERS spectra obtained for 1 × 10 −3 mol dm −3 benzylmercaptan dissolved in the solvents marked using a PolySERS film. (a) Sample applied directly to the film. (b) Film preswelled with 20 μL of water before addition of solution. All spectra in (a) or (b) are shown on the same vertical axis, apart from being offset for clarity.
REFERENCES
(1) Bell, S. E. J.; Sirimuthu, N. M. S. Chem. Soc. Rev. 2008, 37, 1012− 1024. (2) Schlücker, S. In Surface Enhanced Raman Spectroscopy: Analytical, Biophysical and Life Science Applications; John Wiley & Sons: Hoboken, NJ, 2013. (3) Kneipp, K.; Moskovits, M.; Kneipp, H. In Surface-Enhanced Raman Scattering: Physics and Applications; Springer: New York, 2006. (4) http://www.renishawdiagnostics.com/en/klarite-sers-substrates-17076 (accessed March 20, 2014). (5) http://www.q-sers.com/ (accessed March 20, 2014). (6) http://www.rta.biz/Content/SERS_Vials.asp (accessed March 20, 2014). (7) Hankus, M. E.; Holthoff, E. L.; Stratis-Cullum, D. N.; Pellegrino, P. M. Proc. SPIE 8018, Chemical, Biological, Radiological, Nuclear, and Explosives (CBRNE) Sensing XII, 80180P, 2011, DOI:10.1117/ 12.886779. (8) Almaviva, S.; Botti, S.; Cantarini, L.; Fantoni, R.; Lecci, S.; Palucci, A.; Puiu, A.; Rufoloni, A. J. Raman Spectrosc. 2014, 45, 41−46. (9) Lee, V.; Farquharson, S. In Photonic Detection and Intervention Technologies for Safe Food (SPIE Proceedings); SPIE: Bellingham, WA, 2001; Vol. 4206, pp 140−146. (10) Sagmuller, B.; Schwarze, B.; Brehm, G.; Schneider, S. Analyst 2001, 126, 2066−2071. (11) Keating, M.; Chen, Y.; Larmour, I. A.; Faulds, K.; Graham, D. Meas. Sci. Technol. 2012, 23, 1−9. (12) Bishnoi, S. W.; Swarup, V.; Lin, Y.; Tibudan, M.; Huang, Y.; Nakaema, M.; Keiderling, T. A. Anal. Chem. 2011, 83, 4053−4060. (13) Bell, S. E. J.; Spence, S. J. Analyst 2001, 126, 1−3. (14) Bell, S. E. J.; Sirimuthu, N. M. S. Analyst 2004, 129, 1032−1036. (15) Lee, P. C.; Meisel, D. J. Phys. Chem. 1982, 86, 3391−3395. (16) The reverse face (upper side during drying) has some cosmetic defects which are left by residual air bubbles that rise to the surface during drying and leave patches with a cracked appearance when they burst. However, these are surface features which do not affect the distribution of the particles and which disappear when the films are swollen in any case. (17) Bell, S. E. J.; Sirimuthu, N. M. S. J. Phys. Chem. A 2005, 109, 7405−7410. (18) Hu, H.; Larson, R. G. J. Phys. Chem. B 2006, 110, 7090−7094. (19) http://www.nhs.uk/Conditions/Asthma/Pages/ MedicineOverview.aspx?condition=Bronchitis&medicine= Theophylline (accessed March 20, 2014). (20) http://www.nlm.nih.gov/medlineplus/ency/article/003430.htm (accessed March 20, 2014).
interestingly, it was found that, if the films were initially swollen by applying water and, subsequently (2 min), a droplet of the test solution was added, the SERS signal of the analyte could be recorded. Indeed, the intensities of signals recorded using this method for both polar and nonpolar solvents were all within a factor of 2× of each other.
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CONCLUSION The Poly-SERS films developed in this work are highly sensitive and have the potential to transform SERS into a convenient, inexpensive, and reproducible routine analytical technique for a wide range of analytes. The use of a swellable polymer allows the particles to be protected in storage but released when required. Numerous polymers, swellable or nonswellable, could be used for encapsulation; the difficulty is in finding a polymer which when treated with analyte solution releases the particles so they are free to interact with the target compounds. Here, HEC was found to have all the required characteristics of a host material, in that it could be formed into thin films which contained high concentrations of Ag nanoparticle aggregates of the type known to be highly SERS active in solution. These films were easy to cut to the appropriate size and either could be dipped into test solutions or have the solutions applied as small droplets to their surface which caused the films to swell, drawing the solution into the films and allowing the analyte to come into contact with the released particles. The swollen films could be either probed immediately or allowed to dry, which gave a further increase in signal intensity. The drying did not give a noticeable coffee ring effect after evaporation. The absolute signal heights given by the Poly-SERS films were comparable to those given by the parent colloid in normal solution, which are known to be extremely high and were more than an order of magnitude larger than those given by a commercial SERS substrate based on Au particles. These films gave signals 70% of their original value even after being stored in air for 12 months, so they have the potential for genuine offthe-shelf use either for occasional users or for routine analysis, where the low cost and scalability of the preparation method is just as important as the storage lifetime. 8113
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