CN106442701B - Mass spectrum imaging analysis method based on single-pixel solid-phase extraction technology and application thereof - Google Patents
Mass spectrum imaging analysis method based on single-pixel solid-phase extraction technology and application thereof Download PDFInfo
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Abstract
The invention discloses a mass spectrum imaging analysis method based on single-pixel solid phase extraction technology and application thereof, the method comprises (1) spraying solvent for analyzing target molecules in a spray mode to a conical through hole after the purification treatment, wherein the spray passes through the conical through hole to obtain secondary spray containing the target molecules after analyzing the target molecules, the secondary spray enters a sample inlet of a mass spectrometer to complete sample injection of one conical through hole, and sample injection of the next conical through hole is completed by subspecies pushing, so that the mass spectrometer completes mass spectrum analysis of each conical through hole; (2) Sample collection and data analysis are performed by a mass spectrometer, so that a mass spectrum image of the component ions is obtained. The invention creatively adopts a single-pixel solid-phase extraction technology, and effectively avoids the phenomenon of inhibiting target molecular ions by complex matrixes by preprocessing the pixel points, thereby practically solving the influence of complex matrix interference on the accuracy of mass spectrum images and improving the imaging sensitivity of low-abundance components.
Description
Technical Field
The invention belongs to the technical field of mass spectrometry, and particularly relates to a mass spectrometry imaging analysis method based on a single-pixel solid-phase extraction technology and application thereof.
Background
Traditional molecular imaging technology, such as spectroscopy technology using radioisotope or fluorescent label, is time-consuming and labor-consuming, and the label is likely to dissociate in the imaging process, while the label-free imaging spectroscopy method has the defects of weak luminous signal, strong background interference and the like. Mass spectrometry imaging (mass spectrometry imaging, MSI) is a novel imaging technique by which spatial distribution information of a variety of proteins or small molecule metabolites can be obtained directly from the surface of biological tissue sections. The principle of this in situ analysis technique is to ionize molecules on the surface of a tissue slice using a laser or ion beam, then determine the mass-to-charge ratio (ratio of mass to charge, m/z) of these ionized molecules by mass spectrometry, and reconstruct a map of the distribution of the analyte in the tissue by software. Mass spectrometry imaging techniques have the following advantages: (1) As a type of mark-free molecular imaging technology, the mass spectrum imaging technology has relatively high analysis speed, and is time-saving and labor-saving; (2) Simultaneous imaging analysis of a plurality of ionizable components in a sample can be achieved; (3) The spatial distribution information and the molecular structure information of the molecules can be obtained simultaneously, and the recognition of the molecules can be realized. In recent years, mass spectrometry imaging technology has been rapidly developed, and has received a great deal of attention in the fields of biology, medicine, pharmacy, food science and the like.
Currently, ionization techniques for mass spectrometry imaging are well-established, including secondary ion mass spectrometry, matrix-assisted laser desorption mass spectrometry, and the like. Such techniques all need to be performed under vacuum conditions, are complex to operate and have certain limitations on the type, size, etc. of the sample. In addition, novel ionization techniques such as surface desorption atmospheric chemical ionization, low temperature plasma probes, aerodynamic assisted ionization, and other similar ionization methods based on direct liquid phase extraction of sample surfaces are also applied to mass spectrometry imaging techniques. However, the type of extraction liquid, the extraction time, the distance between the probe and the sample surface, etc. have a great influence on the extraction efficiency. In addition, direct contact extraction of the surface through liquid often only can obtain high-abundance or easily-extracted components on the surface of the sample, and the extraction effect of the low-abundance components is difficult to ensure. In summary, with the complexity of the research system, the existing technology still cannot meet the requirement of actual analysis, and development of a mass spectrum imaging technology suitable for low-abundance components on the surface of a sample is beneficial to solving the problems of positioning analysis of the low-abundance components on the surface of a complex biological slice.
Disclosure of Invention
The invention takes the improvement of the sensitivity of mass spectrum imaging analysis as a starting point, establishes a mass spectrum imaging method suitable for plant tissue sections, carries out pretreatment in a single pixel by introducing a solid phase extraction array on the surface of a sample section to be detected, regulates and controls the local chemical environment of target molecules, reduces the influence of surface matrixes or non-target molecules on the ionization process, retains the spatial distribution information of low-abundance components, and realizes the selective microscopic imaging of chemical components of the section; the imaging sensitivity of the mass spectrometry imaging technology on low-abundance components on the surface of the complex matrix is improved.
Aiming at the defects of the prior art, the invention provides a mass spectrum imaging method based on a single-pixel solid-phase extraction technology, so that the problem of low imaging sensitivity of low-abundance components on the surface of a complex sample is effectively solved.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides a single-pixel solid-phase extraction array plate for sample pretreatment in mass spectrometry imaging, which comprises a matrix film, wherein a plurality of conical through holes are arranged on the matrix film according to set intervals, adsorption materials which are adsorbed with target molecules to be subjected to mass spectrometry imaging in plant tissues are loaded in the through holes, a plurality of micro through holes are formed in the adsorption materials, and solvent spray for desorbing the target molecules passes through the micro through holes of the adsorption materials in the conical through holes to form secondary spray which enters a sample inlet of a mass spectrometer.
In order to improve the sensitivity of mass spectrum imaging analysis, the invention firstly designs a single-pixel solid-phase extraction array plate, and introduces a solid-phase extraction array on the surface of a plant tissue slice to be detected, wherein the solid-phase extraction array refers to that conical through holes of the single-pixel solid-phase extraction array plate are arranged according to a certain mode and can be called as an array, and the single pixel in the single-pixel solid-phase extraction array plate can be understood as each conical through hole, chemical components in the plant tissue slice to be detected are preprocessed in the single pixel, the local chemical environment where target molecules are located is regulated and controlled, the influence of surface matrixes or non-target molecules on the subsequent ionization process is reduced, the spatial distribution information of low abundance is reserved through an adsorption material, the selective microscopic imaging of chemical components of the slice is realized, and the imaging sensitivity of a mass spectrum imaging technology on the low-abundance components on the surface of the complex matrixes is improved. Preferably, the substrate film is a silicone adhesive-Teflon film, also known as polytetrafluoroethylene, available from CS Hyde Company, USA, model 15-2S-2-36. The thickness of the matrix film is 0.1 to 0.15mm, preferably 0.12mm. The shape and size of the matrix film are adjusted according to the actual situation, and may be, but not limited to, rectangular, circular, etc.
Preferably, the selection of the adsorption material can be determined according to the property of the target molecule to be imaged in the plant tissue slice, and can be octadecyl bonded silica gel (C18) and the like. The adsorbent is in the form of particles, preferably 2 to 8 μm in size, more preferably 5 μm in size. The mass of the adsorption material loaded in each conical through hole is 0.8-1 mug. The adsorption material cannot fall from the conical through hole, because the adsorption material can be firmly fixed in the conical through hole by adopting a compression means and a Teflon film with silicone adhesive on the surface in the preparation process, and the adsorption material is added with the adsorption material and then is subjected to micro through hole punching, the effect of the micro through hole is that the conical through hole added with the adsorption material is communicated, and both sides of the through hole can be sprayed. The diameter and the number of the micro through holes can be determined according to practical situations, and a person skilled in the art can routinely determine the number of the micro through holes as much as possible for the purpose of enabling the spray to pass, so that the positioning analysis of low-abundance components is facilitated.
Preferably, the set interval is 80-120 μm; further preferably 100 μm, means that the center of the circle of one of the tapered through holes is 80 to 120 μm to the center of the circle of the adjacent one of the tapered through holes.
Preferably, the diameter (L of the tapered through hole at one end thereof 1 ) 70 to 90 μm (preferably 80 μm), and the diameter (L) of the opening at the other end 2 ) 20 to 40 μm (preferably 30 μm). The reason for designing the tapered through hole is that: for carrying the adsorbent material. The adsorption material is carried, and then the micro through hole is formed in the adsorption material, so that the solvent spray can also form spray (secondary spray) when passing through the side with the smaller diameter from the side with the larger diameter of the conical through hole, and then the solvent spray enters the mass spectrometer. Said larger diameter (L 1 ) Opening relative to L 2 Is larger, the smaller diameter (L 2 ) Opening relative to L 1 Smaller in terms of size.
The second object of the present invention is to provide a method for preparing the single-pixel solid-phase extraction array plate, comprising the steps of: firstly, punching holes on the surface of a flaky substrate by adopting laser according to a set interval; then, flattening and fixing the matrix film, and punching tapered through holes on the surface of the matrix film according to the set interval to form a tapered array with fixed depth; and butting the flaky substrate with the substrate film, loading an adsorption material on the surface of the substrate film, compacting the adsorption material, peeling the flaky substrate, and finally punching a micro through hole on the adsorption material in the conical through hole to obtain the single-pixel solid-phase extraction array plate with the adsorption material in the conical through hole.
The sheet-like substrate may be a metal sheet or other hard sheet, such as a copper sheet, an iron sheet, or the like.
The method for loading the adsorption material refers to the following steps: and paving an adsorption material on the surface of the matrix film.
The tapered through holes may be perforated using a needle punch with a tapered needle, which is routinely done by those skilled in the art.
The third object of the invention is to provide a pretreatment method of mass spectrum imaging based on single-pixel solid phase extraction technology, comprising the following steps:
(1) Chemical component transfer and blotting in plant tissue sections: butting and compacting the frozen plant tissue slice with the single-pixel solid-phase extraction array plate, and then stripping the plant tissue slice, wherein chemical components in the plant tissue slice are transferred into the conical through holes on the single-pixel solid-phase extraction array plate, so that chemical component transfer and imprinting in the plant tissue slice are realized;
(2) Purifying in the conical through hole: and eluting the tapered through holes by adopting an organic solvent, eluting part or all of non-target molecules out of the tapered through holes, and retaining the target molecules through an adsorption material.
In the step (1), when chemical component transfer is performed, fresh plant tissue slices are finally adopted and frozen into frozen slices, so that the transfer of the maximum chemical component can be ensured as much as possible.
The thickness of the plant tissue slice is selected to be 4-6 mm, preferably 5mm, so that the treatment and the chemical information transfer are convenient; the slices are too thin and are easy to tear in the transfer process; too thick a slice is detrimental to cleaning to remove some substances that interfere with the ion signal and has poor conductivity.
One specific embodiment of the invention is as follows: the method comprises the steps of (1) quickly butting fresh plant tissue slices with the surface of the single-pixel solid-phase extraction array plate after freezing, then compacting for about several seconds (preferably 5 seconds), and then stripping the plant tissue slices to realize the transfer of chemical components between the plant tissue slices and the single-pixel solid-phase extraction array plate;
in the step (2), during elution, due to complex chemical components in plant tissue slice tissues, organic solvents with different polarities are used for elution respectively, for example, mixed solutions with different volume ratios of methanol or methanol and water or mixed solutions with different volume ratios of acetonitrile or acetonitrile and water are used for elution, and non-target molecules are removed from the conical through holes as much as possible after multiple times of elution. When the chemical components in the plant tissue section are single, only one organic solvent may be used for elution. In any case, it is necessary to use the target molecule according to the specific plant tissue type and the target molecule type.
The adsorbent material may retain low abundance target molecules, but may also retain high abundance target molecules.
The purification in the conical through hole can be realized through a two-axis stepping electric platform, and the organic solvent for purification is adjusted according to the complexity of the sample matrix. The two-axis stepping motor platform is a technology well known to those skilled in the art, is commercially available in the general two-axis motor market, and the structure is not particularly limited, and the method of use is well known to those skilled in the art. The single-pixel solid-phase extraction array plate is loaded on the biaxial stepping motor-driven platform, so that the movement of the single-pixel solid-phase extraction array plate is realized.
A fourth object of the present invention is to provide a mass spectrometry imaging method based on single-pixel solid phase extraction technology, comprising the steps of:
(1) Molecular ion mass spectrum signal acquisition: after the solvent for analyzing the target molecules is sprayed to one conical through hole after the purification treatment in a spray mode, the spray passes through the conical through hole and is analyzed to obtain secondary spray containing the target molecules, the secondary spray enters a sample inlet of a mass spectrometer to complete sample injection of one conical through hole, and sample injection of the next conical through hole is completed by secondary type pushing, so that the mass spectrometer completes mass spectrometry analysis of each conical through hole;
(2) Sample collection and data analysis are performed by a mass spectrometer, so that a mass spectrum image of the component ions is obtained.
In the step (2), analysis software is adopted to analyze imaging data, specifically: and after the coordinates of the conical through holes are obtained by calculating the acquisition time of the target molecules in each conical through hole, the components and the signal intensity in each conical through hole are matched according to the coordinates, so that a mass spectrum image of the component ions is obtained.
Preferably, the molecular ion mass spectrum signal is obtained by adopting a chromatographic mode, the chromatographic time corresponds to the coordinates of each conical through hole, and 10 times of signals are collected in each conical through hole and are subjected to superposition treatment. The invention can adopt a Fourier transform ion cyclotron resonance mass spectrometer for signal acquisition.
A fifth object of the present invention is to provide a mass spectrometry imaging system based on a single-pixel solid-phase extraction technology, which comprises a slice imprinting device for realizing chemical component transfer and imprinting of plant tissue slices, a single-pixel purifying device for purifying and regulating chemical components of plant tissue slices, a spraying device for acquiring target chemical components of each pixel in a mass spectrometry image acquisition process, a single-pixel solid-phase extraction array plate and a mass spectrometer;
the slice imprinting device comprises a pressing piece for realizing chemical component transfer and imprinting of plant tissue slices and a fixing bracket matched with the pressing piece;
the single-pixel purifying device at least comprises a solvent storage tank and a sample injection needle for purifying plant tissue slices;
in the imaging experiment process, a single-pixel solid-phase extraction array plate is arranged between a spraying device and a sample inlet of a mass spectrometer.
Preferably, the shape of the pressing member is not particularly limited so as to be capable of pressing a plant tissue slice, and may be a column having a length of 1cm and diameters of 1,2,5 and 10cm, respectively. The fixing bracket is used in cooperation with the pressing member, an object to be pressed is fixed on the fixing bracket, and the shape of the fixing bracket is not particularly limited and can be a cylindrical base or a cuboid base. During compaction, a tablet press with adjustable pressure can be used for acting on the compaction piece, and the compaction piece can be manually forced. Wherein the pressure-adjustable tablet press is a device conventionally available to a person skilled in the art.
Preferably, the single-pixel purification device further comprises a peristaltic pump, wherein the peristaltic pump is a liquid conveying device capable of controlling flow rate, is a device conventionally known to a person skilled in the art, and can be a laboratory syringe pump, and the model is TYD01-01. The peristaltic pump is characterized in that the input end of the peristaltic pump is connected with the solvent storage tank, and the output end of the peristaltic pump is connected with the sample injection needle.
Further preferably, the single-pixel purification device further comprises a two-axis stepping electric platform, wherein the single-pixel solid-phase extraction array plates are connected, and the two-axis stepping electric platform controls the point-to-point movement of the single-pixel solid-phase extraction array plates.
The single-pixel purifying device realizes the elution and purification process of each conical through hole in the slice imprinting.
The spraying device is a conventional existing device, and its function is to generate a spray for desorbing the target molecule solvent. According to one embodiment of the invention, the spraying device consists of a peristaltic pump, a syringe and a needle, wherein the peristaltic pump is connected with the syringe, and the syringe is connected with the needle. In practice, the needle may be formed by breaking a hollow column of fused silica capillary by heating. The syringe had a volume of 10 μl. The peristaltic pump is an external component on the mass spectrometer, is a laboratory syringe pump, and is a model TYD01-01, and is an applicable syringe: 10 mu L-60 mL. Is a conventional product, and is available to those skilled in the art without any particular limitation.
Preferably, the mass spectrometer can be adjusted according to experimental conditions, a device conventionally available to a person skilled in the art. For example, a fourier transform ion cyclotron resonance mass spectrometer may be used for signal acquisition in the present invention.
Preferably, the imaging system further comprises a camera, which is used for recording a single-pixel extraction process (solvent-resolved target molecules) and/or a mass spectrum signal acquisition process in the imaging test. In the imaging test process, one or more cameras are arranged on one side or two sides of the single-pixel solid-phase extraction array plate.
A sixth object of the invention is to provide a component analysis application of the mass spectrometry imaging method or mass spectrometry imaging system in strawberry sections.
One of the above technical solutions has the following beneficial effects:
(1) The invention creatively adopts a single-pixel solid-phase extraction technology, and effectively avoids the phenomenon of inhibiting the target molecular ions by complex matrixes by carrying out pretreatment in pixel points, thereby practically solving the influence of complex matrix interference on the accuracy of mass spectrum images and improving the imaging sensitivity of low-abundance components; meanwhile, as nano-liter spray is effectively controlled in a single pixel, cross contamination does not exist among all pixels (conical through holes), and the accuracy of mass spectrum images is ensured.
(2) The component structure is simple, the manufacturing cost is lower than that of other related technical methods, the manufacturing method is simple and convenient, and the practicability is very high; in addition, the adsorption material in a single pixel (conical through hole) can be adjusted according to the components of the object to be detected, so that the method has good ductility and popularization and application values.
Drawings
FIG. 1 is a schematic diagram of the structure of a tapered through-hole (loaded with adsorbent material) in a single-pixel solid-phase extraction array plate.
FIG. 2 is a schematic diagram of a single-pixel solid-phase extraction array plate structure.
Fig. 3 is a schematic view of a slice blotting apparatus.
Fig. 4 is a schematic diagram of a single pixel purification device.
Fig. 5 is a schematic view of a spray device.
Fig. 6 is a schematic illustration of a single pixel solid phase extraction plate placed between a nanoliter spray device and a sample inlet of a mass spectrometer.
Fig. 7 is an ion image of rhodamine B of the letter M.
Fig. 8 is a mass spectrum profile of sucrose molecules in a strawberry slice.
FIG. 9 is a mass spectrum distribution diagram of morin molecules in strawberry slices.
FIG. 10a is a schematic flow chart of mass spectrometry imaging; FIG. 10b is a schematic diagram of a solid phase extraction array scan mode.
The device comprises a first solvent storage tank, a first peristaltic pump, a sample injection needle, a second solvent storage tank, a second peristaltic pump, a syringe, a capillary tube needle, a mass spectrum sample injection port and a mass spectrum sample injection port, wherein the first peristaltic pump, the conical through hole, the adsorption material, the capillary tube needle, the first solvent storage tank, the second solvent storage tank, the syringe, the capillary tube needle and the mass spectrum sample injection port are arranged in sequence in the sample injection port, and the mass spectrum sample injection port is arranged in the sample injection port.
Detailed Description
Materials: silicone adhesive-Teflon film was purchased from CS Hyde Company, U.S. A., model 15-2S-2-36, and 0.12mm thick.
Example 1
As shown in fig. 2, a single-pixel solid-phase extraction array plate for sample pretreatment in mass spectrometry imaging comprises a matrix film 3, wherein the matrix film 3 is made of silicone adhesive-teflon film, and the thickness of the matrix film is 0.12mm.
The substrate film 3 is provided with a plurality of tapered through holes 1 arranged at intervals of 100 μm, which means that the center of the circle of one tapered through hole is 100 μm with the center of the circle of the adjacent tapered through hole. As shown in fig. 1, the diameter (L of one end opening of the tapered through hole 1 1 ) 80 μm, diameter of the opening at the other end (L 2 ) 30 μm.
And an adsorption material 2 adsorbed with target molecules to be subjected to mass spectrum imaging in plant tissues is loaded in the conical through hole 1. The selection of the adsorption material 2 may be determined according to the nature of the target molecule to be imaged in the plant tissue section, and octadecyl bonded silica gel (C18) is selected in this embodiment. The shape of the adsorption material is nano particles, and the particle size is 5 mu m. The mass of the supported adsorbent material was 20. Mu.g.
The adsorption material 2 of the conical through hole 1 is provided with a micro through hole (not shown in the figure), and the micro through hole is used for enabling the conical through hole added with the adsorption material to be communicated.
And forming secondary spray after the solvent spray for desorbing the target molecules passes through C18 in the conical through hole, and entering a sample inlet of the mass spectrometer.
Preparation of a single-pixel solid-phase extraction array plate: (1) Firstly, punching holes on the surface of a copper sheet by adopting laser, wherein the hole spacing is set to be 100 mu m. (2) And then, leveling and fixing the silicone adhesive-Teflon film, and punching holes on the surface of the silicone adhesive-Teflon film at equal intervals by adopting a needle punch to form a conical through hole array with fixed depth. (3) And (3) butting the copper sheet with the perforated silicone adhesive-Teflon film, loading an adsorption material C18 on the surface of the composite material, compacting by adopting a copper column, peeling the copper sheet from the copper column, and finally punching a micro through hole on the adsorption material in the conical through hole to form the pixel array plate with the solid phase extraction function, wherein the pixel array plate carries about 20 mug of adsorption material.
Example 2
A pretreatment method of mass spectrum imaging based on single-pixel solid phase extraction technology comprises the following steps:
(1) Chemical component transfer and blotting in plant tissue sections: fresh plant tissue slices with the thickness of 5mm are selected, after freezing, the fresh plant tissue slices are quickly butted and pressed with the surface of the single-pixel solid-phase extraction array plate (the surface with one end of the conical through hole being larger in opening) in the embodiment 1 for about 5 seconds, then the tissue slices are peeled, and chemical components in the plant tissue slices are transferred into the conical through holes on the single-pixel solid-phase extraction array plate, so that transfer and imprinting of the chemical components in the plant tissue slices are realized;
(2) Purifying in the conical through hole: eluting the tapered through holes by adopting an organic solvent (such as a mixed solution of methanol and water), eluting part or all of non-target molecules out of the tapered through holes, and retaining the target molecules by an adsorption material.
The separation of components with different polarities is realized by using a 50 mu L sample injection needle and a peristaltic pump and adopting the speed of 5nL/min, and the target components with low abundance are reserved by an adsorption material (C18); the organic solvent used for purification is adjusted according to the complexity of the sample matrix.
Example 3
A mass spectrometry imaging system based on single-pixel solid-phase extraction technology, wherein the device comprises a nano-liter spraying device, a slice imprinting device, a single-pixel solid-phase extraction array plate in the embodiment 1, a single-pixel purifying device, a camera and a mass spectrometer; the slice imprinting device is used for realizing chemical component transfer and imprinting of plant slices; the single-pixel solid-phase extraction array plate and the single-pixel purification device are used for carrying out in-situ solid-phase extraction on plant tissue slice imprinting and are used for regulating and controlling chemical components in single pixels; the spraying device is used for acquiring target chemical components of each pixel in the mass spectrum image acquisition process; the cameras are arranged on two sides of the solid-phase extraction plate and used for recording a single-pixel extraction process and a mass spectrum signal acquisition process; the mass spectrometer is used for obtaining mass spectrum signals of chemical components of each pixel in a mass spectrum image acquisition process; in the imaging experiment, a single-pixel solid-phase extraction plate is placed between a nanoliter spray device and a sample inlet of a mass spectrometer, and the single-pixel solid-phase extraction plate is not shown in the figure, but is represented by a conical through hole.
As shown in fig. 5, the nano-liter spray device includes: the spraying device comprises a second solvent storage tank 9 for containing a solvent for resolving target molecules, a second peristaltic pump 10, an injector 11 and a capillary needle 12, wherein the second solvent storage tank 9 is connected with the input end of the second peristaltic pump 7, the output end of the second peristaltic pump 10 is connected with the injector 11, and the injector 11 is connected with the capillary needle 12. In practice, the needle may be formed by breaking a hollow column of fused silica capillary by heating. The syringe 11 has a volume of 10. Mu.L.
As shown in fig. 3, the slice imprinting apparatus includes: a pressing piece 4 for realizing chemical component transfer and imprinting of plant tissue slices and a fixing bracket 5 matched with the pressing piece; the shape of the pressing member 4 is not particularly limited, and may be a cylindrical body having a length of 1cm and diameters of 1,2,5 and 10cm, respectively, for the purpose of pressing a plant tissue slice. The fixing bracket 5 is used in cooperation with the pressing member 4, and an object to be pressed is fixed on the fixing bracket, and the shape of the fixing bracket 5 is not particularly limited and may be a cylindrical base or a rectangular base. During compaction, a tablet press with adjustable pressure can be used for acting on the compaction piece, and the compaction piece can be manually forced.
As shown in fig. 4, the single-pixel purification device comprises a first solvent storage tank 6 for containing a purification solvent, a sample injection needle 8, a first peristaltic pump 7 and a two-axis stepping motor platform. The first solvent storage tank 6 is connected with the input end of the first peristaltic pump 7, and the output end of the first peristaltic pump 7 is connected with the sample injection needle 8. The two-axis stepping electric platform controls the single-pixel solid-phase extraction array plate to realize the movement of the single-pixel solid-phase extraction array plate, thereby realizing point-by-point purification. The two-axis stepper motor platform is a well known technology for those skilled in the art, and can be conventionally operated and used by those skilled in the art, and will not be described in detail herein.
Example 4
A mass spectrometry imaging method based on single-pixel solid phase extraction technology, which can be implemented by the device in embodiment 3, comprising the following steps:
(1) Molecular ion mass spectrum signal acquisition: after solvent for analyzing target molecules is sprayed to one conical through hole after purification treatment in the embodiment 2 in a spray mode, analyzing the reserved target molecules in the conical through hole, wherein the spray passes through the conical through hole to obtain secondary spray containing the target molecules after analyzing the target molecules, the secondary spray enters a sample inlet of a mass spectrometer, voltage is applied to complete sample injection of one conical through hole, and the like, so that the mass spectrometer completes mass spectrometry analysis of each conical through hole;
(2) Imaging data analysis: and after the coordinates of the conical through holes are obtained by calculating the acquisition time of the target molecules in each conical through hole, the components and the signal intensity in each conical through hole are matched according to the coordinates, so that a mass spectrum image of the component ions is obtained.
Example 5
Using the apparatus of embodiment 3, a mass spectrometry imaging method based on single-pixel solid phase extraction technology is provided, and the implementation of the method can adopt the apparatus of embodiment 3, including the following specific steps, and the basic flow diagram is shown in fig. 10 a:
(1) Transfer of chemical components between plant tissue sections-single pixel solid phase extraction array plates: the method comprises the steps of adopting a slice imprinting device to realize the butt joint and compaction (about 5 seconds) of fresh plant tissue slices and the surface of the array plate after freezing, and then stripping the tissue slices to realize the transfer of chemical components between the plant slices and the array plate; the compression is to match the compression piece with the fixed bracket, place the plant tissue slice and the array plate after butt joint on the fixed bracket, and apply the tablet press on the compression piece, the compression piece is pressed down to compress the plant tissue slice and the array plate;
(3) Single-pixel intra-purification: the method is realized by adopting a single-pixel purification device, solvents with different polarities are adopted to elute each pixel unit, such as mixed liquid of water and methanol, the separation of components with different polarities is realized by adopting a sample injection needle with the concentration of 50 mu L and a peristaltic pump with the speed of 5nL/min, and the target components with low abundance are reserved by adsorbing nano particles;
(4) Molecular ion mass spectrum signal acquisition: and placing the template which is processed in a single pixel and contains the component imprinting in the slice between a nano liter spraying system and a mass spectrum sample inlet, wherein the distance between the sample inlet and the template is set to be 0.5mm, and the distance between the nano liter spraying array and the template is set to be 0.3mm. The reserved slice chemical components in the desorption template are generated by applying 4.5kv voltage to a sample inlet and nano-liter spraying, and the reserved slice chemical components are moved point by a biaxial stepping motor to obtain mass spectrum signals in each pixel, wherein a schematic diagram of a scanning mode of the solid phase extraction array is shown in fig. 10 b;
(5) Imaging data analysis: and extracting chemical components in the single pixel, calculating the acquisition time to obtain coordinates of the pixels, and matching the components and the signal intensity in each pixel according to the coordinates to obtain a mass spectrum image of the component ions. The molecular ion mass spectrum signal acquisition adopts a chromatographic mode, the chromatographic time corresponds to the coordinates of a single pixel, and 10 times of signals are acquired in each pixel and are subjected to superposition processing.
Experimental example 1 Mass Spectrometry imaging of rhodamine B
(1) Using the single-pixel solid-phase extraction array plate of example 1, red letter "M" was written on the surface of a silicone gel-teflon film (i.e., single-pixel solid-phase extraction array plate) with rhodamine B dye;
(2) Spraying methanol-water (volume ratio of 1:1) serving as a solvent to the conical through holes respectively, placing a single-pixel solid-phase extraction array plate between a sample inlet of a mass spectrometer and a nano-liter spraying device in the embodiment 3, wherein the distance between a spray needle and the single-pixel solid-phase extraction array plate is 0.5mm, the distance between the single-pixel solid-phase extraction array plate and the sample inlet of the mass spectrometer is set to be 0.3mm, and carrying out sample injection analysis;
(3) The method of the invention is applicable to mass spectrometry imaging of target molecules, and is shown in FIG. 7, wherein rhodamine B in the pigment is used as the target molecules, and an ion mass spectrometry image of the rhodamine B (the mass-to-charge ratio of the rhodamine B is 443.23) is extracted as a letter "M".
Experimental example 2 was applied to mass spectrometry image recognition of sucrose molecules and morin molecules in strawberry slices.
(1) Slicing fresh strawberry, rapidly freezing in an ultralow temperature (-80 ℃) refrigerator, placing on the prepared single pixel solid phase extraction array plate in the example 1, pressing for 5 seconds, removing the slice, and purifying;
(2) After sequentially purifying single pixel points by using acetonitrile solution, placing a template in a sample inlet and spraying and desorbing, using water as an analysis liquid, applying 4.5kv voltage, analyzing to obtain a multi-time mass spectrum signal, and then performing superposition treatment, matching pixel coordinates and obtaining mass spectrum signal time to obtain a sucrose molecular ion mass spectrum shown in fig. 8, wherein a schematic diagram of a solid-phase extraction array scanning mode is shown in fig. 10 b.
(3) After the sucrose molecular ion mass spectrum is obtained, the strawberry slice can be used continuously to obtain the morin ion mass spectrum. The specific method comprises the steps of sequentially purifying single pixel points by using water and acetonitrile solution (the volume ratio is 3:1), placing a template in a sample inlet and spraying for desorption, applying 4.5kv voltage to the analysis solution which is ethanol, analyzing to obtain a multi-time mass spectrum signal, performing superposition treatment, matching pixel coordinates and obtaining mass spectrum signal time to obtain a mulberry pigment molecular ion mass spectrum shown in figure 9, wherein a schematic diagram of a solid phase extraction array scanning mode is shown in figure 10 b.
The spray desorption process is realized by adopting a two-axis stepping electric platform through controlling a spray needle, purifying and point-by-point sampling process by a manual three-dimensional moving table, and the two-axis stepping electric platform is controlled by a stepping motor, so that the spray desorption process is a technical means conventionally known to a person skilled in the art.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (14)
1. A single pixel solid phase extraction array plate for sample pretreatment in mass spectrum imaging is characterized in that: the single-pixel solid-phase extraction array plate comprises a matrix film, wherein a plurality of conical through holes are formed in the matrix film and are arranged according to a set interval, an adsorption material which is adsorbed with target molecules to be subjected to mass spectrum imaging in plant tissues is loaded in the through holes, a plurality of micro through holes are formed in the adsorption material, and solvent spray for desorbing the target molecules passes through the micro through holes of the adsorption material in the conical through holes to form secondary spray which enters a sample inlet of a mass spectrometer;
the substrate film is made of silicone adhesive-Teflon film;
the thickness of the matrix film is 0.1-0.15 mm.
2. The single pixel solid phase extraction array plate of claim 1, wherein: the adsorption material is octadecyl bonded silica gel.
3. The single pixel solid phase extraction array plate of claim 1, wherein: the set interval is 80-120 mu m.
4. The single pixel solid phase extraction array plate of claim 1, wherein: the set interval is 100 mu m; the diameter of the opening at one end of the conical through hole is 70-90 mu m, and the diameter of the opening at the other end is 20-40 mu m.
5. The method for preparing the single-pixel solid-phase extraction array plate according to any one of claims 1 to 4, which is characterized in that: the method comprises the following steps: firstly, punching holes on the surface of a flaky substrate by adopting laser according to a set interval; then, flattening and fixing the matrix film, and punching tapered through holes on the surface of the matrix film according to the set interval to form a tapered array with fixed depth; and butting the flaky substrate with a matrix film, loading an adsorption material on the surface of the matrix film, compacting the adsorption material, and then stripping the flaky substrate to obtain the single-pixel solid-phase extraction array plate with the adsorption material in the conical through holes.
6. A pretreatment method of mass spectrum imaging based on single-pixel solid phase extraction technology, which adopts the single-pixel solid phase extraction array plate as claimed in any one of claims 1 to 4, comprising the following steps:
(1) Chemical component transfer and blotting in plant tissue sections: butting and compacting the frozen plant tissue slice with the single-pixel solid-phase extraction array plate, and then stripping the plant tissue slice, wherein chemical components in the plant tissue slice are transferred into the conical through holes on the single-pixel solid-phase extraction array plate, so that chemical component transfer and imprinting in the plant tissue slice are realized;
(2) Purifying in the conical through hole: and eluting the tapered through holes by adopting an organic solvent, eluting part or all of non-target molecules out of the tapered through holes, and retaining the target molecules through an adsorption material.
7. A mass spectrum imaging method based on single-pixel solid phase extraction technology is characterized in that: the method comprises the following steps:
(1) Molecular ion mass spectrum signal acquisition: the solvent for analyzing the target molecules is sprayed to a pretreatment method of mass spectrum imaging based on the single-pixel solid phase extraction technology according to claim 6 in a spray mode, after a conical through hole is purified, the spray passes through the conical through hole and analyzes the target molecules to obtain secondary spray containing the target molecules, the secondary spray enters a sample inlet of a mass spectrometer to complete sample injection of one conical through hole, and sample injection of the next conical through hole is completed by secondary pushing, so that the mass spectrometer completes mass spectrum analysis of each conical through hole;
(2) Sample collection and data analysis are performed by a mass spectrometer, so that a mass spectrum image of the component ions is obtained.
8. The mass spectrometry imaging method of claim 7, wherein: in the step (2), the acquisition time of the target molecules in each conical through hole is calculated, after coordinates of the conical through holes are obtained, components and signal intensity in each conical through hole are matched according to the coordinates, and therefore a mass spectrum image of component ions is obtained.
9. A use of a mass spectrometry imaging method as claimed in claim 7 or 8 in component analysis of a strawberry slice.
10. A mass spectrometry imaging system based on single-pixel solid phase extraction technology, adopting the mass spectrometry imaging method of claim 7 or 8, characterized in that: the system comprises a slice imprinting device for realizing chemical component transfer and imprinting of plant tissue slices, a single-pixel purifying device for purifying and regulating chemical components of the plant tissue slices, a spraying device for acquiring target chemical components of each pixel in a mass spectrum image acquisition process, a single-pixel solid-phase extraction array plate and a mass spectrometer, wherein the single-pixel solid-phase extraction array plate is used for acquiring the target chemical components of each pixel in the mass spectrum image acquisition process;
the slice imprinting device comprises a pressing piece for realizing chemical component transfer and imprinting of plant tissue slices and a fixing bracket matched with the pressing piece;
the single-pixel purifying device at least comprises a solvent storage tank and a sample injection needle, wherein the solvent storage tank and the sample injection needle are used for purifying plant tissue slices;
in the imaging experiment process, a single-pixel solid-phase extraction array plate is arranged between a spraying device and a sample inlet of a mass spectrometer.
11. The system as set forth in claim 10, wherein: the single-pixel purification device further comprises a peristaltic pump, wherein the input end of the peristaltic pump is connected with the solvent storage tank, and the output end of the peristaltic pump is connected with the sample injection needle.
12. The system as set forth in claim 10, wherein: the spraying device consists of a peristaltic pump, an injector and a spray needle, wherein the peristaltic pump is connected with the injector, and the injector is connected with the spray needle.
13. The system as set forth in claim 10, wherein: the imaging system also comprises a camera which is arranged on one side or two sides of the single-pixel solid-phase extraction array plate.
14. A use of a mass spectrometry imaging system according to any of claims 10 to 13 for component analysis in strawberry slices.
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| CN109212010A (en) * | 2018-09-05 | 2019-01-15 | 东南大学 | A kind of sample detection methods of simplicity fast high-flux |
| CN110567786B (en) * | 2019-08-06 | 2020-09-29 | 中南大学 | Spatial resolution enrichment purification sampling method for mass spectrometry imaging |
| CN111208191B (en) * | 2020-01-10 | 2021-05-18 | 中国科学院深圳先进技术研究院 | Sampling head, sampling system, mass spectrum imaging device and sampling method |
| CN111413172A (en) * | 2020-04-17 | 2020-07-14 | 山东省分析测试中心 | In-situ enzymolysis-direct mass spectrometry analysis system and analysis method |
| CN112198217B (en) * | 2020-10-13 | 2022-05-20 | 中南大学 | Absolute quantitative mass spectrometry imaging method based on in-situ liquid extraction |
| CN112730008B (en) * | 2020-12-18 | 2023-03-14 | 山东省分析测试中心 | Ion image mediated compound extraction and identification system and method |
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