Online detection system and method for P-B reaction

Abstract

The invention relates to the technical field of analytical chemistry mass spectrum imaging, in particular to a P-B reaction online detection system and method, and the system comprises a sample fixing unit, a multi-dimensional control platform, a fluid transmission unit, an auxiliary catalysis unit and a mass spectrum acquisition unit. According to the present invention, the P-B reaction and the mass spectrometry imaging technology are combined, the P-B reaction is adopted to generate the oxetane structure at the double bond through the ultraviolet light induced [2 + 2] cycloaddition reaction, and the characteristic fragment is generated after the collision induced dissociation so as to achieve the accurate positioning of the double bond position, and solve the problem of the lipid isomer spatial distribution analysis; meanwhile, accurate positioning and analysis of lipid carbon-carbon double bond positions in a complex biological sample are realized by utilizing high sensitivity and spatial resolution capability of a mass spectrum imaging technology; the method can be applied to the fields of biomedical research, metabonomics and drug development, and an innovative analysis method is provided for lipid molecular structure analysis and function research.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry mass spectrometry imaging technology, and in particular to an online detection system and method for PB reactions. Background Technology

[0002] The PB reaction, or Patrono-Büchi reaction, is a classic photochemical reaction commonly used to study organic molecules containing double bonds, especially in lipid chemistry. This reaction induces [2+2] cycloaddition of double bonds under ultraviolet light irradiation, forming a four-membered ring structure. In lipid research, this reaction is used to investigate the position and type of double bonds in lipid molecules, particularly in complex lipid compounds. Meanwhile, mass spectrometry is a powerful analytical technique used to directly obtain molecular distribution information from the sample surface. In lipid research, mass spectrometry can provide high-resolution molecular localization data, helping to elucidate the distribution and metabolic processes of lipid molecules within organisms.

[0003] However, traditional mass spectrometry imaging has the following limitations in lipid molecular analysis: weak double bond localization capability, as traditional mass spectrometry imaging methods cannot directly determine the position of lipid double bonds; limited isomer differentiation, as traditional mass spectrometry imaging struggles to distinguish lipid isomers with differences in acyl chain length or branching; and limited dynamic range, as traditional methods have a narrow dynamic response range for lipids containing double bonds. Therefore, combining the PB reaction with mass spectrometry imaging technology can reveal the position of carbon-carbon double bonds in lipids more precisely at the molecular level; providing more detailed spatial information for lipid structural analysis and functional studies, which has significant application value in the fields of biomedicine and drug development. Summary of the Invention

[0004] Therefore, the present invention provides an online detection system and method for PB reaction to solve the problem of weak double bond localization ability of mass spectrometry imaging in lipid molecular analysis in the prior art.

[0005] To achieve the above objectives, the present invention provides an online detection system for PB reaction, comprising, Sample fixation unit, used to fix biological tissue sections; The fluid transport unit includes an inlet capillary and a nitrogen transport tube. The inlet capillary is connected to a solvent storage tank to transport the reaction solvent in the solvent storage tank to the biological tissue section. The nitrogen transport tube is sleeved on the outside of the inlet capillary and is connected to an external gas system. The nitrogen pressure in the nitrogen transport tube is adjustable to control the outflow rate of the inlet capillary and to assist in atomization of the outflow. The auxiliary catalytic unit is equipped with a preset wavelength ultraviolet light source to irradiate biological tissue slices and trigger the PB reaction at the biological tissue slices through radiation catalysis. The mass spectrometry acquisition unit, which is a mass spectrometer, is used to capture ion fragments generated by the PB reaction at biological tissue sections in real time and provide mass-to-charge ratio data.

[0006] Furthermore, it also includes a multi-dimensional control platform, which is used to fix the sample fixation unit and drive the sample fixation unit to move in three-dimensional space, assisting the mass spectrometry acquisition unit in realizing the scanning and acquisition of biological tissue sections.

[0007] Furthermore, the mass spectrometry acquisition unit and the multidimensional control platform can also be linked through a data interface. The scanning path can be set by controlling the movement of the multidimensional control platform, and the mass spectrometry acquisition frequency of the mass spectrometry acquisition unit can be set accordingly.

[0008] This invention also provides an online detection method for PB reaction, applied to the above-mentioned online detection system for PB reaction, comprising, Step S1: Install the auxiliary catalytic unit above the sample fixation unit, adjust the vertical distance between the ultraviolet light source and the sample plane to ensure uniform light intensity distribution; connect the liquid inlet capillary to the solvent storage tank, connect the nitrogen transmission tube to the gas path system, and calibrate the liquid outflow rate of the liquid inlet capillary; set the scanning path and mass spectrometry acquisition frequency. Step S2: Attach the pretreated biological tissue slices to the sample fixation unit, which is a glass slide. Step S3: Start the fluid transport unit to continuously spray the reaction solvent onto the surface of the biological tissue slice, and at the same time turn on the nitrogen transport tube to atomize the liquid out of the inlet capillary to form a uniform liquid film. Step S4: The ultraviolet light source of the activated auxiliary catalytic unit is used to irradiate the biological tissue slice, triggering the PB reaction, which causes the double bond site to covalently bind to the reagent. Step S5: Control the pre-set scanning path of the multi-dimensional control platform to perform a moving scan, and the mass spectrometry acquisition unit collects the reaction products after desolvation in real time, and records the intensity distribution of characteristic ions through data acquisition software. Step S6: Use mass spectrometry imaging software to convert ion intensity data into a spatial distribution heatmap, mark double bond sites in biological tissue sections; combine statistical tools to analyze the relative abundance of lipid isomers, and output a quantitative report.

[0009] Furthermore, in step S1, the vertical distance between the ultraviolet light source of the auxiliary catalytic unit and the sample plane is 50-100 mm, and the preset wavelength of the ultraviolet light source of the auxiliary catalytic unit is 254 nm.

[0010] Furthermore, in step S1, the inner diameter of the inlet capillary is 50-100 μm, and the outflow rate of the inlet capillary is calibrated to 2-5 μL / min; the reaction solvent is a carbonyl-containing reagent.

[0011] Further, in step S2, the pretreatment of the biological tissue sections includes, Fresh biological tissue was rapidly embedded and frozen in a cryostat, and continuous sections with a thickness of 10-50 μm were prepared at -20°C. The sections were then attached to a glass slide.

[0012] Furthermore, the embedding solution used for rapid embedding freezing is sodium carboxymethyl cellulose solution or OCT embedding agent.

[0013] Furthermore, in step S4, the irradiation intensity of the ultraviolet light source of the auxiliary catalytic unit is set to 10 mW / cm².

[0014] Furthermore, in step S4, the reaction time for triggering the PB reaction does not exceed 30 seconds.

[0015] Compared with existing technologies, the beneficial effects of this invention are that by combining the PB reaction with mass spectrometry imaging technology, the PB reaction generates an oxobutane structure at the double bond through a UV-induced [2+2] cycloaddition reaction. After collision-induced dissociation, characteristic fragments are generated, thereby achieving precise localization of the double bond position and solving the problem of spatial distribution analysis of lipid isomers. At the same time, the high sensitivity and spatial resolution of mass spectrometry imaging technology enable precise localization and analysis of the carbon-carbon double bond position of lipids in complex biological samples. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the PB reaction online detection system in this embodiment; Figure 2 This is a flowchart of the online detection method for the PB reaction in this embodiment. Detailed Implementation

[0017] To make the objectives and advantages of the present invention clearer, the present invention will be further described below with reference to embodiments; it should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0018] Preferred embodiments of the present invention will now be described with reference to the accompanying drawings. Those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0019] It should be noted that in the description of this invention, the terms "upper", "lower", "left", "right", "inner", "outer", etc., which indicate directions or positional relationships, are based on the directions or positional relationships shown in the accompanying drawings. This is only for the convenience of description and is not intended to indicate or imply that the device or element must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, it should not be construed as a limitation of this invention.

[0020] Furthermore, it should be noted that, in the description of this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0021] Please see Figure 1 As shown, this embodiment provides an online detection system for the PB reaction, including a sample immobilization unit, a multidimensional control platform, a fluid transport unit, an auxiliary catalysis unit, and a mass spectrometry acquisition unit. The sample fixation unit uses a glass slide to fix biological tissue sections of 10-50μm to prevent sample degradation; The multi-dimensional control platform integrates a stepper motor and a piezoelectric ceramic actuator to achieve three-dimensional positioning of biological tissue slices. The scanning height of the sample is adjusted by moving along the Z-axis, and the scanning path and direction are controlled by moving along the X and Y axes to ensure full coverage of the reaction area of ​​the biological tissue slices. The movement control accuracy of the multi-dimensional control platform is ±1μm, and the scanning speed is 0.1-2mm / s. The fluid transport unit includes an inlet capillary and a nitrogen transport tube. The inlet capillary has an inner diameter of 50-100 μm and is connected to a solvent storage tank to deliver the reaction solvent to the biological tissue section. The nitrogen transport tube is fitted outside the inlet capillary and is connected to an external gas system. The nitrogen pressure inside the nitrogen transport tube is adjustable from 0.1 to 0.6 MPa to control the outflow rate of the inlet capillary and to assist in atomization of the outflow. The reaction solvent is selected from carbonyl-containing reagents, such as a mixture of acetone and acetonitrile or a mixture of benzophenone and acetonitrile. The auxiliary catalytic unit is equipped with a UV light source of a preset wavelength. In this embodiment, a 254 nm UV light source is selected to catalyze the PB reaction on the biological tissue slices through radiation, ensuring the efficient photochemical coupling of the double bond and the carbonyl-containing reagent. The mass spectrometry acquisition unit uses an Orbitrap mass spectrometer, employing high-resolution electrostatic orbital trap technology to capture ion fragments generated by the PB reaction at biological tissue sections in real time, providing mass-to-charge ratio data.

[0022] Please continue reading. Figure 2 As shown, this embodiment also provides a method for online detection of PB reaction applied to the above-mentioned online detection system for PB reaction, including: Device assembly and calibration; Install the auxiliary catalytic unit above the sample fixation unit, and adjust the vertical distance between the ultraviolet light source and the sample plane to 50-100mm to ensure uniform light intensity distribution; Connect the inlet capillary to the solvent storage tank, connect the nitrogen transfer tube to the gas path system, and calibrate the outflow rate of the inlet capillary to 2-5 μL / min; The multidimensional control platform is linked with the mass spectrometry acquisition unit data interface, the scanning path is set to a "serpentine trajectory", and the mass spectrometry acquisition frequency and resolution are set. In this embodiment, the mass spectrometry acquisition frequency is set to 1 Hz. Biological sample pretreatment; Fresh biological tissues were rapidly embedded and frozen in a cryostat. Sodium carboxymethyl cellulose solution or OCT embedding agent was selected as the embedding solution. Continuous sections with a thickness of 10-50 μm were prepared at -20 degrees Celsius and attached to glass slides. The glass slide with the attached biological tissue sections is fixed on the sample stage of the multidimensional control platform; Online reaction and detection; The fluid transport unit is activated to continuously spray the reaction solvent onto the surface of the biological tissue slices. At the same time, the nitrogen transport tube is turned on to atomize the liquid output from the inlet capillary, forming a uniform liquid film. The biological tissue slices were irradiated with an ultraviolet light source that activated the auxiliary catalytic unit. The irradiation intensity was set to 10 mW / cm² to trigger the PB reaction. The reaction time was controlled to be less than or equal to 30 s, so that the double bond sites were covalently bound to the reagent. The multidimensional control platform moves and scans in a "serpentine trajectory," while the mass spectrometry acquisition unit collects the reaction products after desolvation in real time and records the intensity distribution of characteristic ions through the data acquisition software Xcalibur. Data analysis and imaging; The mass spectrometry acquisition unit's data was converted from .raw format to .imzML format using MSConvter. Then, the ion intensity data was converted into a spatial distribution heatmap using the mass spectrometry imaging software MSIReader. Double bond sites in biological tissue sections were labeled, and the relative abundance of lipid isomers was analyzed using the statistical tool MATLAB to output a quantitative report.

[0023] In this embodiment, the Patrono-Büchi reaction is combined with mass spectrometry imaging technology for efficient, online analysis of carbon-carbon double bond positions in lipid molecules. The Patrono-Büchi reaction specifically labels the carbon-carbon double bonds in lipid molecules, and combined with the high sensitivity and spatial resolution of mass spectrometry, it enables precise localization and analysis of the carbon-carbon double bond positions in complex biological samples. Traditional mass spectrometry imaging cannot directly resolve lipid double bond positions. The Patrono-Büchi reaction, through a UV-induced [2+2] cycloaddition reaction, generates an oxobutane structure at the double bond position. After collision-induced dissociation, characteristic fragments are generated, thus achieving precise localization of the double bond position and solving the problem of resolving the spatial distribution of lipid isomers.

[0024] Meanwhile, traditional mass spectrometry imaging has difficulty distinguishing lipid isomers with differences in double bond position, chain length, or branching. The PB reaction combined with tandem mass spectrometry can generate specific fragment ions, significantly improving the resolution and reliability of structure identification. It also improves detection sensitivity and selectivity. PB derivatization enhances the ionization efficiency of unsaturated lipids through chemical modification, while reducing matrix interference in complex samples, increasing the detection limit of low-abundance lipids by several times, and effectively alleviating the ion inhibition effect.

[0025] The PB reaction online detection system in this embodiment enhances the signal intensity of unsaturated lipids through targeted derivatization while reducing signal suppression by high-abundance phospholipids, thus broadening the dynamic detection range of the lipidome and improving quantitative accuracy. Furthermore, the PB reaction can be coupled with mass spectrometry imaging online or offline, eliminating the need for complex sample pretreatment and reducing the risk of losing in-situ lipid information.

[0026] In summary, the PB reaction online detection system and method provided in this embodiment can be applied to fields such as biomedical research, metabolomics, and drug development, providing an innovative analytical method for lipid molecular structure analysis and functional research.

[0027] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the scope of protection of the present invention.

[0028] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An online detection system for PB reaction, characterized in that, include, Sample fixation unit, used to fix biological tissue sections; A fluid transport unit includes an inlet capillary and a nitrogen transport tube. The inlet capillary is connected to a solvent storage tank to transport the reaction solvent in the solvent storage tank to the biological tissue section. The nitrogen transport tube is sleeved on the outside of the inlet capillary and is connected to an external gas system. The nitrogen pressure in the nitrogen transport tube is adjustable to control the outflow rate of the inlet capillary and to assist in atomization of the outflow. An auxiliary catalytic unit is equipped with an ultraviolet light source of a preset wavelength for irradiating the biological tissue slices, thereby triggering the PB reaction at the biological tissue slices through radiation catalysis. The mass spectrometry acquisition unit, which is a mass spectrometer, is used to capture and collect ion fragments generated by the PB reaction at the biological tissue slices in real time, providing mass-to-charge ratio data.

2. The PB reaction online detection system according to claim 1, characterized in that, It also includes a multi-dimensional control platform, which is used to fix the sample fixation unit and drive the sample fixation unit to move in three-dimensional space, assisting the mass spectrometry acquisition unit in realizing the scanning acquisition of the biological tissue slices.

3. The PB reaction online detection system according to claim 2, characterized in that, The mass spectrometry acquisition unit and the multidimensional control platform can also be linked through a data interface. The scanning path can be set by controlling the movement of the multidimensional control platform, and the mass spectrometry acquisition frequency of the mass spectrometry acquisition unit can be set accordingly.

4. An online detection method for PB reaction, applied to the online detection system for PB reaction according to any one of claims 1-3, characterized in that, include, Step S1: Install the auxiliary catalytic unit above the sample fixation unit, adjust the vertical distance between the ultraviolet light source and the sample plane to ensure uniform light intensity distribution; connect the liquid inlet capillary to the solvent storage tank, connect the nitrogen transmission tube to the gas path system, and calibrate the liquid outflow rate of the liquid inlet capillary; set the scanning path and mass spectrometry acquisition frequency. Step S2: Attach the pretreated biological tissue slices to the sample fixation unit, which is a glass slide. Step S3: Start the fluid transport unit to continuously spray the reaction solvent onto the surface of the biological tissue slice, and at the same time turn on the nitrogen transport tube to atomize the liquid out of the inlet capillary to form a uniform liquid film. Step S4: The ultraviolet light source of the activated auxiliary catalytic unit is used to irradiate the biological tissue slice, triggering the PB reaction, which causes the double bond site to covalently bind to the reagent. Step S5: Control the pre-set scanning path of the multi-dimensional control platform to perform a moving scan, and the mass spectrometry acquisition unit collects the reaction products after desolvation in real time, and records the intensity distribution of characteristic ions through data acquisition software. Step S6: Use mass spectrometry imaging software to convert ion intensity data into a spatial distribution heatmap and mark double bond sites in biological tissue sections; The relative abundance of lipid isoforms is analyzed using statistical tools, and a quantitative report is generated.

5. The online detection method for PB reaction according to claim 4, characterized in that, In step S1, the vertical distance between the ultraviolet light source of the auxiliary catalytic unit and the sample plane is 50-100 mm, and the preset wavelength of the ultraviolet light source of the auxiliary catalytic unit is 254 nm.

6. The online detection method for PB reaction according to claim 4, characterized in that, In step S1, the inner diameter of the inlet capillary is 50-100 μm, and the outflow rate of the inlet capillary is calibrated to 2-5 μL / min; the reaction solvent is a carbonyl-containing reagent.

7. The online detection method for PB reaction according to claim 4, characterized in that, In step S2, the pretreatment of the biological tissue sections includes, Fresh biological tissue was rapidly embedded and frozen in a cryostat, and continuous sections with a thickness of 10-50 μm were prepared at -20°C. The sections were then attached to a glass slide.

8. The online detection method for PB reaction according to claim 7, characterized in that, The rapid embedding freezing process uses sodium carboxymethyl cellulose solution or OCT embedding agent as the embedding solution.

9. The online detection method for PB reaction according to claim 4, characterized in that, In step S4, the irradiation intensity of the ultraviolet light source of the auxiliary catalytic unit is set to 10 mW / cm².

10. The online detection method for PB reaction according to claim 4, characterized in that, In step S4, the reaction time that triggers the PB reaction does not exceed 30 seconds.

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