Raman scattering spectrometer and Raman scattering spectroscopy

Abstract

A Raman scattering spectroscopic method according to the present invention comprises: a step for preparing a chip having a channel in which a nanostructure is formed; a step for introducing an analysis solution into a part of the channel in the chip; a step for irradiating an interface of the analysis solution with a laser beam; and a step for measuring Raman scattering light generated by irradiation with the laser beam. The irradiation of the laser beam and the measurement of the Raman scattering light may be carried out by fixing the irradiation position of the laser beam in a state in which the interface of the analysis solution is included within the irradiation range of the laser beam or in a state in which the interface of the analysis solution is not included within the irradiation range of the laser beam. The measurement may also be carried out by controlling the irradiation position of the laser beam in accordance with interface displacement resulting from evaporation of the analysis solution so that the state of having the interface of the analysis solution included within the irradiation range of the laser beam is continuously maintained.

Description

[Technical Field]

[0001] The present invention relates to a Raman scattering spectrometer and a Raman scattering spectroscopy. [Background technology]

[0002] Surface-Enhanced Raman Scattering (SERS) is a phenomenon in which, when molecules are adsorbed on the surface of a noble metal with a nanoscale structure, the intensity of Raman scattering is greatly amplified compared to that on a bulk substrate. As a highly sensitive analytical method, it is used in a wide range of fields, including trace substance analysis, pathological diagnosis, environmental measurement, and food safety management. Theoretically, the enhancement of surface-enhanced Raman scattering is 10 times greater than that on a bulk substrate. 12 However, in most cases, the enhancement is 10 6 ~10 8 It is about 2 times more difficult to detect than the femtomolar (fM) level.

[0003] As a method for detecting below fM, a SERS analysis method has been developed that utilizes nanogaps formed by self-organization of metal nanoparticles when metal nanoparticles are dispersed at high density in a solution and the solution evaporates (Non-Patent Documents 1, 2). However, with this method, once the solution has completely evaporated, the metal nanoparticles aggregate completely, and no significant enhancement can be obtained. This method is called Dynamic SERS or Transient SERS because it can detect below fM for a limited period during measurement. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Barmi, MR; Andreou, C.; Hoonejani, MR; Moskovits, M.; Meinhart, CD Aggregation Kinetics of SERS-Active Nanoparticles in Thermally Stirred Sessile Droplets, Langmuir 2013, 29, 13614-13623. [Non-patent document 2] Yan, X.; Li, P.; Zhou, B.; Tang, X.; Li, X.; Weng, S.; Yang, L.; Liu, J. Optimal Hotspots of Dynamic Surface-Enhanced Raman Spectroscopy for Drugs Quantitative Detection, Anal. Chem. 2017, 89, 4875-4881. [Non-patent document 3] Bai, S.; Serien, D.; Hu, A.; Sugioka, K.; 3D Microfluidic Surface-Enhanced Raman Spectroscopy (SERS) Chips Fabricated by All-Femtosecond-Laser-Processing for Real-Time Sensing of Toxic Substances, Adv. Func. Mater. 2018, 1706262. Summary of the Invention [Problem to be solved by the invention]

[0005] In consideration of the above-described current situation, an object of the present invention is to provide a Raman scattering spectroscopy technique that allows continuous measurement with a large enhancement. [Means for solving the problem]

[0006] One aspect of the present invention is an irradiation means for irradiating a laser beam; a measuring means for measuring Raman scattered light generated by irradiation with the laser light; Equipped with A liquid-air interface is formed on the metal nanostructures of an analytical solution introduced into a part of a channel of a chip having the channel on which the metal nanostructures are formed. Before Measurement is performed by irradiating the laser light. stomach, a control unit that controls the irradiation position of the laser light in accordance with the movement of the interface caused by evaporation of the analysis solution; This is a Raman scattering spectrometer.

[0007] In this way, by performing Raman scattering measurement by irradiating the interface of the analysis solution with laser light, a large degree of signal enhancement can be obtained.

[0008] The Raman scattering spectrometer in this aspect may include the chip. The Raman scattering spectrometer in this aspect may also include a solution introducing means for introducing the analysis solution into a part of the channel of the chip.

[0009] In this aspect, the irradiation position of the laser light may be fixed, and the laser light may be irradiated and the Raman scattered light may be measured in a state where the interface of the analysis solution is included in the irradiation area of ​​the laser light, and in a state where the analysis solution is not included in the irradiation area of ​​the laser light.

[0010] When the analytical solution evaporates and is no longer present in the laser beam irradiated area, the enhancement is lower than in measurements taken at an interface, but it is still greater than in conventional Raman scattering spectroscopy, allowing for continuous measurements with a higher signal intensity than conventional methods.

[0011] In this aspect, the Raman scattering spectrometer may further include a control unit that controls the irradiation position of the laser light in accordance with the movement of the interface that accompanies evaporation of the analysis solution.

[0012] According to this method, measurements can be continuously performed at the interface of the solution, thereby enabling measurements to be continuously performed with a large signal intensity.

[0013] In this embodiment, the nanostructure may be a nanodot structure or a nanoripple structure formed in a thin metal film deposited on the channel surface.

[0014] Another aspect of the present invention is a method for Raman scattering spectroscopy, providing a chip having nanostructured channels; introducing an analysis solution into a portion of the channel of the chip; A liquid-air interface formed on the metal nanostructures of the analytical solution Nire irradiating the laser light; measuring Raman scattered light generated by the irradiation of the laser light; The present invention is characterized in that it includes:

[0015] This aspect may further include a step of controlling the irradiation position of the laser beam in accordance with movement of the interface due to evaporation of the analysis solution. Also, this aspect may fix the irradiation position of the laser beam, and irradiate the laser beam and measure the Raman scattered light in a state where the interface of the analysis solution is included in the irradiation area of ​​the laser beam and in a state where the analysis solution is not included in the irradiation area of ​​the laser beam. [Effects of the Invention]

[0016] According to the present invention, continuous Raman scattering spectroscopy can be performed with a large enhancement. [Brief explanation of the drawings]

[0017] [Figure 1] 1 is a diagram showing the overall configuration of a Raman scattering spectrometer according to an embodiment; [Figure 2] FIG. 1 illustrates the fabrication method of a 3D microfluidic SERS chip with microfluidic structures and metal nanostructures. [Figure 3] 1A to 1C are diagrams for explaining a Raman scattering spectroscopy measurement method (LI-SERS) according to an embodiment. [Figure 4]FIG. 4 is a diagram showing the time change in Raman signal intensity obtained by the Raman scattering spectroscopy measurement method according to the embodiment. [Figure 5] 1A and 1B are diagrams showing Raman spectra of R6G solutions of different concentrations obtained by the Raman scattering spectroscopy measurement method according to the embodiment. [Figure 6] 1A and 1B are diagrams showing Raman spectra of R6G solutions of different concentrations obtained by the Raman scattering spectroscopy measurement method according to the embodiment. [Figure 7] FIG. 1 is a diagram illustrating the detection limits of this method (LI-SERS) and other methods. [Figure 8] (A) SEM image of a nanodot structure in a metal thin film, and (B) Raman signal intensity from LI-SERS measurements using this nanodot structure. [Figure 9] FIG. 2 is a graph showing the relationship between the concentration of an analysis solution and the Raman signal intensity in the Raman scattering spectroscopy measurement method according to the embodiment. [Figure 10] 1A and 1B are diagrams showing Raman spectra for crystal violet solutions of different concentrations obtained by a Raman scattering spectroscopy measurement method according to an embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0018] <Summary> The present invention relates to a Raman scattering spectroscopy method capable of performing measurements with improved Raman scattering enhancement. More specifically, the present invention relates to a Raman scattering spectroscopy method capable of achieving detection sensitivity of 10 to 100 attomolar (aM) continuously, not just for a limited period of time.

[0019] This paper provides an overview of the present invention. In this invention, a three-dimensional microfluidic SERS chip with a nanostructured metal film formed in the channel is used. The analytical solution is partially introduced into the channel, and Raman measurements are performed with the liquid-gas interface of the analytical solution positioned at the nanostructure (Figure 3). As long as the analytical solution is in the laser irradiation area, a very large Raman signal is obtained (Dynamic mode). As the laser irradiation proceeds, the analytical solution evaporates, and the liquid interface gradually moves away from the laser irradiation area, resulting in a decrease in signal intensity. However, even after the solution in the laser irradiation area has evaporated, a certain signal intensity continues to be obtained, and this signal intensity is much stronger than when the laser is irradiated into a solution (Static mode).

[0020] Because the Raman scattering measurement method according to the present invention utilizes the liquid interface of the analytical solution, the inventors have named it liquid-interface assisted surface-enhanced Raman scattering (LI-SERS) spectroscopy. In this specification, this method is also referred to as liquid-interface assisted SERS or LI-SERS.

[0021] <Device configuration> As shown in FIG. 1, a Raman scattering spectrometer according to an embodiment of the present invention includes a laser light source 10, a beam splitter 11, an objective lens 12, a spectrometer 13, a CCD detector 14, a sample stage 15, and an information processing device (PC) 16. A three-dimensional microfluidic SERS chip 20 is mounted on the sample stage 15. With an analytical solution introduced into the channels of the three-dimensional microfluidic SERS chip 20, excitation laser light is irradiated from the laser light source 10 and focused by the objective lens 12. The generated Raman scattered light is input to the spectrometer 13 via the objective lens 12 and the beam splitter 11 and dispersed there. The CCD detector 14 is positioned so as to measure a predetermined wavenumber range of Raman shifts. The Raman signal detected by the CCD detector 14 is sent to the information processing device 16 for analysis, display, recording, and so on. The information processing device 16 also controls the laser light source 10, the CCD detector 14, and the sample stage 15.

[0022] The three-dimensional microfluidic SERS chip 20 (hereinafter simply referred to as chip 20) has a metal thin film nanostructure formed in a channel (fluid structure). The structure and manufacturing method of chip 20 have already been disclosed by the present inventor in Non-Patent Document 3, and will be briefly described here. Note that although Non-Patent Document 3 fabricates a metal nanodot structure (two-dimensional structure), a nanoripple structure (line and space pattern, one-dimensional structure) may also be employed.

[0023] Figure 2 illustrates the fabrication method for the chip 20. First, a photosensitive glass 200 is directly written with a femtosecond laser 201 and subjected to a first annealing process (Figure 2(1)). This is followed by hydrofluoric acid etching and a second annealing process, forming a three-dimensional fluidic structure (channel) 202 inside the photosensitive glass 200 (Figure 2(2)). The same femtosecond laser 201 is then used to selectively ablate the interior of the three-dimensional fluidic structure 202 (Figure 2(3)). A thin metal film 203 is then selectively deposited only in the ablated area by electroless plating (Figure 2(4)). The thin metal film 203 is made of, for example, copper (Cu) and silver (Ag) deposited on top of it. Finally, a linearly polarized femtosecond laser is irradiated onto the thin metal film 203 at an intensity close to the ablation threshold to form a periodic nanoripple structure (Figure 2(5)). To make the nanostructures as small as possible, the wavelength of the femtosecond laser was set to 515 nm, the second harmonic. Furthermore, by irradiating the formed nanoripples with a femtosecond laser whose polarization direction was rotated by 90 degrees, a periodic nanodot structure can be formed. Alternatively, a periodic nanodot structure can be formed by rotating the glass substrate by 90 degrees and irradiating it with the femtosecond laser again while maintaining the polarization direction.

[0024] The microfluidic structure fabricated in this embodiment is, for example, 300 μm wide, 90 μm high, and 2000 μm long, and is located 210 μm below the upper surface of the glass substrate and connected to two openings (500 μm × 500 μm × 300 μm each). The nanoripple structure has, for example, an average period of 140 nm and an average groove spacing of 43 nm.

[0025] <Measurement method> The measurement method in this embodiment will be described in more detail with reference to Fig. 3. Fig. 3(a) is a diagram showing the entire three-dimensional microfluidic SERS chip 20, and Fig. 3(b) is an enlarged view showing the vicinity of the laser irradiation region in the channel 202 of the chip 20.

[0026] First, an analytical solution 210 (for example, rhodamine 6G: R6G) is introduced into the channel 202 from one opening of the fabricated three-dimensional microfluidic SERS chip 20. The analytical solution 210 is introduced using, for example, a microsyringe. At this time, the analytical solution 210 is introduced only into a portion of the channel 202, rather than filling the channel 202 with the analytical solution 210. For example, the analytical solution 210 is introduced into the channel 202 in an amount such that the area irradiated with the laser beam 211 is filled with the analytical solution 210 and the liquid interface of the analytical solution 210 is slightly outside the area irradiated with the laser beam 211.

[0027] In this state, the Raman measurement is started by irradiating the sample with an excitation laser beam from the laser source 10. -11 The time evolution of the Raman signal intensity in Raman measurements of the R6G solution of M is shown from the start of the Dynamic mode.

[0028] At the start of the measurement, the analysis solution 210 is present in the entire laser irradiation area, so the same measurement as in Non-Patent Document 3 is performed, and the Raman enhancement is 10 8 Therefore, 10 -11 No Raman signal can be detected from the R6G solution of M.

[0029] As time passes, evaporation of the analytical solution 210 progresses, and the laser light 211 begins to irradiate the liquid-air interface, as shown in Figure 3(b). As a result, the Raman signal intensity increases rapidly, as shown in Figure 4, and the increased Raman signal intensity is stably obtained for a certain period of time (Dynamic mode). In this experiment, it took about 30 seconds for the signal to increase and reach its maximum value, which was maintained for about 3 minutes.

[0030] As time passes, the signal intensity decreases as the liquid interface gradually moves away from the laser irradiation area, but after the analytical solution 210 in the laser irradiation area has completely evaporated, a constant signal intensity (approximately half the intensity in Dynamic mode) continues to be obtained, and this signal intensity is much stronger than when laser light is irradiated into the solution (Static mode).

[0031] Figures 5 and 6 show Raman spectra obtained in dynamic mode when measuring R6G solutions of different concentrations using a three-dimensional microfluidic SERS chip with a nanoripple-structured metal thin film. -12 M to 10 -16 The measurements were performed on a 10 M solution. -14 M to 10 -17 These results indicate that the 10 -16 It can be seen that R6G peaks can be detected up to M (100 aM).

[0032] Figure 7 illustrates the detection limits of several techniques, comparing the detection limits of this technique (LI-SERS), ordinary Raman measurements, and surface-enhanced Raman measurements (SERS). As shown, the detection limit of ordinary Raman measurements is 10 -2 M, whereas our method is 10 -17 It is possible to detect up to M, achieving an improvement of 14 orders of magnitude. Note that "Glass" indicates the results of Raman measurements on a glass substrate, which shows that no Raman signal can be obtained from the glass substrate and does not affect the measurement.

[0033] In this measurement, the enhancement factor (EF) in dynamic mode and static mode was EF dynamic = 3.2 × 10 13 , EF static = 1.5 × 10 12 Here, the enhancement factor is EF = (I SERS / I OR ) / (C SERS / C OR), and I SERS and I OR are the Raman signal intensities for R6G on the SERS substrate and on the glass substrate, respectively, and C SERS and C OR represents the molar concentration of each.

[0034] Similar measurements were also performed using a three-dimensional microfluidic SERS chip with a metal thin film with a nanodot structure. Figure 8(A) shows a scanning electron microscope (SEM) image of the nanodot structure, with dots of an average size of approximately 250 nm formed at intervals of approximately 50 nm. Figure 8(B) shows the results of the SERS measurement of the nanodot structure with a concentration of 10 -17 Raman spectra of R6G with M and a concentration of 10 on the nanoripple structure -16 The Raman spectrum of M for R6G is shown. By using the nanodot structure, the enhancement factor is EF = 1.52 × 10 14 The detection limit is 10 aM or less.

[0035] In addition, the enhancement factor EF in conventional SERS measurements is approximately 10 5 From 10 9 The detection limit is in the range of 1 μM to 1 pM. In the method of Non-Patent Document 3, the enhancement factor EF is 10 8 The enhancement is about 100 aM, with a detection limit of 1 nM. Regarding the transient SERS technique described in Non-Patent Documents 1 and 2, the enhancement level is not reported, but detection of 10 aM is possible for a limited period of time. This method allows continuous Raman measurement with a detection limit of 10 aM to 100 aM. Furthermore, by further narrowing the spacing between the nanostructures of the metal thin film, the enhancement level can be further increased and the detection limit can be improved.

[0036] The mechanism by which the Raman signal intensity is enhanced in this method is not yet fully understood, but it is thought that the R6G molecules decompose due to the heat generated by irradiating the metal with laser light, and that at the same time, the decomposed molecules gather near the metal nanostructure due to Marangoni convection generated in the solution by the heat. It is thought that some of the R6G molecules that gather near the metal nanostructure remain on the thin metal film even after the liquid evaporates, making analysis possible in static mode. In fact, we have confirmed that residual deposits remain in the laser-irradiated area after the liquid evaporates.

[0037] Figure 9 shows the 10 -11 From 10 -15 This figure shows the Raman signal intensity obtained by this method (LI-SERS) from an R6G solution of M. When the R6G concentration is in the range of pM to fM, the coefficient of determination R 2 It can be seen that there is a linear correlation at 93%. Therefore, quantitative measurement of samples is possible using this method.

[0038] This method can also be applied to solutions other than R6G. Figure 10 shows the results of Raman spectroscopy in dynamic mode for a crystal violet solution. The detection limit for crystal violet is approximately 10 -14 I am M.

[0039] From the above, this method (LI-SERS) enables continuous Raman measurements at detection limits from fM to aM, regardless of the sample. This method is also expected to be used in a wide range of fields to achieve ultra-sensitive and rapid material analysis, pathological diagnosis, environmental measurement, and food safety management.

[0040] (Variation) The present invention is not limited to the specific configurations described above, and various modifications are possible within the scope of the technical ideas shown in this disclosure.

[0041] For example, the materials and dimensions of the 3D microfluidic SERS chip or metal nanostructure may be other than those described above. While it has been mentioned that the enhancement can be further increased by making the metal nanostructure finer, it is also possible to perform liquid-interface-assisted SERS with a lower enhancement by using metal nanostructures larger than the above-mentioned sizes. Alternatively, metal nanostructures other than nanoripple structures or nanodot structures, or dispersed metal nanoparticles, may be used. Furthermore, there are no particular limitations on the width, height, or length of the microfluidic channel. While it is desirable to set the height within an appropriate range, the width and length may be large, and the length in the width direction may not be limited.

[0042] In the above example, the laser irradiation position and the position of the three-dimensional microfluidic SERS chip are fixed during measurement, so that the liquid interface of the analysis solution moves out of the laser irradiation area over time. However, the laser irradiation position may be moved relative to the chip in response to the movement of the liquid interface due to evaporation of the analysis solution. To achieve this control, for example, the information processing device 16 can acquire an image of the laser irradiation area and move the sample stage 15 to match the position of the liquid interface. With this configuration, the liquid interface of the analysis solution is always located in the laser irradiation area, allowing continuous measurement in Dynamic mode. [Explanation of symbols]

[0043] 10: Laser light source 11: Beam splitter 12: Objective lens 13: Spectrometer 14: CCD detector 15: Sample stage 20: 3D microfluidic SERS chip 200: Photosensitive glass 201: Femtosecond laser 202: 3D fluid structure (channel) 203: Metal thin film

Claims

1. an irradiation means for irradiating a laser beam; a measuring means for measuring Raman scattered light generated by irradiation with the laser light; Equipped with a chip having a channel on which a metal nanostructure is formed, and a liquid-air interface formed on the metal nanostructure of an analysis solution introduced into a part of the channel is irradiated with the laser light to perform measurement; a control unit that controls the irradiation position of the laser light in accordance with the movement of the interface caused by evaporation of the analysis solution; Raman scattering spectrometer.

2. The chip is provided. The Raman scattering spectrometer according to claim 1 .

3. a solution introducing means for introducing the analysis solution into a part of the channel of the chip; The Raman scattering spectrometer according to claim 2 .

4. Further comprising an acquisition means for acquiring an image of the irradiation position of the laser light, the control means moves the sample stage in accordance with the position of the interface based on the image; The Raman scattering spectrometer according to claim 1 .

5. The metal nanostructure is a nanodot structure or a nanoripple structure. The Raman scattering spectrometer according to claim 1 .

6. providing a chip having channels with metal nanostructures formed therein; introducing an analysis solution into a portion of the channel of the chip; irradiating a liquid-air interface formed on the metal nanostructures of the analysis solution with laser light; measuring Raman scattered light generated by the irradiation of the laser light; Raman scattering spectroscopy, including

7. the irradiation position of the laser light is fixed, and the laser light is irradiated and the Raman scattered light is measured in a state where the interface of the analysis solution is included in the irradiation area of ​​the laser light and in a state where the analysis solution is not included in the irradiation area of ​​the laser light; The Raman scattering spectroscopy method according to claim 6.

8. a step of controlling the irradiation position of the laser light in accordance with the movement of the interface due to evaporation of the analysis solution; The Raman scattering spectroscopy method according to claim 6.

9. The metal nanostructure is a nanodot structure or a nanoripple structure. The Raman scattering spectroscopy method according to any one of claims 6 to 8.

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