EP1226421A1 - Nouveaux substrats actifs (sers) a diffusion raman superficielle amelioree et procede d'interfa age entre spectroscopie raman et electrophorese capillaire - Google Patents

Nouveaux substrats actifs (sers) a diffusion raman superficielle amelioree et procede d'interfa age entre spectroscopie raman et electrophorese capillaire

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Publication number
EP1226421A1
EP1226421A1 EP00970628A EP00970628A EP1226421A1 EP 1226421 A1 EP1226421 A1 EP 1226421A1 EP 00970628 A EP00970628 A EP 00970628A EP 00970628 A EP00970628 A EP 00970628A EP 1226421 A1 EP1226421 A1 EP 1226421A1
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EP
European Patent Office
Prior art keywords
sers
substrate
eluant
solid support
raman
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP00970628A
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German (de)
English (en)
Inventor
Michael J. Natan
Lin He
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Alavita Pharmaceuticals Inc
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Surromed Inc
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Publication of EP1226421A1 publication Critical patent/EP1226421A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites

Definitions

  • the invention is directed to the technique of Raman spectroscopy, specifically to the technique of surface enhanced Raman scattering (SERS)-based Raman spectroscopy.
  • SERS surface enhanced Raman scattering
  • the invention provides novel SERS-active substrates, and also provides a novel interface between SERS-based Raman spectroscopy and capillary electrophoresis (CE).
  • Raman spectroscopy is an ultrasensitive chemical analysis method, well known in the art.
  • the technique relies on the Raman effect, in which the energy of photons that are incident on a molecule is coupled into distinct vibrational modes of the molecule's bonds. Such coupling causes some of the incident photons to be inelastically scattered by the molecule with a range of energies (wavelengths) that differ from the energy of the incident light.
  • the Raman shift can be expressed numerically in wavenumbers (cm "
  • a Raman spectrum is a plot of wavenumber versus intensity. Particular molecular structures deplete the incident photons of specific amounts of energy, thereby causing band(s) to appear at specific wavenumber positions in the Raman spectrum. The intensity value for each band is proportional to the concentration of the molecular structure.
  • Raman spectroscopy can yield structural and quantitative data about samples of unknown molecular composition.
  • the incident illumination for Raman spectroscopy usually provided by a laser, can be concentrated to a small spot if the spectroscope is built with the configuration of a microscope. Since the Raman signal scales linearly with laser power, light intensity at the sample can be very high in order to optimize sensitivity of the instrument.
  • Raman response of a molecule occurs essentially instantaneously (without any long-lived highly energetic intermediate states)
  • photobleaching of the Raman-active molecule by this high intensity light is impossible.
  • Raman spectroscopy in stark contrast to fluorescence spectroscopy, where photobleaching dramatically limits many applications.
  • Raman spectra can be acquired at any excitation frequency, thereby allowing an excitation wavelength to be chosen that minimizes adverse photochemical effects or background fluorescence.
  • the Raman effect can be enhanced at least 10 6 fold by bringing the Raman-active molecule(s) close to a structured noble metal surface (such as gold, silver, or copper), typically through absorption. Enhancement can also be observed when the Raman-active molecule(s) are brought close to structured surfaces of certain other metals as well (such as sodium and potassium).
  • a structured noble metal surface such as gold, silver, or copper
  • Enhancement can also be observed when the Raman-active molecule(s) are brought close to structured surfaces of certain other metals as well (such as sodium and potassium).
  • SERS surface-enhanced Raman scattering
  • SERS-active substrates substrates that demonstrate the SERS effect.
  • SERS substrates SERS-active substrates, or SERS substrates.
  • SERS refers both to the physical phenomenon of surface enhanced Raman scattering, and also to Raman spectroscopy of analytes associated with SERS-active substrates.
  • SERS Raman-active adsorbates
  • SERS exhibits advantages in fluorescence discrimination, limited interference in aqueous media, and the ability to be used in a variety of sensing environments.
  • SERS has also been successfully interfaced with a number of separation techniques, including gas chromatography, liquid chromatography, and flow injection analysis for the quantitative and qualitative analysis of polymers, dyes, environmental and biological molecules.
  • SERS has been an invaluable analytical tool; however, the variability in signal response from SERS-active substrates limits the technique from attaining its enormous potential. It is known in the art that the optical properties of SERS-active substrates depend critically upon the feature size, shape, inter-feature spacing and the extent of coupling between surface features.
  • the art contains reports of potential substrate architectures and preparation techniques. Examples include electrochemically-roughened electrodes, laser ablation of metals, aggregates of colloidal Au or Ag particles, chemically etched metal surfaces, and evaporated metal films.
  • an effective 2-layer SERS substrate comprising a microarray of Ag-coated colloidal Au immobilized on a silanized glass surface is described in Bright, R.
  • CE capillary electrophoresis
  • Analytes within the injected sample are separated from one another based on its charge/size ratio in the capillary eluant at different times.
  • the scope of this technique has been further expanded with the emergence of capillary array electrophoresis, a major technology used for the Human Genome Project, and miniaturized CE, where biological assays can be performed on a single chip.
  • LIF Laser- induced fluorescence
  • derivatization strategies have been developed to enable detection of otherwise non-fluorescent analytes, these processes can be time-consuming and difficult.
  • Other detection approaches including electrochemical, thermo-optical, and chemiluminescence methods have been explored with varying degrees of success.
  • none of the aforementioned techniques can provide qualitative information about the analytes beyond retention times. Since structure determination by retention time alone requires extensive knowledge of the sample beforehand (i.e., a knowledge of the likely components of the sample, coupled with knowledge of the retention time of each component under the particular experimental conditions used), the identification of unknown compounds using these methods is quite complicated.
  • MS mass spectrometry
  • colloidal Ag is a rather crude, variable, and non-optimal SERS substrate, so the full sensitivity potential of SERS cannot be realized.
  • Ag particles gradually build up on the walls of the capillary, leading to a degradation in the SERS response of the analytes.
  • the substrate comprises a submonolayer of microparticles on a solid support, each microparticle comprising a Ag-clad colloidal Au particle, over which microparticles lies a thin, non-continuous layer of Ag film.
  • the 3 layer substrate displays excellent reproducibihty: spectral deviations within different regions of a single 3 layer substrate are of the same magnitude as spectral deviations observed among individual 3 layer substrates from different manufacturing batches.
  • the 3-layer SERS substrate allows the routine detection of analytes in sub-picogram amounts.
  • the invention also provides a novel interface between capillary electrophoresis and SERS that allows the use of the 3-layer SERS substrate for eluant detection.
  • eluant leaving the capillary is deposited onto a moving SERS substrate to form a linear eluant trail.
  • the eluant trail is then analyzed by a Raman microscope, either immediately after each eluant drop is deposited, or after the entire eluant trail is deposited.
  • the eluant trail preserves in a spatial format the temporal separation achieved by CE. Consequently, separation results can be preserved for later examination or for further analysis using a second detection method.
  • Figure 1 depicts schematically stages in the synthesis of Au-Ag-Ag 3-layer SERS- active substrates.
  • Figure 2 illustrates the enhancement in Raman intensity provided by an Au-Ag-Ag
  • Figure 3 shows the detection sensitivity of 1,3,5-Triazine using an Au-Ag-Ag 3- layer SERS-active substrate.
  • Figure 4 depicts schematically the interface between SERS-based Raman spectroscopy and capillary electrophoresis.
  • Figure 5 illustrates the detection by Raman spectroscopy of trans- 1 ,2-bis(4- pyridyl) ethylene (BPE) deposited onto a SERS-active substrate by capillary electrophoresis.
  • Figure 6 shows the time course for separation of BPE and N,N-dimethyl-4- nitrosaniline (p-NDMA) by capillary electrophoresis as monitored by UV-visible spectroscopy (FIGURE 6A) and SERS (FIGURE 6B).
  • Figure 7 shows full Raman spectra for each of points a-f identified in FIGURE 6B.
  • Figure 8 shows a time course for separation of BPE and p-NDMA by capillary electrophoresis as monitored by Raman spectroscopy at wavenumbers where only BPE (curve 2) or p-NDMA (curve 3) show a Raman response.
  • Figure 9 shows a UV-vis spectrograph (FIGURE 9A) and Raman spectra (FIGURES 9B and 9C) for the separation by capillary electrophoresis of a mixture of tyrosine and tryptophan.
  • FIG. 10 shows a UV-vis spectrograph (FIGURE 10A) and Raman spectra (FIGURES 10B and IOC) for the separation by capillary electrophoresis of a mixture of chlorophenol (CP) and dichlorophenol (DCP).
  • CP chlorophenol
  • DCP dichlorophenol
  • the invention provides a 3 layer SERS-active substrate comprising a submonolayer of microparticles attached to a solid support, each microparticle comprising a colloidal particle of first noble metal clad with a second noble metal, and said microparticles overlaid with a thin, non-continuous film of said second noble metal.
  • the substrate can be synthesized by first immobilizing the colloidal metal particles comprising the first noble metal on a solid support, then coating the immobilized colloidal particles with a plating solution containing the second noble metal in a chemically reduced form. Finally, the resulting clad colloidal particles are evaporatively coated with a discontinuous layer of the second noble metal.
  • the first noble metal is Au and the second noble metal is Ag.
  • SERS substrates of this composition are referred to as Au-Ag-Ag substrates.
  • the Au colloid is approximately a 12 nm diameter colloid
  • the Ag film is 10 nm - 30 nm in thickness, most preferably 20 nm in thickness.
  • FIGURE 1 illustrates schematically the stages in the one embodiment of synthesis of Au-Ag-Ag substrates.
  • a glass slide 10 is first derivatized with an organosilane, such as 3-mercaptopropylmethyl-dimethoxysilane (MPMDMS).
  • MPMDMS forms a self- assembled monolayer 11 on the glass surface;
  • Au colloid 12 can bind via the thiol group of MPMDMS to form a Au colloid microarray on the glass slide.
  • Other possible methods and reagents for immobilizing Au colloid on a glass slide are known to those skilled in the art.
  • Example 1 provides an example of a detailed protocol for the synthesis of Au-Ag-Ag substrates.
  • the optical properties of SERS-active substrates depend critically upon the feature size, shape, inter-feature spacing, and the extent of coupling between the surface features.
  • the colloid size, the rate of evaporative deposition, and the length of the evaporative deposition step (thickness of the overlayer of the second noble metal) are varied in order to maximize the SERS enhancement of the 3-layer substrate.
  • these factors are chosen so as to obtain a substrate in which the evaporated second noble metal both forms discrete "islands" between the colloid particles, and also enlarges the size of the colloid particles.
  • this substrate morphology enhances SERS activity by both increasing the surface area for analyte adsorption, and also by increasing the electromagnetic enhancement through the "rod-like" effect, as described in Creighton, J. A. The Selection Rules for Surface- Enhanced Raman Spectroscopy, Clark, R. J. H. and Hester, R. E., Ed.; John Wiley & Sons Ltd., 1988; Vol. 16, pp 37-89, incorporated herein by reference in its entirety.
  • FIGURE 2 illustrates the enhancement in the SERS activity of the 3-layer substrates provided by the invention by comparing the SERS spectra of 2 ⁇ L of 0.1-mM trans- 1 ,2-bis(4-pyridyl) ethylene (BPE) solution drop-coated onto each of the substrates illustrated in FIGURE 1.
  • the 3-layer substrates of the instant invention are useful in any application where it is necessary to detect the presence of a particular analyte(s). Because of their large enhancement factor, the present substrates are especially useful for the detection of analytes that are present at low concentrations. In particular, the substrates are useful for the sensing of biomolecules that are present at low concentrations in biological fluids.
  • the sensitivity of the 3-layer SERS substrates of the instant invention is demonstrated in FIGURE 3 for the environmentally important compound 1,3,5-Triazine. 1,3,5-Triazine is the parent compound of the most prevalently used herbicide family.
  • FIGURE 3 illustrates a plot of the logarithmic concentration value of 1,3,5-Triazine against the normalized peak area of the 926 cm "1 band (from ring breathing) that is characteristic of the compound (using 12 mW of 633 nm excitation illumination).
  • the peak area has been normalized by taking the quotient of the raw peak area and integration time.
  • the inset to FIGURE 3 shows the SERS spectra of 2 ⁇ L of 8 x 10 "3 M (30 second integration) and 8 x 10 "8 M solution (45 seconds) of 1,3,5-Triazine. It can be seen that the 3-layer substrates of the instant invention allow for the detection of picogram quantities of 1,3,5-Triazine, at low excitation powers and in less than a minute of integration time.
  • the 3-layer SERS substrates of the instant invention are a significant improvement over prior art SERS substrates not only because of their increased SERS activity, but also because of the reproducibihty with which they can be fabricated.
  • Table 1 compares the peak areas for the 1010, 1200, and 1610-1640 cm "1 SERS bands of 10 mM BPE taken at five different spots on the same 3-layer substrate. Good reproducibihty between spectra is observed, with all relative average deviations less than 13 %. Similar reproducibihty can be realized for substrates prepared simultaneously and in separate substrate batches; a substrate batch is defined as a group of slides, typically eight due to limitations of evaporation chamber space, who simultaneously undergo each fabrication step.
  • Table 2 gives peak area comparisons for SERS spectra of 10 mM BPE on substrates from six different substrate batches. Again, good reproducibihty is observed, and the average relative deviations is of the same magnitude as a single substrate, with all values less than 12 %. It should also be noted that the data was collected for batches fabricated over an eight-month period, further demonstrating the ability to make reproducible substrates of the instant invention.
  • a method is taught to interface SERS substrates with capillary electrophoresis (CE).
  • CE capillary electrophoresis
  • a SERS substrate is placed underneath the outlet of an electrophoresis capillary such that eluant from the capillary — containing resolved analytes - is deposited onto the SERS substrate.
  • the SERS substrate is associated with translation means to enable the resolved analytes with the eluant to be deposited at different regions of the SERS substrate.
  • FIGURE 4 illustrates an especially preferred embodiment in which the translation means is a computer controlled x-y translation stage 41, wherein the computer controls the position and velocity of substrate 42.
  • the substrate can be translated in such a way that the eluant leaving capillary 43 at outlet 44 is deposited in a linear trail - such as a continuous "S" pattern 45 - that preserves the temporal separation achieved by CE.
  • a marker dye may be included in the eluant.
  • One suitable dye contemplated by the invention is Kiton Red 620.
  • any translation pattern that deposits the eluant in a linear, non-overlapping fashion is contemplated by the invention.
  • any SERS substrate that can be used to deposit a linear eluant trail is also contemplated.
  • eluant can be deposited on a cylindrical SERS substrate as a ring trail or spiral trail. Eluant can be deposited either continuously, or dropwise.
  • Raman microscopy is used to acquire a Raman spectrum at each point along the trail.
  • a Raman spectroscope with a remote fiber-optic probe (often referred to as a Raman microprobe) is employed to retrace the eluant trail.
  • a Raman spectroscope with a remote fiber-optic probe (often referred to as a Raman microprobe) is employed to retrace the eluant trail.
  • Laser excitation light 46 preferably from a He-Ne laser 47 (632.8 nm)
  • SERS light from the substrate is then collected by the microscope objective 49, and directed to the Raman spectrometer 410 by the optical fiber 48.
  • the microscope objective has a focal length of between 3 mm - 8mm, leading to a laser spot size of 5 ⁇ m - 10 ⁇ m on the SERS substrate.
  • a 3 mm objective is used to focus 20 mW of incident illumination, allowing a SERS spectrum to be acquired in ⁇ 5s.
  • FIGURE 5 illustrates data obtained from one embodiment of the invention in which a 10 mM BPE solution was subjected to CE, then deposited onto (a) a 2-layer SERS substrate (Ag-clad Au colloid monolayer) and (b) a blank glass slide, and then examined by Raman microscopy (3 mm objective, 20 mW of 632.8 nm illumination, 5 second integration).
  • the complete lack of Raman response obtained from the BPE on the blank glass slide provides ample evidence that conventional Raman fails as a detection method for these low levels of analyte.
  • the response observed from the Ag-clad Au colloid monolayer substrate clearly demonstrates that SERS is a viable alternative to conventional Raman for identification of analytes separated by CE.
  • Analyte identification based on the SERS-retracing method provided herein is far more accurate than prior art analyte identification techniques that are based on retention time alone. Because the Raman spectrum of each analyte is complex (often consisting of ten or more discrete peaks), it provides an unambiguous and unique "fingerprint" for that analyte. By contrast, prior art UV-vis and fluorescence spectra provide just a single peak that cannot alone identify an analyte without knowledge of retention times.
  • the SERS-retracing method provided herein obviates the need to use analyte retention time as the exclusive basis for analyte identification.
  • the eluant trail preserves, in a spatial format, the temporal resolution achieved by CE.
  • each position on the SERS substrate represents a different elution/retention time point.
  • the Raman spectroscope can examine just those timepoints where the analyte of interest would be expected to be eluted based on prior knowledge of retention time.
  • the eluant trail can be examined first by Raman spectroscopy, and then specific time points on the substrate can be examined by other detection methods, such as mass spectrometry.
  • Raman spectroscopy Raman spectroscopy
  • specific time points on the substrate can be examined by other detection methods, such as mass spectrometry.
  • the SERS-retracing method of the instant invention allows the use of optimized SERS-active substrates.
  • the SERS substrate is the 2- layer substrate comprising Ag-clad Au colloid.
  • the SERS- retracing method is used with the 3-layer substrates provided by the instant invention, most preferably with Au-Ag-Ag 3-layer substrates.
  • FIGURES 6-8 illustrate SERS detection of CE- separated BPE and N,N-dimethyl-4- nitrosaniline (p-NDMA).
  • p-NDMA N,N-dimethyl-4- nitrosaniline
  • FIGURE 6A UV-vis analysis of the separation is presented (FIGURE 6A).
  • the UV- vis elution profile shows two distinct but not fully separated peaks.
  • the constant speed of the translation stage allows the spatial location of both BPE and p- NDMA deposited on the substrate to be converted into a time scale to obtain information on the analyte retention times.
  • FIGURE 6B shows the time course of the changes of integrated SERS intensity in the wavenumber range 1600-1650 cm “1 , a region where both BPE and p-NDMA display Raman response. Inspection of the retention times for both eluants shows agreement with those recorded by UV-vis, illustrating that the CE-SERS interface can perform the same detection function as UV-vis.
  • FIGURE 7 shows SERS spectra corresponding to each of the points indicated in the lower panel of FIGURE 6.
  • Spectra a, e and f represent three examples of SERS spectra of the background, a pH 6.8 phosphate buffer with a low SERS response from 1000 cm “1 to 1700 cm “1 , that were taken along the CE-deposited eluent trace.
  • the only Raman activity observed for these positions were two sizable peaks at 616 cm “1 and 920 cm “1 , which are attributed to components in the LI Ag plating solutions and were present in all SERS spectra collected; their intensities were markedly decreased in the presence of analyte, presumably due to competition for surface sites (data not shown).
  • FIGURE 8 shows a time course of integrated SERS intensity from 1230-1180cm “1 (curve 2), the range in which only BPE showed Raman response, and a plot of SERS intensity from 1180-1130 cm “1 (curve 3) which is unique for p-NDMA response in this experiment.
  • Curve 1 is the base line curve showing SERS intensity at 1600-1650 cm " where both BPE and p-NDMA show a Raman response.
  • FIGURE 9 illustrates SERS data from a CE separation of the amino acids tyrosine and trytophan (1 :1 ratio).
  • FIGURE 9 A shows the UV eluant profile for the separation, and reveals a significant interval in retention times (several minutes between peaks).
  • FIGURE 9 A and FIGURE 9B show the corresponding SERS spectra of tyrosine and tryptophan, respectively.
  • the peak at 837 cm “1 (FIGURE 9B) is characteristic of the ring breathing mode of the p-hydroxyphenyl moiety in tyrosine, while the 1334 cm “1 Raman band (FIGURE 9C) derives from the indole ring of tryptophan .
  • a number of other bands further served to identify, both analytes, namely those that appear at 1176 and 1603 cm “1 for Tyr and at 1012 and 1607 cm “1 for Trp, all of which correspond well to the Raman spectra of the respective amino acids.
  • the data in FIGURE 9 was obtained using the methods of Example 3 and 1 mg/ml solution of tyrosine and trytophan.
  • FIGURE 10 shows data from the application of the SERS-retracing method to a mixture of chlorophenols.
  • FIGURE 10A shows the UV eluant profile of 2-chlorophenol (CP) and 2,4-dichlorophenol (DCP).
  • SERS spectra for the eluted CP and DCP are presented in FIGURE 10B and FIGURE 10C, respectively. Specific bands observed from the SERS spectra can be used to identify each of the compounds.
  • the 562 cm “1 band in FIGURE 10B is a strong indication of the presence of ortho-disubstituted benzene, while the peaks at 851, 1143 and 1285 cm “1 in FIGURE 10C correspond well with the Raman spectrum of pure dichlorophenol. Note that the Raman bands of DCP at 700 and 1028 cm “1 overlapped bands from propanethiol, a compound used to coat the substrate in order to improve the adsorption of hydrophobic compounds.
  • Substrate Preparation Glass slides were cut and cleaned as described in Grabar, K. C. et al, Anal. Chem. 61:135-1 'A3 (1995), or through successively sonicating for 20 minutes in water, methanol, and acetone. Glass slides were functionalized in a 5-10% solution of MPMDMS in methanol for 1 hour. Copious rinsing with methanol and then water were proceeded to remove any unbound MPMDMS. Functionalized substrates were coated with a 12-nm colloidal Au solution for 90 or 95-s, depending on colloid concentration. Substrates were then rinsed with water and partially dried under an Ar stream.
  • Optical spectra were taken after each step above to monitor the surface quality. Film integrity was characterized by UV-visible absorbance spectroscopy, with quality control based on the shape and height of the Au plasmon peak. Suitable substrates were then used to form the 2-layer substrate as described above. The surface plasmon peak in the UV spectrum of the 2-layer substrate is shifted relative to the Au colloid alone, probably due to the optical property of coated Ag. In addition, the 2-layer substrate has a new shoulder around 620 nm caused by the particle coupling effect. After the discontinuous Ag overlayer was evaporated over the 2-layer substrate (3-layer substrate), the shoulder became more pronounced as the result of stronger coupling from increased surface coverage.
  • Trans- 1 ,2-bis(4-pyridyl) ethylene was obtained from Aldrich. BPE was recrystallized several times before use, and a stock 10-mM BPE solution was then prepared in a fresh H 2 O:CH 3 OH (9:1) solvent solution. Dilutions were made as needed to yield 1 mM, 0.1 mM, and 0.01 mM solutions in H 2 O. These solutions were then drop- coated onto the various SERS substrates. SERS spectra were obtained on a Solution 633 micro-Raman spectrometer purchased from Detection Limit, Inc. Excitation was provided by a 20-mW, 632.8-nm HeNe laser. All spectra were taken with a 3-mm focal-length objective ( ⁇ 5 ⁇ m diameter in the spot size). Spectra were collected by a CCD detector with TE-cooling system around ⁇ 8.5°C. The system was operated and monitored by
  • a CE system was constructed similar to those previously described in Tracht et al, Anal. Chem. 66:2382-2389 (1994) and in Olefirowicz, T. M.& Ewing, A. G., J. Chromatogr. 499:713-719 (1990), each of which is incorporated herein by reference in their entirety.
  • Sample injection was accomplished via a hydrostatic method, as described in Landers, J. P. Handbook of capillary electrophoresis; CRC Press (1994), in Engelhardt et al, Angew. Chem., Int. Ed.
  • the CE system was contained within a Plexiglas box secured with interlock; thus the power supply would automatically shut off when the lid was open.
  • the capillary was activated and cleaned subsequently with 1 M NaOH, 18 M ⁇ H 2 O, and 50-mM phosphate buffer at the beginning of every day. Before each injection, the capillary was again flushed with a fresh buffer solution.
  • a linear variable- wavelength UV -visible detector was used for on-line detection in order to compare results with those acquired by SERS.
  • Surface Enhanced Raman Scattering A Solution 633 Fiber-Optic microRaman
  • Spectrometer with a remote probe was used for acquisition of Raman spectra.
  • a He-Ne laser (632.8 nm) was used as the excitation source, and microscope objectives with focal lengths of 3 mm or 8 mm were used to collect spectra.
  • the laser light focusing step was accomplished by adjusting the probe height manually.
  • a light filter was used to adjust the output power from 0 - 20 mW.
  • Spectra were collected by a TE-cooled CCD camera.
  • FIGURE 4 The interface between CE and SERS is presented in FIGURE 4.
  • a 2-layer SERS susbtrate (Ag-clad Au colloid) was placed under the metallized outlet of the capillary.
  • the analyte was deposited directly onto the substrate during separation while the substrate was moving in a continuous 'S' fashion.
  • Peak Mode 3 Integrated Peak Area Standard Relative Average

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Abstract

L'invention porte sur de nouveaux substrats actifs (SERS) à diffusion Raman superficielle améliorée (42) pour spectroscopie Raman à SERS encore accrue et meilleure reproductibilité que les substrats antérieurs. L'invention porte également sur une nouvelle interface (45) entre électrophorèse capillaire et spectroscopie Raman.
EP00970628A 1999-10-06 2000-10-06 Nouveaux substrats actifs (sers) a diffusion raman superficielle amelioree et procede d'interfa age entre spectroscopie raman et electrophorese capillaire Withdrawn EP1226421A1 (fr)

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US15795899P 1999-10-06 1999-10-06
US16893699P 1999-12-03 1999-12-03
US168936P 1999-12-03
US16933999P 1999-12-06 1999-12-06
US169339P 1999-12-06
PCT/US2000/027667 WO2001025757A1 (fr) 1999-10-06 2000-10-06 Nouveaux substrats actifs (sers) a diffusion raman superficielle amelioree et procede d'interfaçage entre spectroscopie raman et electrophorese capillaire
US157958P 2009-03-06

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US20050148098A1 (en) * 2003-12-30 2005-07-07 Xing Su Methods for using raman spectroscopy to obtain a protein profile of a biological sample
WO2005078415A1 (fr) * 2004-02-13 2005-08-25 Omron Corporation Capteur de résonance plasmon de surface
WO2006073117A1 (fr) * 2005-01-07 2006-07-13 Kyoto University Capteur optique et procédé de fabrication idoine
JP4685650B2 (ja) * 2005-02-14 2011-05-18 富士フイルム株式会社 ラマン分光用デバイス、及びラマン分光装置
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