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Near-infrared surface-enhanced Raman scattering (NIR-SERS) of neurotransmitters in colloidal silver solutions
Spectrochirnica Acta, Vol. 51A, No. 3, pp. 481-487, 1995
Pergamon 0584-8539(94)00235-5
Copyright (~) 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved (1584-8539/95 $9.51)+ 0.00
Near-infrared surface-enhanced Raman scattering (NIR-SERS) of neurotransmitters in colloidal silver solutions K A T R I N K N E I P P , ~ Y A N G W A N G , R A M A C H A N D R A R . D A S A R I a n d M I C H A E L S. F E L D George R. Harrison Spectroscopy Laboratory, Massachusetts Institute of Technology, 77 Massachusetts A v e n u e , Cambridge, M A 02139, U.S.A.
(Received 18 August 1994; accepted 28 October 1994) A b s t r a e t - - N I R - S E R S spectra are measured for the neurotransmitters dopamine and norepinephrine at concentrations as low as 5 x 10 ~ M in colloidal silver solutions with accumulation times as short as 25 ms. The detection range and acquisition time are on the order of physiologically relevant concentrations and the time scale of neuronal processes, respectively. The spectra are obtained using a C C D detection system, dye laser at 830 n m for excitation, fiber optic probe and high throughput spectrograph. Mixtures containing the two neurotransmitters are used to demonstrate the capability of extracting quantitative information from SERS spectra. A l b u m i n added to the sample up to 0.5% concentration does not show any influence on the SERS spectra of the neurotransmitters in the silver colloidal solutions. The results demonstrate the potential of N I R - S E R S in probing dopamine and norepinephrine with high sensitivity and specificity. They also suggest that N I R - S E R S from colloidal silver solution can be a powerful tool for the study of neurotransmitters in brain extracts and dialysates.
INTRODUCTION
SURFACE-enhanced Raman scattering (SERS) has been established as a powerful spectroscopic method which can combine very low detection limits with high molecular specificity [1]. In the past several years, SERS spectroscopy has found growing applications in the field of biomedical research [2]. We have shown recently that SERS in the near-infrared region (NIR-SERS) provides strong electromagnetic enhancement factors if the optical fields (excitation laser and Raman scattered radiations) are in resonance with the longitudinal plasmon resonances of aggregated silver or gold colloids [3]. Electromagnetic enhancement factors on the order of 106 could be achieved for optimized colloidal solutions of silver and gold, which is higher than those achievable with visible excitations. In addition, excitations in the near-infrared reduce fluorescence background from biological materials [4]. For these reasons, NIR-SERS is becoming a useful tool to measure low concentration compounds in biological samples. In this work we demonstrate the application of NIR-SERS in colloidal solutions of silver in studying neurotransmitters. The detection of these molecules in brain extracts or in a brain dialysate at very low concentration levels is an important problem in neurochemistry. It is often required, in particular, to measure concentration changes and to correlate those changes to neuronal events, i.e. measurements done in the time scale of neuronal proceses [5]. Studies of SERS of different neurotransmitters on silver electrodes have been reported previously [6-8]. SERS spectra of dopamine at 3 x 10 -7 M were measured in 10 s using a silver electrode [6]. One of the problems in electrode SERS of neurotransmitters seems to be the attenuation of the SERS signal by protein co-adsorption, which could be reduced using polymer-modified electrodes [7]. It has also been shown that artificial neural networks are capable of accurately identifying the Raman spectra of aqueous solutions of neurotransmitters [9]. However, the detection limit with normal Raman scattering (10-z M) is not suitable for most of the neurological applications. We have selected dopamine (I) and norepinephrine (II) in this investigations (see Fig. l(a) for structural formulas). Dopamine is one of the important neurotransmitters. For instance, the relation between neurotransmitter content and neurotransmission or the measurement of a drug-induced change in the release of dopamine in relation to drug? A u t h o r to w h o m correspondence should be addressed. 481
482
K. KNEIPP et al.
(a)
i"
HO
l
H
H
(i) HO
HO
I H
H
H H
(II) (b)
kExe= 829 n m
c~
A
~
B I1)
>
J
r 1200
Raman
i 16'00
Shift (cm -1)
Fig. 1. (a) Molecular formulas for dopamine (I) and norepinephrine (II). (b) Normal Raman scattering spectrum of 7 x 10 1 M dopamine in water (A, accumulation time 5 s) and its SERS spectra in colloidal gold solution (B, accumulation time 5 s) and in colloidal silver solution (C, accumulation time I s), SERS spectrum of norepinephrine in colloidal silver solution (D, accumulation time 1 s). The concentrations of the neurotransmitters in gold and silver colloidal solutions are approximately l0 -4 M and 10 -7 M, respectively.
induced behavior or drug side effects are questions of great interest in neurology. Norepinephrine which is chemically very similar to dopamine was included in the study to obtain quantitative information from the SERS spectra from mixtures of the two neurotransmitters.
NIR-SERS of neurotransminers in colloidal silver solutions
483
MATERIALS AND METHODS
A solution of colloidal silver was prepared following a standard procedure [9] and diluted with water to achieve a maximum SERS signal for a given concentration of the probed molecule. The measured SERS signal from a colloidal sample results from both the surface enhancement from the colloidal solution and its attenuation (scattering and absorption) of the excitation and scattering lights. Most favorable SERS conditions can be achieved with a colloidal concentration which provides just enough surface area for adsorption of all molecules. Higher colloidal concentrations reduce the Raman signal due to scattering and absorption from the excess colloids which do not contribute to the SERS enhancement. An aqueous solution of NaCI at 1 M concentration was added to the colloidal solutions in about 1:15 ratio. The addition of NaCI solution induces special colloidal aggregation and results in colloidal absorption profiles very similar to those of optimized colloidal silver solutions for NIR excitation reported previously [3]. Dopamine and norepinephrine were purchased from Sigma Chemicals and used directly to prepare 10-4-10 -7 M aqueous solutions with or without 10% albumin by weight. The SERS samples were mixtures of one part of these neurotransmitter solutions with 20 parts of the colloidal solutions of silver. The addition of the neurotransmitters to the colloidal solution in this manner did not give rise to any observable changes in the colloidal absorption spectra, allowing accurate subtraction of background from the contributions from the colloidal solution. The Raman system used in this study has been described earlier [4] with minor modifications. The samples were excited with 829 nm light from a dye laser (Coherent 599) pumped by an argon ion laser (Coherent, Innova 90). An imaging spectrograph (Holospec 1.8f, Kaiser) equipped with a CCD detection system (Princeton Instruments) was used for light dispersion and signal detection. The Raman spectra were collected with a resolution of 8 cm-' and accumulation time of 25 ms to 5 s. The average power delivered through a fiber optic probe is about 100 mW. The probe is in contact with the quartz window of the sample container. All the spectra shown were corrected for background contributions of colloidal solutions. The fiber optic probe was fabricated using quartz fibers of 100~m core diameter (Corning), and consisted of 19 fibers. At the probing end, the fibers were bundled in such a way that one central excitation fiber was surrounded by two rings of collection fibers, each with 6 and 12 fibers, respectively. At the other end, all 18 collection fibers were arranged in a linear array parallel to the entrance slit of the spectrograph for maximum throughput. RESULTS AND DISCUSSION
Spectrum A in Fig. 1 shows the normal R a m a n spectrum of 7 x 10-1 M dopamine in aqueous solution. As expected from the chemical structures, the "normal" R a m a n spectra of the two compounds are very similar (data not shown for norepinephrine). Spectra B and C in Fig. 1 display SERS spectra of dopamine in gold and silver colloidal solutions, respectively. Spectrum D is the SERS spectrum of norepinephrine in colloidal silver solution. The strong R a m a n bands at 1277, 1448, and 1618 cm -1 are assigned to C - O , CH2, and phenyl ring stretching modes [6]. The R a m a n cross sections of the dopamine and norepinephrine vibrations were determined by comparison with the R a m a n cross section of the 1030 cm -1 band of methanol [11] which was added to the samples as an internal standard. The normal R a m a n cross sections were found to be about 10 -31 cm 2 mol-~ srfor both dopamine and norepinephrine. The surface-enhanced R a m a n specta of dopamine and norepinephrine on colloidal silver (spectra C and D in Fig. 1) are different from the normal R a m a n spectra of the same molecules in aqueous solution for both band positions and relative intensities. The wave numbers and relative intensities of the SERS spectra of dopamine and norepinephrine on colloidal silver are in agreement with the reported SERS spectra of these molecules measured on silver electrodes [6, 7]. Therefore, we conclude that the molecules are adsorbed on the silver colloids through silver-oxygen bonds as discussed previously [6]. It is interesting to note that SERS spectral features of dopamine in colloidal gold solution are similar to those of the normal R a m a n spectra of the molecule in water (see spectrum B in Fig. 1) indicating that dopamine will not chemically adsorb on a gold
484
K. KNEIPPet NIR SERS S p e c t r a
al.
of N e u r o t r a n s m i t t e r s
69
5x 10-6M Dopamine plus 6 M Methanol
n~ 5x 10-~M D o p a m i n e
Difference
R a m a n Shift ( c m -1) Fig. 2. SERS spectrum of 5 x 10 6 M dopamine in colloidal solution of silver (middle), SERS spectrum of 5 x 10 6M dopamine in colloidal solution of silver with 6 M methanol (top). The contribution of methanol Raman signal, obtained by subtracting the middle spectrum from the top one, is shown in the difference spectrum (bottom).
surface. The observed e n h a n c e m e n t which is about on the order of 103 could be explained by the electromagnetic e n h a n c e m e n t of the R a m a n scattering of dopamine molecules "in the vicinity" (average distance between 10 and 50 nm) of the colloidal gold particles [2]. In spite of their very similar " n o r m a l " R a m a n spectra, clear differences appear between dopamine and norepinephrine SERS spectra in the region of 1271-1325 c m - t . Such differences are probably due to small differences in the adsorption behavior of these molecules. SERS, therefore, can be used to improve the sensitivity and selectivity for similarly structured molecules. We will show later that it is possible to use this difference between the SERS spectra of dopamine and norepinephrine to quantify their relative contents in a mixture. The SERS e n h a n c e m e n t factors of dopamine and norepinephrine can be determined using methanol as an internal standard, as methanol R a m a n lines do not indicate any e n h a n c e m e n t [3]. Figure 2 shows the two SERS spectra of 5 x 10 -6 M dopamine silver colloidal solutions without (middle spectrum) and with 6 M methanol (top spectrum), respectively. The enhancement factors of the R a m a n bands of dopamine can be estimated from the relative intensities of these bands and the methanol line to be on the order of 2 x 10 6, taking into account the ratio of the R a m a n cross sections and the different concentrations of neurotransmitters and methanol in the samples. For norepinephrine the same value could be found in an analogous procedure. These estimations were obtained assuming that all molecules were adsorbed and contribute to the SERS signal. Therefore, the factor of 2 x 106 represents a minimum enhancement factor. Figure 3(a) shows SERS spectra of mixtures of different amounts of dopamine and norepinephrine solutions at 10-6M concentrations. The relative increase of the 1325 cm -I band shows a linear correlation (within 10% accuracy) with the percentage of dopamine concentration (Fig. 3(b)). This demonstrates the capability of a quantitative analytical application of SERS in colloidal solutions.
N I R - S E R S of neurotransmitters in colloidal silver solutions (a)
485
o co
Xexe=829 nm
1200
1600
1400
R a m a n Shift ( e m - ~
(b) 09
A p,d
08
~
07
06 ~" 0
05
~. 04 J e~
J
03
02
01
I0
20
30 40 50 60 70 Percentage of D0pamine Concentration (%)
80
90
100
Fig. 3. (a) SERS spectra of mixtures dopamine/norepinephrine. (b) SERS intensity ratio vs. percentage of dopamine concentration.
Figure 4 demonstrates the detection limits of SERS of neurotransmitters in colloidal silver solutions. Spectra could be measured down to 10 -~ and 5 x 10 -~ M concentration levels in 1 and 5 s, respectively, as shown for dopamine in Fig. 4(a). These levels are similar to those of dopamine for animal brain dialysis experiments where dopamine at a level of 5 f m o l min -j was detected [5]. Assuming a flow rate of 0 . 2 p l m i n -~, the dopamine concentration in the brain dialysate should be on the level of 10-~M concentration. Figure 4(b) illustrates the capability of SERS to measure Raman spectra of neurotransmitters in tens of milliseconds which is close to the time scale of the normal neuronal
486
K. KNEIPPet
al.
(a) XExe=829 n m
o co
/ti
~'~ 5xlO-7 M
J
i
800
i
~
i
1200
i
1600
Raman Shift (cm -1) (b) XExe=829 n m
1000
~
-¢°
]
1500 Raman Shift (em-~
Fig. 4. (a) SERS spectra of 5 × 10 7 and 5 × 10 9 M dopamine (accumulation times 1 and 5 s, respectively). (b) SERS spectra of 5 x 10 -6 M dopamine and 5 × 10 6 M norepinephrine measured in 25 ms.
processes. Spectrum A is the dopamine SERS, spectrum B shows the norepinephrine spectrum, both measured at 25 m. For some of the samples in our experiments (for instance the spectra in Fig. 4) the aqueous solutions of the neurotransmitters contained additionally 10% albumin by weight, which is used to check the influence of proteins on the SERS behavior of these neurotransmitters. We could not find any influence of albumin on the SERS spectra of
NIR-SERS of neurotransmitters in colloidal silver solutions
487
the n e u r o t r a n s m i t t e r s . T h e r e f o r e , we b e l i e v e that p r o t e i n s in b r a i n extract s h o u l d not p r e v e n t the S E R S d e t e c t i o n of n e u r o t r a n s m i t t e r s w h e n silver colloids are used. F l u o r e s e n c e b a c k g r o u n d f r o m o t h e r c o m p o u n d s in real b o d y fluids s h o u l d be drastically r e d u c e d with N I R excitation of the S E R S spectra. T h e p r e s e n t work d e m o n s t r a t e s that N I R - S E R S o n colloidal silver could be a powerful tool to study n e u r o t r a n s m i t t e r s in b r a i n extracts a n d dialysates. Acknowledgement--The research is carried out at the Laser Biomedical Research Center of MIT Spectroscopy Laboratory supported by NIH grant # P41-RR02594. One of the authors (K.K.) is grateful to the Deutsche Forschungsgemeinschaft for the award of the Heisenberg Fellowship and travel grant to come to MIT.
REFERENCES [1] [2] [3] [4] [5] [6] [7] [8]
J. J. Laserna, Analyt. Chem. Acta 283, 607 (1993). I. Nabiev, I. Chourpa and M. Manfait, J. Raman Spectrosc. 25, 13 (1994). K. Kneipp, R. R. Dasari and Yang Wang, Appl. Spectrosc., 48, 951 (1994). J. J. Baraga, M. S. Feld and R. P. Rava, Appl. Spectrosc. 46, 187 (1992). B. H. C. Westerink, G. Damsma, H. Rollema, J. B. de Vries and A. S. Horn, Life Sci. 41, 1763 (1987). N. S. Lee, Y. Z. Hsieh, R. F. Paisley and M. D. Morris, Analyt. Chem. 60, 442 (1988). M. L. Mc(31ashen, K. L. Davis and M. D. Morris, Analyt. Chem. 62, 846 (1990). M. D. Morris, M. L. McGlashen and K. L. Davis, SPIE, Vol. 1201, Optical Fibers in Medicine V, p. 447 (1990). [9] H. G. Schuize, M. W. Blades, A. V. Bree, B. G. Gorzalka, L. S. Greek and R. B. Turner, Appl. Spectrosc. 48, 50 (1994). [10] C. Lee and D. Meisel, J. Phys. Chem. 86, 3391 (1982). [11] H. W. Schroetter and H. W. Kloeckner, in Raman Spectroscopy of Gases and Liquids (Edited by A. Weber, p. 123. Springer, Berlin (1979).
Pergamon 0584-8539(94)00235-5
Copyright (~) 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved (1584-8539/95 $9.51)+ 0.00
Near-infrared surface-enhanced Raman scattering (NIR-SERS) of neurotransmitters in colloidal silver solutions K A T R I N K N E I P P , ~ Y A N G W A N G , R A M A C H A N D R A R . D A S A R I a n d M I C H A E L S. F E L D George R. Harrison Spectroscopy Laboratory, Massachusetts Institute of Technology, 77 Massachusetts A v e n u e , Cambridge, M A 02139, U.S.A.
(Received 18 August 1994; accepted 28 October 1994) A b s t r a e t - - N I R - S E R S spectra are measured for the neurotransmitters dopamine and norepinephrine at concentrations as low as 5 x 10 ~ M in colloidal silver solutions with accumulation times as short as 25 ms. The detection range and acquisition time are on the order of physiologically relevant concentrations and the time scale of neuronal processes, respectively. The spectra are obtained using a C C D detection system, dye laser at 830 n m for excitation, fiber optic probe and high throughput spectrograph. Mixtures containing the two neurotransmitters are used to demonstrate the capability of extracting quantitative information from SERS spectra. A l b u m i n added to the sample up to 0.5% concentration does not show any influence on the SERS spectra of the neurotransmitters in the silver colloidal solutions. The results demonstrate the potential of N I R - S E R S in probing dopamine and norepinephrine with high sensitivity and specificity. They also suggest that N I R - S E R S from colloidal silver solution can be a powerful tool for the study of neurotransmitters in brain extracts and dialysates.
INTRODUCTION
SURFACE-enhanced Raman scattering (SERS) has been established as a powerful spectroscopic method which can combine very low detection limits with high molecular specificity [1]. In the past several years, SERS spectroscopy has found growing applications in the field of biomedical research [2]. We have shown recently that SERS in the near-infrared region (NIR-SERS) provides strong electromagnetic enhancement factors if the optical fields (excitation laser and Raman scattered radiations) are in resonance with the longitudinal plasmon resonances of aggregated silver or gold colloids [3]. Electromagnetic enhancement factors on the order of 106 could be achieved for optimized colloidal solutions of silver and gold, which is higher than those achievable with visible excitations. In addition, excitations in the near-infrared reduce fluorescence background from biological materials [4]. For these reasons, NIR-SERS is becoming a useful tool to measure low concentration compounds in biological samples. In this work we demonstrate the application of NIR-SERS in colloidal solutions of silver in studying neurotransmitters. The detection of these molecules in brain extracts or in a brain dialysate at very low concentration levels is an important problem in neurochemistry. It is often required, in particular, to measure concentration changes and to correlate those changes to neuronal events, i.e. measurements done in the time scale of neuronal proceses [5]. Studies of SERS of different neurotransmitters on silver electrodes have been reported previously [6-8]. SERS spectra of dopamine at 3 x 10 -7 M were measured in 10 s using a silver electrode [6]. One of the problems in electrode SERS of neurotransmitters seems to be the attenuation of the SERS signal by protein co-adsorption, which could be reduced using polymer-modified electrodes [7]. It has also been shown that artificial neural networks are capable of accurately identifying the Raman spectra of aqueous solutions of neurotransmitters [9]. However, the detection limit with normal Raman scattering (10-z M) is not suitable for most of the neurological applications. We have selected dopamine (I) and norepinephrine (II) in this investigations (see Fig. l(a) for structural formulas). Dopamine is one of the important neurotransmitters. For instance, the relation between neurotransmitter content and neurotransmission or the measurement of a drug-induced change in the release of dopamine in relation to drug? A u t h o r to w h o m correspondence should be addressed. 481
482
K. KNEIPP et al.
(a)
i"
HO
l
H
H
(i) HO
HO
I H
H
H H
(II) (b)
kExe= 829 n m
c~
A
~
B I1)
>
J
r 1200
Raman
i 16'00
Shift (cm -1)
Fig. 1. (a) Molecular formulas for dopamine (I) and norepinephrine (II). (b) Normal Raman scattering spectrum of 7 x 10 1 M dopamine in water (A, accumulation time 5 s) and its SERS spectra in colloidal gold solution (B, accumulation time 5 s) and in colloidal silver solution (C, accumulation time I s), SERS spectrum of norepinephrine in colloidal silver solution (D, accumulation time 1 s). The concentrations of the neurotransmitters in gold and silver colloidal solutions are approximately l0 -4 M and 10 -7 M, respectively.
induced behavior or drug side effects are questions of great interest in neurology. Norepinephrine which is chemically very similar to dopamine was included in the study to obtain quantitative information from the SERS spectra from mixtures of the two neurotransmitters.
NIR-SERS of neurotransminers in colloidal silver solutions
483
MATERIALS AND METHODS
A solution of colloidal silver was prepared following a standard procedure [9] and diluted with water to achieve a maximum SERS signal for a given concentration of the probed molecule. The measured SERS signal from a colloidal sample results from both the surface enhancement from the colloidal solution and its attenuation (scattering and absorption) of the excitation and scattering lights. Most favorable SERS conditions can be achieved with a colloidal concentration which provides just enough surface area for adsorption of all molecules. Higher colloidal concentrations reduce the Raman signal due to scattering and absorption from the excess colloids which do not contribute to the SERS enhancement. An aqueous solution of NaCI at 1 M concentration was added to the colloidal solutions in about 1:15 ratio. The addition of NaCI solution induces special colloidal aggregation and results in colloidal absorption profiles very similar to those of optimized colloidal silver solutions for NIR excitation reported previously [3]. Dopamine and norepinephrine were purchased from Sigma Chemicals and used directly to prepare 10-4-10 -7 M aqueous solutions with or without 10% albumin by weight. The SERS samples were mixtures of one part of these neurotransmitter solutions with 20 parts of the colloidal solutions of silver. The addition of the neurotransmitters to the colloidal solution in this manner did not give rise to any observable changes in the colloidal absorption spectra, allowing accurate subtraction of background from the contributions from the colloidal solution. The Raman system used in this study has been described earlier [4] with minor modifications. The samples were excited with 829 nm light from a dye laser (Coherent 599) pumped by an argon ion laser (Coherent, Innova 90). An imaging spectrograph (Holospec 1.8f, Kaiser) equipped with a CCD detection system (Princeton Instruments) was used for light dispersion and signal detection. The Raman spectra were collected with a resolution of 8 cm-' and accumulation time of 25 ms to 5 s. The average power delivered through a fiber optic probe is about 100 mW. The probe is in contact with the quartz window of the sample container. All the spectra shown were corrected for background contributions of colloidal solutions. The fiber optic probe was fabricated using quartz fibers of 100~m core diameter (Corning), and consisted of 19 fibers. At the probing end, the fibers were bundled in such a way that one central excitation fiber was surrounded by two rings of collection fibers, each with 6 and 12 fibers, respectively. At the other end, all 18 collection fibers were arranged in a linear array parallel to the entrance slit of the spectrograph for maximum throughput. RESULTS AND DISCUSSION
Spectrum A in Fig. 1 shows the normal R a m a n spectrum of 7 x 10-1 M dopamine in aqueous solution. As expected from the chemical structures, the "normal" R a m a n spectra of the two compounds are very similar (data not shown for norepinephrine). Spectra B and C in Fig. 1 display SERS spectra of dopamine in gold and silver colloidal solutions, respectively. Spectrum D is the SERS spectrum of norepinephrine in colloidal silver solution. The strong R a m a n bands at 1277, 1448, and 1618 cm -1 are assigned to C - O , CH2, and phenyl ring stretching modes [6]. The R a m a n cross sections of the dopamine and norepinephrine vibrations were determined by comparison with the R a m a n cross section of the 1030 cm -1 band of methanol [11] which was added to the samples as an internal standard. The normal R a m a n cross sections were found to be about 10 -31 cm 2 mol-~ srfor both dopamine and norepinephrine. The surface-enhanced R a m a n specta of dopamine and norepinephrine on colloidal silver (spectra C and D in Fig. 1) are different from the normal R a m a n spectra of the same molecules in aqueous solution for both band positions and relative intensities. The wave numbers and relative intensities of the SERS spectra of dopamine and norepinephrine on colloidal silver are in agreement with the reported SERS spectra of these molecules measured on silver electrodes [6, 7]. Therefore, we conclude that the molecules are adsorbed on the silver colloids through silver-oxygen bonds as discussed previously [6]. It is interesting to note that SERS spectral features of dopamine in colloidal gold solution are similar to those of the normal R a m a n spectra of the molecule in water (see spectrum B in Fig. 1) indicating that dopamine will not chemically adsorb on a gold
484
K. KNEIPPet NIR SERS S p e c t r a
al.
of N e u r o t r a n s m i t t e r s
69
5x 10-6M Dopamine plus 6 M Methanol
n~ 5x 10-~M D o p a m i n e
Difference
R a m a n Shift ( c m -1) Fig. 2. SERS spectrum of 5 x 10 6 M dopamine in colloidal solution of silver (middle), SERS spectrum of 5 x 10 6M dopamine in colloidal solution of silver with 6 M methanol (top). The contribution of methanol Raman signal, obtained by subtracting the middle spectrum from the top one, is shown in the difference spectrum (bottom).
surface. The observed e n h a n c e m e n t which is about on the order of 103 could be explained by the electromagnetic e n h a n c e m e n t of the R a m a n scattering of dopamine molecules "in the vicinity" (average distance between 10 and 50 nm) of the colloidal gold particles [2]. In spite of their very similar " n o r m a l " R a m a n spectra, clear differences appear between dopamine and norepinephrine SERS spectra in the region of 1271-1325 c m - t . Such differences are probably due to small differences in the adsorption behavior of these molecules. SERS, therefore, can be used to improve the sensitivity and selectivity for similarly structured molecules. We will show later that it is possible to use this difference between the SERS spectra of dopamine and norepinephrine to quantify their relative contents in a mixture. The SERS e n h a n c e m e n t factors of dopamine and norepinephrine can be determined using methanol as an internal standard, as methanol R a m a n lines do not indicate any e n h a n c e m e n t [3]. Figure 2 shows the two SERS spectra of 5 x 10 -6 M dopamine silver colloidal solutions without (middle spectrum) and with 6 M methanol (top spectrum), respectively. The enhancement factors of the R a m a n bands of dopamine can be estimated from the relative intensities of these bands and the methanol line to be on the order of 2 x 10 6, taking into account the ratio of the R a m a n cross sections and the different concentrations of neurotransmitters and methanol in the samples. For norepinephrine the same value could be found in an analogous procedure. These estimations were obtained assuming that all molecules were adsorbed and contribute to the SERS signal. Therefore, the factor of 2 x 106 represents a minimum enhancement factor. Figure 3(a) shows SERS spectra of mixtures of different amounts of dopamine and norepinephrine solutions at 10-6M concentrations. The relative increase of the 1325 cm -I band shows a linear correlation (within 10% accuracy) with the percentage of dopamine concentration (Fig. 3(b)). This demonstrates the capability of a quantitative analytical application of SERS in colloidal solutions.
N I R - S E R S of neurotransmitters in colloidal silver solutions (a)
485
o co
Xexe=829 nm
1200
1600
1400
R a m a n Shift ( e m - ~
(b) 09
A p,d
08
~
07
06 ~" 0
05
~. 04 J e~
J
03
02
01
I0
20
30 40 50 60 70 Percentage of D0pamine Concentration (%)
80
90
100
Fig. 3. (a) SERS spectra of mixtures dopamine/norepinephrine. (b) SERS intensity ratio vs. percentage of dopamine concentration.
Figure 4 demonstrates the detection limits of SERS of neurotransmitters in colloidal silver solutions. Spectra could be measured down to 10 -~ and 5 x 10 -~ M concentration levels in 1 and 5 s, respectively, as shown for dopamine in Fig. 4(a). These levels are similar to those of dopamine for animal brain dialysis experiments where dopamine at a level of 5 f m o l min -j was detected [5]. Assuming a flow rate of 0 . 2 p l m i n -~, the dopamine concentration in the brain dialysate should be on the level of 10-~M concentration. Figure 4(b) illustrates the capability of SERS to measure Raman spectra of neurotransmitters in tens of milliseconds which is close to the time scale of the normal neuronal
486
K. KNEIPPet
al.
(a) XExe=829 n m
o co
/ti
~'~ 5xlO-7 M
J
i
800
i
~
i
1200
i
1600
Raman Shift (cm -1) (b) XExe=829 n m
1000
~
-¢°
]
1500 Raman Shift (em-~
Fig. 4. (a) SERS spectra of 5 × 10 7 and 5 × 10 9 M dopamine (accumulation times 1 and 5 s, respectively). (b) SERS spectra of 5 x 10 -6 M dopamine and 5 × 10 6 M norepinephrine measured in 25 ms.
processes. Spectrum A is the dopamine SERS, spectrum B shows the norepinephrine spectrum, both measured at 25 m. For some of the samples in our experiments (for instance the spectra in Fig. 4) the aqueous solutions of the neurotransmitters contained additionally 10% albumin by weight, which is used to check the influence of proteins on the SERS behavior of these neurotransmitters. We could not find any influence of albumin on the SERS spectra of
NIR-SERS of neurotransmitters in colloidal silver solutions
487
the n e u r o t r a n s m i t t e r s . T h e r e f o r e , we b e l i e v e that p r o t e i n s in b r a i n extract s h o u l d not p r e v e n t the S E R S d e t e c t i o n of n e u r o t r a n s m i t t e r s w h e n silver colloids are used. F l u o r e s e n c e b a c k g r o u n d f r o m o t h e r c o m p o u n d s in real b o d y fluids s h o u l d be drastically r e d u c e d with N I R excitation of the S E R S spectra. T h e p r e s e n t work d e m o n s t r a t e s that N I R - S E R S o n colloidal silver could be a powerful tool to study n e u r o t r a n s m i t t e r s in b r a i n extracts a n d dialysates. Acknowledgement--The research is carried out at the Laser Biomedical Research Center of MIT Spectroscopy Laboratory supported by NIH grant # P41-RR02594. One of the authors (K.K.) is grateful to the Deutsche Forschungsgemeinschaft for the award of the Heisenberg Fellowship and travel grant to come to MIT.
REFERENCES [1] [2] [3] [4] [5] [6] [7] [8]
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