JP2003511666A - Novel surface-enhanced Raman scattering (SERS) -active substrate and method of connecting Raman spectroscopy to capillary electrophoresis (CE) - Google Patents

Abstract

Summary of the Invention The present invention provides a novel surface enhanced Raman scattering (SERS) active substrate (42) for Raman spectroscopy that provides higher SERS enhancement and reproducibility than prior art substrates. . The present invention also provides a novel connection (45) between capillary electrophoresis (CE) and Raman spectroscopy.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to Raman spectroscopy techniques, in particular surface-enhanced Raman scattering (SERS)-
Based on Raman spectroscopy technology. The present invention provides a novel SERS-active substrate and also provides a novel connection between SERS-based Raman spectroscopy and capillary electrophoresis (CE). BACKGROUND OF THE INVENTION Raman spectroscopy is an ultrasensitive chemical analysis method well known in the art. The above technique relies on the Raman effect in which the energy of the photons incident on the molecule is coupled to the different modes of vibration of the molecular bond. In such coupling, some of the incident photons have different energy (wavelength) from the energy of the incident light.
Causes non-adaptive scattering by a range of molecules. Incident light quantum (wavelength =
The difference between λ incident) and scattered light quantum (wavelength = λ scattering) is called Raman shift. The Raman shift can be expressed numerically by wave number (cm ⁻¹ ) by calculating 1 / λ incident −1 / λ scattering.

Raman spectra are plots of wavenumber versus intensity. A particular molecular structure robs an incident photon of a certain amount of energy, thereby causing a band to appear at a particular wavenumber position in the Raman spectrum. The intensity value of each band is proportional to the concentration of the molecular structure. Therefore, Raman spectroscopy can yield structural and quantitative data for samples of unknown molecular composition.

The incident illuminance of Raman spectroscopy, which is usually supplied by a laser, can be concentrated to small spots if the spectrograph is constructed with a microscope arrangement. Since the Raman signal is linearly proportional to the laser power, the light intensity in the sample can be very high in order to optimize the sensitivity of the device. Moreover, the Raman response of the molecule occurs essentially instantaneously (without any long-lived, high-energy intermediate states), so photobleaching of Raman-active molecules with this high intensity of light is not possible. This makes Raman spectroscopy in stark contrast to fluorescence spectroscopy, and photobleaching dramatically limits many applications. In addition, Raman spectra can be acquired at any excitation frequency, allowing the excitation wavelength to be selected which minimizes photochemical counteractions or background fluorescence.

The Raman effect is the Raman-active molecule that allows a new metallic surface to be constructed (eg, gold,
Silver, or copper) by at least 1
Can be enhanced by a factor of 0 ⁶ . Enhancement can also be observed when the Raman active molecule is also brought close to the constructed surface of other specific metals (eg sodium and potassium). The mechanism by which this surface-enhanced Raman scattering (SERS) occurs is not well understood, but (i) plasmon resonance of the surface in the metal to enhance the local intensity of light, and (ii) the metal surface It is believed to result from the combination of the formation of a charge transfer complex between the Raman active molecules and the subsequent transfer. Substrates exhibiting the SERS effect are called SERS-active substrates or SERS substrates. As used herein, the term SERS refers to both the physical phenomenon of surface-enhanced Raman scattering and Raman spectroscopy of analytes associated with SERS-active substrates.

The detection of sublayers of Raman-active adsorbates is
It can be easily achieved using SERS. In addition, SERS offers advantages in fluorescence discrimination, limited interference in aqueous media, and the ability to be used in various sensing of the environment. SERS has also been successful in linking many separation techniques, including gas chromatography, liquid chromatography, and flow injection analysis for quantitative and qualitative analysis of polymers, dyes, environmental and biomolecules.

So far, SERS has been a worthless analytical tool; however, SERS
The variability in the signal response from active substrates has limited the technology to achieve its many potentials. It is known in the art that the optical properties of SERS-active substrates are critically dependent on the size of features, morphology, mutual feature space and extent of coupling between surface properties. The art includes a report on the dominant substrate structures and manufacturing techniques. Examples include electrochemically roughened electrodes, laser ablation of metals, agglomeration of colloidal gold or silver particles, chemically etched metal surfaces, and evaporated metal films. In addition, an effective bilayer SERS substrate comprising silver coated colloidal gold microarrays immobilized on a silanized glass surface is described in Bright, R. et al. M et al, “Chemi
cal and Electrochemical Ag Depositio
nonto Preformed Au Colloid Properti
es "Langmuir 12: 810-817 (1996). This two-layer SERS substrate has the favorable properties of gold-monodispersity and ease of operation-and the excellent SERS enhancer of silver. Combine.

Among these prior art, SERS with vapor deposited silver film
-The production of active substrates is popular due to its stability and ease of handling. The dependence of SERS activity on the morphological and optical properties of vapor deposited thin metal films has been extensively studied in the art as a function of deposition rate, bonding structure, and temperature. Some attempts have also been reported by pre-deposition immobilization of microparticles such as latex beads, evaporated silica, and alumina powder on the surface for better control of the surface roughness. Substrate enhancement improved to a certain level,
The reproducibility of substrate fabrication remains an important limiting factor in many SERS applications. It is an object of the present invention to provide new SERS-active substrates that approach these limitations of the prior art. In particular, it is an object of the present invention to provide SERS substrates with increased enhancement factors and increased manufacturing reproducibility.

It is also an object of the present invention to extend its application as a SERS detection method. In particular, it is an object of the present invention to connect SERS with capillary electrophoresis (CE) technology. CE is known in the art for its rapid, efficient, and high-resolution analysis of nanoliter volume samples and ranges from simple metal ions to large molecules such as proteins and DNA fragments. It has become a dominant separation technique used for analytes. CEs are typically fused silica capillaries (in some applications up to 100 cm long, 10 μm long in some applications).
This is accomplished by injecting the sample at one end (inside diameter <m) and then applying a very high potential difference across the ends of the capillary. The analyte in the injected sample is separated from the other based on its charge / size ratio in the capillary eluate at another time. The scope of this technology is the urgency of capillary array electrophoresis,
The key techniques and biological assays used for the human genome project have been further extended by minimized CE when performed on a simple chip.

Despite its low sensitivity, UV absorption detection on a column is by far its most common CE separation monitoring tool because of its low cost and flexibility. Laser induced fluorescence (LIF) offers much higher sensitivity but requires a fluorescent analyte. Derivatization strategies have been developed to allow the detection of non-fluorescent analytes by other methods, but these processes are time consuming and difficult. Other detection approaches, including electrochemistry, temperature optimization, and chemiluminescence, have been explored with varying degrees of success. However, none of the above techniques can provide qualitative information about the analyte beyond the retention time. Structure determination based only on retention time requires a wide range of sample information in advance (
The identification of unknown components using these methods is quite complex, i.e. the information on the likely components of the sample combined with the information on the retention time of each component under the particular experimental conditions used.

To circumvent this drawback, mass spectroscopy (MS) was developed as another detection method for CE. MS provided molecular mass data and structural information based on the fragmentation pattern and was successfully connected to CE. However, this technique often requires complex CE / MS connections and presents challenges in device design.

Morris and co-workers have developed an online convention for CE (non-SERS
) Proved Raman detection. For example, they use 10 μM nitrate (620 ppb)
And reported detection of perchlorate (1 ppm), but Raman bands of nitrate (1047 cm ^-1 ) and perchlorate (934 cm ^-1 ) were measured with a separation time of less than 3 minutes. Unfortunately, the weak signal inherent in Raman scattering makes detection of sub-micromolar concentrations extremely difficult. The limit of detection is typically in the range of about 10 ^-7 M with the aid of isocratic sample concentration.

[0012] Nirode and coworkers have demonstrated SERS detection on columns directly in CE using colloidal silver as a SERS substrate in running buffer, which is incorporated herein by reference in its entirety in Anal. Chem. 72:18
66-1871 (2000). In this technology,
Raman spectra were obtained through the wall of the capillary during electrophoresis ("on the fly" SERS detection) by using a Raman microprobe device. The limit of detection using this approach was found to be about 10 ^-9 M for the model analyte studied (Rhodamine 6G). Despite the obvious promise of this method, there are many drawbacks. First, the presence of colloidal silver in the running buffer can be expected to interfere with the electrophoretic separation of the same sample. Second, colloidal silver is a somewhat rough, variable, and non-optimal SERS substrate, so the full sensitivity capability of SERS cannot be realized. Third, only a small fraction of the analyte appears to bind to colloidal silver during CE. Fourth, to minimize the artifacts caused by performing SERS on moving analytes,
The electrophoretic voltage must be kept low (ie the electrophoretic separation time extended) and a short SERS spectral integration time must be used (leading to low sensitivity). Finally, silver particles are gradually deposited on the walls of the capillary leading to a decline in the SERS response of the analyte.

It is an object of the present invention to provide a connection between CE and SERS that allows a SERS substrate with the highest enhancement factor to be used, the novel SERS substrate provided by this invention. including. SUMMARY OF THE INVENTION The present invention provides a novel SERS substrate. In a preferred embodiment, the substrate comprises a sublayer of microparticles on a solid support, each microparticle consisting of silver colloidal gold particles, the microparticles being on a thin, discontinuous layer of silver film. Located in. The above three-layer substrate shows excellent reproducibility:
Spectral deviations in different regions of a single 3-layer substrate are of the same order of magnitude as spectral deviations observed between individual 3-layer substrates from different manufacturing batches. Furthermore, the above three layers S
The ERS substrate allows for routine detection of subpicogram amounts of analytes.

The present invention also provides a novel connection between capillary electrophoresis and SERS, which allows the use of a three-layer SERS substrate for eluate detection. In a preferred embodiment, the eluate leaving the capillary is deposited on a moving SERS substrate to form a trace of linear elution. The eluate traces are then analyzed by Raman microscopy, either immediately after each eluate drop is deposited, or after all eluate traces have been deposited. The eluate traces, in spatial format, retain the chronological separation achieved by CE. Consequently, the separation results can be saved for later testing or for further analysis with the second detection method. Since the electrophoretic and SERS steps are performed separately, the above two steps can be optimized separately, allowing the full sensitivity of SERS to be exploited. In addition, "off-line" SERS detection can use the entire length of the capillary for separation, thereby maximizing the potential for CE analysis. Detailed Description of the Preferred Embodiments Three Layer SERS Substrate In one embodiment, the present invention comprises a subbing monolayer of microparticles deposited on a solid support, each microparticle comprising a first inert metal coating ( clad) and second
To provide a novel SERS-active substrate comprising colloidal particles of an inert metal of, wherein the microparticles are coated with a thin, non-continuous film of a second inert metal.

In a preferred embodiment, the substrate first immobilizes colloidal metal particles comprising a first inert metal on a solid support, and then chemically immobilizes the immobilized colloidal particles. It can be synthesized by coating with a plating solution containing the second inert metal in a chemically reduced form. Finally, the resulting coated colloidal particles are vapor coated with a discontinuous layer of a second inert metal.

In a preferred embodiment, the first inert metal is gold and the second inert metal is silver. The SERS substrate of this composition is called an Au-Ag-Ag substrate. Preferably, the gold colloid is a colloid of about 12 nm diameter and the silver film is about 10 nm-30 nm thick, most preferably 20 nm thick.

[0017]   Figure 1 schematically depicts the steps in one embodiment of the synthesis of Au-Ag-Ag substrate.
Make a copy. The glass slide 10 is first placed on an organosilane, such as 3-mercaptope.
Derivatize with ropylmethyl-dimethoxysilane (MPMDMS). MPMDMS
Form a self-assembled monolayer 11 on the glass surface; colloidal gold 12 is MPMD
By attaching the thio group of MS to the colloid of gold on the glass slide,
A black array can be formed. Immobilize colloidal gold on a glass slide
Other possible methods and reagents for are known to those skilled in the art. After fixing, gold colloid
And then a plating solution, such as LI Ag plating solution (Na
noprobes, Inc. (Commercially available from ⁺ To form a silver coating 13 on the gold colloid. last
Then, a discontinuous layer of silver 14 is deposited on the silver-coated gold colloid in the thermal evaporation system.
Evaporate by placing a glass slide. Example 1 is Au-Ag-Ag.
An example of a detailed protocol for substrate synthesis is provided.

It is known in the art that the optical properties of SERS active substrates are critically dependent on the size of the features, morphology, mutual feature space and extent of coupling between surface properties. In a preferred embodiment, the colloid size, evaporation rate, and evaporation step length (second inert metal thickness) are modified to maximize SERS enhancement of the trilayer substrate. In a particularly preferred embodiment, the choice of these factors is such that the deposited second inert metal forms discrete "islands" between the colloidal particles, as well as increasing the size of the colloidal particles. , A substrate is obtained. Without being bound by a single theory or hypothesis, the substrate is increased by increasing the surface area for analyte adsorption and increasing the electromagnetic enhancement through a "rod-like" effect. The morphology is believed to enhance SERS activity and is incorporated herein by reference in its entirety in Creighton, J. et al. A. The S
selection Rules for Surface-Enhanced
Raman Spectroscopy, Clark, R.M. J. H. and H
ester, R.A. E. , Ed., John Wiley & Sons Ltd. ，
1988; Vol. 16, pp 37-89. 12n
For the Au-Ag-Ag substrate using m colloid, a coating of 20nm thick silver, it is deposited at a rate of 0.1nm sec ^-1 -0.5 nm s ^-1 by evaporation. SER
The image of the S substrate is Atomic Force Mi in tapping mode.
crosscopy (AFM) or Field Emission Scanni
ng Microscopy (FE-SEM).

FIG. 2 illustrates 2 μL of 0.1% drop coated onto each of the substrates shown in FIG.
Figure 3 shows the enhanced SERS activity of a three layer substrate provided by comparing the SERS spectra of 1-mM trans-1,2-bis (4-pyridyl) ethylene (BPE) solutions. No SERS signal was observed on the simply silanized glass surface (trace a) or the colloidal gold microarray surface (trace b). A significant SERS signal can be observed on the bilayer substrate (silver-coated gold colloid) (curve c). Three layers of Au-Ag-Ag substrate (20 nm thick silver coating) (curve d) is about 20.
It has a% fluorescence background reduction and a signal enhancement greater than 3-fold compared to the bilayer substrate (measured by integrating the peak areas from 1071 cm ^-1 to 1271 cm ^-1 ). Example 2 provides the experimental protocol used to obtain the data presented in FIG.

[0020]   The three-layer substrate of the present invention can be used to detect the presence of specific analytes.
It is useful in all applications. Due to their large enhancement factors, the substrate is low in concentration.
It is particularly useful for detecting analytes that are present in degrees. In particular, the substrate is a biological material.
It is useful for detecting biomolecules present in low concentrations in the body. Three-layer SERS of the present invention
The sensitivity of the substrate depends on the environmentally important compound 1,3,5-triazine in FIG.
Will be shown. 1,3,5-triazine is the most widely used herbicide fu
Amilly's parent compound. FIG. 3 shows the characteristic of the compound, 926 cm.^-1Standard
Of the 1,3,5-triazine relative to the band of the peak area (ring blushing)
A plot of logarithmic concentration is shown. The above peak area is the raw peak area and total
It was standardized by using the quotient between. Inset to Figure 3 is 2 μL of 8x10 ^-3 M (30 seconds in total) and 8x10^-8M (45 seconds) of 1,3,5-triazine
3 shows a SERS spectrum. The three-layer substrate of the present invention has picogram amounts of 1,3,5-toluene.
It is expected that riazine detection can be performed with low excitation power and less than the integration time.
I can guess.

[0021]   The three-layer SERS substrates of the present invention represent a significant improvement over prior art SERS substrates.
However, due to their increased SERS activity,
This is because of the reproducibility obtained. Table 1 shows 5 different spots on the same 3-layer substrate.
10 mM BPE 1010, 1200, and 1610-1640 cm taken ^-1 Compare the peak areas for the SERS band. Good reproducibility between spectra
Are observed and all relative mean deviations are less than 13%. Similar reproducibility made at the same time
Can be realized both on the manufactured substrate and in separate substrate batches;
A body batch is defined as a group of slides, which limits the deposition chamber space.
Therefore, the number is typically eight, and each manufacturing process is performed at the same time. Table 2 shows 6 different groups
Peaks for SERS spectra of 10 mM BPE on substrate from body batch
Gives a comparison of areas. Again, good reproducibility was observed and the average relative deviation was single.
It is in the same order of magnitude as one substrate and all values are less than 12%. Made over a period of 8 months
It should also be noted that the data collected for the batch made was
In particular, the ability to make reproducible substrates of the present invention is demonstrated. Capillary electrophoresis and SERS connection   In another aspect of the invention, the SERS substrate is subjected to capillary electrophoresis (CE).
A method of connecting is taught. In a preferred embodiment, the key containing the resolved analyte is
The SERS substrate is placed so that the eluate from the capillary is deposited on the SERS substrate.
Place below the exit of the electrophoresis capillary. The SERS substrate will dissolve with the eluate.
A transposition that allows the revealed analyte to be deposited on different regions of the SERS substrate.
A translation means is attached. FIG. 4 shows an xy translation scan in which the translation means is computer-controlled.
Is a particularly preferred embodiment of the tage 41, in which the computer determines the position of the substrate
And speed. At the outlet 44, the substrate has a direct flow of eluate passing through the capillary 43.
Can be translated in a manner that is deposited in the traces of lines, eg achieved by CE
Is a continuous "S" pattern that retains the separation in chronological order. Traces of effluent
Marker dyes may be included in the eluate to improve visibility. According to the present invention
One suitable dye illustrated is Kiton Red 620.

Any translation modality that deposits the eluate in a linear, non-overlapping fashion is contemplated by the present invention. In addition, any SER that can be used to deposit traces of linear eluate
S substrates are also contemplated. For example, the eluate can be deposited on the cylindrical SERS substrate as an annular trace or a spiral trace. The eluate can be deposited either continuously or dropwise.

In one embodiment, after deposition of the eluate, Raman spectroscopy is used to capture the Raman spectrum at each point along the trace. In a preferred embodiment, Raman spectroscopy with a remote fiber-optical probe (often referred to as Raman microprobe) is used to retrace the traces of the eluate. Such a device is depicted in FIG. Laser excitation light 46, preferably laser excitation light from a He-Ne laser 47 (632.8 nm), is introduced by optical fiber 48 into the back focal plane of microscope objective 49 and focused onto SERS substrate 42. The SERS light from the substrate is then collected by microscope objective 49 and directed by optical fiber 48 to Raman spectrometer 410. Preferably the microscope objective is 3 mm
With a focal length of from 8 mm to a laser spot size of 5 μm-10 μm on the SERS substrate. Most preferably, a 3 mm objective lens is used to focus the 20 mW incident illumination, allowing the SERS spectrum to be captured in <5 seconds. For longer acquisition times, it is preferable to use larger spot sizes. For those skilled in the art, determining the appropriate spot size, laser power, and integration time for a particular application of the method of the invention is routine experimentation. Those skilled in the art will realize that the use of higher laser flux may cause damage to the sample at longer exposure times, but will give an increased SERS signal compared to low laser flux.

FIG. 5 shows that a 10 mM BPE solution was subjected to CE and then deposited on (a) a two-layer SERS substrate (silver-coated gold colloid monolayer) and (b) a blank glass slide, and Raman spectroscopy. (3mm objective, 20mW 632)
． 8 nm illumination, 5 second integration), data from one aspect of the invention are shown.
The complete lack of Raman response selected from BPE on blank glass slides is
It provides ample evidence that conventional Raman does not act as a detection method for these low levels of analyte. In contrast, the response observed from the silver-coated gold colloidal monolayer substrate makes it clear that SERS is a viable alternative to conventional Raman for the identification of analytes separated by CE. Prove.

In prior art UV-visible spectroscopic or fluorescence detection of CE, initial migration of each analyte in purified form is required to select individual retention times. Analytes in a mixture can only be identified by comparing retention times to previously established standards, based on the hypothesis that the presence of other analytes in the mixture does not affect individual migration rates. Furthermore, if the eluate contains a previously run analyte as a standard, determining the composition of the eluate can be extremely difficult. Therefore, prior art methods are useful only if (i) the likely analyte composition of the eluate is known; and (ii) purified amounts of the individual analytes are available. is there. The SERS elution retrace method provided herein, however, has sufficient Raman spectra recorded for each point along the elution passage.
That is, a single trace on the eluate-deposited substrate is sufficient to determine the nature of the analytes based solely on the characteristic Raman spectra of each analyte. Therefore, it is not necessary to measure the prior retention time of each analyte detected in the eluate. In fact, unlike prior art detection methods, the SERS retrace method does not require prior knowledge of the nature of the analytes contained in the eluate. This allows the composition of the eluate to be determined by using the methods provided herein without any prior knowledge of the likely composition of the eluate.

Analyte identification based on the SERS retrace method provided herein is
It is much more accurate than prior art analyte identification techniques based solely on retention time. Because the Raman spectrum of each analyte is complex (often more than 10 individual peaks), a unique and unquestionable "fingerprint" for that analyte
I will provide a. In contrast, prior art UV-vis and fluorescence spectra provide just a single peak that cannot uniquely identify an analyte without retention time information.

The SERS retrace method provided herein successfully bypasses the requirement to use analyte retention time as the only basis for analyte identification. However, it is important to note that the eluate signature retains the chronological analysis achieved by CE in the spiral format. In particular, each position on the SERS substrate represents a different elution / retention time point. In some embodiments, rather than retrace the traces of total elution, the exact time points at which the analytes of interest would be expected to elute based on prior knowledge of retention time were Raman tested. Spectroscopy can be inspected. In other embodiments, elution signatures can be first examined by Raman spectroscopy, and then specific time points on the substrate can be examined by other detection methods, such as mass spectroscopy. This is SE in the running buffer
There are significant advantages over prior art attempts to connect CE to SERS using colloidal silver as the RS active substrate. In summary, the creation of elution traces on SERS substrates provides an extremely flexible, accessible and data-rich source for eluate analysis.

Using prior art UV-vis and fluorescence detection techniques, a substantial amount of effort is often required to reach the baseline separation to pick a quantitative result for a mixture of analytes. Become. In some examples, different analytes may have similar capillary retention times, causing them to be analyzed less well than the other. As a result, the UV-vis or fluorescence peaks of such analytes partially or substantially overlap, making quantification difficult or impossible. Using the SERS retrace method provided herein, it is possible to perform a quantitative analysis of a mixture of analytes by monitoring the change in intensity in the wavenumber range where only one analyte exhibits a Raman response. it can. As a result, less effort is required to achieve baseline separation and even poorly analyzed analytes can be accurately quantified.

Unlike prior art attempts to connect CE to SERS using colloidal silver as the SERS active substrate, the SERS retrace method of the present invention is optimized for SERS.
Allows the use of active substrates. In some embodiments, the SERS substrate is a bilayer substrate that includes a silver-coated gold colloid. In a preferred embodiment, the above SERS
The retrace method is a three layer substrate provided by the present invention, most preferably Au-.
For use with Ag-Ag tri-layer substrates.

The utility of the methods provided herein is demonstrated by the data shown in FIGS. 6-8, CE-separated BPE and N, N-dimethyl-4-nitrosaniline (
p-NDMA) SERS detection. A UV-vis analysis of the separation is presented as a criterion for the effect of the SERS retrace method (Fig. 6A). The UV-vis elution profile shows two distinct but not completely resolved peaks. In SERS detection, a constant rate translation step is used for the deposition of BPE and p-N on the substrate.
Allows the spatial position of both DMAs to be converted to a time scale to obtain information on analyte retention time. FIG. 6B shows the wave number range 1600-1650c.
The time course of the change of the integrated SERS intensity at m ⁻¹ is shown, and BPE and p−N
Both DMAs are areas that show Raman response. Retention time tests for both eluates show agreement with those recorded by UV-vis, thus CE-SERS
Connection can perform the same detection function as UV-vis.

FIG. 7 shows the SERS spectrum corresponding to each of the points shown in the bottom panel of FIG. Spectrum a, e and f are the pH6.8 buffer having a low SERS response of 1700 cm ^-1 from 1000 cm ^-1, background S
Three examples of ERS spectra are shown, taken with a CE-deposited elution trace. The only Raman activities observed for these positions were two fairly large peaks at 616 cm ⁻¹ and 920 cm ⁻¹ , but due to the components in the LI Ag plating solution and all recovered. Were present in the SERS spectra; their intensities were significantly reduced in the presence of analyte, probably due to competition for surface sites (data not shown). Spectrum B is BPE
Taken at the position corresponding to the elution ((t = 946 seconds). As expected,
Characteristics distinct BPE is observed (e.g., C = C-derived band at 1010 cm ^-1 derived Blessing bands and rings in 1200 cm ^-1) of. The spectrum c recorded at the position corresponding to t = 949 seconds at which the eluate separated incompletely shows the SERS profile of both analytes. The spectrum d (t = 952 seconds) is p−N
Structure for CE separation of the SERS retrace method provided herein, showing a spectrum and features corresponding to DMA, including the 1166 cm ⁻¹ band from fail nitroso stretch on a silver-coated surface. Again, its utility as a specific detection method is confirmed.

[0032]   FIG. 8 is a range in which only BPE shows a Raman response, which is 1230-1180 cm. ^-1 In the time course of the integrated SERS intensity from (curve 2) and in this experiment
1180-1130 cm, unique for p-NDMA response^-1From (curve 3)
3 shows a plot of the SERS intensity of. Curve 1 shows that both BPE and p-NDMA
1600-1650 cm showing Raman response^-1La showing the SERS intensity at
It is an in curve. This data shows how the method of the present invention can be used in only one analysis.
By monitoring the intensity change in the wavenumber range where the object exhibits a Raman response,
Prove whether it allows quantitative analysis of a mixture of unanalyzed analytes.

The method of the present invention is, in essence, well adapted for biological assay applications in which the analyte is typically present in low concentrations. As an example of such an application, FIG. 9 shows SERS data from CE separations of amino acids tyrosine and tryptophan (1: 1 ratio). FIG. 9A shows the UV elution profile for the separation and reveals significant intervals in retention time (minutes between peaks). 9A and 9B show
The corresponding SERS separations of tyrosine and tryptophan, respectively, are shown. For example, 8
The peak at 37 cm ^-1 (Figure 9B) is characteristic of the ring blessing mode of p-hydroxyphenyl moieties in tyrosine, while the Raman band at 1334 cm ^-1 (Figure 9C) is derived from the tryptophan indole ring. A number of other bands were also found at 1176 and 1603 cm ^{−1 for} both analytes, Tyr,
It then serves to identify those that appear at 1012 and 1607 cm ⁻¹ for Trp, all of which correspond well to the Raman spectrum of each amino acid. The data in FIG. 9 shows the method of Example 3 and 1 mg / tyrosine and tryptophan.
Obtained using a ml solution.

[0034]   The method of the invention is also well suited for the isolation and identification of environmental pollutants. Of this role
By way of example, FIG. 10 shows the SERS retrace method for a mixture of chlorophenols.
Shows data from an application of. Figure 10A shows 2-chlorophenol (CP) and 2,4
-Shows the UV elution profile of dichlorophenol (DCP). CP eluted
And SERS data for DCP are presented in Figures 10B and 10C, respectively.
Identify each of the compounds using a specific band obtained from the SERS spectrum
be able to. 562 cm in FIG. 10B^-1Band of ortho-substituted benzene
It is a strong indication of existence, while 851, 1143 and 1285 cm in FIG. 10C.^-1 The peak of corresponds well to the Raman spectrum of pure dichlorophenol. 7
00 and 1028 cm^-1The Raman band of DCP in
Derived from propanethiol used to coat substrates to improve adhesion
Note that it overlapped with the band. Example Example 1 Manufacture of 3-layer substrates Material The glass slide used is Fisher Premium Micros.
It was the Cope Slides. The water used for all experiments was Barnstea.
It was 18.2 MΩ distilled by dNanoPure system. 3-mercapto
Ropylmethyldimethoxysilane (MPMDMS) United Chem
purchased from al. HAuCl_Four・ 3H₂O, AgNO₃, Trisodium citrate
Dihydrate, NaOH was purchased from Aldrich. 12 nm colloidal gold
HAuCl reduced with citric acid_Four・ 3H₂Manufactured from O, but by quoting that
Grabar, K. et al., Which is incorporated herein in its entirety. C. et al, Anal. Ch
em. 67: 735-743 (1995). In this text
And all references to "12 nm gold" particles are nearly spherical, 2
Standard deviation of less than nm. The size of the particles is measured by NIH Image Software (w
ww. nih. (available on the internet at gov)
It was measured by a transmission electron micrograph. Li Ag solution is Nanoprob
Purchased from es and used as directed. Concentrated HCl, HNO ₃ , H₂SO_FourAnd 30% H₂O₂J. T. Purchased from Baler Inc. Minute
Light metering grade CH₃OH was purchased from EM Science. Used for vapor deposition
Shot with silver (99.99%) is Johnson-Matthey Corp.
Got from Manufacture of Substrates Glass slides were cut into pieces by Grabar, K. et al. C. et al,
Anal. Chem. 67: 735-743 (1995).
Or wash through continuous sonication for 20 minutes in water, methanol, and acetone
It was Glass slides in a 5-10% solution of MPMDMS in methanol for 1 hour
Functionalized. Depending on the colloid concentration, the functionalized substrate is treated with 12 nm colloidal gold solution.
The solution was coated for 90-95-seconds. The substrate is then rinsed with water and Ar flow
Partially dried under. After 24 hours of exposure to LI Ag solution (two-layer SER
S-active substrate, ie yielding a silver-coated gold colloid), discontinuous, 20 nm silver foil
The film is thermally evaporated onto the top of the substrate so that three layers of Au-Ag-
This produced an Ag substrate. Thermal evaporation with the Edwards Auto 306 evaporation system
It carried out using. Metal deposition is 2x10^-6For pressures of mbar and various deposition rates
With constant sample rotation to ensure even evaporation. All substrates are steamed
Used immediately after departure.

Optical spectra were examined after the above steps for monitoring surface quality. Film integrity was characterized by UV-visible absorption spectroscopy, with quality control based on the shape and height of the gold plasmon peak. The appropriate substrate was then used to form a two layer substrate as described above. The surface plasmon peak of the UV spectrum of the bilayer substrate shifts relative to the gold colloid only, probably due to the optical properties of the coated silver. In addition, the bilayer substrate has a new shoulder near 620 nm caused by the particle coupling effect. After the discontinuous silver coating layer evaporated on the two-layer substrate (three-layer substrate), the shoulder became more pronounced as a result of the strong coupling from the increased surface coating. Example 2 SERS Spectra of Various Substrates Trans-1,2-bis (4-pyridyl) ethylene (BPE) is Aldric
Obtained from h. BPE was recrystallized several times before use, and then a 10 mM BPE stock solution was prepared in fresh H ₂ O: CH ₃ OH (9: 1) solvent solution. Dilutions were performed as needed to obtain 1 mM, 0.1 mM and 0.01 mM in H ₂ O.
A solution of mM was produced. These solutions were then drop coated onto various SERS substrates. The SERS spectrum was detected by Detection Limited, In
Obtained on a Solution 633 micro-Raman spectrometer purchased from c. Excitation was supplied by a 20-mV, 632.8-nm HeNe laser. All spectra were obtained with a 3 mm focal length objective (~ 5 μm diameter at spot size). The spectrum is about 8. by a CCD detector with TE-cooling system.
It was recovered at around 5 ° C. The above system is a Monorail laptop (Monora
il Corp. ) Operated and monitored by Labview software above.
The SERS response was optimized by manually adjusting the height of the probe attached to the translation stage. Spectral analysis is based on Grams 32 or Igor Pr
o Manipulated by either of the software packages. Example 3 CE-SERS CE systems are each incorporated herein by reference in their entirety, Tracht.
et al, Anal. Chem. 66: 2382-2389 (1994) and Olefirewicz, T .; M. & Ewing, A.A. G. J. Chro
matgr. 499: 713-719 (1990) as previously described. Fused silica capillaries with an inner diameter (id) of 50 μm with a total length of 75 cm and an effective length of 65 c for online UV detection.
m was used. Injection of samples is accomplished by the hydrostatic method, each of which is incorporated herein by reference in its entirety, Landers, J. et al. P. Capillary Electrophoresis Handbook; CRC Press (1994); Engelha
rdt et al, Angew. Chem. , Int. Edited by Engl. 32:
629-649 (1993), and Weinberger, R .; Practical capillary electrophoresis; as described in Academic Press: Boston (1993). In particular, a 10 cm height difference between the two ends of the capillaries was maintained by injection immersing the ends in sample vials for 10 seconds (calculated 3 × 10 ⁻¹¹ molar analyte injection). Running buffer is 50 mM, p
It was H6.8 phosphate buffer. 15k on the capillary while keeping the current at 40μA using a Spelman CZE 100OR power supply
A V potential was applied. As a safe measure, the CE system was contained within a Plexiglas box that was securely secured by an internal lock; that is, the power supply should shut off automatically when the lid is opened. The capillaries were activated with 1 M NaOH, 18 MΩ H ₂ O, and 50 mM phosphate buffer at the beginning of each day and subsequently washed. Before each injection, the capillary was flushed again with fresh buffer solution. A linear variable wavelength UV visible detector was used for on-line detection and compared to the results obtained by SERS.

Surface-enhanced Raman scattering: A Solution 633 Fiber-Optic micro-Raman spectrometer with a remote probe was used for the acquisition of Raman spectra. He-Ne laser (632.8n
m) was used as the excitation source and spectra were collected using a microscope objective with a focal length of 3 mm or 8 mm. The laser beam focusing process was accomplished by manually adjusting the height of the probe. Output power of 0-20 mW was adjusted by using a light filter. Spectra are TE-cooled CCD
It was collected by a camera.

The connection between CE and SERS is shown in FIG. A two-layer SERS substrate (silver coated gold colloid) was placed under the above metalized outlet of the capillary. Two 850
The x-axis and y-axis were moved at 0.8 mm / sec by using a translation stage controlled by a -B actuator (Newport Corp.). The analyte was deposited directly on the substrate during separation, which moved the substrate in a continuous "S" fashion.

For time course experiments (plot integrated SERS intensities for a particular wavenumber range plotted against elution time), the substrate was translated after deposition of the eluate in the position below the micro-Raman probe and CE elution At the same speed, the course of precipitation of the material is SERS
Retraced on data acquisition. Next, SERS intensity within the wave number range indicated in the text was obtained using a 1 second integration time with a 1 second delay.
For the measurement of integrated SERS intensity, the background for normalization was obtained by integrating the SERS intensity in the wave number range where no analyte of interest showed a Raman response.

[0039]

[Table 1]

[0040]

[Table 2]

[Brief description of drawings]

FIG. 1 schematically depicts the steps in the synthesis of a Au-Ag-Ag trilayer SERS active substrate.

FIG. 2 illustrates the Raman intensity enhancement provided by a Au-Ag-Ag trilayer SERS-active substrate.

FIG. 3 shows the detection sensitivity of 1,3,5-triazine using a Au-Ag-Ag trilayer SERS-active substrate.

FIG. 4 schematically depicts the SERS-based Raman spectroscopy and capillary electrophoresis connection.

FIG. 5 illustrates Raman spectroscopy detection of trans-1,2-bis (4-pyridyl) ethylene (BPE) deposited on SERS-active substrates by capillary electrophoresis.

FIG. 6 shows BPE and N, N-dimethyl-4-nitrosaniline (by capillary electrophoresis monitored by UV-visible spectroscopy (FIG. 6A) and SERS (FIG. 6B)).
(p-NDMA) separation time course.

FIG. 7 shows a complete Raman spectrum for each of the points af identified in FIG. 6B.

FIG. 8: Time for separation of BPE and p-NDMA by capillary electrophoresis monitored by Raman spectroscopy at wavenumbers where only BPE (curve 2) or p-NDMA (curve 3) shows Raman response. Show progress.

FIG. 9 shows a UV-vis spectrograph (FIG. 10A) and Ramansulfolobus (FIGS. 9B and 9C) for separation by capillary electrophoresis of a mixture of tyrosine and tryptophan.

FIG. 10 shows UV-vis spectrograph (FIG. 10A) and Raman spectra (FIGS. 10B and 10C) for separation by capillary electrophoresis of a mixture of chlorophenol (CP) and dichlorophenol (DCP).

─────────────────────────────────────────────────── ─── Continued front page    (31) Priority claim number 60 / 169,339 (32) Priority date December 6, 1999 (December 1999) (33) Priority claiming countries United States (US) (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A, CH, CN, CR, CU, CZ, DE, DK, DM , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, K E, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW F-term (reference) 2G043 AA01 BA14 CA03 DA05 EA03                       EA19 GA08 GB01 GB21 HA01                       HA05 KA02 KA09 NA13

Claims (8)

[Claims] 1. A solid support; a subbing monolayer of microparticles attached to a solid support, each microparticle comprising a colloidal particle of a first inert metal and a second inert metal. A surface enhanced Raman scattering (SERS) active substrate comprising the subbing monolayer with an outer coating; and a discontinuous film of a second inert metal located on the support and the microparticles. 2. The SERS active substrate of claim 1, wherein the first inert metal is gold and the second inert metal is silver. 3. The discontinuous film is 10 nm to 30 nm thick.
A SERS-active substrate as described. 4. A solid support; a subbing monolayer of microparticles connected to a solid support, each microparticle comprising silver-coated colloidal gold particles; and A surface enhanced Raman scattering (SERS) active substrate comprising a discontinuous film of a second inert metal located on the support as well as the microparticles. 5. The SERS-active substrate of claim 1, wherein a discontinuous film is deposited on the support and microparticles. 6. Step: preparing gold colloidal particles, preparing a solid phase support capable of binding to the gold colloidal particles, and forming an undercoat monolayer of the gold colloidal particles bonded to the solid phase support. Contacting the gold colloidal particles with the solid support under predetermined conditions to allow the gold colloidal particles bound to the solid support to be contacted with the silver plating solution,
A method for forming a SERS-active substrate comprising thereby coating gold colloid with silver and depositing a discontinuous film of silver on a solid support. 7. A device for depositing an analyte contained in an eluate analyzed by capillary electrophoresis (CE) on a surface-enhanced Raman scattering (SERS) active substrate, which is eluted during CE. A CE unit having a capillary with an output through which a substance is discharged; a SERS substrate arranged adjacent to said output so that the eluate is deposited on the SERS during CE, said SERS substrate being defined A SERS substrate optionally connected to a translation means that can be moved in a pattern; provided that the movement of the SERS substrate in the above defined pattern during CE is different along the traces of the eluate. An apparatus as described above, which leads to the formation of a linear eluate trace such that another analyte deposits at the location. 8. A method for Raman spectroscopy of an analyte contained in an eluate analyzed by capillary electrophoresis (CE), the capillary having an output during which the eluate is ejected. A CE unit containing different amounts of different analytes is ejected into the eluate at different times; the eluate is surface-enhanced placed adjacent to the output so as to deposit on the SERS during CE. A Raman scattering (SERS) substrate optionally connected to a translation means capable of moving the SERS substrate in a defined pattern.
Prepare an RS substrate; perform CE and simultaneously move the SERS substrate in the pattern defined above, provided that a linear trace of the eluate is deposited on the SERS substrate and along the trace of the eluate. And another analyte is deposited in the eluate at different locations; and inspecting the eluate for traces by Raman spectroscopy.