Disaccharide isomer distinguishing method and device based on gas-liquid interface reaction and application

Abstract

The invention provides a gas-liquid interface reaction-based disaccharide isomer distinguishing method, a gas-liquid interface reaction-based disaccharide isomer distinguishing device and application. According to the distinguishing method, a disaccharide aqueous solution serves as a sample solution, N2-containing gas serves as atomizing gas, the flowing disaccharide aqueous solution is atomized into micro-droplets under the air pressure larger than 50 psi, meanwhile, a gas-liquid interface with an electric field of 108-109 V / m is formed, N2 molecules are activated at the normal temperature under the action of the electric field of the gas-liquid interface, and the micro-droplets are separated from the disaccharide aqueous solution. Combining with hydrogen free radicals in the disaccharide water solution to synthesize NH3, and combining NH3 with disaccharide molecules to generate a [M + NH4] < + > adduct; then through mass spectrometry, [M + NH4] < + > of different disaccharide isomers generates unique ion signals in an MS / MS fragment mode, and the unique ion signals can be used as effective fingerprint information for distinguishing the disaccharide isomers; moreover, the signals are obviously enhanced in a micro-droplet interface environment, so that qualitative and quantitative analysis of the disaccharide isomer can be quickly and efficiently realized, and the method can be further applied to food detection, medicine research and development or biomics research.

Description

Technical Field

[0001] The present invention relates to the fields of green chemistry technology and biomass omics, and particularly to a method, device and application for distinguishing disaccharide isomers through the reaction at the gas-liquid interface of water microdroplets. Background Art

[0002] Ammonia (NH 3 ) is the cornerstone of modern agriculture and industry, and is widely used in agricultural fertilizers, pharmaceutical manufacturing and fine chemical fields, supporting the development of the global modern industry. At the same time, NH 3 is also expected to become an energy carrier and hydrogen storage molecule for future sustainable development. In the chemical process of the origin of life, NH 3 also plays a crucial role. It is not only a precursor of key biomolecules such as amino acids and nucleotides, but also plays an important role in many primitive metabolic pathways. Currently, the industrial production of ammonia mainly relies on the Haber-Bosch process. Even though this process has greatly increased the nitrogen fertilizer production in the past century, due to the need to react high-purity nitrogen gas (N 2 ) with hydrogen gas (H 2 ) to produce ammonia under high temperature (300–500 °C) and high pressure (100–300 atmospheres) conditions with the assistance of a catalyst. However, this process has significant problems such as high energy consumption, large carbon emissions and strong dependence on fossil fuels, making it difficult to meet the requirements of sustainable development and restricting its long-term application in the context of sustainable development. Therefore, the development of low-energy-consuming, environmentally friendly and renewable ammonia synthesis technologies has become the focus of common concern in the current scientific and industrial communities.

[0003] In recent years, with the rapid development of chemical reactions in microdroplets, ammonia synthesis technology based on the gas-liquid interface has gradually become a research hotspot. In particular, the unique reaction environment at the gas-liquid interface of water microdroplets provides new ideas for ammonia synthesis. Due to the ultra-high surface area-to-volume ratio, spontaneous high electric field (up to 10 9 V / m) and the significant increase in reaction rate occurring at the interface in the gas-liquid interface of microdroplets, some reactions that are difficult to carry out or have extremely slow reaction rates in the bulk solution can be efficiently carried out at the microdroplet interface. These technologies use water as the hydrogen source, replacing the need for high-purity hydrogen gas in traditional processes, significantly reducing the complexity of raw material preparation and transportation. Microdroplet technology not only provides a new tool for basic research at the laboratory scale, but also lays an important theoretical foundation and technical support for the popularization of future industrial ammonia synthesis technology. Currently, on this basis, researchers have found that water microdroplets are sprayed onto catalyst coating materials (such as Fe 3 O 4, on the surface of Nafion, etc., nitrogen molecules are effectively activated under the action of the catalyst, and at the same time, water molecules provide a hydrogen source to further promote the efficient synthesis of ammonia. In the contact electrification mechanism, an aqueous solution and solid particles such as polytetrafluoroethylene (PTFE) form a strong electric field at the interface, and the reduction reaction of nitrogen molecules is realized through the charge transfer process, thereby generating ammonia.

[0004] In addition, CN 118125468 A discloses a method for synthesizing ammonia based on the interface reaction of plasma / inorganic solvent. This method uses a nanoliter electrospray device as an interface microreactor. During the application of negative high voltage, corona discharge occurs at the tip of the capillary. The plasma excitation generated by the corona discharge can generate reactive nitrogen species (such as N*, N 2 *, N 2 ⁺) at the gas-liquid interface. These reactive nitrogen species rapidly generate ammonia in the interaction with water molecules; the method disclosed in this invention patent application requires an additional applied voltage and is applicable to plasma-assisted ammonia synthesis reactions, and does not involve its application in the discrimination analysis of disaccharide isomers such as maltose, isomaltose, sucrose, palatinose, melibiose, and turanose. Summary of the Invention

[0005] In view of this, to solve the above problems, the main object of the present invention is to provide a simple, rapid, and efficient method, device, and application for discriminating disaccharide isomers. The present invention is mainly based on the gas-liquid interface reaction of water microdroplets, which can achieve efficient synthesis of NH at room temperature without the need for additional auxiliary means such as adding a catalyst or external conditions (temperature, voltage, etc.). 3 , NH 3 As a basic molecule, NH has a strong binding ability with oxygen-containing molecules, especially forming a stable adduct ([M+NH 4 ) with disaccharides. In the microdroplet interface environment, the binding effect of ammonia with disaccharide isomers is significantly enhanced. Using these characteristics, qualitative and quantitative analysis of disaccharide isomers can be achieved quickly and efficiently. +

[0006] To achieve the above object, the present invention provides the following technical solutions: A method for discriminating disaccharide isomers based on the gas-liquid interface reaction, comprising the steps of: [M+NH 4 + Adduct formation: Using an aqueous solution of disaccharide as a sample solution and a gas containing N 2 as an atomizing gas, the aqueous solution of disaccharide is injected into a capillary and continuously flows in the capillary. The aqueous solution of disaccharide in the capillary is atomized into microdroplets at a gas pressure greater than 50 psi and ejected from the ejection port of the capillary, and at the same time, a self-generated 10 8 -10 9 ​The gas-liquid interface of an electric field of V / m, under the action of the electric field at the gas-liquid interface, N 2 molecules are activated at room temperature and combine with the hydrogen radicals in the H 2 O molecules in the disaccharide aqueous solution to synthesize NH 3 molecules. The NH 3 molecules combine with the disaccharide molecules in the disaccharide aqueous solution to form [M + NH 4 + adducts; Mass spectrometry detection: Using a mass spectrometer to fragment the adduct [M + NH 4 + to form characteristic fragment ions, and detecting the intensity and abundance distribution of the characteristic fragment ions; Disaccharide isomer discrimination: According to the intensity and abundance distribution rules of the characteristic fragment ions of different disaccharide isomers, distinguish the disaccharide isomers.

[0007] A relative quantitative detection method for disaccharide isomers in a binary mixture system based on the above discrimination method, comprising the steps of: Preparing a series of binary mixed aqueous solutions with different proportions of disaccharide isomers, and the total concentrations of the two disaccharide isomers in the binary mixed aqueous solution are the same; Using the microdroplet gas-liquid interface reaction to generate ammonia molecules, which react with the disaccharide isomers in the binary mixed aqueous solution to form corresponding [M + NH 4 + adducts; Using a mass spectrometer to fragment the adduct [M + NH 4 + in the binary mixed aqueous solution to form characteristic fragment ions; Using a mass spectrometer to detect the intensity of the characteristic fragment ions in the binary mixed aqueous solution and calculate the relative ratio of the intensity of the characteristic fragment ions; Establish a calibration curve according to the following formula and calculate the relative molar content of the disaccharide isomers in the binary mixed aqueous solution: , , ; wherein, RI mix is the relative ratio of the intensity of the characteristic fragment ions in the binary mixed aqueous solution; RI a and RI b respectively represent the relative intensities of the corresponding characteristic fragment ions when the disaccharide isomers a and b exist alone; α a and α ​​​​b are the molar (or mass) percentages of disaccharide isomers a and b in the binary mixed aqueous solution, respectively, and satisfy α a + α b = 1, C a and C b are the configuration ratios of disaccharide isomers a and b at a known concentration ratio, and k and t are constants obtained by linear fitting.

[0008] Convert the characteristic fragment ion signal of the unknown sample into the relative content of the isomer by using the calibration curve.

[0009] A disaccharide isomer detection and analysis device, comprising: a microliter liquid supply unit, an N 2 gas source, a three-way spray assembly and a mass spectrometer. Among them, the microliter liquid supply unit includes a capillary, and can continuously provide a disaccharide aqueous solution to the three-way spray assembly through the capillary; The N 2 gas source is used to provide N 2 gas with a pressure greater than 50 psi to the three-way spray assembly, and atomize the disaccharide aqueous solution into microdroplets; The three-way spray assembly includes a liquid inlet end, a gas inlet end and an outlet end through which N 2 gas can escape. The gas inlet end is connected to the N 2 gas source; one end of the capillary is inserted into the three-way spray assembly from the liquid inlet end and penetrates through it, and extends out from the outlet end to form a spray port for spraying the microdroplets; the microdroplets form a gas-liquid interface with an electric field of 10 8 -10 9 V / m while being sprayed. Under the action of the electric field at this gas-liquid interface, N 2 is activated at room temperature and combines with the hydrogen radicals in the H 2 O molecules in the disaccharide aqueous solution to synthesize NH 3 molecules. The NH 3 molecules combine with the disaccharide molecules in the disaccharide aqueous solution to generate [M+NH 4 + adducts; The mass spectrometer includes a sampling port oppositely arranged with the spray port, and is used to monitor in real time the mass spectrometry detection signal of the [M+NH 4 + adducts generated by the reaction at the gas-liquid interface of the microdroplets.

[0010] ​​Based on the above detection and analysis device, it further includes an XYZ three-axis moving platform, which is used to fixedly install the three-way spray assembly and adjust the relative position between the ejection port of the capillary and the sample injection port; so that the ejection port of the capillary in the three-way spray assembly is coaxially arranged with the sample injection port of the mass spectrometer, that is, at the same height, realizing adjustable and controllable reaction distance. To ensure the optimal reaction distance of nitrogen at the gas-liquid interface, the position of the platform can be precisely adjusted through the XYZ three-axis moving platform, which can optimize the desolvation efficiency of the sample and reaction conditions, thereby significantly improving the accuracy and repeatability of the detection of disaccharide isomers.

[0011] Application of the above disaccharide isomer differentiation method, relative quantitative detection method or disaccharide isomer detection and analysis device in food detection, pharmaceutical research and development or biomics research.

[0012] Compared with the prior art, the above technical solutions protected by the present invention have the following characteristics: 1) For the disaccharide isomer differentiation method and relative quantitative detection method provided by the present invention, by using high-pressure N 2 gas as the atomizing gas, the aqueous disaccharide solution is atomized into micro-droplets. Under normal temperature conditions, without any catalyst and external conditions (such as temperature, voltage, etc.), the water micro-droplets will have a high electric field at the gas-liquid interface. N 2 can be activated at this interface and further react with the H radicals generated by water to complete the synthesis of NH 3 . NH 3 forms a stable adduct [M+NH 4 + with the disaccharide; through mass spectrometry analysis, the [M+NH 4 + of different disaccharide isomers (such as maltose, isomaltose, sucrose, etc.) generates unique ion signals in the MS / MS fragmentation mode, and these signals can be used as effective fingerprint information to distinguish disaccharide isomers; moreover, these signals are significantly enhanced in the micro-droplet interface environment, so qualitative and quantitative analysis of disaccharide isomers can be achieved quickly and efficiently.

[0013] 2) For the above differentiation method or detection method provided by the present invention, neither a catalyst nor a complex electrochemical or photochemical device needs to be used. By atomizing with high-pressure gas, NH 3 can be generated at the water micro-droplet gas-liquid interface, simplifying the synthesis process of NH 3 , and further simplifying the differentiation or detection process of disaccharide isomers and reducing the operation cost.

[0014] 3) The above disaccharide isomer detection and analysis device provided by the present invention includes a microliter liquid supply unit, N 2 ​​The gas source, three-way spray assembly and mass spectrometer have a simple structure and are easy to operate. There is no need to perform complex modifications on the existing mass spectrometry device, reducing equipment investment and being easy to promote and apply.

[0015] 4) The above-mentioned disaccharide isomer discrimination and detection technology provided by the present invention can be used in food detection, pharmaceutical research and development, and bioinformatics research.

[0016] Therefore, the present invention provides a new technical path for efficient, simple and energy-saving separation of disaccharide isomers, showing broad application potential in the fields of green chemistry and food science. Brief Description of the Drawings

[0017] Figure 1 It is a schematic diagram of the water micro-droplet atomization device provided in Embodiment 1 of the present invention.

[0018] Figure 2 It is the disaccharide isomer detection and analysis device provided in Embodiment 2 of the present invention.

[0019] Figure 3 It is a high-resolution mass spectrum of maltose reacting with ammonia at the water micro-droplet gas-liquid interface by using the method provided by the present invention.

[0020] Figure 4 It is the influence result diagram of the signal intensity and yield of [M+NH 4 + generated at the gas-liquid interface of water micro-droplets according to the reaction distance and the pressure of the atomizing gas nitrogen by using the device provided in Embodiment 2 of the present invention.

[0021] Figure 5 It is the overall device structure of the experimental characterization of the nitrogen source in the micro-droplet gas-liquid interface by the mixed gas system of the present invention.

[0022] Figure 6 It is for six disaccharide isomers [M+NH 4 + ( m / z is 360.15) in the MS / MS mass spectrum at a collision energy of 20 eV in the gas-liquid interface formed by the spray of the water micro-droplet atomization device provided in Embodiment 4 of the present invention.

[0023] Figure 7 It is the MS / MS fragment ion multivariate data PCA and OPLS-DA analysis of six disaccharide isomers provided in Embodiment 4 of the present invention.

[0024] Figure 8 It is the linear calibration curve constructed for the relative quantification of disaccharide isomers in a binary mixed system based on the fragment ion peak intensity ratio provided in Embodiments 5-7 of the present invention.

[0025] ​​Among them, the component symbols in the above figures are as follows: sample injection needle 1, quartz capillary 2, XYZ three-axis moving platform 3, X-axis 3-1, Z-axis 3-2, Y-axis 3-3, three-way spray assembly 4, gas inlet end 4-1, liquid inlet end 4-2, outlet end 4-3, reaction distance 5, mass spectrometer 6, gas supply unit 7, CO 2 gas source 7-1, N 2 gas source 7-2, mixed gas 7-3, reaction chamber 8. Specific embodiments

[0026] To make the objectives, technical solutions and advantages of the present invention clearer, the following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention.

[0027] Unless otherwise specified, the terms used in the present invention are all common terms in the art. The technical means adopted in the embodiments, such as preparation processes, analysis methods, etc., are all conventional means well-known to those skilled in the art. The reagents and products used are also commercially available. The sources, trade names of the reagents used, and those that are necessary to list their components are indicated when they first appear.

[0028] At the gas-liquid interface of water microdroplets, the present invention first discovered that microdroplets can effectively activate N 2 molecules at the gas-liquid interface and directly react with the protons provided by water molecules to generate ammonia molecules. Compared with the traditional Haber-Bosch process, it can be carried out under normal temperature and pressure conditions, avoiding the high energy consumption and safety hazards brought by high temperature and pressure. The high electric field of water microdroplets can not only promote the synthesis of ammonia, but also be used for the characterization of complex organic molecules such as disaccharides. As a basic molecule, ammonia has a strong binding ability with oxygen-containing molecules, especially forming stable adducts with disaccharides ([M+NH 4 + ). Through mass spectrometry analysis, ammonia will show characteristic fragment ion distributions after binding to disaccharides. The [M+NH 4 + of different disaccharide isomers (such as maltose, isomaltose, and sucrose, etc.) generate unique ion signals in the MS / MS fragmentation mode, and these signals can be used as effective fingerprint information to distinguish disaccharide isomers. Research shows that in the microdroplet interface environment, the binding effect of ammonia with disaccharide isomers is significantly enhanced, and these characteristics can be used to quickly and efficiently achieve the qualitative and quantitative analysis of disaccharide isomers.

[0029] The first aspect of the present invention provides a method for synthesizing ammonia (NH 3 ) based on gas-liquid interface reaction, including: using an aqueous solution and a gas containing N 2 as raw materials, and when the aqueous solution is in a flowing state, it is subjected to the gas containing N 2 ​​The gas is atomized into micro-droplets at an atomization air pressure greater than 50 psi, and at the same time, a gas-liquid interface with an electric field of 10 8 -10 9 V / m is formed. Under the action of the electric field at the gas-liquid interface, N 2 molecules are activated at room temperature and combine with hydrogen free radicals in the H 2 O molecules in the aqueous solution to synthesize ammonia molecules.

[0030] In this way, the ammonia synthesis method uses high-pressure N 2 -containing gas as the atomizing gas to atomize the aqueous solution into micro-droplets. Under room temperature conditions, without any catalyst and external conditions (temperature, voltage, etc.), the water micro-droplets will have a high electric field at the gas-liquid interface. N 2 At this interface, activation can be achieved, and further reaction with H free radicals generated by water can be carried out to complete the synthesis of ammonia; that is, the synthesis of NH 3 can be achieved at the gas-liquid interface with a high electric field, providing a new technical path for the synthesis of NH 3 and breaking through the limitations of traditional processes in terms of energy consumption, temperature, pressure, etc., showing significant advantages of green, low-carbon, and sustainable, and having the characteristics of environmental protection and high energy efficiency.

[0031] Among them, the aqueous solution described in the present invention includes tap water, distilled water, inorganic aqueous solutions, etc.

[0032] In the present invention, the atomization air pressure is 60 - 120 psi, such as 60 psi, 70 psi, 80 psi, 90 psi, 100 psi, 110 psi, 120 psi. In this way, water can be fully atomized into droplets, and further efficient and energy-saving synthesis of ammonia can be achieved.

[0033] In the present invention, the N 2 -containing gas is air or nitrogen with a purity of 99.99%.

[0034] In the present invention, the flow rate of the aqueous solution is 10 - 15 µL / min, such as 10 µL / min, 11 µL / min, 12 µL / min, 13 µL / min, 14 µL / min, 15 µL / min, etc. In this way, it is convenient to form uniform micro-droplets at the gas-liquid interface and achieve efficient and continuous synthesis of ammonia.

[0035] The second aspect of the present invention provides a water micro-droplet atomization device for synthesizing ammonia. This device can generate NH 3 at the water micro-droplet gas-liquid interface without applying external high temperature and external high voltage, and has a simple structure and is easy to operate.

[0036] Specifically, a water micro-droplet atomization device includes: a microliter liquid supply unit, N2 A gas source and a tee spray assembly, wherein the microliter liquid supply unit includes a capillary tube, and an aqueous solution can be continuously supplied to the tee spray assembly through the capillary tube; the N 2 The gas source is used to supply a gas containing N with a gas pressure greater than 50 psi to the tee spray assembly 2 and atomize the aqueous solution into micro-droplets; the tee spray assembly includes a liquid inlet end, a gas inlet end, and an outlet end through which the gas containing N can escape, and the gas inlet end is connected to the N 2 gas source. One end of the capillary tube is inserted into the tee spray assembly from the liquid inlet end and penetrates through it, and extends out from the outlet end to form a spray port for spraying the micro-droplets; when the micro-droplets are sprayed out, a gas-liquid interface with an electric field of 10 2 -10 8 V / m is formed. Under the action of the electric field at the gas-liquid interface, N 9 is activated at room temperature and combines with the hydrogen radicals in the H 2 O molecules in the aqueous solution to synthesize ammonia. 2

[0037] The microliter liquid supply unit further includes a sampling needle and an injection pump installed on the capillary tube. The capillary tube is respectively connected to the sampling needle and the tee spray assembly. Among them, the injection pump can accurately control the flow rate of the aqueous solution to ensure that the aqueous solution in the sampling needle flows through the capillary tube outlet to form uniform micro-droplets.

[0038] The inner diameter of the outlet end is larger than the outer diameter of the capillary tube. In particular, the inner diameter of the outlet end is larger than the outer diameter of the spray port of the capillary tube, which can ensure that nitrogen gas escapes from the outlet end and provides raw materials for synthesizing ammonia at the gas-liquid interface.

[0039] The inner diameter of the capillary tube can be 50-100 µm, such as 50 µm, 75 µm, 100 µm, etc., to ensure that micro-droplet sprays with smaller particle sizes can be generated to form a gas-liquid interface. The material of the capillary tube can be glass, quartz, etc. Quartz capillary tubes are preferably used to reduce impurities and improve product purity.

[0040] The third aspect of the present invention provides a method for synthesizing ammonia using the above water micro-droplet atomization device, including supplying an aqueous solution to the tee spray assembly at a flow rate of 10-15 µL / min through the microliter liquid supply unit, and simultaneously supplying a gas containing N with a gas pressure greater than 50 psi to the tee spray assembly through the N 2 gas source. The tee spray assembly atomizes the aqueous solution therein into micro-droplets and sprays them out from the capillary tube outlet at the outlet end, forming a gas-liquid interface with an electric field of 10 2 -10 8 -10 9 ​The gas-liquid interface of an electric field of V / m. Under the action of the electric field at this gas-liquid interface, N 2 molecules are activated at room temperature and combine with hydrogen free radicals in H 2 O molecules to synthesize ammonia, which is ejected from the capillary outlet at the outlet end. Preferably, the gas pressure of the gas containing N 2 is 60 - 120 psi.

[0041] Ammonia, as a basic molecule, has a strong binding ability with oxygen-containing molecules, especially forming stable adducts ([M + NH 4 + ) with disaccharides. Through mass spectrometry analysis, ammonia will show characteristic fragment ion distributions after binding with disaccharides. The [M + NH 4 + of different disaccharide isomers (such as maltose, isomaltose, and sucrose, etc.) generates unique ion signals in the MS / MS fragmentation mode, and these signals can be used as effective fingerprint information to distinguish disaccharide isomers. Research shows that in the micro-droplet interface environment, the binding effect between ammonia and disaccharide isomers is significantly enhanced, and these characteristics can be used to quickly and efficiently achieve the qualitative and quantitative analysis of disaccharide isomers.

[0042] Therefore, the present invention further combines the adduct reaction of ammonia with target molecules and develops a method for distinguishing and relatively quantifying disaccharide isomers based on the micro-droplet gas-liquid interface technology. By atomizing an aqueous solution with high-purity N 2 to generate NH 3 , NH 3 molecules bind to disaccharide molecules at the gas-liquid interface to form stable [M + NH 4 + adducts. Subsequently, the characteristic fragment ion abundances of [M + NH 4 + adducts are analyzed in the tandem secondary mass spectrometry MS / MS mode, and six common disaccharide isomers, including maltose, isomaltose, sucrose, palatinose, melibiose, and turanose, are successfully distinguished. In the relative quantification of disaccharide isomers, the present invention establishes a calibration curve based on the change law of the signal intensity ratio of target fragment ions by regulating the mixing ratio of disaccharides for the quantitative analysis of disaccharide isomers in a binary mixed system.

[0043] Specifically, the fourth aspect of the present invention provides a method for distinguishing disaccharide isomers, including the steps of: [M + NH 4 + Adduct generation: Using an aqueous solution of disaccharide as the sample solution and using a gas containing N 2 ​​​​​A gas is used as the atomizing gas. The disaccharide aqueous solution is injected into a capillary and continuously flows therein. Under a gas pressure greater than 50 psi, the disaccharide aqueous solution in the capillary is atomized into micro-droplets and ejected from the ejection orifice of the capillary. Meanwhile, a gas-liquid interface with an electric field of 10 8 -10 9 V / m is formed. Under the action of the electric field at the gas-liquid interface, N 2 molecules are activated at room temperature and combine with the hydrogen radicals in the H 2 O molecules in the disaccharide aqueous solution to synthesize NH 3 molecules. The NH 3 molecules combine with the disaccharide molecules in the disaccharide aqueous solution to generate an [M+NH 4 + adduct; Mass spectrometry detection: The adduct [M+NH 4 + is fragmented to form characteristic fragment ions, and the intensities and abundance distributions of the characteristic fragment ions are detected; Disaccharide isomer discrimination: Different disaccharide isomers are distinguished according to the intensity and abundance distribution rules of the characteristic fragment ions of different disaccharide isomers.

[0044] Among them, the steps for generating the [M+NH 4 + adduct include: The disaccharide aqueous solution flows at a flow rate of 10-20 µL / min and is atomized into the micro-droplets by the N 2 -containing gas under a pressure of 60-120 psi.

[0045] In the gas-liquid interface environment, the generation of ammonia and the binding effect with disaccharide molecules are significantly enhanced. The mass spectrometry signal intensity of the [M+NH 4 + conjugate is closely related to the spatial structure of the disaccharide isomer. The distance between the inlet of the mass spectrometer and the ejection orifice of the capillary (the formation outlet of the micro-droplets) is adjustable, and this distance is defined as the reaction distance. By optimizing the reaction distance and the gas pressure of the atomizing gas, the mass spectrometry signal intensity and the discrimination accuracy are further improved; preferably, the reaction distance is 5-20 mm.

[0046] The method for forming the characteristic fragment ions includes: For higher ion selectivity and less interference, the target ion [M+NH 4 + can be analyzed more precisely. The isolation width is set to a narrow width, such as 1-2 m / z , and a collision energy of 10-30 eV is used to fragment the adduct [M+NH 4 + ​​​​​Fragmentation can be carried out. The disaccharides in the aqueous disaccharide solution include but are not limited to: maltose, isomaltose, sucrose, palatinose, melibiose, turanose, and their mixtures. The characteristic fragment ions include but are not limited to m / z 342.14, 325.11, 163.06, 145.05, 127.04, 97.03, 85.03, etc.

[0047] Among them, the step of identifying the disaccharide isomers is to distinguish the disaccharide isomers by comparing the peak values and relative abundances of the characteristic fragment ions.

[0048] The step of identifying the disaccharide isomers further includes: performing multivariate statistical analysis on the distribution of the characteristic fragment ions by combining principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) to further verify the accuracy of the disaccharide isomer discrimination.

[0049] The fifth aspect of the present invention provides a method for relative quantitative detection of disaccharide isomers in a binary mixed system. Based on the above method for distinguishing disaccharide isomers, this detection method can quickly and efficiently achieve quantitative detection and analysis of disaccharide isomers.

[0050] Specifically, a method for relative quantitative detection of disaccharide isomers in a binary mixed system based on the above distinguishing method includes the steps: Preparing a series of binary mixed aqueous solutions with different ratios of disaccharide isomers, and the total concentrations of the two disaccharide isomers in the binary mixed aqueous solution are the same; Using the micro-droplet gas-liquid interface reaction to generate ammonia molecules, which react with the disaccharide isomers in the binary mixed aqueous solution to form corresponding [M+NH 4 + adducts; Using a mass spectrometer to fragment the adduct [M+NH 4 + in the binary mixed aqueous solution to form characteristic fragment ions; using a mass spectrometer to detect the intensities of the characteristic fragment ions in the binary mixed aqueous solution and calculate the relative ratios of the intensities of the characteristic fragment ions therein; Establishing a calibration curve according to the following formula and calculating the relative molar contents of the disaccharide isomers in the binary mixed aqueous solution: , , ; Among them, RI mix is the relative ratio of the intensities of the characteristic fragment ions in the binary mixed aqueous solution; RI a and​​RI b respectively represent the relative intensities of the corresponding characteristic fragment ions of disaccharide isomers a and b when they exist alone; α a and α b are the molar (or mass) percentages of disaccharide isomers a and b in the binary mixed aqueous solution respectively, and satisfy α a + α b = 1, C a and C b are the configuration ratios of disaccharide isomers a and b at a known concentration ratio, and k and t are constants obtained by linear fitting.

[0051] Convert the characteristic fragment ion signal of the unknown sample into the relative content of the isomer by using the calibration curve.

[0052] Among them, the mass ratio or molar ratio of disaccharide isomers a and b in the binary mixed aqueous solution is 20:1 - 1:100, and the relative intensity ratios of the characteristic fragment ions of different disaccharide isomer combinations include but are not limited to: palatinose and turanose: m / z 145 / 259, isomaltose and palatinose: m / z 145 / 325, sucrose and melibiose: m / z 145 / 163.

[0053] The sixth aspect of the present invention provides a disaccharide isomer detection and analysis device, which includes a water microdroplet atomization device. Without high temperature and external high voltage, [M+NH 4 + can be generated at the water microdroplet gas-liquid interface, and the operation is simple and easy; there is no need to add any additional derivatization reagents to react with the sample compound, nor to introduce metal or non-metal ligands with complex structures to form complexes with sugars. Only the sample solution to be tested needs to be directly injected, omitting the complicated pretreatment steps in the traditional method; the isomers can be quickly distinguished by tandem mass spectrometry technology, and the time of collision-induced dissociation is in the millisecond level. There is no need for separation equipment such as liquid chromatography or ion mobility spectrometry, thus making the separation method of disaccharide isomers efficient and simple.

[0054] Specifically, a disaccharide isomer detection and analysis device includes: The above-mentioned water microdroplet atomization device is used to generate ammonia molecules by using the reaction at the microdroplet gas-liquid interface and react with the disaccharide isomer to form [M+NH 4 + adduct; ​​A mass spectrometer, including a sampling port disposed opposite to the ejection port of the capillary, for real-time monitoring of the mass spectrometry detection signal of the [M+NH 4 + adduct generated by the gas-liquid interface reaction of micro-droplets.

[0055] Based on the above detection and analysis device, it further includes an XYZ three-axis moving platform, which is used to fixedly install the three-way spray assembly and adjust the relative position of the ejection port of the capillary and the sampling port; so that the ejection port of the capillary in the three-way spray assembly is coaxially arranged with the sampling port of the mass spectrometer, that is, at the same height, realizing adjustable and controllable reaction distance. To ensure the optimal reaction distance of nitrogen at the gas-liquid interface, the position of the platform can be precisely adjusted through the XYZ three-axis moving platform, which can optimize the desolvation efficiency of the sample and the reaction conditions, thereby significantly improving the accuracy and repeatability of the detection of disaccharide isomers.

[0056] Among them, the mass spectrometer is an existing mass spectrometry detection device, preferably a high-resolution mass spectrometer, which can perform MS / MS analysis of [M+NH 4 + adduct in real time.

[0057] In short, ammonia combines with disaccharide molecules in the solution at the gas-liquid interface of micro-droplets to form a stable [M+NH 4 + adduct, which shows characteristic fragment ion distribution in the tandem secondary mass spectrometry MS / MS mode. Using these characteristic ions, rapid differentiation and quantitative analysis of disaccharide isomers of maltose, isomaltose, sucrose, palatinose, melibiose and turanose can be achieved.

[0058] Therefore, the seventh aspect of the present invention provides an application of any one of the above ammonia synthesis methods, water micro-droplet atomization devices, disaccharide isomer differentiation methods, relative quantitative detection methods and disaccharide isomer detection and analysis devices in ammonia synthesis, carbohydrate isomer differentiation and quantitative analysis, food detection, pharmaceutical research and development or biomics research.

[0059] The above method provided by the present invention realizes the efficient synthesis of NH 3 under normal temperature and catalyst-free conditions by adopting a gas-liquid interface reaction device of micro-droplets and adjusting the optimal reaction distance on the XYZ three-axis moving platform, without relying on traditional high-temperature and high-pressure processes. This process not only avoids the need for high-purity hydrogen in traditional ammonia synthesis methods, but also significantly reduces carbon dioxide emissions and energy consumption generated by hydrogen preparation, storage and transportation, reflecting the characteristics of green and sustainable. In addition, the present invention does not require the use of catalysts or complex electrochemical and photochemical devices, simplifies the process flow of ammonia synthesis, and reduces equipment investment and operating costs. With the help of the high-intensity electric field formed at the gas-liquid interface of micro-droplets (~10​​​9 (V / m), significantly improving the ammonia production efficiency and the binding ability with disaccharides. By optimizing the gas-liquid interface conditions of the microdroplets, the present invention realizes the efficient in-situ generation and enrichment of ammonia, and provides a unique technical means for the rapid separation and quantitative analysis of disaccharide isomers.

[0060] The above-mentioned water microdroplet atomization device and disaccharide isomer detection and analysis device provided by the present invention have a simple structure, do not require complex modification of the existing mass spectrometry device, are easy to operate, and are easy to promote and apply. This technology provides a new technical path for efficient, simple and energy-saving ammonia synthesis and disaccharide isomer separation, showing broad application potential in the fields of green chemistry and food science, etc.

[0061] The technical solutions of the present invention will be further described in detail below through specific embodiments. Among them, the water microdroplet atomization device and the disaccharide isomer detection and analysis device adopted in the following embodiments are shown in Embodiment 1 and Embodiment 2 respectively.

[0062] Embodiment 1 Water Microdroplet Atomization Device and Ammonia Synthesis As Figure 1 shown, this embodiment provides a water microdroplet atomization device for synthesizing ammonia (NH 3 ), which mainly consists of a microliter liquid supply unit, an N 2 gas source 7-2 and a tee spray assembly 4. The microliter liquid supply unit is mainly used to provide an aqueous solution to the tee spray assembly 4, including a sampling needle 1, a capillary 2 for connecting the sampling needle 1 and the tee spray assembly 4, and an injection pump installed on the capillary 2. The inner diameter of the capillary 4 can be 50-100 µm, and its material can be glass, quartz, etc. In this embodiment, the capillary 4 is a quartz capillary with an inner diameter of 75 µm.

[0063] The tee spray assembly 4 includes a liquid inlet end 4-2 connected to the sampling needle 1, an outlet end 4-3, and an air inlet end 4-1 connected to the N 2 gas source 7-2, with the capillary 2 running through it internally. One end of the capillary 2 is inserted into the tee spray assembly 4 from the liquid inlet end 4-2 and runs through it, and the outlet end 4-3 extends out to form a spray nozzle for spraying microdroplets. The injection pump can accurately control the flow rate of the aqueous solution to ensure that the aqueous solution in the sampling needle 1 forms uniform microdroplets at the outlet of the capillary 2. The N 2 gas source 7-2 is used to provide a gas containing N 2 with a pressure greater than 50 psi to the tee spray assembly. Among them, the N 2 gas source 7-2 includes air or pure nitrogen (purity 99.99%). In this embodiment, the N 2 gas source 7-2 is pure nitrogen.

[0064] The three-way spray assembly 4 can atomize the aqueous solution into micro-droplets, and at the same time form a gas-liquid interface with an electric field of 10 8 -10 9 V / m. Under the action of the electric field at this gas-liquid interface, N 2 is activated at room temperature and combines with the hydrogen radicals in the hydrogen atoms of the H 2 O molecules in the aqueous solution to synthesize ammonia.

[0065] This embodiment also provides a method for synthesizing NH 3 based on the above device. The specific method includes the following steps: S1) Use a syringe pump to deliver pure water H 2 O to the three-way spray assembly 4 through the injection needle 1 at a flow rate of 10-15 μL / min; at the same time, connect the gas inlet end 4-1 of the three-way spray assembly 4 to the N 2 gas source 7-2 through a gas pipeline to provide a pure nitrogen gas source for the pure water H 2 O; the nitrogen gas enters the three-way spray assembly 4 under a pressure of 60-120 psi and atomizes the pure water H 2 O flowing in the capillary 2 it passes through into micro-droplets; S2) Under normal temperature, without a catalyst and without applying an external voltage to the capillary 2 and other auxiliary components, N 2 with a pressure of 60-120 psi is ejected from the outlet end 4-3 and forms a high-speed gas-liquid interface together with the micro-droplets formed by the atomization of purified H 2 O; the micro-droplets have a strong electric field of up to 10 9 V / m at the interface, the activation energy of the N≡N bond is reduced, so that N 2 molecules are more likely to break and be activated at this interface; the activated nitrogen atoms (or nitrogen-containing free radicals) react with the hydrogen atoms (or hydrogen free radicals) provided by the water molecules to generate NH 3 and are enriched at the gas-liquid interface. Among them, in the process of synthesizing NH 3 , the flow rate of the pure water H 2 O can be 10, 11, 12, 13, 14, 15 μL / min, etc., and the pressure of the nitrogen gas can be 60 psi, 70 psi, 80 psi, 90 psi, 100 psi, 110 psi, 120 psi, etc.

[0066] Therefore, the above-mentioned water micro-droplet atomization device for synthesizing ammonia provided in this embodiment is an efficient system for generating a micro-droplet gas-liquid interface. The core design of this device includes a three-way connector (three-way spray assembly 4), whose two ends are connected by precisely machined capillary tubes. The upper end is connected to a nitrogen source, which serves as the atomizing gas. By precisely controlling the nitrogen gas flow rate, it is ensured that the solution in the injection needle forms uniform micro-droplets when flowing through the capillary tube outlet, thereby realizing the rapid, efficient, and green synthesis of ammonia.

[0067] Example 2 Disaccharide Isomer Detection and Analysis Device Please refer to Figure 2 , this embodiment provides a disaccharide isomer detection and analysis device, which includes the water micro-droplet atomization device provided in Example 1, an XYZ three-axis moving platform 3, and a mass spectrometer 6. The water micro-droplet atomization device is used to generate ammonia molecules through the reaction at the micro-droplet gas-liquid interface and react with disaccharide isomers to form [M+NH 4 + adducts; the XYZ three-axis moving platform 3 is mainly used to precisely adjust the relative positions of the three-way spray assembly 4 and the mass spectrometer; the mass spectrometer is mainly used to real-time monitor the mass spectrometry detection signals of the ammonia molecules and [M+NH 4 + adducts generated by the reaction at the micro-droplet gas-liquid interface. In this embodiment, the mass spectrometer 6 is a high-resolution mass spectrometer.

[0068] The capillary tube 2 passing through the inside of the three-way spray assembly 4 extends from the outlet end 4-3 to form a jet orifice facing the inlet of the mass spectrometer 6. The distance between the jet orifice of the capillary tube 2 and the inlet of the mass spectrometer 6 is defined as the reaction distance 5. The three-way spray assembly 4 is fixedly installed on an acrylic plate with dimensions of 5 cm × 8 cm, and this acrylic plate is perpendicular to the XY plane of the XYZ three-axis moving platform 3. By adjusting the positions of the X-axis 3-1, Z-axis 3-2, and Y-axis 3-3 in the XYZ three-axis moving platform 3, the desolvation efficiency of the detection sample and the reaction conditions, as well as the optimal reaction conditions for ammonia synthesis, can be optimized, thereby significantly improving the accuracy and repeatability of disaccharide isomer discrimination detection.

[0069] NH 3 Preliminary Mass Spectrometric Characterization of NH Synthesis Experiments found that NH generated at the micro-droplet gas-liquid interface 3 , which shows high solubility in aqueous solution and is prone to combine with protons to form ammonium ions under non-alkaline conditions. In addition, NH 3 tends to form stable bindings with oxygen atom-containing molecules, especially sugar compounds, through non-covalent interactions such as hydrogen bonds. Therefore, maltose (MT) was selected as the marker for NH 3 , and high-resolution mass spectrometry technology was used to analyze the generated NH 3 ​​Qualitative analysis is carried out to verify the synthesis of ammonia.

[0070] Combined with Figure 2 , the specific verification method of ammonia includes the following steps: S1) Use a syringe pump to deliver a 100 μmol / L aqueous solution of maltose to the three-way spray assembly 4 through the injection needle 1 at a flow rate of 15 μL / min; at the same time, connect the gas inlet end 4-1 of the three-way spray assembly 4 to the N 2 gas source 7-2 through a gas pipeline to provide a pure nitrogen gas source for the aqueous solution of maltose; the nitrogen gas enters the three-way spray assembly 4 at a pressure of 100 psi, atomizing the sugar aqueous solution flowing in the capillary 2 it passes through into micro-droplets; S2) Under the conditions of normal temperature, no catalyst, and no external voltage applied to the capillary 2, N 2 with a pressure of 100 psi passes through the capillary and sprays out from the outlet end 4-3, and forms a high-speed gas-liquid interface together with the micro-droplets formed by the atomization of the aqueous solution of maltose; the micro-droplets have a strong electric field of up to 10 9 V / m at the interface, reducing the activation energy of the N≡N bond, so that the nitrogen molecules are more likely to break and be activated at this interface; the activated nitrogen atoms (or nitrogen-containing free radicals) react with the hydrogen atoms (or hydrogen free radicals) provided by water molecules to generate NH 3 and accumulate at the gas-liquid interface; S3) Precisely adjust the relative position between the injection port of the capillary 2 and the injection port of the mass spectrometer 6 through the XYZ three-axis moving platform 3 to achieve precise adjustment of the reaction distance 5. The adjustment precision in the X direction 3-1, Y direction 3-3, and Z direction 3-2 is all 10 μm.

[0071] Furthermore, under the conditions of the optimal reaction distance and atomizing gas pressure, the mass spectrometer can accurately detect the generated ammonia and the related mass spectrometry signals of its adduct ions with maltose, significantly improving the reproducibility and accuracy of the ammonia synthesis process.

[0072] Based on the above, a pure aqueous solution of maltose without any added ions forms micro-droplets under the action of N 2 and enters the mass spectrometer for detection. As shown in the high-resolution mass spectrum Figure 3 , an obvious m / z signal peak of 360.1499 can be observed, and this signal peak is considered to be [MT + NH 4 + , and the calculated exact mass number is 360.1500. The deviation (Δ m ) between the two values is only 0.28 ppm, which fully meets the error range allowed by the high-resolution mass spectrometer. In addition, a small amount of [MT + Na] + ( m / z ​365.1053) and [MT + K] + ( m / z 381.0794) signal peaks. [MT + NH 4 + The results of Figure 6 a) were further verified using tandem mass spectrometry in positive ion mode at a collision energy of 20 eV, and the fragment ions m / z produced were 342.14, 325.11, 163.06, 145.05, 127.04, 97.03, 85.03, etc. The generation of these fragments was mainly due to the stepwise hydrolysis of the disaccharide and the consecutive ring-opening reactions common in the carbohydrate fragmentation pathway, among which m / z the peak at 325.11 was produced by m / z the loss of one molecule of NH 3 (17 Da) from the fragment ion at 342.14, further verifying the generation of NH 3 . Therefore, the device water microdroplet gas-liquid interface method successfully achieved the synthesis of NH 3 .

[0073] Effect test of atomizing gas pressure on [MT + NH 4 + The following combines Figure 4 the results shown in (left) and the relevant test data to illustrate the effect of atomizing gas pressure on the generation of [MT + NH 4 + . Those skilled in the art can understand the regulation principle of the microdroplet gas-liquid interface reaction efficiency in the present invention according to the following content, but it is not limited to this embodiment.

[0074] To evaluate the generation efficiency of [MT + NH 4 + , based on the ion adducts Figure 3 generated, the present invention defines the product yield as: (1) where the content in the parentheses of formula (1) represents the peak intensity of the corresponding ion in the mass spectrum.

[0075] Basically, the specific verification method of ammonia provided in "Preliminary Mass Spectral Characterization of NH 3 Synthesis" was adopted, and the main difference was that the atomization pressure in this test was between 60 - 100 psi, and the other method steps were the same. The results are as shown in Figure 4 (left). When the atomizing gas pressure was gradually increased from 60 psi to 100 psi, [MT + NH 4 + ​​​​​The peak intensity and product yield have both increased significantly. The main reason is that as the atomizing gas pressure increases, the particle size of the micro-droplets becomes smaller, and the surface area to volume ratio increases accordingly, which strengthens the water-gas interface interaction, making the ammonia synthesis and its addition process with target molecules (such as maltose) more efficient. Thus, it can be seen that by increasing the atomizing gas pressure, the reaction efficiency at the gas-liquid interface of the micro-droplets can be effectively enhanced.

[0076] The influence test of the reaction distance on [MT + NH 4 + The influence test Basically, the specific verification method of ammonia provided in "Preliminary Mass Spectrometry Characterization of the Synthesis of NH 3 " is adopted. The main difference is that in this test, the injection distance (i.e., the reaction distance 5) of the gas-liquid interface of the micro-droplets is further adjusted within the range of 5 mm to 20 mm, and other method steps are the same, to evaluate the influence of the reaction distance on [MT + NH 4 + generated. The results are as shown in Figure 4 (right). As the reaction distance increases from 5 mm to 10 mm, both the ion signal intensity and the product percentage show an upward trend and reach a peak at 10 mm, with the highest generation efficiency approaching 95%. When the distance exceeds 10 mm, both of them decrease significantly, and the product percentage drops to about 60% at 20 mm. This phenomenon indicates that the micro-droplets need sufficient flight and gas-liquid interface reaction time within a certain range to complete effective desolvation and ammonia addition reactions; but when the distance is too large, the micro-droplets are prone to dispersion or secondary coalescence, resulting in a weakened gas-liquid interface, and at the same time, the long distance affects the transmission efficiency of mass spectrometry for its detection, ultimately reducing the generation efficiency of [MT + NH 4 + Therefore, choosing an appropriate reaction distance, such as 10 mm, is crucial for achieving high ionization efficiency and product yield.

[0077] Experimental characterization of the nitrogen source The following combines Figure 5 with the specific experimental process to elaborate in detail on the experimental characterization of the nitrogen source in the embodiments of the present invention. It should be understood that the disclosed embodiments are only used to illustrate the principle of the present invention and are not used to limit the protection scope of the present invention.

[0078] As shown in Figure 5, in order to confirm that the nitrogen atoms in the synthesized NH 3 come from the atomizing gas N 2 ​​​, a sealed reaction chamber 8 that can be connected to the inlet of the mass spectrometer 6 was designed and constructed. The capillary 2 is inserted from the front end of the reaction chamber 8 to ensure that the sample can be accurately transported to the gas-liquid interface of the micro-droplet for reaction. To maintain the pressure balance in the reaction chamber, a gas outlet 8-1 is provided at the bottom of the reaction chamber 8 to allow excess gas to escape. The gas supply unit 7 includes a flow regulating valve for the N 2 gas source 7-2 and the CO 2 gas source 7-1. The mixed gas 7-3 mixed in a predetermined ratio is introduced into the reaction chamber 8 through the intake end 4-1 of the tee spray assembly 4 by the gas mixing device, so as to establish a controllable gas environment during the experiment. Except for the gas composition, other experimental conditions (including sample concentration, temperature, distance between the sprayer and the mass spectrometer inlet, sheath gas pressure, etc.) are kept consistent, so as to systematically characterize and compare the generation process of NH 3 under different gas environments. The experiment was carried out under six gas conditions: synthetic air, pure N 2 , and 80% N 2 + 20% CO 2 , 50% N 2 + 50% CO 2 , 20% N 2 +80% CO 2 and pure CO 2 mixed gases.

[0079] The mass spectrometry results show that the formation of [MT + NH 4 + significantly depends on the presence of N 2 atomizing gas. Under pure N 2 conditions, the product percentage of [MT + NH 4 + is slightly higher than that under synthetic air conditions. In the mixed gas of N 2 and CO 2 , with the increase of the CO 2 proportion, the formation of [MT + NH 4 + gradually decreases. At the same time, the peak intensities of [MT + Na] + and [MT + K] + gradually increase, indicating that ionization or adduct formation is enhanced in a high CO 2 environment. When pure CO 2 is used as the atomizing gas, no signal of [MT + NH 4 + is observed. This result shows that without the participation of N 2 , nitrogen molecules cannot be effectively activated to synthesize ammonia, and the adduct reaction cannot form [MT + NH​​​​4 + This further confirmed that N 2 is the key source for the formation of NH 3 .

[0080] The experimental results provide important evidence for the ammonia synthesis mechanism at the gas-liquid interface of microdroplets in the present invention, and also provide a reference basis for further optimizing the reaction atmosphere and exploring the effects of other gas components on ammonia synthesis. The above embodiments do not constitute a limitation to the present invention. Those skilled in the art can make various improvements and deformations to the present invention without departing from the spirit and scope of the present invention, and still fall within the protection scope of the present invention.

[0081] Example 3 Method for distinguishing disaccharide isomers This example provides a method for distinguishing disaccharide isomers, including the steps of: first, using an aqueous disaccharide solution as a sample solution, and using a gas containing N 2 as an atomizing gas, and synthesizing NH 3 through a gas-liquid interface reaction. The NH 3 molecules combine with the disaccharide molecules in the aqueous disaccharide solution to form [M+NH 4 + adducts; using a mass spectrometer to detect the [M+NH 4 + adducts to obtain the mass spectrometry signal information of characteristic fragment ions; distinguishing disaccharide isomers according to the mass spectrometry signal information of characteristic fragment ions.

[0082] That is, on the basis of generating ammonia at the gas-liquid interface of microdroplets, this example of the present invention further utilizes the binding characteristics of ammonia and saccharide substances to realize the distinction and analysis of disaccharide isomers. The specific implementation steps are as follows: S4) Inject the sample solution from the end of the capillary 2 through the injection needle 1. The sample solution is an aqueous disaccharide solution; in this example, disaccharides such as maltose, isomaltose, sucrose, palatinose, melibiose, and turanose are respectively prepared into aqueous solutions with a concentration of 100 µmol / L, and are transported into the three-way connector 4 through the injection needle 1 by using an injection pump. The atomization pressure and the aqueous solution flow rate in this step are the same as those in step S1) of the "Preliminary mass spectrometry characterization of NH 3 synthesis" mentioned above; S5) Under normal temperature conditions, without any catalyst and applying an external voltage to the capillary 2, a high electric field at the gas-liquid interface of microdroplets is formed after the aqueous disaccharide solution is atomized by N 2 . At this time, NH 3 is synthesized in real time at the interface, and further NH 3 combines with the disaccharide molecules to form [M+NH 4 + ion adduct peaks, which can be directly observed in mass spectrometry detection;​​​​ S6) By optimizing the reaction distance and the nebulizing gas pressure, the 4 + signal peak reaches the optimal intensity; in this embodiment, by adjusting the XYZ three-axis moving platform 3 to change the reaction distance 5, and finely tuning the flow rate or nebulizing pressure of the disaccharide aqueous solution, the 4 + peak intensity and yield reach the optimal values; S7) Select 4 + as the parent ion, perform MS / MS fragmentation through collision-induced dissociation to form characteristic fragment ions, and detect the intensity and abundance distribution characteristics of the characteristic fragment ions; in this embodiment, the isolation width is set to 1 m / z , the collision energy is 20 eV, record the characteristic fragment ion spectrum and its relative abundance, and the results are as Figure 6 shown; S8) By comparing the distribution of characteristic fragment ions generated after collision dissociation of different disaccharides, rapid discrimination of disaccharide isomers is achieved.

[0083] From Figure 6 it can be seen that: the typical fragment ions detected for 6 disaccharide isomers in this embodiment mainly include m / z 342.14, 343.12, 325.11, 163.06, 145.05, 127.04, 97.03, 85.03, etc. Among them, m / z 342.14 breaks m / z 325.11 observes a mass loss of about 17 Da, showing an obvious 3 NH loss process, which conforms to the common multi-step hydrolysis and stepwise ring-opening fragmentation rules of carbohydrates. For melibiose, isomaltose, and sucrose, obvious + peak ( m / z 343.12) can also be detected in the experiment. Research shows that glycosidic bond types, such as the α(1→6) bond of melibiose and isomaltose and the α(1→2) non-reducing bond of sucrose, are more likely to undergo linear protonation or 3 NH loss under these conditions to generate relatively stable protonated ions. Due to its special non-reducing structure, sucrose will show relatively abundant fragment peaks at m / z 198.10 and 180.09 during the collision-induced dissociation process, showing that it first forms monosaccharide fragment ions ( 4 + 198.10) in cooperation with m / z NH, and then further dehydrates to generate m / z 180.09. ​​​

[0084] The fragment ions of six disaccharides were visualized using principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). The results are as follows Figure 7 shown, and different disaccharide isomers can be clearly distinguished. The relative abundances of each ion signal and the fragmentation pathways together constitute the differential discriminant features. According to Figure 7 the PCA results shown in a, the cumulative interpretation rate R 2 X reaches 0.995, indicating that the model can explain 99.5% of the original variable information and has a good data fitting degree; while Q 2 is 0.976, indicating that the prediction ability of the model is also relatively reliable during the cross-validation process. In the Figure 7 OPLS-DA model shown in b, the cumulative interpretation rate R 2 X of the model is 1, and R 2 Y is 0.999, indicating that the model has extremely high fitting degree for data and interpretation degree for classification variables; at the same time, Q 2 can reach 0.998, indicating that the prediction ability for unknown samples is also extremely excellent.

[0085] Therefore, by performing MS / MS collision-induced dissociation on the [MT + NH 4 + ions formed by the adduct of disaccharides and ammonia in the high electric field environment of the microdroplet gas-liquid interface, and combining multivariate statistical analyses including PCA and OPLS-DA, this example can achieve rapid and efficient differentiation of six common disaccharide isomers without the need for complex derivatization or harsh conditions such as high temperature and high pressure. This method has the advantages of high sensitivity, high specificity and good reproducibility, and can provide important technical support for the identification and analysis of carbohydrate isomers in the fields of food, medicine and biology.

[0086] Those skilled in the art can reasonably adjust the collision energy, microdroplet size and other parameters according to actual needs, and it still falls within the protection scope of the present invention.

[0087] Example 4-6 Relative Quantitative Detection Method for Disaccharide Isomers in Binary Mixture Systems Since disaccharide isomers are highly similar in molecular structure and physical and chemical properties, traditional analytical methods often face great challenges in quantitative differentiation. To solve this problem, this example uses the [MT +NH 4 + ions generated by the high electric field at the microdroplet gas-liquid interface, and combines the change law of the intensity ratio of the characteristic fragmentation ion peaks of MS / MS to construct a relative quantitative method for binary mixture systems suitable for disaccharide isomers.

[0088] ​​Specifically, a method for relative quantitative detection of disaccharide isomers in a binary mixture system includes the following steps: First, two disaccharide isomers were separately prepared and mixed in the following different ratios and dissolved in pure water to obtain mixed aqueous solutions: 20:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:20, and 1:100, and it was ensured that the total concentration of the binary mixture system was consistent, all being 100 µmol / L; among them, the two disaccharide isomers in the binary mixture system provided in Examples 5-7 were palatinose and turanose, isomaltose and palatinose, and sucrose and melibiose, respectively. Second, using the method for distinguishing disaccharide isomers provided in Example 4, ammonia molecules were generated by the microdroplet gas-liquid interface reaction and reacted with the disaccharide isomers in the binary mixed aqueous solution to form corresponding 4 + adducts; the adducts 4 + in the binary mixed aqueous solution were fragmented by a mass spectrometer to form characteristic fragment ions; the intensities of the characteristic fragment ions in the binary mixed aqueous solution were detected by a mass spectrometer, and the relative ratio of the intensities of the characteristic fragment ions was calculated. Then, according to the above detection results, a corresponding calibration curve was constructed. The following gives the relevant mathematical expressions for relative quantification of disaccharide isomers in the embodiments of the present invention. It should be understood that the shown equations are only used to illustrate the principle of the method and do not limit the protection scope of the present invention.

[0089] Relationship formula (2) between relative intensity ratio and proportion of isomers in the binary system: , where RI mix is the relative ratio of the intensities of the characteristic fragment ions in the binary mixed aqueous solution; RI a and RI b respectively represent the relative signal intensities of the corresponding characteristic fragment ions when disaccharide isomers a and b exist alone; α a and α b are the molar (or mass) percentages of disaccharide isomers a and b in the binary mixed aqueous solution, respectively, and satisfy α a + α b = 1.

[0090] Derivation of the isomer ratio from the relative intensity ratio of the mixed system, formula (3): , and can be measured through formula (3)​​RI mix With the known C a and C b being the relative proportions of disaccharide isomers a and b in a binary mixture system at a known concentration ratio α a / α b .

[0091] Linear relationship formula (4) between the isomer ratio and the known concentration ratio: , where in this formula C a and C b are the preparation ratios of disaccharide isomers a and b at a known concentration ratio, and k and t are constants obtained by linear fitting. Thus, a calibration curve can be established based on the measured values at multiple known ratios, and the relative content of the isomers in the unknown sample can be deduced.

[0092] In Example 5, palatinose (Glcα1→6Fru) and turanose (Glcα1→3Fru): Both have the same monosaccharide composition, containing glucose and fructose, but differ in the glycosidic bond positions α1→6 and α1→3. Using m / z the characteristic ions of 145 / 259 for quantitative evaluation, a calibration curve as shown in Figure 8 a is established according to the linear relationship formula (4) within the concentration range of 0.02 - 100 µmol / L, where y is α 帕拉金糖 / α 松二糖 , and x is C 帕拉金糖 / C 松二糖 , and R² = 0.9964.

[0093] In Example 6, isomaltose (Glcα1→6Glc) and palatinose (Glcα1→6Fru): Both have the same glycosidic bond position α1→6, but different monosaccharide compositions (glucose and fructose); using m / z the intensity ratio of the fragment ion peak of 145 / 325 to establish a calibration curve as shown in Figure 8 b according to the linear relationship formula (4) within the concentration range of 0.01 - 10 µmol / L, where y is α 异麦芽糖 / α 帕拉金糖 , and x is C 异麦芽糖 / C帕拉金糖 , R² = 0.9982.

[0094] In Example 7, sucrose (Glcα1→2Fru) and melibiose (Galα1→6Glc): These two isomers not only differ in glycosidic bond position and monosaccharide type, but also include the non-reducing end characteristics of sucrose. Based on m / z the ion ratio of 145 / 163 for fitting, a calibration curve as shown in Figure 8 c was established according to the linear relationship formula (4) within the linear range of 0.01 - 80 µmol / L, y = 0.415x - 0.1528, where y is α 蔗糖 / α 蜜二糖 , and x is C 蔗糖 / C 蜜二糖 , R² = 0.9996.

[0095] Therefore, the embodiments of the present invention utilize the microdroplet gas-liquid interface technology and the MS / MS fragmentation analysis method, which can not only identify the fine structures of disaccharide isomers, but also achieve high-precision relative quantification through the calibration curve in a binary mixed system. This method has the advantages of a wide linear range, strong adaptability, high selectivity, etc., and can be widely applied to fields such as food detection, pharmaceutical research and development, and glycobiology research. Those skilled in the art can make necessary adjustments to the selected fragment ions, flow rates, and other operating parameters according to actual needs.

[0096] In summary, the ammonia synthesis and disaccharide isomer differentiation method and device provided by the embodiments of the present invention are mainly based on the gas-liquid interface reaction of water microdroplets in a high electric field environment, and utilize the activation of nitrogen (N 2 ) and hydrogen radicals provided by water molecules for efficient ammonia synthesis, without the need for catalysts, high temperature or high pressure conditions, realizing green ammonia production at normal temperature and pressure. At the same time, the embodiments of the present invention have developed an adduct reaction method for ammonia and disaccharide molecules to generate a stable adduct [M+NH 4 + ​, and combined with tandem mass spectrometry (MS / MS) to analyze the distribution of its characteristic fragment ions, successfully achieving the rapid differentiation and quantitative detection of disaccharide isomers. The device in the embodiments of the present invention can precisely control parameters such as the reaction distance and the pressure of the atomizing gas, optimizing the reaction efficiency and the signal intensity of the adduct. Therefore, the device provided by the present invention has a simple structure, and the method significantly simplifies the experimental process, reduces energy consumption and environmental pollution, provides an efficient, green and economical solution for ammonia synthesis and sugar isomer detection, opens up a new application direction for the detection of complex sugars in food chemistry and omics analysis, is expected to become an important development direction for nitrogen fixation, ammonia synthesis and biomass analysis in the future, contributes to the global energy structure transformation and sustainable development, and has broad industrial and scientific research application potential.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: modifications can still be made to the specific embodiments of the present invention or equivalent replacements can be made to some technical features; without departing from the spirit of the technical solutions of the present invention, they should all be covered within the scope of the technical solutions claimed by the present invention.

Claims

1. A method for distinguishing disaccharide isomers based on gas-liquid interface reaction, comprising the steps of: [M+NH4] + Adduct formation: A disaccharide aqueous solution is used as a sample solution, and a N2-containing gas is used as an atomizing gas. The disaccharide aqueous solution is injected into a capillary and continuously flows in the capillary. Under a gas pressure greater than 50 psi, the disaccharide aqueous solution in the capillary is atomized into micro droplets and ejected from the ejection port of the capillary, and a self-contained 10 8 -10 9 At the gas-liquid interface of a V / m electric field, under the action of the electric field at the gas-liquid interface, the N2 molecules are activated at room temperature and combine with the hydrogen free radicals in the H2O molecules in the disaccharide aqueous solution to synthesize NH3 molecules, and the NH3 molecules combine with the disaccharide molecules in the disaccharide aqueous solution to generate [M+NH4] + adducts; Mass spectrometry detection: The adduct [M+NH4] is detected by mass spectrometry. + Fragmenting to form characteristic fragment ions, and detecting the intensity and abundance distribution of the characteristic fragment ions; Disaccharide isomer identification: Disaccharide isomers are distinguished based on the intensity and abundance distribution of the characteristic fragment ions of different disaccharide isomers.

2. The distinguishing method according to claim 1, characterized in that: The [M+NH4] + The step of forming the adduct comprises: the disaccharide aqueous solution flows at a flow rate of 10-20 μL / min and is atomized into the micro droplets by the N2-containing gas at a pressure of 60-120 psi.

3. The distinguishing method according to claim 1 or 2, characterized in that: The distance between the injection port of the mass spectrometer and the injection port of the capillary is defined as the reaction distance, and the reaction distance is 5-20 mm.

4. The distinguishing method according to claim 3, characterized in that: The method for forming the characteristic fragment ions comprises: using [M+NH4] + The adduct [M+NH4] was detected using a collision energy of 10-30 eV. + Just break it up.

5. The distinguishing method according to claim 4, characterized in that: The disaccharide in the disaccharide aqueous solution includes one or a mixture of maltose, isomaltose, sucrose, palatinose, melibiose and turanose, and the characteristic fragment ions include m / z 342.14, 325.11, 163.06, 145.05, 127.04, 97.03 or 85.

03.

6. A relative quantitative detection method for disaccharide isomers in a binary mixed system based on the differentiation method according to any one of claims 1 to 5, comprising the steps of: preparing a series of binary mixed aqueous solutions with different ratios of disaccharide isomers, wherein the total concentrations of the two disaccharide isomers in the binary mixed aqueous solutions are consistent; Repeat the differentiation method of any one of claims 1 to 5 wherein [M+NH4] + The step of adduct formation is to generate ammonia molecules by micro-droplet gas-liquid interface reaction, and react with disaccharide isomers in the binary mixed aqueous solution to form corresponding [M+NH4] + adducts; The adduct [M+NH4] in the binary mixed aqueous solution is first analyzed by mass spectrometry. + Fragmenting to form characteristic fragment ions; then detecting the intensity of the characteristic fragment ions in the binary mixed aqueous solution, and calculating the relative ratio of the intensity of the characteristic fragment ions therein; Wherein, a calibration curve is established according to the following formula to calculate the relative molar content of disaccharide isomers in the binary mixed aqueous solution; in the formula, RI mix is the relative ratio of the characteristic fragment ion intensities in the binary mixed aqueous solution; RI a and RI b They represent the relative intensities of the corresponding characteristic fragment ions of disaccharide isomers a and b when they exist alone; α a and α b are the molar (or mass) percentages of disaccharide isomers a and b in the binary mixed aqueous solution, and satisfy α a + α b = 1, C a and C b is the configuration ratio of disaccharide isomers a and b at a known concentration ratio, k and t are constants obtained by linear fitting: ， ， ； The calibration curve is used to convert the characteristic fragment ion signals of the unknown sample into the relative contents of the isomers.

7. The relative quantitative detection method according to claim 6, characterized in that: The mass ratio or molar ratio of the disaccharide isomers in the binary mixed aqueous solution is 20:1 - 1:100, and the relative ratios of the characteristic fragment ion intensities of the disaccharide isomer combination include: palatinose and turanose: m / z 145 / 259, Isomaltose and Palatinose: m / z 145 / 325, sucrose and melibiose: m / z 145 / 163.

8. A disaccharide isomer detection and analysis device, characterized in that: include: A microliter liquid supply unit, a N2 gas source, a three-way spray assembly and a mass spectrometer, wherein the microliter liquid supply unit comprises a capillary tube, through which a disaccharide aqueous solution can be continuously provided to the three-way spray assembly; The N2 gas source is used to provide N2 gas with a pressure greater than 50 psi to the three-way spray assembly and atomize the disaccharide aqueous solution into micro droplets; The three-way spray assembly comprises a liquid inlet end, an air inlet end and an outlet end from which N2 gas can escape, wherein the air inlet end is connected to the N2 gas source; one end of the capillary is inserted into the three-way spray assembly from the liquid inlet end and penetrates therein, and extends from the outlet end to form an injection port for spraying the micro-droplets; the micro-droplets are sprayed out and form a self-contained 10 8 -10 9 At the gas-liquid interface of a V / m electric field, under the action of the electric field at the gas-liquid interface, N2 is activated at room temperature and combines with hydrogen free radicals in H2O molecules in the disaccharide aqueous solution to synthesize NH3 molecules, and NH3 molecules combine with disaccharide molecules in the disaccharide aqueous solution to generate [M+NH4] + adducts; The mass spectrometer includes an injection port arranged opposite to the injection port, and is used to monitor in real time the [M+NH4] generated by the micro-droplet gas-liquid interface reaction. + Mass spectrometry detection signal of the adduct.

9. The disaccharide isomer detection and analysis device according to claim 8, characterized in that: It also includes an XYZ three-axis moving platform, which is used to fix the three-way spray assembly and adjust the relative position of the micro-droplet outlet and the sample inlet.

10. Use of the disaccharide isomer differentiation method according to any one of claims 1 to 5, the relative quantitative detection method according to any one of claims 6 to 7, or the disaccharide isomer detection and analysis device according to any one of claims 8 to 9 in food testing, pharmaceutical research and development, or bio-omics research.

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