Method for raman analysis of polysaccharides, device and use thereof

Abstract

The application discloses a method for analyzing a polysaccharide by Raman, a device thereof and application, and relates to the field of polysaccharide analysis. The method comprises the following steps: (1) obtaining a Raman spectrum of the polysaccharide by high-resolution spatial scanning of the polysaccharide by probe coupling Raman; (2) analyzing the stretching vibration characteristic peaks of the chemical bonds of the Raman spectrum to preliminarily determine the monosaccharide molecular formula of the polysaccharide at different spatial positions; (3) analyzing the non-stretching vibration characteristic peaks of the functional groups of the Raman spectrum to accurately determine the monosaccharide molecular formula and the monosaccharide molecular configuration of the polysaccharide at different spatial positions; and (4) determining the arrangement sequence of the monosaccharide molecules in the polysaccharide based on the monosaccharide molecular formula and the monosaccharide molecular configuration of the polysaccharide at different spatial positions to obtain the spatial structure of the polysaccharide. Thus, the monosaccharide molecular formula and the monosaccharide molecular configuration of the polysaccharide at different spatial positions are distinguished, and the accurate detection of the spatial structure of the polysaccharide is realized, which is beneficial to the further application and research of the polysaccharide.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharide detection technology, specifically relating to a method, apparatus, and application of Raman spectroscopy for polysaccharide analysis. Background Technology

[0002] Polysaccharides possess extremely high medicinal value, but the unclear pharmacological mechanisms of most polysaccharide drugs severely hinder their further application. In fact, apart from the anticoagulant heparin pentasaccharide, the mechanisms of action of other polysaccharides have not been definitively established. Research on the relationship between polysaccharide structure and its pharmaceutically active components has progressed slowly because polysaccharides are extremely complex; most are mixtures lacking fixed molecular formulas. Furthermore, glycosyl groups have numerous isomers, and the composition, arrangement, branching and position of glycan chains, and derivatives of glycosyl groups make it difficult to confirm the structure of polysaccharides. An oligosaccharide composed of three monosaccharides can produce 1056 isomers, while under the same conditions, oligopeptides (proteins) have only 6 isomers. The amount of information contained in polysaccharides far exceeds that of proteins and nucleic acids. Moreover, existing methods for polysaccharide structure analysis are still imperfect, and research methods applicable to proteins and nucleic acids cannot be directly applied to polysaccharides.

[0003] Existing polysaccharide detection methods can be broadly categorized into two types. One is bottom-up detection, which involves completely degrading long-chain polysaccharides into monosaccharides or disaccharides, and then using methods such as NMR, chromatography, and mass spectrometry to determine the monosaccharide or disaccharide composition. However, this method results in the loss of the polysaccharide's spatial structure, making it impossible to determine the effective medicinal structure. The other type is top-down detection, which uses NMR and liquid chromatography-optical detection to determine the molecular weight information of polysaccharides. For example, the latest MHC-2DLC-MS (size exclusion-two-dimensional liquid chromatography-mass spectrometry) method can separate oligosaccharides with different degrees of polymerization through molecular sieve size exclusion, and then use liquid chromatography-mass spectrometry to infer the possible structure of the oligosaccharides. However, because this method has low sensitivity and poor specificity for separating structures with similar molecular weights, it still cannot achieve accurate structural characterization.

[0004] As can be seen from the above, existing polysaccharide detection technologies struggle to detect the spatial structure of polysaccharides and accurately determine their molecular configuration, thus posing significant obstacles to polysaccharide structure research and the elucidation of the mechanisms of action of polysaccharide drugs. Furthermore, the extremely small size of individual polysaccharide molecules presents a critical challenge in the measurement process: how to rapidly locate and capture their positions. Summary of the Invention

[0005] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, one objective of this invention is to provide a Raman spectroscopy method, apparatus, and application for the analysis of polysaccharides. This enables the differentiation of monosaccharide molecular formulas and configurations at different spatial positions of polysaccharide molecules, thereby achieving precise detection of the spatial structure of polysaccharide molecules, which is beneficial for further applications and research of polysaccharide molecules.

[0006] In one aspect of the present invention, a method for Raman spectroscopy analysis of polysaccharides is provided. In an embodiment of the present invention, the method includes:

[0007] (1) High-resolution spatial scanning of polysaccharide molecules was performed using probe-coupled Raman spectroscopy to obtain the Raman spectra of the polysaccharide molecules at different spatial positions;

[0008] (2) Based on the characteristic peaks of stretching vibrations of chemical bonds, the Raman spectrum is analyzed to determine the chemical bonds of the polysaccharide molecule at different spatial positions, so as to preliminarily determine the monosaccharide molecular formula of the polysaccharide molecule at different spatial positions;

[0009] (3) Based on the characteristic peaks of the non-stretching vibration of the functional group, the Raman spectrum is analyzed. According to the shift of the characteristic peak position and the change of the characteristic peak intensity caused by the non-stretching vibration of the functional group, the precise determination of the monosaccharide molecular formula is completed. The functional groups of the isomers of the polysaccharide molecule in different spatial positions are distinguished so as to determine the monosaccharide molecular configuration of the polysaccharide molecule in different spatial positions.

[0010] (4) Based on the monosaccharide molecular formula and the monosaccharide molecular configuration of the polysaccharide molecule at different spatial positions, determine the arrangement order of the monosaccharide molecules within the polysaccharide molecule to obtain the spatial structure of the polysaccharide molecule.

[0011] According to the Raman analysis method for polysaccharides of the present invention, during Raman detection, chemical bonds undergo stable stretching vibrations. Based on the characteristic peaks of these stretching vibrations, the chemical bonds in different spatial positions of the polysaccharide molecule can be determined by analyzing the detected Raman spectrum, thereby preliminarily determining the monosaccharide molecular formula of the polysaccharide molecule in different spatial positions. During Raman detection, adjacent functional groups affect each other's non-stretching vibrations such as wobbling and bending, causing shifts in the Raman peak positions and / or changes in intensity of the non-stretching vibrations of the functional groups. Based on the characteristic peaks of these non-stretching vibrations, the Raman spectrum is analyzed, and the monosaccharide molecular formula of the polysaccharide molecule in different spatial positions is preliminarily determined. The shifts in the peak positions and intensity changes of characteristic peaks caused by the non-stretching vibrations of functional groups determine the relative positions of functional groups within a monosaccharide molecule. This allows for the differentiation of isomers corresponding to the same molecular formula, the identification of monosaccharide types, and the determination of the monosaccharide molecular configurations of polysaccharides in different spatial positions. Simultaneously, based on the shifts in the peak positions and / or intensity changes of characteristic peaks caused by the non-stretching vibrations of functional groups, the monosaccharide molecular formula of a polysaccharide molecule in different spatial positions can also be accurately determined. Furthermore, based on the monosaccharide molecular formulas and configurations of polysaccharides in different spatial positions, the arrangement order of monosaccharide molecules within the polysaccharide molecule can be determined, thus obtaining the spatial structure of the polysaccharide molecule. Therefore, it is possible to distinguish the monosaccharide molecular formulas and configurations of polysaccharides in different spatial positions, thereby achieving precise detection of the spatial structure of polysaccharides, which is beneficial for further applications and research of polysaccharides.

[0012] In addition, the Raman analysis method for polysaccharides according to the above embodiments of the present invention may also have the following additional technical features:

[0013] In some embodiments of the present invention, the polysaccharide molecules are pre-detected before the probe is coupled to the Raman spectroscopy to perform high-resolution spatial scanning of the polysaccharide molecules.

[0014] In some embodiments of the present invention, the detection device for pre-detecting the polysaccharide molecules includes at least one of nuclear magnetic resonance, X-ray, mass spectrometry and chromatography, preferably nuclear magnetic resonance.

[0015] In some embodiments of the present invention, before using the probe to couple the Raman spectroscopy to perform high-resolution spatial scanning of the polysaccharide molecules, the method further includes: preparing a substrate with a surface coated with a nanostructured metal; and adsorbing the polysaccharide molecules onto the substrate with the surface coated with the nanostructured metal.

[0016] In some embodiments of the present invention, the nanostructured metal includes at least one of silver nanoparticles, gold nanoparticles, and copper nanoparticles, preferably silver nanoparticles.

[0017] In some embodiments of the present invention, the method of adsorbing the polysaccharide molecules onto the substrate coated with a nanostructured metal is impregnation adsorption.

[0018] In some embodiments of the present invention, in step (1), the device for providing the probe is an AFM or an SPM.

[0019] In some embodiments of the present invention, step (2) further includes: judging the chemical bonds of the polysaccharide molecule at different spatial positions; when multiple characteristic peaks of the chemical bonds are present, it is determined that the chemical bonds exist; integrating all chemical bonds and referring to the distribution of chemical bonds in the surrounding spatial positions, so as to determine the monosaccharide molecular formula of the polysaccharide molecule at different spatial positions.

[0020] In some embodiments of the present invention, during the analysis of the Raman spectrum, the intensity of the stretching vibration characteristic peak of the chemical bond is greater than the intensity of the non-stretching vibration characteristic peak of the functional group.

[0021] In some embodiments of the present invention, during the analysis of the Raman spectrum in step (2), the intensity of the characteristic peak of the stretching vibration of the chemical bond is greater than twice the standard deviation of the spectral noise intensity at that spatial location.

[0022] In some embodiments of the present invention, in step (2), the method for determining the characteristic peak of the stretching vibration of the chemical bond includes at least one of pre-experimental calibration and first-principles calculation.

[0023] In some embodiments of the present invention, during the analysis of the Raman spectrum in step (3), the intensity of the non-stretching vibration characteristic peak of the functional group is greater than 1.5 times the standard deviation of the spectral noise intensity at that spatial location.

[0024] In some embodiments of the present invention, in step (3), the method for determining the non-stretching vibration characteristic peak of the functional group includes at least one of pre-experimental calibration and first-principles calculation.

[0025] In some embodiments of the present invention, in step (2), the Raman spectra are analyzed in batches.

[0026] In some embodiments of the present invention, in step (2), the batch parsing method includes at least one of matrix masking and matrix normalization.

[0027] In some embodiments of the present invention, the chemical bonds include single bonds, double bonds, triple bonds, and sugar ring vibrations formed by any combination of carbon, oxygen, hydrogen, nitrogen, sulfur, and phosphorus elements.

[0028] In some embodiments of the present invention, in step (3), the Raman spectra are analyzed in batches.

[0029] In some embodiments of the present invention, in step (3), the batch parsing method includes at least one of matrix masking and matrix normalization.

[0030] In some embodiments of the present invention, in step (3), the functional group is an atom or group of atoms that affects the configuration of the monosaccharide, including atoms or groups of atoms formed by any combination of acyl, hydroxyl, carboxyl, ether bond, aldehyde, carbonyl, sulfonyl, amino and halogen elements.

[0031] In some embodiments of the present invention, in step (4), the spatial structure of the polysaccharide molecule includes: the composition and configuration of the monosaccharide glycosyl groups inside the polysaccharide molecule, and the arrangement order of the monosaccharide glycosyl groups inside the polysaccharide molecule.

[0032] In a second aspect, the present invention provides a Raman analysis apparatus for implementing the Raman analysis method for polysaccharides described in the above embodiments, the Raman analysis apparatus comprising:

[0033] A probe-spectral measurement system includes a laser, a probe, a probe displacement platform, a sample displacement platform, a mirror, an objective lens, a filter, and a spectral detection device. The laser generates laser light to detect polysaccharide molecules. The probe control platform is connected to the probe and controls its movement. The sample displacement platform carries the polysaccharide molecules and controls their movement. The mirror reflects the laser or Raman scattering signal. The objective lens focuses the laser light onto the surface of the polysaccharide molecules and collects the Raman scattering signal. The filter removes Rayleigh scattering from the Raman scattering signal collected by the objective lens. The spectral detection device detects the Raman scattering signal. The laser light irradiates the polysaccharide molecules, and the Raman scattering signal obtained by the polysaccharide molecules is transmitted to the spectral detection device.

[0034] The control system includes a probe control module, a sample displacement platform control module, and a spectral detection control module. The probe control module controls the movement of the probe by controlling the probe control platform. The sample displacement platform control module controls the movement of the polysaccharide molecules by controlling the sample displacement platform. The spectral detection control module controls the spectral detection device to detect the Raman scattering signal.

[0035] The data analysis system includes a monosaccharide molecular formula analysis module, a monosaccharide configuration analysis module, and a polysaccharide structure analysis module. The monosaccharide molecular formula analysis module is used to determine the monosaccharide molecular formula of the polysaccharide molecule at different spatial positions. The monosaccharide configuration analysis module is used to determine the monosaccharide configuration of the polysaccharide molecule at different spatial positions. The polysaccharide structure data analysis module is used to determine the arrangement order of monosaccharide molecules within the polysaccharide molecule, thereby obtaining the spatial structure of the polysaccharide molecule.

[0036] The Raman analysis apparatus according to embodiments of the present invention performs high-resolution spatial scanning of polysaccharide molecules using a probe-spectroscopy measurement system, thereby obtaining Raman spectra of polysaccharide molecules at different spatial positions. The control system controls the probe-spectroscopy measurement system to perform high-resolution spatial scanning of polysaccharide molecules, improving the convenience and accuracy of the detection process. The data analysis system performs batch analysis of the Raman spectra of the polysaccharide molecules at different spatial positions, greatly improving the efficiency of polysaccharide molecule analysis. Therefore, the Raman analysis apparatus enables precise detection of the spatial structure of polysaccharide molecules, which is beneficial for further applications and research of polysaccharide molecules. Furthermore, the aforementioned Raman analysis apparatus allows for batch and automated detection of polysaccharide molecules, significantly improving detection efficiency.

[0037] In addition, the Raman analysis apparatus according to the above embodiments of the present invention may also have the following additional technical features:

[0038] In some embodiments of the present invention, the probe-spectral measurement system further includes a narrowband pass filter, which is used to improve the laser quality of the laser.

[0039] In some embodiments of the present invention, the sample displacement platform carries the polysaccharide molecules via a substrate with a surface coated with a nanostructured metal.

[0040] In some embodiments of the present invention, the wavelength of the laser generated by the laser is 500nm-1600nm, preferably 600nm-800nm.

[0041] In a third aspect, the present invention provides a method for studying the pharmacology of polysaccharide drugs. According to embodiments of the present invention, the method for studying the pharmacology of polysaccharide drugs includes the Raman spectroscopy method for polysaccharides described in the above embodiments. This facilitates the detection of the spatial structure of polysaccharide drugs, and further investigation of the detected spatial structure and effects of the polysaccharide drugs, thereby helping to reveal the pharmacology of polysaccharide drugs.

[0042] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0043] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0044] Figure 1 This is a flowchart of a Raman spectroscopy method for polysaccharides according to an embodiment of the present invention;

[0045] Figure 2 This is a schematic diagram of the Raman spectra of two isomers of glucose and galactose according to an embodiment of the present invention;

[0046] Figure 3 This is a schematic diagram of a Raman analysis apparatus according to an embodiment of the present invention;

[0047] Figure 4 This is a schematic diagram of the Raman spectroscopy measurement results obtained after tip-enhanced scanning Raman of the polysaccharide molecules in Example 1 of the present invention;

[0048] Figure 5 This is a schematic diagram of the chemical bond distribution of the polysaccharide molecule in Example 1 of the present invention and possible monosaccharide molecules at spatial positions (2, 5); wherein, (a) is a CC bond, (b) is a CO bond, (c) is a CN bond, (d) is an SN bond, (e) is an SO bond and (f) is a possible monosaccharide molecule at spatial positions (2, 5);

[0049] Figure 6 The functional group movement of the polysaccharide molecule in Example 1 of the present invention and the monosaccharide configuration at spatial position (2, 5) are shown; wherein (a) is a -SO2 functional group, (b) is a -OH functional group, (c) is a -COOR functional group, (d) is a -CCH3 functional group and (f) is a monosaccharide configuration at spatial position (2, 5);

[0050] Figure 7 This is a schematic diagram of the spatial structure of a polysaccharide molecule according to an embodiment of the present invention.

[0051] Figure label:

[0052] 1-Laser; 2-Probe displacement platform; 3-Sample displacement platform; 4-Reflector; 5-Objective lens; 6-Filter; 7-Spectroscopic detection device; 8-Probe control module; 9-Sample displacement platform control module; 10-Spectroscopic detection control module; 11-Monosaccharide molecular formula analysis module; 12-Monosaccharide configuration analysis module; 13-Polysaccharide structure analysis module; 14-Narrow bandpass filter. Detailed Implementation

[0053] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0054] In one aspect of the invention, a method for Raman spectroscopy analysis of polysaccharides is provided. In embodiments of the invention, refer to the appendix... Figure 1 The above methods include:

[0055] S100: High-resolution spatial scanning of polysaccharide molecules is performed using probe-coupled Raman spectroscopy to obtain the Raman spectra of polysaccharide molecules.

[0056] In this step, a probe-coupled Raman spectroscopy is used to perform high-resolution spatial scanning of the polysaccharide molecules in order to obtain the Raman spectra of the polysaccharide molecules at different spatial positions; using the tip of the probe to couple Raman spectroscopy for detection can significantly increase the resolution of Raman detection.

[0057] According to some specific embodiments of the present invention, before performing high-resolution spatial scanning of polysaccharide molecules using probe-coupled Raman spectroscopy, the polysaccharide molecules are pre-detected. Thus, the elemental information and / or possible monosaccharide and oligosaccharide structures in the polysaccharide molecules can be preliminarily determined through pre-detection, which is beneficial for providing a reference for the chemical bonds to be analyzed and the possible monosaccharide molecular configurations in step S200.

[0058] In the embodiments of the present invention, the apparatus for performing the above-mentioned pre-detection on the polysaccharide molecules includes, but is not limited to, at least one of nuclear magnetic resonance, X-ray, mass spectrometry and chromatography, preferably nuclear magnetic resonance. Thus, by means of nuclear magnetic detection, the elemental information in the polysaccharide molecules and the monosaccharide and oligosaccharide structures that may exist in the polysaccharide molecules can be preliminarily determined, which is further beneficial to provide a reference for the chemical bonds to be analyzed and the possible monosaccharide molecule configurations in step S200.

[0059] According to some specific embodiments of the present invention, before performing high-resolution spatial scanning of the polysaccharide molecules using the probe coupled with the Raman spectroscopy, the method further includes: preparing a substrate with a surface coated with a nanostructured metal; adsorbing the polysaccharide molecules onto the substrate with the surface coated with the nanostructured metal. Thus, by adsorbing the polysaccharide molecules onto the substrate with the surface coated with the nanostructured metal, it is beneficial to quickly locate the polysaccharide molecules to be detected, solving the problem that small-sized polysaccharide molecules are not easy to capture.

[0060] In embodiments of the present invention, the nanostructured metal includes, but is not limited to, at least one of silver nanoparticles, gold nanoparticles, and copper nanoparticles, preferably silver nanoparticles.

[0061] According to some specific embodiments of the present invention, the above-mentioned nanostructured metal is subjected to electrochemical treatment or a specific substance is modified on the surface of the above-mentioned nanostructured metal. This can significantly increase the adsorption strength of polysaccharide molecules on the surface coated with nanostructured metal, which is beneficial for rapidly locating the polysaccharide molecules to be detected.

[0062] According to some specific embodiments of the present invention, the method of adsorbing the above-mentioned polysaccharide molecules onto the substrate coated with a nanostructured metal is impregnation adsorption, thereby further increasing the adsorption strength of polysaccharide molecules on the surface coated with a nanostructured metal, and further facilitating the rapid localization of the polysaccharide molecules to be detected.

[0063] In an embodiment of the present invention, in step S100, the device for providing the probe includes, but is not limited to, AFM or SPM.

[0064] S200: Analyze the characteristic peaks of stretching vibrations of chemical bonds in Raman spectra to preliminarily determine the monosaccharide molecular formulas of polysaccharide molecules in different spatial positions.

[0065] In this step, during Raman detection, chemical bonds undergo stretching vibrations. The characteristic peaks of stretching vibrations have large and stable vibrational intensities. Based on the calculated characteristic peaks of non-stretching vibrations of functional groups, the chemical bonds of polysaccharide molecules at different spatial positions can be determined by analyzing the detected Raman spectra, thereby preliminarily determining the monosaccharide molecular formulas of polysaccharide molecules at different spatial positions.

[0066] According to some specific embodiments of the present invention, step S200 further includes: judging the chemical bonds of the polysaccharide molecule at different spatial positions; when multiple sets of characteristic peaks of the chemical bond are present, it is determined that the chemical bond exists; integrating all chemical bonds and referring to the distribution of chemical bonds in the surrounding spatial positions, so as to determine the monosaccharide molecular formula of the polysaccharide molecule at different spatial positions. Since the stretching vibration mode of a single chemical bond can be further subdivided according to direction and symmetry, vibration modes with different vibration directions and symmetries correspond to characteristic peaks at different peak positions. When only one set of characteristic peaks of a certain chemical bond is detected, it may be an error characteristic peak caused by the shift of other chemical bonds in the surrounding area. Therefore, only when multiple sets of characteristic peaks of a chemical bond are detected can the existence of the corresponding chemical bond be determined. During Raman scanning, due to the scanning step size, when integrating all chemical bonds, it is necessary to refer to the distribution of chemical bonds in the surrounding spatial positions in order to obtain the accurate monosaccharide molecular formula of the polysaccharide molecule at different spatial positions.

[0067] According to further embodiments of the present invention, during the analysis of the Raman spectra, the intensity of the stretching vibration characteristic peaks of the chemical bonds is greater than the intensity of the non-stretching vibration characteristic peaks of the functional groups. Chemical bonds undergo stretching vibrations, resulting in a larger amplitude, while functional groups undergo non-stretching vibrations, resulting in a smaller amplitude. Therefore, it is possible to better distinguish between chemical bond characteristic peaks and functional group characteristic peaks, which is beneficial for detecting the spatial structure of polysaccharide molecules. It should be noted that the intensities of the stretching vibration chemical bond characteristic peaks and the non-stretching vibration functional group characteristic peaks in this application are both characteristic peak intensities selected during the Raman spectroscopy analysis process.

[0068] According to some specific embodiments of the present invention, in step S200, during the analysis of the Raman spectrum, if the intensity of the characteristic peak of the stretching vibration of the chemical bond is greater than twice the standard deviation of the spectral noise intensity at that spatial position, it can be determined that the characteristic peak of the chemical bond exists at that location. This avoids both setting the preset detection intensity threshold for the characteristic peak of the stretching vibration of the chemical bond too high, which would cause the chemical bonds that need to be detected to be missed during the analysis of the Raman spectrum, and setting the preset detection intensity threshold for the characteristic peak of the stretching vibration of the chemical bond too low, which would cause a large amount of unwanted spectral noise to be detected during the analysis of the Raman spectrum, thereby reducing the efficiency of the analysis process.

[0069] According to some specific embodiments of the present invention, in step S200, the above-mentioned Raman spectra are analyzed in batches, thereby realizing the batch and automated detection of chemical bonds in polysaccharide molecules, which greatly improves the efficiency of the analysis work.

[0070] In an embodiment of the present invention, in step S200, the method for determining the characteristic peaks of the stretching vibrations of the chemical bonds includes, but is not limited to, at least one of pre-experimental calibration and first-principles calculation.

[0071] In an embodiment of the present invention, in step S200, the above-mentioned batch parsing method includes, but is not limited to, at least one of matrix masking processing and matrix normalization processing.

[0072] In an embodiment of the present invention, in step S200, the aforementioned chemical bonds include, but are not limited to, single bonds, double bonds, triple bonds, and sugar ring vibrations formed by any combination of carbon, oxygen, hydrogen, nitrogen, sulfur, and phosphorus elements.

[0073] S300: Analyzes the characteristic peaks of non-stretching vibrations of functional groups in Raman spectra to accurately determine the monosaccharide formula and configuration of polysaccharide molecules at different spatial positions.

[0074] In this step, the Raman spectrum is analyzed based on the characteristic peaks of the non-stretching vibrations of the functional groups. The peak position shift and intensity change of the characteristic peaks caused by the non-stretching vibrations of the functional groups are used to accurately determine the formula of the monosaccharide molecule. Furthermore, the functional groups of the isomers of the polysaccharide molecule in different spatial positions are distinguished in order to determine the configuration of the monosaccharide molecule in different spatial positions.

[0075] Specifically, adjacent functional groups influence each other's oscillations and in-plane bending. Therefore, the vibrational amplitude of functional groups is small and unstable. By measuring the shift and intensity changes of the Raman peaks of the oscillations and bending of functional groups, it is possible to determine whether other functional groups around this functional group have an influence, thereby determining the configuration of the monosaccharide molecule. (Using a molecular formula of C6H...) 12 Taking O6 monosaccharides as an example, refer to the appendix. Figure 2 Glucose and galactose have the same molecular formula, but due to differences in monosaccharide molecular configuration, galactose has a homo-oriented -COH structure at the -CH2OH ortho position, hence its higher molecular weight at 703 cm⁻¹. -1 and 780cm -1 The characteristic peaks generated at this location are caused by the co-vibration of -CH2OH and -COH, while glucose does not have significant characteristic peaks at this location. Therefore, characteristic peaks can be used as a criterion for the configuration of monosaccharide molecules.

[0076] According to some specific embodiments of the present invention, in step S300, during the analysis of the Raman spectrum, the intensity of the non-stretching vibration characteristic peak of the functional group is greater than 1.5 times the standard deviation of the spectral noise intensity at that spatial location. This avoids both the preset detection intensity threshold of the non-stretching vibration characteristic peak of the functional group being too high, which would cause the functional groups that need to be detected to be missed during the analysis of the Raman spectrum, and the preset detection intensity threshold of the non-stretching vibration characteristic peak of the functional group being too low, which would cause a large amount of unwanted spectral noise to be detected during the analysis of the Raman spectrum, thereby reducing the efficiency of the analysis work.

[0077] According to some specific embodiments of the present invention, in step S300, the above-mentioned Raman spectra are analyzed in batches, thereby realizing the batch detection and automated detection of functional groups of isomers of polysaccharide molecules at different spatial positions, which greatly improves the efficiency of the analysis work.

[0078] In an embodiment of the present invention, in step S200, the method for determining the non-stretching vibration characteristic peak of the functional group includes, but is not limited to, at least one of pre-experimental calibration and first-principles calculation.

[0079] In an embodiment of the present invention, in step S300, the above-mentioned batch parsing method includes, but is not limited to, at least one of matrix masking processing and matrix normalization processing.

[0080] In an embodiment of the present invention, in step S300, the functional group is an atom or group of atoms that affects the configuration of the monosaccharide, including but not limited to atoms or groups of atoms formed by any combination of acyl, hydroxyl, carboxyl, ether bond, aldehyde, carbonyl, sulfonyl, amino and halogen elements.

[0081] S400: Based on the monosaccharide formulas and configurations of polysaccharide molecules at different spatial positions, to obtain the spatial structure of polysaccharide molecules.

[0082] In this step, based on the monosaccharide molecular formulas and configurations of the polysaccharide molecules at different spatial positions, the arrangement order of the monosaccharide molecules within the polysaccharide molecules is determined to obtain the spatial structure of the polysaccharide molecules.

[0083] In an embodiment of the present invention, in step S400, the spatial structure of the polysaccharide molecule includes: the molecular formula of the monosaccharide molecule inside the polysaccharide molecule, the configuration of the monosaccharide molecule, and the arrangement order of the monosaccharide molecule.

[0084] In an embodiment of the present invention, when the polysaccharide molecule sample is a mixture, a molecular sieve is used to sieve the polysaccharide molecule sample, and then the above method is used to detect the polysaccharide molecules. Thus, the detection of polysaccharide mixture molecule samples can be achieved by the above method.

[0085] In embodiments of the present invention, when the degree of polymerization of polysaccharide molecular samples in the same batch is inconsistent, the above-described Raman analysis method for polysaccharides can be used to detect polysaccharide molecular samples with different degrees of polymerization.

[0086] The Raman spectroscopy method for polysaccharide analysis according to embodiments of the present invention has the following advantages:

[0087] (1) It has achieved precise detection of the spatial structure of monosaccharide molecules in polysaccharide molecules;

[0088] (2) The precise determination of the monosaccharide molecular formula of polysaccharide molecules in different spatial positions was achieved; by distinguishing the functional groups of isomers of polysaccharide molecules in different spatial positions, the monosaccharide molecular configuration of polysaccharide molecules in different spatial positions was determined, and the precise detection of the spatial structure of polysaccharide molecules was finally achieved.

[0089] (3) When the polysaccharide molecule sample is a mixture, molecular sieves are used to sieve the polysaccharide molecule sample, which can realize the detection of polysaccharide mixture molecule samples;

[0090] (4) When the degree of polymerization of polysaccharide molecules in the same batch is inconsistent, the above method can be used to detect polysaccharide molecules with different degrees of polymerization.

[0091] In a second aspect, the present invention provides a Raman analysis apparatus for implementing the Raman analysis method for polysaccharides described in the above embodiments, as shown in the attached drawing. Figure 3 The aforementioned Raman analysis apparatus includes:

[0092] The probe-spectral measurement system includes a laser 1, a probe, a probe displacement platform 2, a sample displacement platform 3, a mirror 4, an objective lens 5, a filter 6, and a spectral detection device 7. The laser 1 generates laser light to detect polysaccharide molecules. The probe control platform 2 is connected to the probe and controls its movement. The sample displacement platform 3 carries and controls the movement of the polysaccharide molecules. The mirror 4 reflects the laser or Raman scattering signal. The objective lens 5 reflects the laser light. The light generated by the optical device 1 is focused on the surface of the polysaccharide molecule and used to collect the Raman scattering signal. The filter 6 is used to remove Rayleigh scattering in the Raman scattering signal collected by the objective lens 5. The spectral detection device 7 is used to detect the Raman scattering signal. The laser generated by the laser device 1 irradiates the polysaccharide molecule. The Raman scattering signal obtained by the scattering of the polysaccharide molecule is transmitted to the spectral detection device 7. Thus, the probe-spectral measurement system performs high-resolution spatial scanning of the polysaccharide molecule to obtain the Raman spectrum of the polysaccharide molecule at different spatial positions.

[0093] The control system includes a probe control module 8, a sample displacement platform control module 9, and a spectral detection control module 10. The probe control module 8 controls the movement of the probe by controlling the probe control platform 2, the sample displacement platform control module 9 controls the movement of the polysaccharide molecule by controlling the sample displacement platform 3, and the spectral detection control module 10 controls the spectral detection device 7 to detect the Raman scattering signal. Thus, the control system controls the probe-spectral measurement system to perform high-resolution spatial scanning of the polysaccharide molecule, improving the convenience and accuracy of the detection work.

[0094] The data analysis system includes a monosaccharide molecular formula analysis module 11, a monosaccharide configuration analysis module 12, and a polysaccharide structure analysis module 13. The monosaccharide molecular formula analysis module 11 determines the monosaccharide molecular formula of the polysaccharide molecule at different spatial positions. The monosaccharide configuration analysis module 12 determines the monosaccharide configuration of the polysaccharide molecule at different spatial positions. The polysaccharide structure analysis module 13 determines the arrangement order of monosaccharide molecules within the polysaccharide molecule, thereby obtaining the spatial structure of the polysaccharide molecule. Therefore, by performing batch analysis of the Raman spectra of the polysaccharide molecules at different spatial positions using the data analysis system, the efficiency of polysaccharide molecule analysis is greatly improved. The Raman analysis device enables precise detection of the spatial structure of polysaccharide molecules, which is beneficial for further applications and research of polysaccharide molecules. Furthermore, the Raman analysis device can perform batch and automated detection of polysaccharide molecules.

[0095] According to some specific embodiments of the present invention, see attached drawing. Figure 3 The probe-spectroscopy measurement system also includes a narrow bandpass filter 14, which is used to improve the laser quality of the laser 1 and further facilitates high-resolution spatial scanning of polysaccharide molecules, thereby obtaining the Raman spectra of polysaccharide molecules at different spatial positions.

[0096] According to some specific embodiments of the present invention, see attached drawing. Figure 3 The sample displacement platform 3 mentioned above carries the polysaccharide molecules on a substrate coated with a nanostructured metal. Thus, by adsorbing the polysaccharide molecules onto the substrate coated with a nanostructured metal, it is beneficial to quickly locate the polysaccharide molecules to be detected, solving the problem that small-sized polysaccharide molecules are not easy to capture.

[0097] According to some specific embodiments of the present invention, see attached drawing. Figure 3 The wavelength of the laser generated by the laser 1 is 500nm-1600nm, preferably 600nm-800nm. This avoids both the problem of the wavelength being too short, which would prevent the probe-coupled Raman detection of polysaccharide molecules, and the problem of the wavelength being too long, which would result in the Raman scattering intensity being too low.

[0098] In a third aspect, the present invention provides a method for studying the pharmacology of polysaccharide drugs. According to embodiments of the present invention, the method for studying the pharmacology of polysaccharide drugs includes the Raman spectroscopy method for polysaccharides described in the above embodiments. This facilitates the detection of the spatial structure of polysaccharide drugs, and allows for the exploration of the detected spatial structure and effects of polysaccharide drugs, thereby contributing to the elucidation of the pharmacology of polysaccharide drugs.

[0099] The embodiments of the present invention are described in detail below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. In addition, unless otherwise specified, all reagents used in the following embodiments are commercially available or can be synthesized according to the methods described herein or known to others. For reaction conditions not listed, they are also readily available to those skilled in the art.

[0100] Example 1

[0101] A method for Raman spectroscopy analysis of polysaccharides, the steps of which are as follows:

[0102] (1) Pre-detection: Nuclear magnetic resonance detection is performed on polysaccharide molecule samples to determine the chemical element types of polysaccharide molecules, including H, C, N, O, and S. The known possible monosaccharide types are GlcNS6S, GlcA, GlcNS6S3S, IdoA2S, and MeO-GlcNS6S. Pre-detection can quickly narrow down the possible monosaccharide glycosyl range within the polysaccharide, which helps to determine the polysaccharide structure more accurately and efficiently. The polysaccharide molecule used in this embodiment is the polysaccharide fondaparinux sodium composed of five monosaccharides.

[0103] (2) Raman detection of polysaccharides: The polysaccharide molecules to be analyzed are adsorbed onto an electrochemically treated nanostructured metal substrate, and Raman detection is enhanced by the probe tip of AFM. The Raman spectra of the polysaccharide molecules at different spatial positions (x, y) are obtained by high-resolution spatial scanning. Step (2) can be specifically divided into the following three steps:

[0104] (2-1) Preparation of a rough silver substrate with nanostructure: The electrochemical cell was cleaned with anionic surfactant, sodium linear alkylbenzene sulfonate, and dilute acid solution to remove impurities and dirt remaining on the surface of the electrochemical cell. The silver substrate was polished to a mirror finish with sandpaper and polishing machine. Then, it was ultrasonically cleaned in ethanol and water for 15 minutes. A three-electrode system was used to electrochemically roughen the silver sheet until a uniform nanoscale rough structure was produced on the surface. The minimum size of the nanoscale rough silver substrate should be less than 5 nm. The rough silver substrate was then cleaned with distilled water, alcohol, and distilled water in sequence to ensure that there was no electrolyte residue on the surface, thus obtaining a rough silver substrate with nanostructure.

[0105] (2-2) Adsorbing the polysaccharide molecules to be analyzed onto the nanostructured metal substrate: First, dissolve the polysaccharide molecules in distilled water with a solution concentration of 1 mmol / L. Then, immerse the rough silver substrate with nanostructure prepared in step (2-2) into the polysaccharide solution so that the polysaccharide molecules can be naturally adsorbed onto the silver substrate. The immersion time is 24 hours. After the adsorption is completed, the sample is slowly dried in a low-speed airflow to ensure that the polysaccharide molecules are adsorbed onto the substrate as freely as possible.

[0106] (2-3) Combining tip-enhanced Raman detection, the Raman spectra of the molecule to be analyzed at different spatial positions (x, y) are obtained through high-resolution spatial scanning: An atomic force microscope (AFM) probe with a silver-plated metal surface is selected. The substrate is irradiated with a laser and Raman signals are collected using a spectral detection device. A sample displacement platform is used to move the rough silver substrate to determine the approximate area where the polysaccharide molecule to be analyzed is adsorbed. The probe is moved to the vicinity of this area using a probe displacement platform. The probe is scanned with a laser. The tip position is determined based on the Rayleigh scattering intensity of the metal. The center of the laser spot is moved to the tip. The tapping mode of the atomic force microscope is used to make the rough silver substrate contact the probe. The relative position of the laser spot center and the probe tip is finely adjusted until the strongest tip-enhanced Raman scattering is obtained. The relative position of the laser spot center and the probe tip is fixed. The rough silver substrate is moved and tip-enhanced Raman scanning is performed. The Raman spectra of the molecule to be analyzed at different spatial positions are obtained through high-resolution spatial scanning. The high spatial resolution refers to a scanning step size of 0.5 nm, a scanning range of 10 nm × 10 nm, and a laser wavelength of 633 nm.

[0107] The final Raman spectrum obtained is shown in the attached figure. Figure 4 As shown, a total of 400 Raman spectra were obtained within the measurement range;

[0108] (3) Analysis of monosaccharide molecular formula and monosaccharide configuration, wherein step (3) can be specifically divided into the following two steps:

[0109] (3-1) Monosaccharide molecular formula analysis: The monosaccharide molecular formula analysis module was used to analyze the Raman spectra in batches. The chemical bonds of the polysaccharide molecules obtained by the preliminary detection were C-C bonds, CO bonds, CN bonds, SN bonds and SO bonds. Therefore, the stretching vibrations of C-C bonds, CO bonds, CN bonds, SN bonds and SO bonds were selected for analysis.

[0110] Specifically, the characteristic peak position of the C / C bond is selected as 885 cm⁻¹. -1 1050cm -1 and 1121cm -1 ;

[0111] The characteristic peak position for the CO bond is selected at 737 cm⁻¹. -1 1014cm -1 and 1142cm -1 ;

[0112] The characteristic peak position of the CN bond is selected at 1030 cm⁻¹. -1 and 1094cm -1 ;

[0113] The characteristic peak position of the SN bond was selected at 818 cm⁻¹. -1 ;

[0114] The characteristic peak position for SO bond selection is 897 cm⁻¹. -1 1126cm -1 and 1348cm -1 ;

[0115] The method for determining the presence of characteristic peaks of chemical bonds is that, for vibrational characteristic peaks of chemical bonds, the intensity of the characteristic peak must be no less than twice the standard deviation of the spectral noise intensity at that position;

[0116] Appendix Figure 5 (a)-(e) show the distribution of CC bonds, CO bonds, CN bonds, SN bonds and SO bonds in this embodiment. Spatial position (2, 5) is selected for judgment. There is an obvious SN bond at spatial position (2, 5), and CC bonds, CO bonds, CN bonds and SO bonds are present at this position and its surroundings. Among the possible monosaccharides determined in step (1), GlcA and IdoA2S do not have SN bonds. Therefore, the monosaccharide type at spatial position (2, 5) may be GlcNS6S3S, GlcNS6S and MeO-GlcNS6S.

[0117] (3-2) Monosaccharide configuration analysis: -SO2, -OH, -COOR and -CH3 were selected for analysis, where R represents any group;

[0118] Specifically, the swing characteristic peak position of the -SO2 functional group is selected at 501 cm⁻¹. -1 and 533cm -1 ;

[0119] The oscillating characteristic peak position of the -OH functional group was selected at 1119 cm⁻¹. -1 1239cm -1 and 1470cm -1 ;

[0120] -The COOR functional group was selected with a characteristic peak position at 557 cm⁻¹. -1 and 600cm -1 ;

[0121] The characteristic peak position of the CH3 functional group was selected at 1256 cm⁻¹. -1 ;

[0122] The method for determining the presence of functional group characteristic peaks is that, for non-vibrational characteristic peaks (i.e., oscillating characteristic peaks) of functional groups, the intensity of the characteristic peak is not less than 1.5 times the standard deviation of the spectral noise intensity at that position;

[0123] Appendix Figure 6(a)-(d) show the wobbling behavior of the -SO2, -OH, -COOR, and -CH3 groups in this embodiment, respectively. Spatial positions (2,5) are used for illustration. No -CH3 functional group signal is present at spatial position (2,5), therefore it is not MeO-GlcNS6S. Simultaneously, while the stretching vibration characteristic peak of the SO bond is present at spatial position (2,5), no wobbling characteristic peak is present, indicating that other large groups near the -SO2 functional group suppress the wobbling of the -SO2 functional group. In GlcNS6S3S and GlcNS6S, GlcNS6S contains a freely wobbling -SO2 functional group, while GlcNS6S3S does not. Therefore, as shown in the attached diagram... Figure 6 As shown in (e), the monosaccharide configuration at spatial position (2, 5) is GlcNS6S3S;

[0124] (4) Analysis of the monosaccharide order:

[0125] By following steps (3-1) and (3-2), the molecular formula and configuration of monosaccharides at different spatial locations can be determined, thus obtaining the spatial structure information of the corresponding polysaccharides, as shown in the attached figure. Figure 7 As shown, the spatial structure of polysaccharide molecules can be analyzed within the x range (1, 3) and y range (1, 9).

[0126] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for Raman analysis of polysaccharides, characterized in that, include: (1) High-resolution spatial scanning of polysaccharide molecules was performed using probe-coupled Raman spectroscopy to obtain the Raman spectra of the polysaccharide molecules at different spatial positions; (2) Based on the characteristic peaks of stretching vibrations of chemical bonds, the Raman spectrum is analyzed to determine the chemical bonds of the polysaccharide molecules at different spatial positions, so as to preliminarily determine the monosaccharide molecular formula of the polysaccharide molecules at different spatial positions; (3) Based on the characteristic peak of the non-stretching vibration of the functional group, the Raman spectrum is analyzed. According to the shift of the characteristic peak position and the change of the characteristic peak intensity caused by the non-stretching vibration of the functional group, the precise determination of the monosaccharide molecular formula is completed. The functional groups of the isomers of the polysaccharide molecule in different spatial positions are distinguished so as to determine the monosaccharide molecular configuration of the polysaccharide molecule in different spatial positions. (4) Based on the monosaccharide molecular formula and the monosaccharide molecular configuration of the polysaccharide molecule at different spatial positions, determine the arrangement order of the monosaccharide molecules within the polysaccharide molecule to obtain the spatial structure of the polysaccharide molecule; Step (2) further includes: judging the chemical bonds of the polysaccharide molecule at different spatial positions; when multiple characteristic peaks of the chemical bond are present, it is determined that the chemical bond exists; integrating all chemical bonds and referring to the distribution of chemical bonds in the surrounding spatial positions, so as to determine the monosaccharide molecular formula of the polysaccharide molecule at different spatial positions. During the analysis of the Raman spectrum, the intensity of the stretching vibration characteristic peak of the chemical bond is greater than the intensity of the non-stretching vibration characteristic peak of the functional group. In step (2), during the analysis of the Raman spectrum, the intensity of the characteristic peak of the stretching vibration of the chemical bond is greater than twice the standard deviation of the spectral noise intensity at that spatial location; In step (2), the method for determining the characteristic peak of the stretching vibration of the chemical bond includes at least one of pre-experimental calibration and first-principles calculation; In step (3), during the analysis of the Raman spectrum, the intensity of the non-stretching vibration characteristic peak of the functional group is greater than 1.5 times the standard deviation of the spectral noise intensity at that spatial location; In step (3), the method for determining the non-stretching vibration characteristic peak of the functional group includes at least one of pre-experimental calibration and first-principles calculation.

2. The method according to claim 1, characterized in that, Before performing high-resolution spatial scanning of the polysaccharide molecule using the probe coupled with the Raman spectrometer, the polysaccharide molecule is pre-detected; The detection device for pre-detecting the polysaccharide molecules includes at least one of nuclear magnetic resonance, X-ray, mass spectrometry, and chromatography.

3. The method according to claim 1, characterized in that, Before using the probe coupled with Raman to perform high-resolution spatial scanning of the polysaccharide molecule, the method further includes: A substrate with a surface coated with a nanostructured metal was prepared. The polysaccharide molecules are adsorbed onto the substrate whose surface is coated with a nanostructured metal; The nanostructured metal includes at least one of silver nanoparticles, gold nanoparticles, and copper nanoparticles; The method of adsorbing the polysaccharide molecules onto the substrate coated with a nanostructured metal is called impregnation adsorption. In step (1), the device that provides the probe is an AFM or an SPM.

4. The method according to any one of claims 1-3, characterized in that, In step (2), the Raman spectra are analyzed in batches; In step (2), the batch parsing method includes at least one of matrix masking and matrix normalization. The chemical bonds include single bonds, double bonds, triple bonds, and sugar ring vibrations formed by any combination of carbon, oxygen, hydrogen, nitrogen, sulfur, and phosphorus elements. In step (3), the Raman spectra are analyzed in batches; In step (3), the batch parsing method includes at least one of matrix masking and matrix normalization. In step (3), the functional group is an atom or group of atoms that affects the configuration of the monosaccharide, including atoms or groups of atoms formed by any combination of acyl, hydroxyl, carboxyl, ether bond, aldehyde, carbonyl, sulfonyl, amino and halogen elements; In step (4), the spatial structure of the polysaccharide molecule includes: the molecular formula of the monosaccharide molecule inside the polysaccharide molecule, the configuration of the monosaccharide molecule, and the arrangement order of the monosaccharide molecules.

5. A Raman analysis apparatus for implementing the method of Raman analysis of polysaccharides according to any one of claims 1-4, characterized in that, The Raman analysis device includes: A probe-spectral measurement system includes a laser, a probe, a probe displacement platform, a sample displacement platform, a mirror, an objective lens, a filter, and a spectral detection device. The laser generates laser light to detect polysaccharide molecules. The probe control platform is connected to the probe and controls its movement. The sample displacement platform carries the polysaccharide molecules and controls their movement. The mirror reflects the laser or Raman scattering signal. The objective lens focuses the laser-generated light onto the surface of the polysaccharide molecules and collects the Raman scattering signal. The filter removes Rayleigh scattering from the Raman scattering signal collected by the objective lens. The spectral detection device detects the Raman scattering signal. The laser light irradiates the polysaccharide molecules, and the Raman scattering signal obtained by the polysaccharide molecules is transmitted to the spectral detection device. The control system includes a probe control module, a sample displacement platform control module, and a spectral detection control module. The probe control module controls the movement of the probe by controlling the probe control platform. The sample displacement platform control module controls the movement of the polysaccharide molecules by controlling the sample displacement platform. The spectral detection control module controls the spectral detection device to detect the Raman scattering signal. The data analysis system includes a monosaccharide molecular formula analysis module, a monosaccharide configuration analysis module, and a polysaccharide structure analysis module. The monosaccharide molecular formula analysis module is used to determine the monosaccharide molecular formula of the polysaccharide molecule at different spatial positions. The monosaccharide configuration analysis module is used to determine the monosaccharide configuration of the polysaccharide molecule at different spatial positions. The polysaccharide structure data analysis module is used to determine the arrangement order of monosaccharide molecules within the polysaccharide molecule, thereby obtaining the spatial structure of the polysaccharide molecule.

6. The Raman analysis apparatus according to claim 5, characterized in that, The probe-spectral measurement system also includes a narrowband pass filter, which is used to improve the laser quality of the laser.

7. The Raman analysis apparatus according to claim 5 or 6, characterized in that, The sample displacement platform supports the polysaccharide molecules via a substrate with a surface coated with a nanostructured metal. The laser generates laser light with a wavelength of 500nm-1600nm.

8. A method for studying the pharmacology of polysaccharide drugs, characterized in that, The method for Raman analysis of polysaccharides as described in any one of claims 1-6.

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