Liquid sample inspection method, non-woven fabric sheet for raman spectroscopic analysis, and inspection plate for raman spectroscopic analysis

Abstract

The present invention provides: a liquid sample inspection method capable of stably acquiring a Raman spectrum with excellent reproducibility from a very small amount of a liquid sample by simpler operation; and a non-woven fabric sheet for Raman spectroscopic analysis used in the method to hold a liquid sample.　Provided is a liquid sample inspection method by Raman spectroscopic analysis including a step for dropping a liquid sample on a non-woven fabric sheet (1) for Raman spectroscopic analysis, and a step for focusing excitation light on the upper surface of the non-woven fabric sheet (1) for Raman spectroscopic analysis and acquiring a Raman spectrum of the liquid sample. Also provided is a non-woven fabric sheet (1) for Raman spectroscopic analysis used in the inspection method to hold the liquid sample. Further provided is an inspection plate for Raman spectroscopic analysis used in the inspection method.

Description

Method for inspecting liquid samples, nonwoven fabric sheet for Raman spectroscopic analysis, and inspection plate for Raman spectroscopic analysis

[0001] The present disclosure relates to a method for testing a liquid sample, a nonwoven fabric sheet for Raman spectroscopic analysis, and a test plate for Raman spectroscopic analysis.

[0002] Raman spectroscopy is an analytical method for investigating the molecular and crystalline structure of a substance by irradiating the substance with light and analyzing the Raman scattered light generated. The Raman scattered light waveform (Raman spectrum) contains various information about the molecules in the sample, and analyzing the Raman spectrum can be used to identify the substance and investigate its crystallinity, orientation, etc. Raman spectroscopy can be applied to any sample, regardless of its nature, including solids, liquids, gases, and gels. Liquid samples are typically measured by sealing the sample in a glass container (cell) if the sample is relatively large (several mL or more), or by dropping the sample onto a glass plate (slide) if the sample is very small.

[0003] Meanwhile, cancer is a serious disease that accounts for a large number of causes of death worldwide. Early detection of cancer and early initiation of appropriate treatment improves treatment outcomes. Currently, most cancer diagnoses are made by imaging diagnostics using ultrasound, CT, MRI, etc., followed by a definitive histopathological diagnosis through microscopic observation of tissue samples.

[0004] Blood tests are one of the most basic medical tests and are widely used because they are useful and minimally invasive. One method for diagnosing cancer using blood tests is to measure tumor markers, but satisfactory results have not been achieved in terms of both sensitivity and specificity, and so this method remains a secondary testing method.

[0005] The present inventors have developed a new cancer testing method that applies Raman spectroscopy to analyze the Raman spectrum of serum to determine whether the patient has cancer (see Patent Document 1). Patent Document 1 discloses a method (needle method) in which a serum sample is used as a droplet to form a convex curved surface, excitation light is focused on the apex of the convex curved surface, and the Raman spectrum of the serum sample is acquired using a micro-Raman spectrometer.

[0006] Patent No. 7129732 specification

[0007] Raman spectroscopy is expected to be useful because it is a non-destructive, non-contact, versatile, and simple analytical technique. However, conventional Raman spectroscopy has problems, such as insufficient detection sensitivity making it difficult to detect trace substances, and the spectrum is significantly affected by the sample's autofluorescence, making it difficult to reliably analyze biological samples. Furthermore, when obtaining Raman spectra of trace liquid samples, the method of measuring by dropping the sample onto a glass plate is often chosen. However, this method can lead to changes in component concentration, such as solidification or dissipation into the atmosphere, which can cause the Raman spectrum to become unstable and potentially compromise the reliability of the measurement results.

[0008] The needle method of Patent Document 1 obtains Raman spectra directly from droplets of serum samples, making the sample less susceptible to change and enabling stable Raman spectra to be obtained from small amounts of biologically derived liquid samples. However, the needle method requires careful attention to ensure that the liquid sample always maintains a convex curve during Raman spectrum acquisition and that the focus of the excitation laser light coincides with the apex of the convex curve of the liquid sample.

[0009] In view of the above-mentioned problems, the present disclosure aims to provide a liquid sample testing method that enables reproducible and stable acquisition of Raman spectra from extremely small amounts of liquid sample through simpler operations, as well as a nonwoven fabric sheet for Raman spectroscopic analysis that is used in the method and that is used to hold the liquid sample, and a Raman spectroscopic analysis testing plate that includes the nonwoven fabric sheet.

[0010] As a result of extensive research to achieve the above-mentioned object, the inventors of the present application discovered that by dropping a very small amount of liquid sample onto quartz fiber filter paper, which is originally used for sampling air pollutants, and measuring it by irradiating it with an excitation laser, it is possible to obtain the Raman spectrum of the sample with good reproducibility and stability. They also discovered that Raman spectra of liquid samples can be obtained even when using laboratory qualitative filter paper made of materials other than quartz fiber or nonwoven fabric sheets such as paper towels. Based on these findings, the present disclosure has been completed.

[0011] That is, the present disclosure provides a method for inspecting a liquid sample by Raman spectroscopy, the inspection method including the steps of dropping the liquid sample onto a nonwoven fabric sheet and focusing excitation light on the upper surface of the nonwoven fabric sheet to obtain the Raman spectrum of the liquid sample.

[0012] The present disclosure also provides a nonwoven fabric sheet for holding a liquid sample, which is used in the testing method, and has a basis weight of 80 to 185 g / m ２ and a thickness of 0.15 to 1 mm.

[0013] The present disclosure also provides a test plate for use in the test method, the test plate comprising: a plate; and one or more nonwoven fabric sheets placed on the plate for holding a liquid sample, the one or more nonwoven fabric sheets having a basis weight of 80 to 185 g / m ２ and a thickness of 0.15 to 1 mm.

[0014] According to the present disclosure, it is possible to obtain Raman spectra from very small amounts of liquid samples with good reproducibility and stability through the simple operation of dropping the liquid sample onto a nonwoven fabric sheet and obtaining the Raman spectrum. The present disclosure also provides a nonwoven fabric sheet for Raman spectroscopy that can hold a liquid sample. Furthermore, the present disclosure also provides a Raman spectroscopy test plate that includes the nonwoven fabric sheet for Raman spectroscopy.

[0015] 9 is a schematic diagram illustrating the surface tension and adsorptive force acting on a droplet of a liquid sample on a nonwoven fabric sheet.

[0034] FIG. 9 is a diagram comparing Raman spectra measured under the same conditions using quartz fiber filter paper of different shapes.

[0035] FIG. 9 is a top view of a test plate for Raman spectroscopy according to one embodiment.

[0036] FIG. 9 is a top view of a test plate for Raman spectroscopy according to one embodiment.

[0037] FIG. 9 is a perspective view and a front view of a test plate for Raman spectroscopy according to one embodiment.

[0038] FIG. 9 is a schematic diagram illustrating an example of the configuration of a micro-Raman spectrometer for carrying out a testing method according to the present embodiment.

[0039] FIG. 9 is a diagram mapping the difference in Raman scattered light intensity at measurement sites on quartz fiber filter paper of different shapes.

[0039] FIG. 9 is a graph showing the results of Raman spectroscopy analysis in Test Example 1.

[0039] FIGS. 9(A) to 9(C) are Raman spectra measured using quartz fiber filter paper for aqueous solutions of rhodamine 6G at concentrations of 5 M, 6 M, and 7 M, respectively.

[0039] FIG. 9 is a graph showing the results of Raman spectroscopy analysis in Test Example 1. Figures 10(A) to 10(C) show Raman spectra measured using quartz fiber filter paper for aqueous solutions of rhodamine 6G at concentrations of 8M, 9M, and 10M, respectively. This graph shows the results of Raman spectroscopy in Test Example 1. Figures 11(A) to 11(C) show Raman spectra measured two months after dropwise application of rhodamine 6G solutions at concentrations of 5M, 6M, and 7M to quartz fiber filter paper. This graph shows the results of Raman spectroscopy in Test Example 1. Figures 12(A) to 12(C) show Raman spectra measured two months after dropwise application of rhodamine 6G solutions at concentrations of 8M, 9M, and 10M to quartz fiber filter paper. This graph shows the results of Raman spectroscopy in Test Example 1. Figures 13(A) to 13(C) show Raman spectra (positive control) measured using the needle method for aqueous solutions of rhodamine 6G at concentrations of 5M, 6M, and 7M, respectively. This graph shows the results of Raman spectroscopy in Test Example 1. Figures 14(A) to 14(C) show Raman spectra (positive controls) measured using the needle method for aqueous solutions of rhodamine 6G at concentrations of 8 M, 9 M, and 10 M, respectively. ... quartz slide only, and Figure 15(B) shows the Raman spectrum (negative control) measured using the quartz slide and quartz fiber filter paper.Graphs showing the results of Raman spectroscopy in Test Example 1. Figures 16(A) and 16(B) show Raman spectra measured twice at the center (droplet site) of a rhodamine 6G aqueous solution dispensed onto a paper towel, while Figure 16(C) shows a Raman spectrum measured at a peripheral location 5 mm from the center. Photographs showing measurement points on QM-B and qualitative filter paper in Test Example 2. Photographs showing measurement points on QM-A in Test Example 2. Graphs showing the results of Raman spectroscopy in Test Example 2. Figure 19(A) shows the Raman spectrum measured at the center (droplet site) of a rhodamine 6G aqueous solution dispensed onto quartz fiber filter paper QM-B, and Figure 19(B) shows the Raman spectrum measured near the center. Figure 19(C) shows the Raman spectrum (positive control) of a 10 M rhodamine 6G aqueous solution dispensed using the needle method. Graphs showing the results of Raman spectroscopy in Test Example 2. Figure 20(A) shows the Raman spectrum measured at the center (droplet site) when a rhodamine 6G aqueous solution was dropped onto qualitative filter paper, Figure 20(B) shows the Raman spectrum measured near the center, and Figure 20(C) shows the Raman spectrum measured at a peripheral area 4 mm from the center. Figure 21(A) shows the Raman spectrum measured at the center (droplet site) when a rhodamine 6G aqueous solution was dropped onto quartz fiber filter paper QM-A, and Figure 21(B) shows the Raman spectrum measured near the center. Figure 22(A) shows the Raman spectrum measured at the center (droplet site) when a propylene glycol aqueous solution was dropped onto quartz fiber filter paper QM-B, and Figure 20(B) shows the Raman spectrum measured near the center. Figure 20(C) shows the Raman spectrum (positive control) of a propylene glycol aqueous solution measured using the needle method. 23A and 23B are graphs showing the results of Raman spectroscopy in Test Example 3. Fig. 23A shows the Raman spectrum measured at the center (droplet location) when a propylene glycol aqueous solution was dropped onto quartz fiber filter paper QM-A, and Fig. 23B shows the Raman spectrum measured near the center. Fig. 23B is a graph showing the results of Raman spectroscopy in Test Example 4.Figure 24(A) shows the Raman spectrum measured at the center (drop site) when human serum was dropped onto quartz fiber filter paper QM-B, and Figure 24(B) shows the Raman spectrum measured near the center. Figure 24(C) shows the Raman spectrum (positive control) of human serum measured using the needle method. This graph shows the results of Raman spectroscopy in Test Example 5. When a rhodamine 6G aqueous solution was dropped onto quartz fiber filter paper QM-B, Figure 25(A) shows the Raman spectrum measured at the center 7 days after dropping, and Figure 25(B) shows the Raman spectrum measured near the center. This graph shows the results of Raman spectroscopy in Test Example 5. When a rhodamine 6G aqueous solution was dropped onto qualitative filter paper, Figure 26(A) shows the Raman spectrum measured at the center 7 days after dropping, Figure 26(B) shows the Raman spectrum measured near the center, and Figure 26(C) shows the Raman spectrum measured near the periphery. This graph shows the results of Raman spectroscopy in Test Example 5. Figure 27(A) shows the Raman spectrum measured at the center of quartz fiber filter paper QM-A seven days after the instillation of a rhodamine 6G aqueous solution, and Figure 27(B) shows the Raman spectrum measured near the center. Figure 28(A) shows the Raman spectrum measured near the center of quartz fiber filter paper QM-B seven days after the instillation of human serum. Figure 28(B) shows the Raman spectrum measured near the center. Figure 28(A) shows the Raman spectrum measured near the center. Figure 28(B) shows the Raman spectroscopic analysis results of human serum samples in Test Example 6. The coordinates of the measurement sites on a circular (3.5 mm diameter) quartz fiber filter paper QM-B are shown on the right, and the Raman spectrum at each measurement site is shown on the left. Figure 28(B) shows the Raman spectroscopic analysis results of human serum samples in Test Example 6. The coordinates of the measurement sites on a square (3 mm side) quartz fiber filter paper QM-B are shown on the right, and the Raman spectrum at each measurement site is shown on the left. This figure shows the results of Raman spectroscopy near the apex of each angle when human serum was dropped onto triangular quartz fiber filter paper QM-B with different angles as shown in the center of the figure in Test Example 7. For angles of 30° (upper left), 45° (upper right), 60° (lower left), and 90° (lower right), the upper row shows Raman spectra measured at a point 0.1 mm away from the apex toward the center, and the lower row shows Raman spectra measured at a point 0.5 mm away from the apex toward the center.In Test Example 7, human serum was dropped onto circular and isosceles triangular quartz fiber filter paper QM-B with a 120° apex angle, as shown in the top of the figure, and the results of Raman spectroscopy were obtained near the edge or apex. For the circular (left) and 120° (right) shapes, the top row shows Raman spectra measured 0.1 mm from the edge or apex, and the bottom row shows Raman spectra measured 0.5 mm from the edge or apex. In Test Example 8, human serum was dropped onto square quartz fiber filter paper QM-B of different sizes, and the results of Raman spectroscopy were obtained at each measurement site. For filter paper with side lengths of 2 mm (left), 3 mm (center), and 4 mm (right), the Raman spectra were measured at 0.1 mm, 0.5 mm, and 1.0 mm from the apex, as well as at the center, from top to bottom. In Test Example 8, human serum was dropped onto square quartz fiber filter paper QM-B of different sizes, and the results of Raman spectroscopy were obtained at each measurement site. The Raman spectra measured at 0.1 mm, 0.5 mm, 1.0 mm from the apex, and the center are shown for filter papers with side lengths of 5 mm (left) and 6 mm (right), respectively. This figure shows the shape and measurement range of the quartz fiber filter paper used in Test Example 9. In Test Example 9, the Raman spectroscopy results for each measurement site when a 1M aqueous solution of rhodamine 6G was dropped onto quartz fiber filter paper QM-B of different shapes are shown. For the fan-shaped (left) filter paper, the Raman spectra measured at 2.7 mm, 1.8 mm, 0.9 mm, and 0 mm from the center are shown, from top to bottom. For the circular (center) and square (right) filter paper, the Raman spectra measured at 0.1 mm, 0.5 mm, 1.0 mm from the edge or apex, and the center are shown, from top to bottom. In Test Example 10, the Raman spectroscopy results for each measurement site when a 1M aqueous solution of rhodamine 6G was dropped onto qualitative filter paper of different shapes are shown. The Raman spectra measured at points 0.1 mm, 0.5 mm, and 1.0 mm from the edge or apex, and at the center, are shown for each of the circular (left) and square (right) filter papers, respectively, from top to bottom. In Test Example 11, a 1M aqueous solution of rhodamine 6G was dropped onto experimental paper towels of different shapes, and the results of Raman spectroscopy at each measurement point are shown.The Raman spectra measured for the circular (left) and square (right) filter papers are shown, from top to bottom, at points 0.1 mm, 0.5 mm, and 1.0 mm from the edge or apex, as well as at the center.

[0016] The inspection method according to this embodiment is a method for inspecting a liquid sample by Raman spectroscopic analysis, and is characterized by including the steps of dropping the liquid sample onto a nonwoven fabric sheet and focusing excitation light on the upper surface of the nonwoven fabric sheet to obtain the Raman spectrum of the liquid sample.

[0017] The nonwoven fabric sheet for Raman spectroscopic analysis according to this embodiment is a nonwoven fabric sheet for holding a liquid sample used in the above-mentioned testing method, and has a basis weight of 80 to 185 g / m ２ and a thickness of 0.15 to 1 mm. With this configuration, the nonwoven fabric sheet according to this embodiment can concentrate the sample by diffusing moisture while retaining sample components, thereby increasing the intensity of Raman scattered light from the sample. It is also believed that the internal structure of the nonwoven fabric sheet causes diffuse reflection of excitation light, which is one of the reasons for the increased intensity of Raman scattered light.

[0018] Furthermore, a Raman spectroscopic analysis test plate according to this embodiment is a test plate used in the test method, and includes a plate and one or more nonwoven fabric sheets placed on the plate for holding a liquid sample, the one or more nonwoven fabric sheets having a basis weight of 80 to 185 g / m ２ and has a thickness of 0.15 to 1 mm. With this configuration, the inspection method according to this embodiment can be easily carried out.

[0019] The nonwoven fabric sheet preferably has a basis weight of 80 to 185 g / m ２ , more preferably 85 to 180 g / m ２ , more preferably 90 to 177 g / m ２ , particularly preferably 100 to 175 g / m ２ , particularly preferably 110 to 174 g / m ２ , 120-173g / m ２ , 130-172g / m ２, 140-171g / m ２ , 150-170g / m ２ , 160-170g / m ２ is.

[0020] The nonwoven fabric sheet preferably has a thickness of 0.15 to 1 mm, more preferably 0.2 to 1 mm, even more preferably 0.3 to 1 mm, particularly preferably 0.4 to 1 mm, and especially preferably 0.5 to 0.99 mm, 0.6 to 0.98 mm, 0.7 to 0.97 mm, 0.8 to 0.96 mm, or 0.9 to 0.95 mm.

[0021] The size of the nonwoven fabric sheet is not particularly limited as long as it is large enough to hold a liquid sample. Specifically, it is preferable that the length and width are each within 1 cm, preferably within 9 mm, more preferably within 8 mm, even more preferably within 7 mm, even more preferably within 6 mm, even more preferably within 5 mm, even more preferably within 4 mm, and particularly preferably within 3.5 mm. Limiting the size of the nonwoven fabric sheet to the above ranges can prevent diffusion of sample components, concentrate the sample, and increase the Raman scattering light intensity of the sample. Furthermore, the lower limit of the size of the nonwoven fabric sheet is not particularly limited as long as it does not cause operational inconvenience. Specifically, it is preferable that the length and width are each 1 mm or more, more preferably 1.5 mm or more, even more preferably 2 mm or more, and particularly preferably 2.5 mm or more. More specifically, it is particularly preferable that the length and width are each 3.5 mm or less, and that the length and width are each 2.5 mm or more.

[0022] FIG. 1 is a schematic diagram illustrating the surface tension and adsorption force acting on a droplet of a liquid sample on a nonwoven fabric sheet. Here, the surface tension (γ ＳＧThe force that causes the sample components to diffuse due to the surface tension (surface tension; left-pointing thick arrow in the figure) is greater than the force that causes the sample components to adsorb to the nonwoven fabric sheet (adsorption force; downward-pointing thick arrow in the figure). Therefore, the sample components diffuse on the nonwoven fabric sheet, resulting in dilution. The smaller the molecular weight of the sample components and the lower the concentration of the liquid sample, the more likely diffusion occurs. Therefore, by limiting the size of the nonwoven fabric sheet to the above range, sample dilution due to diffusion can be prevented and stable Raman spectra can be obtained, even for samples with relatively low concentrations of sample components, such as biological samples. Note that the optimal size of the nonwoven fabric sheet can be determined appropriately by those skilled in the art depending on the amount of liquid sample to be dropped. In other words, it is preferable to select a size that allows the liquid sample to spread throughout the entire nonwoven fabric sheet piece, even to the edges, without overflowing from the nonwoven fabric sheet.

[0023] The shape of the nonwoven fabric sheet is not particularly limited. Specifically, the shape of the nonwoven fabric sheet can be polygonal or circular. Here, the polygon may be a triangle, a square, a pentagon, or a hexagon, preferably a triangle, a square, a pentagon, or a hexagon having one or more angles of 90° or less, more preferably a rectangle, and even more preferably a square.

[0024] FIG. 2 compares Raman spectra measured under the same conditions (measurement point: 0.3 mm from the edge toward the center for the circular nonwoven fabric sheet, 0.3 mm from the apex toward the center for the square nonwoven fabric sheet) after dropping a human serum sample onto quartz fiber filter paper of different shapes. As shown in FIG. 2 , it was confirmed that the Raman spectra measured using a square nonwoven fabric sheet had higher Raman scattered light intensity (as indicated by the horizontal bar in the figure) and a higher S / N ratio (as indicated by the circled area in the figure) than the circular nonwoven fabric sheet. By using a polygonal or circular nonwoven fabric sheet having the above-mentioned dimensions, it is possible to obtain Raman spectra with sufficiently high Raman scattered light intensity and S / N ratio. When a rectangular (especially square) nonwoven fabric sheet is used, it is possible to obtain Raman spectra with even higher Raman scattered light intensity and S / N ratio than when a circular nonwoven fabric sheet is used.

[0025] The material of the nonwoven fabric sheet is not particularly limited as long as it does not interfere with the acquisition of Raman spectra, but it is preferable that the nonwoven fabric sheet be made of a material with low autofluorescence, which acts as noise in Raman spectra. Preferred materials for nonwoven fabric sheets with low autofluorescence include quartz glass fiber (hereinafter also referred to as "quartz fiber") and glass fiber, but this embodiment is not limited to these. Furthermore, paper products such as laboratory paper towels, qualitative filter paper, and quantitative filter paper made from cellulose can also be used as nonwoven fabric sheets for Raman spectroscopy analysis in this embodiment.

[0026] Next, one embodiment of a Raman spectroscopy test plate will be described with reference to FIGS. 3 and 4. The Raman spectroscopy test plate 10a is primarily composed of a plate 12a supporting a nonwoven fabric sheet and one or more nonwoven fabric sheets 1a placed on the plate. The material of the plate is not particularly limited as long as it does not interfere with the acquisition of a Raman spectrum, but it is preferably made of a material with low autofluorescence, which acts as noise in the Raman spectrum. Preferred plate materials with low autofluorescence include, but are not limited to, quartz glass, glass, precious metals, and metals (stainless steel, etc.). The shape of the plate is not particularly limited as long as it does not interfere with the acquisition of a Raman spectrum. Specifically, the plate shape can be similar in shape (rectangular) and thickness (approximately 2 mm to 5 mm) to glass slides commonly used in Raman spectroscopy devices. The number of nonwoven fabric sheets placed on the plate can be one or more, and preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 or more. The spacing between the nonwoven fabric sheets is preferably at least about 5 mm to prevent liquid sample spilling over from the nonwoven fabric sheet from contaminating other samples. The plate 12a and the nonwoven fabric sheet 1a may or may not be fixed with an adhesive or the like.

[0027] In another embodiment, as shown in FIG. 5 , a Raman spectroscopy test plate 10b is primarily composed of a stainless steel plate 12b, one or more recesses 14b formed on one side of the plate 12b for holding nonwoven fabric sheets, and one or more nonwoven fabric sheets 1b placed on the bottom of the recesses 14b. The plate 12b is configured as a rectangular or square plate with a thickness of approximately 4 mm to 10 mm. The plate 12b and the recesses 14b may be integrally molded or formed by combining multiple parts. The recesses 14b for holding the nonwoven fabric sheets are formed so that the entire nonwoven fabric sheet remains horizontal when placed on its bottom, and the depth to the bottom can be approximately 1 mm to 3 mm. This configuration reliably prevents contamination of other samples even if a liquid sample overflows from the nonwoven fabric sheet, and reduces the need to secure the nonwoven fabric sheet with adhesive or the like. If the nonwoven fabric sheet 1b is not fixed to the recess 14b, the plate can be easily washed and reused after measurement, which is economical.

[0028] In another embodiment, as shown in FIG. 6 , a Raman spectroscopy test plate 10c is primarily composed of a stainless steel plate 12c, one or more recesses 14c formed on one side of the plate 12c for holding nonwoven fabric sheets, and one or more nonwoven fabric sheets 1c placed on the bottom of the recesses 14c. The recesses 14c are rectangular and surrounded by a partition extending from the plate 12c. The plate 12c and the recesses 14c may be integrally molded or formed by combining multiple components. The recesses 14c for holding the nonwoven fabric sheets are formed so that the entire nonwoven fabric sheet is kept horizontal when placed on the bottom surface, and the depth to the bottom surface can be approximately 1 mm to 3 mm. The nonwoven fabric sheets 1c do not need to be fixed to the recesses 14c.

[0029] In this embodiment, the liquid sample is not particularly limited as long as it can be analyzed by Raman spectroscopy. For example, the liquid sample may be a biopsy sample collected from a subject. More specifically, examples of the liquid sample include serum, plasma, blood, cerebrospinal fluid, lymph, urine, saliva, milk, amniotic fluid, and semen collected from a subject. The subject may be a human or a non-human mammal. Examples of non-human mammals include primates (monkeys, chimpanzees, gorillas, etc.), rodents (mice, hamsters, rats, etc.), rabbits, dogs, cats, cows, goats, sheep, and horses. Furthermore, for example, the liquid sample may be a liquid containing environmental substances, such as water from rivers, oceans, lakes, ponds, agricultural water, or street drains, or a liquid containing a toxic substance.

[0030] In the step of dripping a liquid sample onto a nonwoven fabric sheet, the nonwoven fabric sheet can be placed on a suitable support such as a quartz glass slide, and the sample can be dripped using, for example, a micropipette. In another embodiment, the sample can be dripped onto a nonwoven fabric sheet placed on a Raman spectroscopic analysis test plate. After dripping the sample, the process can immediately proceed to the step of acquiring a Raman spectrum using a Raman spectrometer. As described above, a stable Raman spectrum can be acquired even after a certain period of time has passed since dripping the sample.

[0031] The period from the time when the liquid sample is dropped onto the nonwoven fabric sheet until the time when the sample is subjected to Raman spectroscopic analysis is not particularly limited, but analysis can be performed immediately after dropping, and may be performed thereafter, for example, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, etc. However, analysis can be performed even after a longer period if the sample is stored in a dark place at room temperature. In this specification, room temperature means a temperature in the range of about 20 to 25°C.

[0032] The amount of liquid sample to be dropped onto the nonwoven fabric sheet is preferably 1 to 19 μL, more preferably 2 to 18 μL, even more preferably 3 to 17 μL, particularly preferably 4 to 16 μL, and even more preferably 5 to 15 μL, 6 to 14 μL, 7 to 13 μL, 8 to 12 μL, or 9 to 11 μL. The optimal amount of liquid sample to be dropped can be determined appropriately by those skilled in the art depending on the size of the nonwoven fabric sheet. In other words, it is preferable to use an amount that allows the liquid sample to spread over the entire nonwoven fabric sheet piece, including its edges, without overflowing from the nonwoven fabric sheet.

[0033] Next, the step of acquiring the Raman spectrum of the liquid sample can be performed by a micro-Raman spectrometer such as a Raman microscope. Fig. 7 is a schematic diagram showing an example of the configuration of a micro-Raman spectrometer for carrying out the inspection method according to this embodiment.

[0034] The micro-Raman spectrometer 100 acquires a Raman spectrum of a liquid sample. The micro-Raman spectrometer 100 includes a stage 120, a light source device 130, a dichroic filter 140, a beam splitter 141, a long-pass filter 142, lenses 143 and 144, an objective lens 150, a spectroscope 160, a cooled CCD camera 170, and an observation CCD camera 171.

[0035] A nonwoven fabric sheet 1 for Raman spectroscopy, which holds a liquid sample, is placed on a slide glass 110. The slide glass 110 is placed on a stage 120. The stage 120 is coupled to a stage scanner 121. The stage scanner 121 drives the stage 120 parallel and perpendicular to the surface on which the slide glass 110 is placed, as indicated by the arrows x-y-z in the figure. This allows the focus of the excitation light to be adjusted to a desired position on the nonwoven fabric sheet 1 for Raman spectroscopy.

[0036] The light source device 130 generates excitation light to be irradiated onto the liquid sample. The light source device 130 is preferably a laser light source that generates laser light with a wavelength of 785 nm or 1064 nm, and more preferably a laser light source that generates laser light with a wavelength of 1064 nm. Using a laser light source with such wavelengths allows for the acquisition of a clearer Raman spectrum. The light source device 130 may be equipped with two or more types of laser light sources (e.g., a 785 nm laser light source and a 1064 nm laser light source), and these laser light sources may be switchable. The maximum output of the laser light source may be 50 to 500 mW. Specific examples of laser light sources include a laser light source capable of generating laser light with a wavelength of 1064 nm and a maximum output of 200 mW, or a laser light source capable of generating laser light with a wavelength of 785 nm and a maximum output of 50 mW.

[0037] The dichroic filter 140 reflects the excitation light from the light source device 130 and guides the excitation light to the beam splitter 141. The dichroic filter 140 also transmits the Raman scattered light from the liquid sample and guides the Raman scattered light to the long-pass filter 142.

[0038] The beam splitter 141 transmits the excitation light from the dichroic filter 140. The beam splitter 141 also splits the Raman scattered light from the liquid sample and guides it to the dichroic filter 140, a lens 143, and an observation CCD camera 171.

[0039] The objective lens 150 focuses the excitation light transmitted through the beam splitter 141 onto the liquid sample. The objective lens 150 is preferably a lens capable of observation in the near-infrared region corresponding to the excitation light from the light source device 130. The magnification of the objective lens 150 is preferably 10 to 50 times, more preferably 10 to 30 times, even more preferably 10 times or 20 times, and particularly preferably 20 times. The numerical aperture (NA) of the objective lens 150 is preferably 0.2 or more, more preferably 0.2 to 0.65, even more preferably 0.3 to 0.6, and particularly preferably 0.4 to 0.5. By setting the magnification and NA of the objective lens within the above ranges, a clearer Raman spectrum can be obtained.

[0040] The long-pass filter 142 cuts out the short wavelength region of the Raman scattered light from the liquid sample and transmits the long wavelength region. The lens 144 collects the Raman scattered light that has passed through the long-pass filter 142 and guides it to the spectrometer 160.

[0041] The spectroscope 160 separates the Raman scattered light. The cooled CCD camera 170 detects the light of each wavelength separated by the spectroscope 160. In this way, the Raman spectrum of the liquid sample is obtained.

[0042] The observation CCD camera 171 detects Raman scattered light from the liquid sample that has been split by the beam splitter 141 and guided to the lens 143. The observation CCD camera 171 is used to focus the excitation light at a desired position on the nonwoven fabric sheet for Raman spectroscopic analysis 1. The excitation light is focused on the upper surface of the nonwoven fabric sheet for Raman spectroscopic analysis 1, preferably near the center of the portion where the liquid sample has been dropped.

[0043] However, as mentioned above, even if the size of the nonwoven fabric sheet is limited to a predetermined range, diffusion of the sample components cannot be completely prevented if the molecular weight of the sample components is relatively small or the concentration of the liquid sample is relatively low. Therefore, the location where the excitation light is focused, i.e., the measurement point, can be the center of the liquid sample drip site, or can be a location 0.1 mm or more, further 0.2 mm or more, further 0.3 mm or more, further 0.4 mm or more, further 0.5 mm or more, further 0.6 mm or more, further 0.7 mm or more, further 0.8 mm or more, further 0.9 mm or more, further 1.0 mm or more, further 1.1 mm or more, further 1.2 mm or more, further 1.3 mm or more, further 1.4 mm or more, or even 1.5 mm or more away from the liquid sample drip site. Furthermore, since the edges of a nonwoven fabric sheet may not be suitable for measurement due to the state of the fibers, the measurement point can be at least 0.05 mm, preferably at least 0.1 mm, away from the edge or apex toward the center.

[0044] Figure 8 is a mapping diagram of the difference in Raman scattered light intensity depending on the measurement site when a human serum sample was dropped onto quartz fiber filter paper of different shapes. As shown in Figure 8, for a circular nonwoven fabric sheet, the Raman scattered light intensity increased with increasing distance from the center (droplet site), while for a square nonwoven fabric sheet, the Raman scattered light intensity increased with increasing distance from the center (droplet site) and closer to the apex. Thus, depending on the components and concentration of the liquid sample, focusing the excitation light at a position some distance from the liquid sample drop site, as described above, may result in a higher Raman scattered light intensity than at the center of the drop site, resulting in a more stable Raman spectrum. Preferably, the measurement point can be located 0.01 mm to 1 mm, preferably 0.05 mm to 0.8 mm, even more preferably 0.1 mm to 0.6 mm, and particularly preferably 0.2 mm to 0.4 mm from the edge of the nonwoven fabric sheet (especially the apex in the case of a polygonal nonwoven fabric sheet).

[0045] The acquired Raman spectrum information of the liquid sample is sent to the processing device 190. The processing device 190 analyzes the Raman spectrum information and outputs the data.

[0046] Next, an example of a process for acquiring a Raman spectrum will be described.

[0047] When acquiring a Raman spectrum, a liquid sample is held in a nonwoven fabric sheet for Raman spectroscopy 1 and placed on a glass slide 110. The glass slide 110 is further placed on a stage 120. Excitation light (e.g., laser light with a wavelength of 1064 nm) generated from a light source device 130 is reflected by a dichroic filter 140, transmitted through a beam splitter 141, and focused by an objective lens 150 before being irradiated onto the liquid sample. At this time, the focus of the excitation light is adjusted to the upper surface of the nonwoven fabric sheet for Raman spectroscopy 1, preferably near the location where the liquid sample has been dropped on the upper surface of the nonwoven fabric sheet for Raman spectroscopy 1. Raman scattered light generated from the liquid sample by irradiation with the excitation light passes through the objective lens 150, the beam splitter 141, and the dichroic filter 140, has its short wavelength region cut off by a long-pass filter 142, is focused by a lens 144, and is then dispersed by a spectrometer 160. The separated light of each wavelength is detected by a cooled CCD camera 170, and the Raman spectrum of the liquid sample is obtained.

[0048] The light source device 130 preferably generates excitation light at an output of 50 to 500 mW, more preferably 100 to 300 mW, and even more preferably 150 to 250 mW. 200 mW is particularly preferable. When the output is within this range, a clearer Raman spectrum can be obtained. The irradiation time of the excitation light is preferably 1 to 30 seconds, more preferably 1 to 20 seconds, and even more preferably 3 to 15 seconds. When the irradiation time is within this range, a clearer Raman spectrum can be obtained.

[0049] According to the present embodiment described above, it is possible to quickly and inexpensively obtain a high-resolution Raman spectrum from a minute amount of liquid sample. In addition, there is no significant change in the Raman spectrum when the liquid sample is measured immediately after being dropped onto the nonwoven fabric sheet for Raman spectroscopic analysis and when a certain period of time has elapsed, and stable measurement results can be obtained even over time.

[0050] Furthermore, this embodiment can also measure liquid biopsy samples. Because Raman spectra can be obtained from extremely small amounts of liquid biopsy samples, the invasiveness to the subject from which the sample is collected can be minimized, allowing repeated testing as needed. Therefore, applying this embodiment to the testing method using serum samples described in Patent Document 1 is effective not only for the early diagnosis of various diseases, including cancer, but also for assessing treatment efficacy and diagnosing recurrence. Furthermore, because similar Raman spectra can be obtained even after a certain period of time has passed since sample preparation, the collected liquid biopsy sample can be dropped onto the nonwoven fabric sheet for Raman spectroscopy analysis of this embodiment and then transported to a remote location for testing. Furthermore, since this embodiment can also be used to measure small amounts of liquid samples containing environmental substances or toxic substances, it can be widely applied outside the medical field, and is highly versatile and useful, and is expected to contribute widely to society.

[0051] (Test Example 1) Raman Spectroscopic Analysis of Rhodamine 6G Aqueous Solution Using Quartz Fiber Filter Paper High-purity quartz (SiO ２ ) fiber, and quartz fiber filter paper (Whatman, QM-B, weight: 165 g / m ２ Raman spectroscopic analysis of a liquid sample was performed using a 0.95 mm thick PET film. The liquid sample used was an aqueous solution of rhodamine 6G (concentration: 5M-10M), which is one of the standard substances for Raman spectroscopic evaluation. The Raman spectroscopic analysis was performed using a Raman microscope under the conditions shown in Table 1 below.

[0052]

[0053] A quartz fiber filter was placed in the center of a quartz glass slide, and 10 μL of liquid sample was dropped onto the center of the filter. The slide with the filter was then placed in a Raman microscope. Five minutes after the drop, the excitation laser was focused on the filter surface to obtain a Raman spectrum of the drop. The quartz fiber filter used in the above measurements was also stored in a dark place at room temperature (20-25°C, hereinafter the same) for two months, and then measured in the same manner as above.

[0054] As a positive control, Raman spectra of liquid samples were obtained using the needle method described in Patent Document 1. In the needle method, a convex surface of the liquid sample was formed at the tip of an injection needle, and measurements were performed by focusing an excitation laser on the convex surface. As negative controls, measurements were similarly performed on a quartz glass slide alone and a quartz glass slide with quartz fiber filter paper (neither of which had a sample dripped onto it).

[0055] Measurements were also conducted in the same manner as above, except that a laboratory paper towel (Elleair Prowipe Soft Towel, manufactured by Daio Paper Co., Ltd.) was used instead of the quartz fiber filter paper. When the sample was dropped onto the paper towel, the sample spread over an area of ​​approximately 7 mm from the drop site, so measurements were taken at the center (the drop site) and the periphery (a point approximately 5 mm away from the drop site).

[0056] The measurement results are shown in Figures 9 to 16. Figures 9(A) to 9(C) and Figures 10(A) to 10(C) show Raman spectra of rhodamine 6G solutions at concentrations of 5M, 6M, 7M, 8M, 9M, and 10M, respectively, measured using quartz fiber filter paper. Figures 11(A) to 11(C) and Figures 12(A) to 12(C) show Raman spectra of rhodamine 6G solutions at concentrations of 5M, 6M, 7M, 8M, 9M, and 10M, respectively, measured two months after application to quartz fiber filter paper. Figures 13(A) to 13(C) and Figures 14(A) to 14(C) show Raman spectra (positive control) of rhodamine 6G solutions at concentrations of 5M, 6M, 7M, 8M, 9M, and 10M, respectively, measured using the needle method. Figure 15(A) shows the Raman spectrum of the quartz slide alone, and Figure 15(B) shows the Raman spectrum of the quartz slide and quartz fiber filter paper (negative control). Figures 16(A) to 16(C) show the Raman spectra of a 10M aqueous solution of rhodamine 6G measured using a laboratory paper towel. Figures 16(A) and 16(B) show two measurements taken at the center (application site), and Figure 16(C) shows a measurement taken at the periphery. In all of the above graphs, the vertical axis represents the Raman scattered light intensity, and the horizontal axis represents the Raman shift.

[0057] A comparison of the Raman spectra obtained using quartz fiber filter paper (Figures 9A-9C and 10A-10C) with those obtained using the needle method (Figures 13A-13C and 14A-14C) demonstrates that the method according to this embodiment provides sufficient Raman scattered light intensity for analysis and enables the acquisition of high-resolution Raman spectra with a high S / N ratio. In particular, for a highly concentrated (10 M) aqueous solution of rhodamine 6G, the use of quartz fiber filter paper tended to provide higher scattered light intensity than the use of the needle method (Figure 10C vs. Figure 14C).

[0058] Figures 15(A) and 15(B) show that the background quartz slide glass and quartz fiber filter paper are made of materials with low autofluorescence and do not affect the Raman spectrum of the sample. Furthermore, comparing the Raman spectra (Figures 9(A) to 9(C) and 10(A) to 10(C)) measured 5 minutes after sample application with the Raman spectra (Figures 11(A) to 11(C) and 12(A) to 12(C)) measured 2 months later, the original spectra were maintained even after 2 months. This indicates that if the quartz fiber filter paper is stored under appropriate conditions for the sample after sample application, it is likely to be able to withstand evaluation over long periods of time.

[0059] As shown in Figures 16(A) to 16(C), a large difference was observed in the Raman spectrum between the center of the experimental paper towel (the drop location; Figures 16(A) to 16(B)) and the periphery (Figure 16(C)). Furthermore, the results of two measurements taken at the center (Figures 16(A) to 16(B)) indicated that the spectrum itself was very unstable. It was thought that the Raman spectrum of rhodamine 6G became unstable because rhodamine 6G did not spread evenly on the lightly weighed, thin paper towel.

[0060] These results demonstrate that Raman spectroscopy using quartz fiber filter paper can obtain Raman spectra with high resolution and a good signal-to-noise ratio, comparable to or even better than the needle method. This is presumably because the sample molecules are held in the voids within the quartz fiber filter paper, while the water molecules diffuse, resulting in a localized increase in the concentration of sample molecules at the drop site, resulting in an increase in the intensity of Raman scattered light. It is also possible that the irradiated light is scattered within the quartz fiber filter paper, amplifying the irradiated light and Raman scattered light. Furthermore, it was demonstrated that the quartz fiber filter paper after sample application can obtain stable Raman spectra with good reproducibility even after a long period of time. In contrast, when using laboratory paper towels, the liquid sample does not spread evenly, making it impossible to obtain stable Raman spectra.

[0061] (Test Example 2) Raman Spectroscopic Analysis of Rhodamine 6G Aqueous Solution Using Quartz Fiber Filter Paper and Qualitative Filter Paper Raman spectroscopic analysis of a liquid sample was performed using the same quartz fiber filter paper as in Test Example 1. The liquid sample used was a 10 M rhodamine 6G aqueous solution. The Raman spectroscopic analysis was performed using a micro-Raman spectrometer under the conditions shown in Table 2 below.

[0062]

[0063] A quartz fiber filter was placed in the center of a quartz glass slide, and 10 μL of liquid sample was dropped onto the center of the filter. The slide with the filter was then placed in a Raman microscope. Five minutes after the drop, the excitation laser was focused on the filter surface, and Raman spectra were acquired at the drop site. As shown in Figure 17, when the sample was dropped onto the quartz fiber filter, the sample spread over an area of ​​approximately 2 mm from the drop site. Therefore, measurements were taken at the center (the drop site) and near the center (a point approximately 1 mm away from the drop site) (each measurement point is indicated by a circle in Figure 17).

[0064] As a positive control, Raman spectra of liquid samples were obtained by the needle method (analysis conditions are shown in Table 2). In addition, qualitative filter paper (manufactured by AS ONE Corporation, product code: 2-870-04, material: cellulose, weighing: 80±4 g / m) was used instead of quartz fiber filter paper. ２Measurements were performed in the same manner as above, except that a filter paper with a thickness of approximately 0.18 mm was used. As shown in Figure 17, when a sample was dropped onto the qualitative filter paper, the sample spread over an area of ​​approximately 7 mm from the drop site, so measurements were taken at the center (drop site), near the center (a point approximately 1 mm away from the drop site), and at the periphery (a point approximately 4 mm away from the drop site) (each measurement point is indicated by a circle in Figure 17).

[0065] Furthermore, a Whatman QM-A (weighing: 85 g / m) quartz fiber filter was used. ２ Measurements were performed in the same manner as above using a quartz fiber filter paper QM-A (thickness: 0.47 mm). As shown in Figure 18, when a sample was dropped onto the quartz fiber filter paper QM-A, the sample spread over a slightly wider area, approximately 4 mm from the drop site, compared to QM-B. Therefore, as with QM-B, measurements were performed at the center (drop site) and near the center (a point approximately 1 mm away from the drop site) (each measurement point is indicated by a circle in Figure 18).

[0066] The measurement results are shown in Figures 19 to 21. Figures 19(A) and 19(B) show Raman spectra of a 10 M rhodamine 6G aqueous solution measured using quartz fiber filter paper QM-B. Figure 19(A) shows the spectrum measured at the center (droplet location), and Figure 19(B) shows the spectrum measured near the center. Figure 19(C) shows the Raman spectrum (positive control) of a 10 M rhodamine 6G aqueous solution measured using the needle method. Figures 20(A) to 20(C) show Raman spectra of a 10 M rhodamine 6G aqueous solution measured using qualitative filter paper. Figure 20(A) shows the spectrum measured at the center (droplet location), Figure 20(B) shows the spectrum measured near the center, and Figure 20(C) shows the spectrum measured near the periphery. Figures 21(A) and 21(B) show Raman spectra measured using a quartz fiber filter paper QM-A for a 10 M rhodamine 6G aqueous solution, with Figure 21(A) showing the measurement at the center (the drop location) and Figure 21(B) showing the measurement near the center. In both graphs, the vertical axis represents the Raman scattered light intensity, and the horizontal axis represents the Raman shift.

[0067] From Figures 19(A)-(C) and Figure 20(A), it can be seen that the Raman spectra for the quartz fiber filter paper QM-B (Figures 19(A)-(B)), the needle method (Figure 19(C)), and the center of the qualitative filter paper (Figure 20(A)) are all similar, suggesting that stable measurements were possible. On the other hand, near the center and peripheral areas of the qualitative filter paper (Figures 20(B)-(C)), the sample was not spread evenly, resulting in a decrease in the Raman scattered light intensity and an unstable spectrum.

[0068] Furthermore, a comparison of Figures 19(A) and 19(C) indicates that, as in Test Example 1, higher scattered light intensity was obtained using the quartz fiber filter paper QM-B than using the needle method. Furthermore, a comparison of Figures 19(A)-(B) and Figures 21(A)-(B) indicates that, although some variation in Raman scattered light intensity was observed when using QM-A, the Raman spectrum remained intact, demonstrating that a fairly stable measurement was possible. Furthermore, while similar Raman spectra were obtained in the center and its vicinity with QM-A, the sample spread was less uniform than with QM-B (Figure 17), resulting in variation in Raman scattered light intensity.

[0069] These results demonstrate that not only quartz fiber filter paper but also qualitative filter paper made from cellulose fiber can be used as nonwoven fabric sheets for Raman spectroscopy to hold liquid samples, provided that measurements are limited to the drop site, and that Raman spectra with high resolution and a good S / N ratio comparable to those obtained with the needle method can be obtained. Furthermore, it was demonstrated that even when using quartz fiber filter paper QM-A, which is about half the thickness of QM-B, a fairly stable Raman spectrum can be obtained.

[0070] (Test Example 3) Raman Spectroscopic Analysis of Propylene Glycol Aqueous Solution Using Quartz Fiber Filter Paper Raman spectroscopic analysis of a liquid sample was performed using the quartz fiber filter paper (Whatman, QM-A and QM-B) used in Test Example 2. A propylene glycol aqueous solution (99.0%) was used as the liquid sample. As in Test Example 2, the measurement points were the center (the dripping site) and near the center (a point approximately 1 mm away from the dripping site). Raman spectroscopic analysis was performed under the same conditions as Test Example 2. As a positive control, a Raman spectrum of the liquid sample was obtained using the needle method.

[0071] The measurement results are shown in Figures 22 and 23. Figures 22(A) and 22(B) show Raman spectra of a propylene glycol aqueous solution measured using quartz fiber filter paper QM-B. Figure 22(A) shows the spectrum measured at the center (drop site), and Figure 22(B) shows the spectrum measured near the center. Figure 22(C) shows the Raman spectrum (positive control) of a propylene glycol aqueous solution measured using the needle method. Figures 23(A) and 23(B) show the Raman spectrum of a propylene glycol aqueous solution measured using quartz fiber filter paper QM-A. Figure 23(A) shows the spectrum measured at the center (drop site), and Figure 23(B) shows the spectrum measured near the center. In all of the above graphs, the vertical axis represents Raman scattered light intensity, and the horizontal axis represents Raman shift.

[0072] As can be seen from Figures 22(A)-(C), the Raman spectrum of the quartz fiber filter paper QM-B (Figures 22(A)-(B)) has a similar spectral shape to that obtained using the needle method, suggesting that stable measurements were possible even with propylene glycol aqueous solution. Comparing Figures 22(A)-(B) with Figures 23(A)-(B) indicates that QM-A, like QM-B, can also obtain good Raman spectra. While the QM-A sample tended to be more spread out than QM-B, the spectrum at and near the center was good. These results demonstrate that stable Raman spectra can be obtained using quartz fiber filter paper even with propylene glycol aqueous solution.

[0073] (Test Example 4) Raman Spectroscopic Analysis of Human Serum Using Quartz Fiber Filter Paper Raman spectroscopic analysis of a liquid sample was performed using quartz fiber filter paper QM-B. Human serum was used as the liquid sample. As in Test Example 2, the measurement points were the center (the drop site) and near the center (a point approximately 1 mm away from the drop site). Raman spectroscopic analysis was performed under the same conditions as in Test Example 2. As a positive control, the Raman spectrum of the liquid sample was obtained using the needle method.

[0074] The measurement results are shown in Figure 24. Figures 24(A) and 24(B) show Raman spectra of human serum measured using quartz fiber filter paper QM-B. Figure 24(A) shows the spectrum measured at the center (droplet site), and Figure 24(B) shows the spectrum measured near the center. Figure 24(C) shows the Raman spectrum (positive control) of human serum measured using the needle method. In the graph, the vertical axis represents Raman scattered light intensity, and the horizontal axis represents Raman shift.

[0075] 24(A) to (C), the Raman spectrum of the quartz fiber filter paper (FIGS. 24(A) to (B)) has a similar spectral shape to that of the needle method, suggesting that a fairly stable measurement was possible for serum. These results demonstrate that a fairly stable Raman spectrum can also be obtained for human serum using quartz fiber filter paper.

[0076] (Test Example 5) Raman spectroscopic analysis of various liquid samples 7 days after dropwise addition For the various liquid samples measured in Test Examples 2 and 4, the quartz fiber filter paper and qualitative filter paper after dropwise addition were stored in a dark place at room temperature for 7 days, and then measured again in the same manner as in the original test example.

[0077] The measurement results are shown in Figures 25 to 28. Figures 25(A) and 25(B) show Raman spectra of a 10 M rhodamine 6G aqueous solution measured using quartz fiber filter paper QM-B 7 days after the solution was dropped. Figure 25(A) shows the spectrum measured at the center (the site of the drop, hereinafter the same) and Figure 25(B) shows the spectrum measured near the center (a point approximately 1 mm away from the site of the drop, hereinafter the same). Figures 26(A) to 26(C) show Raman spectra of a 10 M rhodamine 6G aqueous solution measured using qualitative filter paper 7 days after the solution was dropped. Figure 26(A) shows the spectrum measured at the center, Figure 26(B) shows the spectrum measured near the center, and Figure 26(C) shows the spectrum measured at the periphery (a point approximately 4 mm away from the site of the drop, hereinafter the same). Figures 27(A) and (B) show Raman spectra of a 10 M aqueous solution of rhodamine 6G measured using quartz fiber filter paper QM-A seven days after instillation. Figure 27(A) shows the spectrum measured at the center, and Figure 27(B) shows the spectrum measured near the center. Figures 28(A) and (B) show Raman spectra of human serum measured using quartz fiber filter paper QM-B six days after instillation. Figure 28(A) shows the spectrum measured at the center, and Figure 28(B) shows the spectrum measured near the center. In each of the graphs, the vertical axis represents the Raman scattered light intensity, and the horizontal axis represents the Raman shift.

[0078] 25 to 28 show that the measurement results for all nonwoven fabric sheets tended to be similar to the measurement results 5 minutes after application in Test Example 2 (FIGS. 19 to 21) and Test Example 4 (FIG. 24). These results indicate that, depending on the type of sample, the quality of the sample can be maintained and repeated analysis is possible by storing the nonwoven fabric sheet under appropriate conditions, even after the liquid sample has been applied to it. Furthermore, because nonwoven fabric sheets are easier to transport than the liquid sample itself, they can be moved from the point where the liquid sample was collected and analyzed, suggesting the possibility of expanding the scope of application of Raman spectroscopy.

[0079] (Test Example 6) Raman Spectroscopic Analysis of Human Serum Using Circular and Square Quartz Fiber Filters. Quartz fiber filter paper QM-B cut into circular (3.5 mm diameter) and square (3 mm side) shapes was used as a nonwoven fabric sheet to perform Raman spectroscopic analysis of a liquid sample. The liquid sample used was human serum (sample No. P-268). The Raman spectroscopic analysis was performed using a micro-Raman spectrometer under the conditions shown in Table 3 below.

[0080]

[0081] A cut piece of quartz fiber filter paper was placed on a quartz glass slide, and 10 μL of liquid sample was dropped onto the center of the filter paper. The slide with the filter paper on it was then set into a Raman microscope. Five minutes after the drop, the excitation laser was focused on the positions indicated by small circles on the filter paper surface in Figures 29 and 30, and Raman spectra were obtained at each measurement point. The position of each measurement point is represented by the X coordinate axis in the horizontal direction and the Y coordinate axis in the vertical direction, as shown in the figures, with the center of the filter paper designated as (X,Y) = (0,0) (unit: mm).

[0082] The measurement results are summarized in Figures 29 and 30. The right side of Figure 29 shows a map of the measurement points on the circular filter paper, and the left side of Figure 29 shows the Raman spectra at (X,Y) = (0,0.1), (0,0.5), (0,1.0), and (0,1.75). The right side of Figure 30 shows a map of the measurement points on the square filter paper, and the left side of Figure 30 shows the Raman spectra at (X,Y) = (0,0.1), (0,0.5), (0,1.0), (0,2.0), (0.4,0.5), and (0.9,1.0). In both graphs, the vertical axis represents the Raman scattered light intensity, and the horizontal axis represents the Raman shift.

[0083] A comparison of the Raman spectra of each measurement point on the circular filter paper (3.5 mm diameter) in Figure 29 (the top graph is closest to the edge, and the graphs below are closer to the dripping site) shows that the Raman scattered light intensity is higher near the edge, resulting in a Raman spectrum with a high S / N ratio. A comparison of the Raman spectra of each measurement point on the square filter paper (3 mm on each side) in Figure 30 (the top graph is closest to the apex, and the graphs below are closer to the dripping site; the bottom and second-to-bottom graphs are farther from the apex but closer to the edge) shows that the Raman scattered light intensity is higher near the apex, resulting in a Raman spectrum with a high S / N ratio. Furthermore, the Raman scattered light intensity is higher near the apex than the edge, resulting in a Raman spectrum with a high S / N ratio.

[0084] These results indicate that for circular nonwoven fabric sheets of a given size, the Raman scattered light intensity increases with increasing distance from the center (droplet site), while for square nonwoven fabric sheets of a given size, the Raman scattered light intensity increases with increasing distance from the center (droplet site) and toward the apex. Specifically, particularly strong Raman scattered light intensity was obtained at measurement points approximately 0.1 to 0.5 mm away from the edge or apex of the nonwoven fabric sheet. This is thought to be because, in the case of serum with a relatively low sample concentration, surface tension causes serum components to diffuse across the nonwoven fabric sheet, resulting in a local increase in the concentration of sample molecules near the edge or apex, resulting in high Raman scattered light intensity.

[0085] (Test Example 7) Comparison of Raman Spectra of Human Serum Near Vertices and Edges of Circles with Different Angles. Liquid samples were analyzed using triangular and circular (3 mm diameter) quartz fiber filter paper QM-B cut into nonwoven fabric sheets. Four types of triangular filter paper were prepared, as shown in Figures 31 and 32: right-angled triangles at 30°, 60°, and 90° (with sides of the right angle of 4 mm and 6.9 mm), equilateral triangles with sides of 4 mm, right-angled isosceles triangles (with sides of the right angle of 4 mm), and isosceles triangles with sides of 120°, 30°, and 30° (with sides of the obtuse angle of 5 mm). The same human serum as in Test Example 6 was used as the liquid sample. Raman spectroscopic analysis was performed under the same conditions as in Test Example 6.

[0086] A cut piece of quartz fiber filter paper was placed on a quartz glass slide, and 10 μL of liquid sample was dropped onto the center of the filter paper. The slide with the filter paper on it was then placed in a Raman microscope. Five minutes after the drop was dropped, the excitation laser was focused on the surface of the filter paper, and Raman spectra were obtained at each measurement point. The measurement points were 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, and 0.5 mm from the apex (or edge in the case of circular filter paper) toward the center.

[0087] The measurement results are summarized in Figures 31 and 32. Figure 31 shows triangular quartz fiber filter papers with different angles and the Raman spectra near the apex of each angle (the upper row shows a point 0.1 mm from the apex, and the lower row shows a point 0.5 mm from the apex toward the center). Figure 32 shows circular and triangular quartz fiber filter papers with a 120° angle and the Raman spectra near the edge of the circular filter paper or the apex of the 120° angle (the upper row shows a point 0.1 mm from the edge or apex, and the lower row shows a point 0.5 mm from the edge or apex toward the center). In both graphs, the vertical axis represents the Raman scattered light intensity, and the horizontal axis represents the Raman shift.

[0088] A comparison of Raman spectra near the apex at different angles in Figure 31 shows that Raman spectra can be recorded at any angle, and that Raman scattering intensity and S / N ratio tend to be higher the closer to the apex. Furthermore, the shape of the Raman spectra was similar at all angles. Thus, no differences in Raman spectra were observed at angles below 90°. On the other hand, as shown in Figure 32, for the 120° obtuse angle and circular filter paper, the Raman scattering intensity and S / N ratio at the apex or edge were good, but compared to angles below 90° (Figure 31), the Raman scattering intensity and S / N ratio tended to decrease with increasing distance from the apex.

[0089] These results show that for polygonal nonwoven fabric sheets of a given size, the vertex angles (i.e., the number of vertices) have no effect, and spectra with good Raman scattered light intensity and S / N ratios are obtained at all measurement points close to the vertices.

[0090] (Test Example 8) Comparison of Raman Spectra of Human Serum on Square Quartz Fiber Filter Paper of Different Sizes Quartz fiber filter paper QM-B cut into squares (2 mm, 3 mm, 4 mm, 5 mm, and 6 mm on a side) was used as a nonwoven fabric sheet to perform Raman spectroscopic analysis of a liquid sample. The same human serum as in Test Example 6 was used as the liquid sample. The Raman spectroscopic analysis was performed under the same conditions as in Test Example 6.

[0091] A cut quartz fiber filter paper was placed on a quartz glass slide, and a predetermined amount of liquid sample was dropped onto the center of the filter paper. The drop volume was set according to the size of the filter paper, so that the liquid sample reached the edge of the filter paper but did not overflow: 10 μL (2 mm, 3 mm), 15 μL (4 mm), or 20 μL (5 mm, 6 mm). The slide with the filter paper was then placed in a Raman microscope. Five minutes after the drop, the excitation laser was focused on the filter paper surface, and Raman spectra were obtained at each measurement point. The measurement points were 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, and 1.0 mm from the apex toward the center, as well as the center (the drop location).

[0092] The measurement results are summarized in Figures 33 and 34. Figure 33 shows Raman spectra measured at 0.1 mm, 0.5 mm, and 1.0 mm from the apex toward the center, and at the center, for quartz fiber filter papers with side lengths of 2 mm (left), 3 mm (center), and 4 mm (right), respectively, while Figure 34 shows Raman spectra measured at 0.1 mm, 0.5 mm, and 1.0 mm from the apex toward the center, and at the center, respectively, for quartz fiber filter papers with side lengths of 5 mm (left) and 6 mm (right). In both graphs, the vertical axis represents the Raman scattered light intensity, and the horizontal axis represents the Raman shift.

[0093] A comparison of the Raman spectra for quartz fiber filter papers of different sizes in Figures 33 and 34 shows that, for any size, Raman spectra with high S / N ratios could be recorded from the apex to 1.0 mm (the third graph from the top), and that the closer to the apex, the higher the Raman scattered light intensity and S / N ratio of the Raman spectrum obtained. For squares with sides of 2 mm and 3 mm, Raman spectra with high S / N ratios were recorded even in the center (bottom graph), but the S / N ratios of the Raman spectra in the center were poor for squares with sides of 4 mm, 5 mm, and 6 mm.

[0094] These results indicate that for a square nonwoven fabric sheet of a given size, a good Raman spectrum with a high S / N ratio can be obtained regardless of the size, as long as the measurement point is within 1.0 mm from the apex.

[0095] Test Example 9: Raman Spectroscopic Analysis of Rhodamine 6G Aqueous Solution and Propylene Glycol Aqueous Solution on Nonwoven Fabric Sheets of Different Shapes. Quartz fiber filter papers QM-A and QM-B were used as nonwoven fabric sheets, cut into sectors (100 mm diameter circular filter paper cut into quarters), circles (3.5 mm diameter), and squares (3 mm sides), as shown in Figure 35. Raman spectroscopic analysis of liquid samples was performed using a Raman microscope under the conditions shown in Table 4. The liquid samples used were a 1 M rhodamine 6G aqueous solution and a 99% propylene glycol aqueous solution. Raman spectroscopic analysis was performed using a Raman microscope under the conditions shown in Table 4.

[0096]

[0097] A cut quartz fiber filter paper was placed on a quartz glass slide, and 10 μL of liquid sample was dropped onto the center of the filter paper. The slide with the filter paper was then placed in a Raman microscope. Five minutes after the drop, the excitation laser was focused on the filter paper surface, and Raman spectra were acquired at each measurement point. Measurement points were located within the area indicated by the arrows in Figure 35. For the fan-shaped filter paper, measurements were taken at 11 points, including each of the 10 equal points dividing the 3 mm from the center (droplet point) to the edge of the sample, as well as both ends. For the circular and square filter papers, measurements were taken at eight points: 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 1.0 mm, and 1.5 mm from the edge or apex, respectively, and the center (droplet point).

[0098] An overview of the measurement results for QM-B (Rhodamine 6G) is shown in Figure 36. The left side of Figure 36 shows the Raman spectra of the fan-shaped filter paper, measured at points 2.7 mm, 1.8 mm, 0.9 mm, and 0 mm from the center, from top to bottom. The center and right sides of Figure 36 show the Raman spectra of the circular and square filter paper, measured at points 0.1 mm, 0.5 mm, and 1.0 mm from the edge or apex, respectively, toward the center, from top to bottom.

[0099] In the sector shape, only a Raman spectrum with an extremely low S / N ratio was recorded, with the peak characteristic of rhodamine 6G barely observed near the edge (the top graph is closest to the edge) (Figure 36, left). On the other hand, in the circular and square shapes, the Raman scattered light intensity was high, and Raman spectra with a high S / N ratio were recorded. In particular, it was shown that the Raman scattered light intensity was higher near the edge or apex, and decreased the closer to the center. A similar trend was observed in QM-A. A similar trend was also observed in the measurement of propylene glycol.

[0100] These results demonstrate that, for any liquid sample, not just serum, using a nonwoven fabric sheet of an appropriate size (a size that allows the sample to reach the edge of the sheet without overflowing) according to the amount of sample dropped can produce a good Raman spectrum with high Raman scattered light intensity and a high S / N ratio. This is thought to be because, particularly when the sample concentration is relatively low or the molecular weight of the sample components is relatively small, a large nonwoven fabric sheet would cause the sample components to diffuse and dilute the sample. Therefore, using a nonwoven fabric sheet of a specific size prevents the sample components from diffusing and produces a strong Raman spectrum.

[0101] Test Example 10: Raman Spectroscopic Analysis of Rhodamine 6G Aqueous Solution and Propylene Glycol Aqueous Solution in Nonwoven Fabric Sheets of Different Shapes and Materials. The qualitative filter paper used in Test Example 2 was cut into circles (4 mm diameter) and squares (4 mm sides) and used as nonwoven fabric sheets to perform Raman spectroscopic analysis of liquid samples. The liquid samples used were a 1 M rhodamine 6G aqueous solution and a 99% propylene glycol aqueous solution. The Raman spectroscopic analysis was performed using a micro-Raman spectrometer under the conditions shown in Table 5 below.

[0102]

[0103] A cut piece of qualitative filter paper was placed on a quartz glass slide, and 10 μL of liquid sample was dropped onto the center of the filter paper. The slide with the filter paper was then placed in a Raman microscope. Five minutes after the drop, the excitation laser was focused on the filter paper surface, and Raman spectra were obtained at each measurement point. The measurement points were 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, and 1.0 mm from the edge or apex, respectively, toward the center, and at the center (the drop location).

[0104] An overview of the measurement results for Rhodamine 6G is shown in Figure 37. Figure 37 shows Raman spectra measured at points 0.1 mm, 0.5 mm, and 1.0 mm from the edge or apex toward the center, and at the center, for circular and square qualitative filter papers, respectively, from top to bottom.

[0105] Significant Raman spectra were recorded for both the circle and square shapes, with the Raman scattered light intensity being particularly high near the edge or apex (the upper graph is close to the edge or apex), resulting in Raman spectra with a high S / N ratio (Figure 37). A similar trend was observed in measurements of an aqueous propylene glycol solution.

[0106] These results show that, by using not only quartz fiber filter paper but also qualitative filter paper of a certain size, it is possible to obtain good Raman spectra with high Raman scattered light intensity and a high S / N ratio. However, because the cellulose from which qualitative filter paper is made itself emits strong Raman scattered light, the Raman scattered light from the material cannot be completely removed even by background correction, making it unsuitable for measuring samples with low Raman scattered light intensity.

[0107] (Test Example 11) Raman Spectroscopic Analysis of Rhodamine 6G Aqueous Solution in Nonwoven Fabric Sheets of Different Shapes and Materials Raman spectroscopic analysis of liquid samples was performed using the same paper towels used in Test Example 1, cut into circles (4 mm diameter) and squares (4 mm sides). The liquid sample used was a 1 M Rhodamine 6G aqueous solution. The Raman spectroscopic analysis was performed using a micro-Raman spectrometer under the same conditions as in Test Example 9.

[0108] A cut paper towel was placed on a quartz glass slide, and 10 μL of liquid sample was dropped onto the center of the filter paper. The slide with the filter paper was then placed in a Raman microscope. Five minutes after the drop, the excitation laser was focused on the filter paper surface, and Raman spectra were obtained at each measurement point. The measurement points were 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 1.0 mm, and 1.5 mm from the edge or apex, respectively, and at the center (the drop location).

[0109] An overview of the measurement results is shown in Figure 38. Figure 38 shows Raman spectra measured at points 0.1 mm, 0.5 mm, and 1.0 mm from the edge or apex toward the center, and at the center, for the circular and square qualitative filter papers, respectively, from top to bottom.

[0110] Significant Raman spectra were recorded for both the circular and square shapes, and relatively stable Raman spectra were obtained at all measurement points, regardless of the distance from the edge, although the S / N ratio was low (Figure 38). This is thought to be due to the heterogeneous structure of the paper towel, which makes it difficult for sample components to concentrate near the edge or apex.

[0111] These results demonstrate that stable Raman spectra can be obtained even with laboratory paper towels of a specified size. In Experiment 1, significant Raman spectra were not obtained with laboratory paper towels. This is likely due to the fact that the use of laboratory paper towels of a specified size prevented the diffusion of the liquid sample. However, because the cellulose material used in paper towels generates strong Raman scattering, background correction cannot completely eliminate the Raman scattering from the material, making them unsuitable for measuring samples with low Raman scattering intensity.

[0112] Although the embodiments of the present invention have been described in detail above, the above-mentioned embodiments are merely examples of the present invention, and the present invention is not limited to the configurations of the above-mentioned embodiments. Of course, even if there are design changes within the scope of the present invention, they are included in the present invention. CROSS-REFERENCE TO RELATED APPLICATIONS

[0113] This application claims priority based on Japanese Patent Application No. 2023-199664, filed with the Japan Patent Office on November 27, 2023, the entire disclosure of which is incorporated herein by reference in its entirety.

[0114] 1 Nonwoven fabric sheet for Raman spectroscopic analysis 100 Microscopic Raman spectroscopic device 110 Slide glass 120 Stage 150 Objective lens

Claims

1. A method for inspecting a liquid sample by Raman spectroscopy, comprising: ２ and a step of obtaining a Raman spectrum of the liquid sample by focusing excitation light on an upper surface of the nonwoven fabric sheet.

2. The inspection method according to claim 1, wherein the nonwoven fabric sheet is circular or polygonal with dimensions of no more than 1 cm in length and width.

3. The inspection method according to claim 1, wherein the excitation light is focused at a point on the upper surface of the nonwoven fabric sheet that is 0.01 mm or more and 1 mm or less away from an edge or apex of the nonwoven fabric sheet, and a Raman spectrum of the liquid sample is obtained.

4. The inspection method according to claim 1, wherein the nonwoven fabric sheet contains quartz glass fiber.

5. The testing method according to any one of claims 1 to 4, wherein the amount of the liquid sample dropped onto the nonwoven fabric sheet is 1 to 19 μL.

6. A nonwoven fabric sheet for holding a liquid sample, used in the testing method according to claim 1, having a basis weight of 80 to 185 g / m ２ and a thickness of 0.15 to 1 mm.

7. The nonwoven fabric sheet for Raman spectroscopic analysis according to claim 6, which is circular or polygonal with dimensions of 1 cm or less in length and width.

8. The nonwoven fabric sheet for Raman spectroscopy according to claim 6, which contains quartz glass fibers.

9. A test plate for use in the test method according to claim 1, comprising a plate and one or more nonwoven fabric sheets placed on the plate for holding a liquid sample, the one or more nonwoven fabric sheets having a basis weight of 80 to 185 g / m ２ and having a thickness of 0.15 to 1 mm.

10. The Raman spectroscopy test plate of claim 9, wherein said one or more nonwoven fabric sheets are circular or polygonal with dimensions within 1 cm in each direction.

Images