Anal. Chem. 2005, 77, 8151-8154
Profiling an Electrospray Plume Using Surface-Enhanced Raman Spectroscopy Douglas Davis, Erik Portelius, Yu Zhu, Charles Feigerle, and Kelsey D. Cook*
Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996-1600
We report the use of silver nanoparticles to obtain surfaceenhanced Raman spectra of Crystal Violet in an electrospray plume. Surface enhancement allowed detection at low concentrations with the high specificity afforded by vibrational spectroscopy. SERS spectra were used to obtain an axial concentration profile closely matching that obtained in previous fluorescence experiments. SERS can provide more analyte structural information than has been obtainable from fluorescence studies of the plume.
into an electrospray. Electrolyte-induced clustering of these particles can further strengthen the enhancement. Enhancements as large as 1014 have been reported for colloidal clusters, allowing SERS to be used for single-molecule detection in small volumes of 10-12-10-14 M solutions.7-11 In the current work, we sought to assess whether SERS enhancement can be exploited for analysis of the dilute solutions encountered in the electrospray plume. We used Crystal Violet as an analyte, due to its excellent SERS enhancement and low fluorescence interference as seen in the single-molecule detection experiments of Kneipp et al.7-11
Electrospray (ES) mass spectrometry has been used widely to study solution-phase chemistry (e.g., noncovalent interactions in biomolecular systems) as well as molecular structure. However, it has been shown that ES may in some instances be an invasive probe due to chemical changes taking place as a consequence of the spray process. For example, Kiselev et al.1 used a special probe linked to a gas chromatograph to investigate solvent fractionation in the spray plume. We have studied similar chemistry, as well as axial and lateral changes in pH and temperature, using fluorescence probes.2-4 Rodriguez-Cruz et al.5 and Ideue et al.6 also used laser fluorescence to study the spray plume, characterizing spray-induced changes in protein conformations. Fluorescence and chromatography provide little if any structural information. In principle, Raman spectroscopy could offer a powerful alternative for noninvasive determination of the structure of molecules in the spray. However, the very low signal levels ordinarily associated with Raman (relative to those obtained by fluorescence) would be expected to limit its applicability in the dilute plume environment. One way to overcome this difficulty is through the use of surface-enhanced Raman spectroscopy (SERS), whereby analyte adsorption onto a metal substrate (typically a silver or gold film or colloidal suspension) can increase Raman signals by many orders of magnitude, depending on the substrate used and the molecule being examined. In a SERS colloidal suspension, the signal enhancement is also dependent on the particle size. SERS-active particles are typically tens of nanometers in diameter, suggesting that they might readily be incorporated
EXPERIMENTAL SECTION Materials. Reagent grade AgNO3, Crystal Violet (95%), and sodium citrate were obtained from Aldrich Chemical Co. and used as received, as was NaCl from Mallinckrodt. Burdick and Jackson HPLC grade methanol (UN1230) was obtained from Baxter. All aqueous solutions were made using 18 MΩ deionized water obtained from a Millipore purification system. Crystal Violet solutions were prepared by dilution of a 10-4 M aqueous stock solution. Silver colloid solutions (sols) were prepared by citrate reduction of AgNO3 using the procedure of Lee and Meisel.12 A UVvisible spectrum showed that the sol had a λmax of 444 nm, similar to that reported by Lee and Meisel.12 The SERS activity of the solutions was checked by recording the Raman spectrum of an aqueous solution containing 10-7 M Crystal Violet, 10-2 M NaCl, and 7.5 × 10-4 M Ag (silver concentration is based on the concentration of AgNO3 initially used in making the sol and therefore represents the total concentration of both Ag+ and Ag). A strong SERS spectrum of Crystal Violet was obtained from a 1-cm quartz cuvette containing this solution, indicating good SERS enhancement (>104; data not shown). For electrospray experiments, Crystal Violet was mixed with the silver sol, NaCl, and methanol (water/methanol 72:28 v/v to mimic typical ES conditions) immediately before spraying. Methanol is known to show no SERS enhancement; it therefore has been used for comparisons in measuring SERS enhancement factors.13
* To whom correspondence should be addressed. E-mail:
[email protected]. (1) Kiselev, P.; Rosell, J.; Fenn, J. B. Ind. Eng. Chem. Res. 1997, 36, 30813084. (2) Zhou, S.; Edwards, A. G.; Cook, K. D.; Van Berkel, G. J. Anal. Chem. 1999, 71, 769-776. (3) Zhou, S.; Prebyl, B. S.; Cook, K. D. Anal. Chem. 2002, 74, 4885-4888. (4) Zhou, S.; Cook, K. D. Anal. Chem. 2000, 72, 963-969. (5) Rodriguez-Cruz, S. E.; Khoury, J. T.; Parks, J. H. J. Am. Soc. Mass Spectrom. 2001, 12, 716-725. (6) Ideue, S.; Sakamoto, K.; Honma, K.; Clemmer, D. E. Chem. Phys. Lett. 2001, 337, 79-84.
(7) Kneipp, K.; Wang, Y.; Kneipp, H.; Perelman, L. T.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Phys. Rev. Lett. 1997, 78, 1667-1670. (8) Kneipp, K.; Kneipp, H.; Deinum, G.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Appl. Spectrosc. 1998, 52, 175-178. (9) Kneipp, K.; Roth, E.; Engert, C.; Kiefer, W. Chem. Phys. Lett. 1993, 207, 450-454. (10) Kneipp, K.; Kneipp, H.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Chem. Rev. 1999, 99, 2957-2975. (11) Kneipp, K.; Kneipp, H.; Manoharan, R.; Itzkan, I.; Dasari, R. R.; Feld, M. S. J. Raman Spectrosc. 1998, 29, 743-747. (12) Lee, P. C.; Meisel, D. J. Phys. Chem. 1982, 86, 3391-3395.
10.1021/ac0511039 CCC: $30.25 Published on Web 11/09/2005
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Because they are not SERS enhanced, the methanol peaks were not prominent in SERS spectra and did not constitute a significant interference. Salt and dye concentrations represented something of a compromise between optimum SERS and ES conditions. SERS experiments aimed at single-molecule detection employ up to ∼10-2 M salt (NaCl) to enhance the SERS signal by promoting aggregation of the colloidal silver particles.7-9 Such high salt concentrations are atypical in electrospray, where total ionic strengths are usually on the order of 10-4 M or less. To more closely approximate normal electrospray conditions, a salt concentration of ∼7 × 10-5 M was employed, necessitating a Crystal Violet concentration of ∼10-6 M to achieve a good signal. This is a typical ES analyte concentration and was low enough to avoid sol or dye aggregation8 and concomitant plugging of the ES capillary. The final concentrations of the mixture components were therefore as follows: 7.5 × 10-4 M Ag, 1.1 × 10-6 M Crystal Violet, 6.9 × 10-5 M NaCl, and 28% MeOH. Apparatus. The custom-built electrospray apparatus was the same as that used in previous studies.2 Briefly, it consisted of concentric stainless steel tubes, carrying sample solution (7.7 µL/ min from a Harvard Apparatus model 11 syringe pump) through the inner tube (152 µm i.d. × 267 µm o.d.) and dry nitrogen (National Welders Supply, UN1066, 3.6 L/min) through the outer tube (508 µm i.d. × 762 µm o.d.). High voltage (4.5 kV) was applied via a Valco “tee” fitting that supported the two capillaries. Raman measurements employed a Dilor XY spectrometer (a triple 1200 grooves/mm grating instrument) equipped with an 800 × 2000 back-thinned CCD detector. Spectra were obtained using the double subtractive mode of operation, which yielded a spectral window on the CCD of ∼1200 cm-1. This was sufficient to encompass the key region of the Crystal Violet Raman spectrum in a single window. Except where noted, 300 mW (measured at the entrance to the spectrometer) of 514.5-nm excitation from a Coherent Innova 200 argon ion laser was used. The electrospray apparatus was positioned in front of a 75mm focal length collection lens at the macrostage sampling port of the Raman spectrometer. Spectra were recorded in a 180° backscattering geometry. A confocal aperture limited the collection depth of field to ∼1.5 mm. As previously described,2 the electrospray apparatus was mounted on a two-axis translation stage (The L. S. Starrett Co., No. 63), which allowed for vertical (z-axis) and horizontal (x-axis) positioning of the spray with respect to the lens. The stage position in the x- and z-dimension was initially zeroed using the tip of the electrospray emitter as a reference. Distances reported in axial profiles are z-displacements relative to this point. The collection lens focal point (along the y-axis) was adjusted to maximize the intensity of Raman scattered light from the spray. Data Acquisition. To generate axial profiles, spectra were recorded from the spray beginning at z ) 1.9 mm (to avoid the Taylor cone) and extending typically to z ) 5.2 mm, or until the signal-to-noise ratio (S/N) fell below 3.5. The spectrum obtained for each z-point was an average of nine scans. Peak areas for baseline-subtracted spectra were evaluated using automatic peak detection and fitting algorithm 1 from PeakFit (v. 4.11, SYSTAT Software Inc.). Within a given profile, areas were normalized to that at z ) 1.9 mm. Four replicate profiles were averaged to give a composite profile for fitting and comparison with profiles (13) Kneipp, K.; Dasari, R. R.; Wang, Y. Appl. Spectrosc. 1994, 48, 951-955.
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Figure 1. Raman spectra of 1.1 × 10-6 M Crystal Violet dissolved in ∼72:28 (v/v) water/methanol. Trace A is normal Raman, i.e., without silver sol (9-mW laser power, average of 10 × 10 s acquisitions, sample in a cuvette). Trace B is from an electrosprayed SERS solution (300-mW laser power, average of 3 × 75 s acquisitions). Trace C is from a SERS solution in a cuvette (9-mW laser power, average of 10 × 10 s acquisitions). Traces are offset as needed for clarity.
obtained from fluorescence measurements of Eosin Y2 and Rhodamine B. For a given profile, the spectral accumulation time for a scan was either 75 or 100 s, depending on the initial signal levels at z ) 1.9 mm. Error bars represent the standard deviation of the four replicates. RESULTS AND DISCUSSION Normal and SERS Raman spectra of Crystal Violet in a cuvette are compared in Figure 1, curves A and C. The laser power (9 mW), analyte concentration (1.1 × 10-6 M), and acquisition times (10 scan average of 10-s acquisitions) for these two spectra are the same. Under these conditions, Crystal Violet Raman bands are not apparent in the conventional spectrum (curve A); the barely discernible peak near 1020 cm-1 is due to methanol solvent. As noted earlier, methanol peaks are not enhanced by SERS conditions (curve C). In contrast, the rich SERS spectrum of Crystal Violet clearly stands out in curve C, which closely matches published SERS spectra of Crystal Violet.14,15 For example, the prominent features at 910 and 1174 cm-1 (discussed further below) can be assigned to ring skeletal vibrations of radial orientation and in-plane ring C-H bends, respectively. As might be anticipated, low concentrations in the electrospray plume result in considerable lowering of absolute signal levels relative to those in Figure 1, curve C. Some of the consequent reduction in S/N, can be regained by increasing the acquisition time and laser power. The SERS spectral features obtained from the spray (3-scan average of 75-s acquisitions, 300-mW laser power; Figure 1, curve B) closely match those obtained with the cuvette (10 × 10 s acquisition, 9-mW laser power; Figure 1, curve C). It should be noted that the y-axis scaling is invariant for the three curves of Figure 1; the absolute signal (and resulting S/N) is only reduced by a factor of ∼4 in the spray. When the sol was omitted (14) Persaud, I.; Grossman, W. E. L. J. Raman Spectrosc. 1993, 24, 107-112. (15) Lueck, H. B.; Daniel, D. C.; McHale, J. L. J. Raman Spectrosc. 1993, 24, 363-370.
Table 1. Comparison of Parameters Obtained by Fitting the Equation y ) azb to the profiles of Figure 2a
a
Figure 2. (a) Axial concentration profiles of the electrospray plume obtained using Crystal Violet SERS. (b) Comparison of data and best fits for Crystal Violet SERS [, Eosin Y fluorescence 9, and Rhodamine B fluorescence b.
from the spray sample, neither the dye spectrum nor the weak methanol peak at 1020 cm-1 could be detected (data not shown). Figure 2a shows an axial SERS intensity profile of Crystal Violet in the spray based on the combined areas of the peaks at 910 and 1174 cm-1. The 910-cm-1 peak was chosen for both its narrow width and high intensity, while the 1174-cm-1 peak was chosen because it had the highest intensity. Combining these signals improved S/N slightly. To determine whether the profile of Figure 2a is a reasonable representation of the density of species in the plume, it was compared with a fluorescence profile for Eosin Y obtained previously2 (Figure 2b). Such a comparison should ideally use a single analyte. However, neither dye could be analyzed by both techniques. Crystal Violet (a cationic dye) was chosen for its strong SERS enhancement and its lack of fluorescence with 514-nm excitation. Eosin Y was chosen for the earlier studies2 due to its strong fluorescence; it shows no SERS enhancement under the conditions of this experiment, probably because this anionic dye does not interact strongly with the sol.16 The stronger fluorescence signals allowed sampling of the spray out to 12 mm from the electrospray tip before S/N reached 3.5, whereas the weaker SERS signal levels limited sampling out to only 5.2 mm. When fit to a curve of the form y ) azb (Figure 2b and Table 1), the two profiles agree within experimental error. (16) Wang, K.; Li, Y.-S. J. Raman Spectrosc. 1996, 27, 385-389.
analyte
a
b
Crystal Violet Eosin Y Rhodamine B
2.0 ( 0.1 1.947 ( 0.008 1.70 ( 0.04
-1.10 ( 0.07 -1.037 ( 0.004 -0.84 ( 0.02
Uncertainties represent one standard deviation.
In contrast, there is a significant difference between these two profiles and that obtained for the fluorescence of Rhodamine B (Figure 2b and the last row of Table 1). The latter has been shown17-19 to be strongly temperature dependent in contrast to either the fluorescence of Eosin Y or the Raman Stokes lines of Crystal Violet. Thus, the curve for Rhodamine B in Figure 2b reflects both the decreasing dye concentration at longer z and the increasing fluorescence yield of Rhodamine B as the plume cools. Finally, it should be considered whether the ∼ 1/z dependence obtained for the axial profiles of Eosin Y and Crystal Violet is reasonable. Assuming a conical shape for the plume, the concentration should decrease in the plume as the inverse square of the distance from the emitter tip (z-axis). However, for that to be manifest in the data would require point sampling by the laser beam and Raman collection optics. Experimentally, the plume has been shown to have a relatively low divergence (spreading to a width of only ∼4 mm at z ) 12 mm).2 Considering the width of the laser beam when it intercepts the plume (∼1 mm) and the sampling depth near the laser focus (∼1.5 mm), a significant slice of the plume is sampled for each z-displacement. For signals collected by integrating over a cylindrical slice of the plume, a 1/z dependence is expected. Therefore, the ∼ 1/z decrease in intensity evident from the exponents in Table 1 is reasonable. CONCLUSIONS This preliminary study clearly establishes the viability of SERS as a technique for examining an electrospray plume. Although the signal-to-noise ratios are not as good as those from fluorescence, they are certainly adequate to obtain structural information. Moreover, there is no evidence in this or previous studies of laser damage to the analyte, probably due to the brief exposure times within the plume. It should therefore be possible to achieve increased sensitivity through increased laser power in cases where higher concentrations are not practical. The ability to examine the structure of molecules as they move through the spray has many practical applications. In particular it would be valuable to probe chemical and structural changes within the spray. For example, if a peak can be associated with a suitable vibrational mode, it can be monitored to see the loss or addition of a proton due to changing pH. With respect to biological molecules, significant insight into protein folding, assembly, and aggregation has been obtained from classical Raman investigations.20 Extension to SERS has been less common due in part to (17) Gallery, J.; Gouterman, M.; Callis, J.; Khalil, G. Rev. Sci. Instrum. 1994, 65, 712-720. (18) Erickson, D.; Sinton, D.; Li, D. Lab Chip 2003, 3, 141-149. (19) Lavielle, P.; Lemoine, F.; Lavergne, G.; Lebouche´, M. Exp. Fluids 2001, 31, 45-55.
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concerns over potential protein denaturation.21 However, cytochrome c has been widely studied by SERS and the degree of protein-sol interactions has been shown to be dependent on the SERS substrate.21-24 The best substrate to date for SERS of this protein has been aggregates of colloidal Ag particles made by citrate reduction,22-24 the same substrate used in the present study. SERS spectra of cytochrome c using colloidal Ag show that the protein retains its native spin states with no alteration of frequency in the principal bands; it was therefore concluded that this protein does not denature upon interaction with these SERS substrates.22 SERS also has been applied to detection of a wide range of trace (20) (21) (22) (23)
Tuma, R. J. Raman Spectrosc. 2005, 36, 307-319. Smulevich, G.; Spiro, T. G. J. Phys. Chem. 1985, 89, 5168-5173. Macdonald, I. D. G.; Smith, W. E. Langmuir 1996, 12, 706-713. Keating, C. D.; Kovaleski, K. M.; Natan, M. J. J. Phys. Chem. 1998, 102, 9404-9413. (24) Delfino, I.; Bizzarri, A. R.; Cannistraro, S. Biophys. Chem. 2005, 113, 4151. (25) Dou, X.; Yamaguchi, Y.; Doi, S.; Ozaki, Y. J. Raman Spectrosc. 1998, 29, 739-742.
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biological molecules including assay of an immune reaction without separating bound and free antigen.25 Combined with the results of the present study demonstrating the feasibility of spray SERS, these earlier results suggest that SERS may be a valuable probe of structural changes of biomolecules in an electrospray plume; SERS detection and analysis of a protein in an electrospray plume will be a subject of a future investigation. ACKNOWLEDGMENT This work was supported in part by the University of Tennessee Measurement and Control Engineering Center (a National Science Foundation Industry/University Cooperative Research Center; Grant ENG 0432387) and by the National Institutes of Health (Grant R01AG18927-01). Received for review June 21, 2005. Accepted October 7, 2005. AC0511039
