CN106404885B - Spraying system and method for matrix assisted laser desorption ionization mass spectrometry imaging - Google Patents
Spraying system and method for matrix assisted laser desorption ionization mass spectrometry imaging Download PDFInfo
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- CN106404885B CN106404885B CN201510452507.5A CN201510452507A CN106404885B CN 106404885 B CN106404885 B CN 106404885B CN 201510452507 A CN201510452507 A CN 201510452507A CN 106404885 B CN106404885 B CN 106404885B
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Abstract
The invention discloses the method for the spraying system being imaged for matrix assisted laser desorption ionization mass spectrometry and analysis biological sample, which includes: injection device, and injection device is suitable for supply matrix solution；Capillary, capillary are connected with injection device, and are suitable for supplying matrix solution to capillary；Power supply unit, power supply unit are connected with capillary, and are suitable for being atomized the matrix solution in capillary, to obtain matrix solution droplet；Sheath tracheae, sheath tracheae are set on capillary；Sheath air feed system, sheath air feed system are connected with sheath tracheae, and are suitable for spraying matrix solution droplet using sheath gas；Sample stage, sample stage are movably disposed at the lower end of capillary；And sample stage control device, sample stage control device are connected with sample stage, and are suitable for controlling the movement of the sample stage.The even application of biological sample surface matrix may be implemented in the system, and the system structure is simple, accessory is cheap and easy to get, and controllability is strong.
Description
Technical field
The invention belongs to field of biotechnology, specifically, the present invention relates to one kind for Matrix Assisted Laser Desorption electricity
The method of spraying system and analysis biological sample from mass spectrum imaging.
Background technique
Matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is that one kind directly realizes region from biological sample
The technology of specific molecular detection.Currently, the technology has been widely used in determining for protein in biological tissue section, lipid etc.
Property, quantitative, in-situ study and exploration, the pathogenic mechanism research of biomarker etc., and in biochemistry, clinical medicine etc.
Field is playing an increasingly important role.
In matrix assisted laser desorption ionization mass spectrometry imaging process, the preparation of sample is highly important link, wherein
The spraying of biological sample surface matrix is the most key step, determines the success or failure of mass spectrum imaging.There are many commercial at present
Matrix, which sprays instrument, to be occurred, these instruments substantially can be realized matrix and form uniform crystallizing layer, such as cloth in tissue sample surface
The ImagePre imaging of tissue matrix atomizer of Luke dalton company, μM atrix Spotter of HST etc..But these
Equipment purchase is sufficiently expensive, needs ten thousand U.S. dollar of 4-10, and all more severe for the alternative condition of operation requirement, reagent and matrix
It carves, it is difficult to meet the needs of routine experimentation.
Therefore, develop the instrument in sample tissue surface simplicity, fast spraying uniform high quality matrix crystallization coating, it is non-
It is often significant.
Summary of the invention
The present invention is directed to solve at least some of the technical problems in related technologies.For this purpose, of the invention
One purpose is to propose a kind of spraying system for matrix assisted laser desorption ionization mass spectrometry imaging and analysis biological sample
Method, which may be implemented the even application of biological sample surface matrix, and the system structure is simple, accessory is inexpensively easy
, controllability is strong.
In one aspect of the invention, the invention proposes it is a kind of for matrix assisted laser desorption ionization mass spectrometry imaging
Spraying system, according to an embodiment of the invention, the system includes:
Injection device, the injection device are suitable for supply matrix solution；
Capillary, the capillary are connected with the injection device, and are suitable for supplying the matrix solution to the hair
Tubule；
Power supply unit, the power supply unit are connected with the capillary, and are suitable for the matrix solution in the capillary
It is atomized, to obtain matrix solution droplet；
Sheath tracheae, the sheath tracheae are set on the capillary；
Sheath air feed system, the sheath air feed system are connected with the sheath tracheae, and are suitable for using sheath gas to the base
Matter solution droplet is sprayed；
Sample stage, the sample stage are movably disposed at the lower end of the capillary；And
Sample stage control device, the sample stage control device are connected with the sample stage, and are suitable for controlling the sample
The movement of platform.
The spraying system according to an embodiment of the present invention for matrix assisted laser desorption ionization mass spectrometry imaging passes through as a result,
Using electron spray principle, available superfine matrix solution is spraying, smaller so as to form partial size on biological sample surface
Matrix crystallization, and then be conducive to matrix assisted laser desorption ionization mass spectrometry and analysis for biological sample be imaged, and this is
Structure of uniting is simple, accessory is cheap and easy to get, and controllability is strong.
In addition, the spraying system according to the above embodiment of the present invention for matrix assisted laser desorption ionization mass spectrometry imaging
It can also have the following additional technical features:
In some embodiments of the invention, the sheath tracheae includes connected the first pipeline section and the second pipeline section.
In some embodiments of the invention, first pipeline section is polyfluortetraethylene pipe, and second pipeline section is plastics
Pipe, rubber tube, glass tube or ceramic tube.Thus, it is possible to significantly improve system run all right.
In some embodiments of the invention, the matrix solution is the mixed solution for matrix and organic solvent.
In some embodiments of the invention, the matrix is selected from 2,5-dihydroxybenzoic acid, alpha-cyano -4- hydroxyl meat
Cinnamic acid, alpha-cyano -4- chloro-cinnamic acid, 4- hydroxyl -3,5- dimethoxy-cinnamic acid, 9-aminoacridine, indole-3-acetic acid, to nitre
Base aniline, 3- hydroxyl -2- pyridine carboxylic acid, 2-mercaptobenzothiazole, 2,4,6- trihydroxy-acetophenone, 2,5- resacetophenone,
At least one of 2,6- resacetophenones, N- (5- nitro -2- pyridyl group) -1,2- ethylenediamine, 3- aminoquinoline, it is described to have
Solvent is acetonitrile solution, and the volume ratio of acetonitrile and water is 8:2 in the acetonitrile solution.As a result, not only it is possible to prevente effectively from spray
The blocking of pipeline during painting, and the quality of matrix crystallization can be significantly improved.
In some embodiments of the invention, the flow velocity of the sheath gas is 30~70 ls/h.Thus, it is possible to further
Improve the quality of matrix crystallization.
In some embodiments of the invention, the flow velocity of the sheath gas is 50 ls/h.Thus, it is possible to further increase
The quality of matrix crystallization.
In some embodiments of the invention, the mass concentration of the matrix solution is 1~50 mg/ml.As a result, may be used
To further increase the quality of biological sample surface matrix crystallization.
In some embodiments of the invention, the mass concentration of the matrix solution is 30 mg/mls.Thus, it is possible to
Further increase the quality of biological sample surface matrix crystallization.
In some embodiments of the invention, the distance between the capillary lower end and the sample stage be 4.5~
10.5 centimetres.Thus, it is possible to further increase the quality of biological sample surface matrix crystallization.
In some embodiments of the invention, the distance between the capillary lower end and the sample stage are 8.5 centimetres.
Thus, it is possible to further increase the quality of biological sample surface matrix crystallization.
In some embodiments of the invention, the voltage of the power supply unit is 3500~6000 volts.Thus, it is possible into one
Step improves the quality of biological sample surface matrix crystallization.
In some embodiments of the invention, the voltage of the power supply unit is 4500 volts.Thus, it is possible to further increase
The quality of biological sample surface matrix crystallization.
In some embodiments of the invention, the flow velocity of the matrix solution is 120~300 micro- ls/h.As a result, may be used
To further increase the quality of biological sample surface matrix crystallization.
In some embodiments of the invention, the flow velocity of the matrix solution is 250 micro- ls/h.Thus, it is possible into one
Step improves the quality of biological sample surface matrix crystallization.
In some embodiments of the invention, the internal diameter of the capillary is 50~150 microns, the outer diameter of the capillary
It is 150~250 microns.Thus, it is possible to further increase the quality of biological sample surface matrix crystallization.
In some embodiments of the invention, the internal diameter of the capillary is 100 microns, and the outer diameter of the capillary is
200 microns.Thus, it is possible to further increase the quality of biological sample surface matrix crystallization.
In some embodiments of the invention, the capillary is conductive metal pipe.
In some embodiments of the invention, the sheath gas is selected from least one of nitrogen and argon gas.
In another aspect of the invention, according to the present invention the invention proposes a kind of method for analyzing biological sample
Embodiment, this method comprises:
(1) matrix solution is sprayed on biological sample surface, to form matrix crystallization on the biological sample surface；With
And
(2) biological sample obtained to step (1) is analyzed,
Wherein, the step (1) is using the spray for matrix assisted laser desorption ionization mass spectrometry imaging described above
What mist system carried out.
The method of analysis biological sample according to an embodiment of the present invention is used for ground substance assistant laser by using above-mentioned as a result,
The spraying system of desorption ionization mass spectrum imaging carries out spraying matrix solution to biological sample surface, can be in biological sample surface shape
It is crystallized at evenly dispersed tiny matrix, and then is conducive to the further analysis for biological sample, and this method analysis knot
Fruit is accurate, experimental result reproducibility with higher.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Detailed description of the invention
Above-mentioned and/or additional aspect of the invention and advantage will become from the description of the embodiment in conjunction with the following figures
Obviously and it is readily appreciated that, in which:
Fig. 1 is the spraying system according to an embodiment of the invention for matrix assisted laser desorption ionization mass spectrometry imaging
Result schematic diagram；
Fig. 2 is that resulting host crystal is micro- when the distance between capillary lower end and sample stage are 4.5cm in experimental example 1
Figure；
Fig. 3 is that resulting host crystal is micro- when the distance between capillary lower end and sample stage are 6.5cm in experimental example 1
Figure；
Fig. 4 is that resulting host crystal is micro- when the distance between capillary lower end and sample stage are 8.5cm in experimental example 1
Figure；
Fig. 5 is that resulting host crystal is aobvious when the distance between capillary lower end and sample stage are 10.5cm in experimental example 1
Micro- figure；
Fig. 6 is the resulting host crystal micrograph of experimental example 2；
Fig. 7 is the resulting host crystal micrograph of experimental example 3；
Fig. 8 is the resulting host crystal micrograph of experimental example 4；
Fig. 9 is the resulting host crystal micrograph of experimental example 5；
Figure 10 is the resulting Mice brain tissues sample micrograph of experimental example 6；
Map is imaged in the ionization mass spectrometry of the resulting Mice brain tissues sample of Figure 11 experimental example 6.
Specific embodiment
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings, wherein from beginning to end
Same or similar label indicates same or similar element or element with the same or similar functions.Below with reference to attached
The embodiment of figure description is exemplary, it is intended to is used to explain the present invention, and is not considered as limiting the invention.
In the description of the present invention, it is to be understood that, term " center ", " longitudinal direction ", " transverse direction ", " length ", " width ",
" thickness ", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outside", " up time
The orientation or positional relationship of the instructions such as needle ", " counterclockwise ", " axial direction ", " radial direction ", " circumferential direction " be orientation based on the figure or
Positional relationship is merely for convenience of description of the present invention and simplification of the description, rather than the device or element of indication or suggestion meaning must
There must be specific orientation, be constructed and operated in a specific orientation, therefore be not considered as limiting the invention.
In addition, term " first ", " second " are used for descriptive purposes only and cannot be understood as indicating or suggesting relative importance
Or implicitly indicate the quantity of indicated technical characteristic.Define " first " as a result, the feature of " second " can be expressed or
Implicitly include at least one this feature.In the description of the present invention, the meaning of " plurality " is at least two, such as two, three
It is a etc., unless otherwise specifically defined.
In the present invention unless specifically defined or limited otherwise, term " installation ", " connected ", " connection ", " fixation " etc.
Term shall be understood in a broad sense, for example, it may be being fixedly connected, may be a detachable connection, or integral；It can be mechanical connect
It connects, is also possible to be electrically connected；It can be directly connected, can also can be in two elements indirectly connected through an intermediary
The interaction relationship of the connection in portion or two elements, unless otherwise restricted clearly.For those of ordinary skill in the art
For, the specific meanings of the above terms in the present invention can be understood according to specific conditions.
In the present invention unless specifically defined or limited otherwise, fisrt feature in the second feature " on " or " down " can be with
It is that the first and second features directly contact or the first and second features pass through intermediary mediate contact.Moreover, fisrt feature exists
Second feature " on ", " top " and " above " but fisrt feature be directly above or diagonally above the second feature, or be merely representative of
First feature horizontal height is higher than second feature.Fisrt feature can be under the second feature " below ", " below " and " below "
One feature is directly under or diagonally below the second feature, or is merely representative of first feature horizontal height less than second feature.
In one aspect of the invention, the invention proposes it is a kind of for matrix assisted laser desorption ionization mass spectrometry imaging
Spraying system.According to an embodiment of the invention, the system includes: injection device, the injection device is suitable for supply matrix solution；
Capillary, the capillary are connected with the injection device, and are suitable for supplying the matrix solution to the capillary；Power supply
Device, the power supply unit are connected with the capillary, and are suitable for being atomized the matrix solution in the capillary, so as to
Obtain matrix solution droplet；Sheath tracheae, the sheath tracheae are set on the capillary；Sheath air feed system, the sheath gas supply
It is connected to device with the sheath tracheae, and is suitable for spraying the matrix solution droplet using sheath gas；Sample stage, the sample
Sample platform is movably disposed at the lower end of the capillary；And sample stage control device, the sample stage control device and institute
It states sample stage to be connected, and is suitable for controlling the movement of the sample stage.Inventors have found that by applying voltage to matrix solution, it can
So that matrix solution is dispersed to be formed tiny matrix solution droplet, and under the action of sheath gas even application to biological sample
This surface to form the tiny matrix crystallization of partial size on biological sample surface, and then is conducive to Matrix Assisted Laser Desorption electricity
The mass spectrum imaging of analysis from to(for) biological sample, while the injection rate of the system mesostroma solution, sheath gas, application voltage
It can be adjusted, so as to the more flexible preparation for realizing the sample required different analyses, and due to capillary
The distance between lower end spout and sample stage are adjustable, so as to realize the control to matrix spray area, next system knot
Structure is simple, accessory is cheap and easy to get, so as to significantly reduce equipment cost, in addition the system can be used under extreme condition for
The spraying of complex matrices solution, such as the spraying of nanocrystalline, highly basic, chloroform system, to improve the applicability of the system.
Below with reference to Fig. 1 to the spraying system for matrix assisted laser desorption ionization mass spectrometry imaging of the embodiment of the present invention
It is described in detail.According to an embodiment of the invention, the system includes:
Injection device 100: according to an embodiment of the invention, injection device 100 is suitable for supply matrix solution.According to the present invention
One embodiment, matrix solution can be matrix and organic solvent mixed solution.Still another embodiment in accordance with the present invention,
The concrete type of matrix is not particularly restricted, and those skilled in the art can select according to actual needs, according to this hair
Bright specific embodiment, matrix can be for selected from 2,5-dihydroxybenzoic acid, alpha-cyano -4- hydroxycinnamic acid, alpha-cyano -4- chlorine
Cinnamic acid, 4- hydroxyl -3,5- dimethoxy-cinnamic acid, 9-aminoacridine, indole-3-acetic acid, paranitroanilinum, 3- hydroxyl -2-
Pyridine carboxylic acid, 2-mercaptobenzothiazole, 2,4,6- trihydroxy-acetophenone, 2,5- resacetophenone, 2,6- resacetophenone,
At least one of N- (5- nitro -2- pyridyl group) -1,2- ethylenediamine, 3- aminoquinoline.Another implementation according to the present invention
Example, the concrete type of organic solvent are not particularly restricted, and those skilled in the art can select according to actual needs, root
According to specific embodiments of the present invention, organic solvent can be acetonitrile solution.Inventors have found that the acetonitrile solution of matrix can be dry
The crystallization that smaller size is formed during dry, to can be formed carefully on biological sample surface in biological sample spraying process
Small matrix crystallization, and then be conducive to the subsequent analysis to biological sample.
According to another embodiment of the invention, the proportion of acetonitrile and water is not particularly restricted in acetonitrile solution, ability
Field technique personnel can select according to actual needs, according to a particular embodiment of the invention, acetonitrile and water in acetonitrile solution
Volume ratio can be 8:2.Inventors have found that although the acetonitrile solution of matrix can form the crystallization of fine size, if
Acetonitrile concentration is excessively high, since acetonitrile has stronger volatility, is easy to cause the blocking of system pipeline, to influence the matter of spraying
Amount and efficiency, and add water that can slow down the formation of crystallization into acetonitrile, pipeline blockage is prevented, but the presence of water will affect knot again
Brilliant quality, causes crystal size to become larger, and the present inventor is had been surprisingly found that by many experiments, when the body of acetonitrile and water
When product is than being 8:2, it can not only guarantee to form the crystallization of the matrix of better quality, but also the blocking of system pipeline can be effectively prevented.
According to still another embodiment of the invention, the concentration of matrix solution is not particularly restricted, those skilled in the art
It can be selected according to actual needs, according to a particular embodiment of the invention, the mass concentration of matrix solution can be 1~50
Mg/ml.Inventors have found that if matrix solution solubility is excessively high, so that in the matrix knot of biological surface bulk easy to form
Crystalline substance, so that matrix coating quality is influenced, and matrix solution concentration is too low, so that the matrix crystallization that biological sample surface is formed is more
Dispersion can significantly improve matrix coating quality using the matrix solution of concentration range of the present invention as a result,.It is according to the present invention again
One embodiment, the mass concentration of matrix solution can be 30 mg/mls.Thus, it is possible to further increase matrix spraying matter
Amount.
According to still another embodiment of the invention, the flow velocity of matrix solution is not particularly restricted, those skilled in the art
It can be selected according to actual needs, according to a particular embodiment of the invention, the flow velocity of matrix solution is 120~300 microlitres/
Hour.Inventors have found that if the flow velocity of matrix solution is lower, so that host crystal is unevenly distributed on biological sample surface, and
If matrix solution flow velocity is higher, so that the nebulization efficiency of subsequent matrix solution reduces, to reduce matrix coating quality.According to
Another embodiment of the invention, the flow velocity of matrix solution can be 250 micro- ls/h.Thus, it is possible to further increase biology
The matrix coating quality of sample surfaces.
Another specific embodiment according to the present invention, injection device can be syringe pump.
Capillary 200: according to an embodiment of the invention, capillary 200 is connected with injection device 100, and it is suitable for matrix
Solution is supplied into capillary.According to one embodiment of present invention, injection device 100 can pass through nut and capillary 200
Realize seamless connection.
Still another embodiment in accordance with the present invention, capillary can be conductive metal pipe, such as can be copper pipe or steel pipe.
Thus, it is possible to significantly improve the nebulization efficiency of subsequent matrix solution.
According to still another embodiment of the invention, the size of capillary is not particularly restricted, and those skilled in the art can
To be selected according to actual needs, according to a particular embodiment of the invention, the internal diameter of capillary can be 50~150 microns,
Outer diameter can be 150~250 microns.Inventors have found that the capillary of the size can not only significantly improve the mist of matrix solution
Change efficiency, and the matrix crystalline quality of subsequent spraying process can be significantly improved.According to still another embodiment of the invention, hair
The internal diameter of tubule can be 100 microns, and outer diameter can be 200 microns.Thus, it is possible to further increase matrix coating quality.
Power supply unit 300: according to an embodiment of the invention, power supply unit 300 is connected with capillary 200, and it is suitable for hair
Matrix solution in tubule is atomized, so as to obtain matrix solution droplet.Inventors have found that by applying to capillary
Voltage can make the matrix solution in capillary be dispersed to be formed tiny matrix solution droplet, and in subsequent sheath gas
Lower even application is acted on to biological sample surface, to form the tiny matrix crystallization of partial size, Jin Eryou on biological sample surface
The analysis for biological sample is imaged conducive to matrix assisted laser desorption ionization mass spectrometry.
According to one embodiment of present invention, the voltage of power supply unit is not particularly restricted, and those skilled in the art can
To be selected according to actual needs, according to a particular embodiment of the invention, the voltage of power supply unit can be 3500~6000
Volt.Inventors have found that the nebulization efficiency of matrix solution can be significantly improved under the condition of high voltage, thus in biological sample surface shape
At host crystal it is thinner, and host crystal is evenly distributed on biological sample surface.Still another embodiment in accordance with the present invention,
The voltage of power supply unit can be 4500 volts.Thus, it is possible to further increase biological sample surface matrix coating quality.
According to one embodiment of present invention, power supply unit can be high-voltage DC power supply.
Sheath tracheae 400: according to an embodiment of the invention, sheath tracheae 400 is set on capillary 200.It is according to the present invention
One embodiment, sheath tracheae may include connected the first pipeline section and the second pipeline section, wherein the first pipeline section is set in capillary
Upper semisection, i.e. the first pipeline section are connected with injection device, and the second pipeline section is set in the lower semisection of capillary, and the first pipeline section passes through
Nut is connected with the second pipeline section.According to a particular embodiment of the invention, the first pipeline section and the second pipeline section can be adopted independently
Polyfluortetraethylene pipe can be used with insulation tube, such as the first pipeline section, the second pipeline section can use plastic tube, rubber tube, glass
Pipe or ceramic tube.Thus, it is possible to significantly improve system run all right.
Sheath air feed system 500: according to an embodiment of the invention, sheath air feed system 500 is connected with sheath tracheae 400, and
Suitable for being sprayed using sheath gas to matrix solution droplet.According to one embodiment of present invention, sheath gas can use indifferent gas
Body, such as nitrogen or argon gas can be used.
The flow velocity of still another embodiment in accordance with the present invention, sheath gas is not particularly restricted, and those skilled in the art can be with
It is selected according to actual needs, according to a particular embodiment of the invention, the flow velocity of sheath gas can be 30~70 ls/h.Hair
Bright people's discovery, if the flow velocity of sheath gas is too low, so that reunion is easy to happen in the host crystal that biological sample surface is formed, and if sheath
Gas velocity is excessively high, and the host crystal for causing biological sample surface to be formed is unevenly distributed, and thus selects the sheath gas of the range,
The thinner host crystal of partial size can be formed on biological sample surface, and host crystal distribution is more uniform.According to this hair
Another bright specific embodiment, the flow velocity of sheath gas can be 50 ls/h.Thus, it is possible to further increase biological sample table
Face matrix coating quality.
Sample stage 600: according to an embodiment of the invention, sample stage 600 is movably disposed at the lower end of capillary 200,
So as to realize the control to matrix spray area as needed.Specifically, sample stage is grounded.A reality according to the present invention
Example is applied, the distance between sample stage and the lower end of capillary can be 4.5~10.5 centimetres.Inventors have found that when sample stage with
When the distance of the lower end of capillary is too low, in the host crystal partial size that biological sample surface is formed, larger (partial size is greater than 40 micro-
Rice), and with when sample stage is excessively high at a distance from the lower end of capillary, in the more bulk base that biological sample surface is formed
Matter crystallization, and host crystal dispersion is uneven.Distance range of the invention is used as a result, it not only can be in biological sample table
Face forms the lower host crystal of partial size, and host crystal is more uniform in biological sample Dispersion on surface, thus after being conducive to
The analysis for biological sample is imaged in matrix assisted laser desorption ionization mass spectrometry during continuous.Specific implementation according to the present invention
Example, the distance between capillary and sample stage can be 8.5 centimetres.Inventor is had been surprisingly found that by many experiments, under the distance
Acquired host crystal size smaller (partial size is 10 microns), host crystal are uniformly dispersed in sample surfaces, and spray-coating surface
Product is suitble to.
Sample stage control device 700: according to an embodiment of the invention, sample stage control device 700 and 600 phase of sample stage
Connect, and is suitable for controlling the movement of sample stage 600.Specifically, sample stage may be implemented in two-dimensional directional in sample stage control device
It accurately at the uniform velocity moves, so as to significantly improve the flexibility of system operatio.
It is according to an embodiment of the present invention for matrix assisted laser desorption ionization mass spectrometry imaging spraying system by using
Electron spray principle, available superfine matrix solution is spraying, so as to form the smaller base of partial size on biological sample surface
Matter crystallization, and then be conducive to analysis of the matrix assisted laser desorption ionization mass spectrometry imaging for biological sample, and the system knot
Structure is simple, accessory is cheap and easy to get, and controllability is strong.
In order to facilitate understanding, below to being imaged for matrix assisted laser desorption ionization mass spectrometry using the embodiment of the present invention
Spraying system technique that biological sample surface is sprayed be described in detail.
The ito glass for being loaded with biological tissue section sample is placed on sample stage first, sample stage control device is opened, makes
Section sample can at the uniform velocity move back and forth in specific two-dimentional section, then adjust between sample stage and capillary lower end away from
From, unlatching sheath air feed system, and sheath gas is adjusted as needed, it is then turned on power supply unit, and adjust supply voltage, most
After open injection device so that the matrix solution in capillary is atomized to form tiny matrix solution droplet under hyperbaric environment,
And the matrix solution droplet is dispersed during spraying from capillary lower end by sheath gas, can along with the movement of sample stage
Coating is crystallized to form uniform matrix on histotomy surface.
In the second aspect of the invention, the invention proposes a kind of methods for analyzing biological sample.It is according to the present invention
Embodiment, this method comprises: (1) sprays matrix solution on biological sample surface, to be formed on the biological sample surface
Matrix crystallization；And the biological sample that (2) obtain step (1) is analyzed, wherein the step (1) is using above-mentioned institute
What the spraying system for matrix assisted laser desorption ionization mass spectrometry imaging stated carried out.Inventors have found that by using above-mentioned
Spraying system for matrix assisted laser desorption ionization mass spectrometry imaging carries out spraying matrix solution to biological sample surface, can be with
Evenly dispersed tiny matrix crystallization is formed on biological sample surface, and then is conducive to the further analysis for biological sample,
And this method analysis result is accurate, experimental result reproducibility with higher.Specifically, the analysis method can be biological group
Knit qualitative, quantitative, in-situ study and biomarker the analysis of protein, lipid etc. in slice.On it should be noted that
It states and is equally applicable to for feature and advantage described in the spraying system for matrix assisted laser desorption ionization mass spectrometry imaging
The method of the analysis biological sample, details are not described herein again.
Below with reference to specific embodiment, present invention is described, it should be noted that these embodiments are only to describe
Property, without limiting the invention in any way.
Experimental example 1
Determination of distance between capillary lower end and sample stage:
Experiment condition: biological tissue samples use normal mouse brain tissue slice, and matrix uses 2,5-dihydroxybenzoic acid
(DHB), sheath gas (nitrogen) flow velocity is 50L/h, and acetonitrile and water volume ratio are 8:2 in acetonitrile solution, and the concentration of matrix solution is
30mg/mL, matrix solution flow velocity are 250 μ L/h, and power supply unit voltage is 4500V, spray time 12min, capillary lower end
The distance between sample stage is respectively 4.5cm, 6.5cm, 8.5cm and 10.5cm.
The ito glass for being loaded with Mice brain tissues slice: being placed on sample stage by operating procedure first, opens sample stage control
Device, moves back and forth section sample at the uniform velocity in specific two-dimentional section, then adjust sample stage and capillary lower end it
Between distance, open sheath air feed system, and adjust sheath gas, be then turned on power supply unit, and adjust supply voltage, finally
Injection device is opened, so that the matrix solution in capillary is atomized to form tiny matrix solution droplet under hyperbaric environment, and
And the matrix solution droplet is dispersed during spraying from capillary lower end by sheath gas, it, can be with along with the movement of sample stage
Uniform matrix is formed in slice surface and crystallizes coating, and gained host crystal micrograph is respectively such as Fig. 2 (4.5cm), Fig. 3
Shown in (6.5cm), Fig. 4 (8.5cm), Fig. 5 (10.5cm).
With the variation of distance between capillary lower end and sample stage it can be seen from Fig. 2-5, the pattern of DHB crystallization has
The difference of conspicuousness, when distance is 4.5cm, crystalline size is 40 μm or so；When distance is 6.5cm, crystalline size is decreased to
Less than 10 μm；When distance continues to increase to 8.5cm, crystalline size is 10 μm or so；When distance continues to increase to 10.5cm, from
5 as can be seen that there is more bulk DHB crystallization in sample surfaces in figure, and the dispersion of DHB crystal is not uniform enough, most of
Crystal particle diameter is greater than 20 μm.Comprehensive Experiment result and actual spray area as a result, confirmation is when distance is 8.5cm, gained
To crystalline size it is smaller, be uniformly dispersed, spray area be suitble to, therefore between capillary lower end and sample stage distance be 8.5cm
For the best spacing for carrying out the spraying of DHB matrix.
Experimental example 2
Influence of the high voltage environment to host crystal quality:
Experiment condition: biological tissue samples use normal mouse brain tissue slice, and matrix uses 2,5-dihydroxybenzoic acid
(DHB), sheath gas (nitrogen) flow velocity is 50L/h, and acetonitrile and water volume ratio are 8:2 in acetonitrile solution, and the concentration of matrix solution is
30mg/mL, matrix solution flow velocity are 250 μ L/h, spray time 12min, and the distance between capillary lower end and sample stage are
8.5cm, the voltage of power supply unit are 0V.
Operating procedure: with experimental example 1, gained host crystal micrograph difference is as shown in Figure 6.
As seen from Figure 6, it under other conditions same case, closes the resulting DHB crystal size of high pressure and obviously becomes larger,
General 1 times is increased compared to crystal (Fig. 4) size for applying high pressure, but does not influence the homogeneity of the dispersion of DHB crystallization.By
This, the atomization of matrix solution can be conducive to by applying high pressure to matrix solution, be conducive to obtain thinner drop, and then formed more
The host crystal of small size.
Experimental example 3
Influence of the matrix solution flow velocity to host crystal quality:
Experiment condition: biological tissue samples use normal mouse brain tissue slice, and matrix uses 2,5-dihydroxybenzoic acid
(DHB), sheath gas (nitrogen) flow velocity is 50L/h, and acetonitrile and water volume ratio are 8:2 in acetonitrile solution, and the concentration of matrix solution is
The voltage of 30mg/mL, power supply unit are 4500V, spray time 12min, and the distance between capillary lower end and sample stage are
8.5cm, matrix solution flow velocity are 125 μ L/h, and gained host crystal micrograph difference is as shown in Figure 7.
Operating procedure: with experimental example 1, gained host crystal micrograph difference is as shown in Figure 7.
It as shown in Figure 7, is 250 μ L/h compared to matrix solution flow velocity under the conditions of matrix solution flow velocity is 125 μ L/h
(Fig. 4), DHB crystal size are obviously reduced, but the dispersion homogeneity of crystal is 250 μ L/h when institutes significantly lower than matrix solution flow velocity
Obtain host crystal.Therefore, comprehensively consider, matrix solution flow velocity is that 250 μ L/h are the matrix solution flow velocity most preferably sprayed.
Experimental example 4
Influence of the organic solvent to host crystal quality:
Experiment condition: biological tissue samples use normal mouse brain tissue slice, and matrix uses 2,5-dihydroxybenzoic acid
(DHB), sheath gas (nitrogen) flow velocity is 50L/h, and organic solvent uses acetonitrile, and the concentration of matrix solution is 30mg/mL, matrix solution
Flow velocity is 250 μ L/h, spray time 12min, and the distance between capillary lower end and sample stage are 8.5cm, power supply unit
Voltage is 4500V.
Operating procedure: with experimental example 1, gained host crystal micrograph is as shown in Figure 8.
As seen from Figure 8, use is significantly less than as DHB crystalline size obtained by organic solvent using 100% acetonitrile
Organic solvent is acetonitrile solution (acetonitrile and water volume ratio are 8:2 in acetonitrile solution) gained host crystal (Fig. 4), but is used
100% acetonitrile is as while smaller szie crystal, the distribution of DHB crystal is very uneven obtained by organic solvent, or even goes out
Show bulk DHB flaky crystal group, it is with the naked eye i.e. distinguishable, matrix coating quality is seriously affected, reason may be due to height
The presence of piezoelectric field, so that DHB is also ionized while atomization, the DHB of ionization is reunited again, acetonitrile body
This effect is particularly evident in system, affects dispersibility, or even forms crystallization group.In summary it analyzes, final choice acetonitrile/
The system of water=8/2 (V/V) carries out the spraying of DHB matrix.And all the time, influence of the host solvents for matrix spraying effect
It is all a highly important factor.The matrix spraying instrument of any commercialization in use, is required for host solvents
It is explored, so as to the matrix spraying effect being optimal, and the selection of solvent will follow the principle of " different because of matrix ".
Experimental example 5
Experiment condition: biological tissue samples use normal mouse brain tissue slice, and matrix uses 2,5-dihydroxybenzoic acid
(DHB)。
Operating procedure: the spray equipment (HST uMatrix Spotter) of laboratory apparatus commercialization, using routine operation to just
Normal Mice brain tissues slice is sprayed, and gained host crystal micrograph is as shown in Figure 9.
As shown in Figure 9, compared with existing commercial apparatus, using spraying matrix crystallization obtained by paint finishing of the invention
(Fig. 4) is smaller, more uniform, and easy to operate, and acid and alkali resistance, salt are not easy to plug, convenient for safeguarding.
Experimental example 6
Experiment condition: biological tissue samples use normal mouse brain tissue slice, and matrix uses 2,5-dihydroxybenzoic acid
(DHB), sheath gas (nitrogen) flow velocity is 50L/h, and acetonitrile and water volume ratio are 8:2 in acetonitrile solution, and the concentration of matrix solution is
30mg/mL, matrix solution flow velocity are 250 μ L/h, and power supply unit voltage is 4500V, spray time 12min, capillary lower end
The distance between sample stage is 8.5cm.
Operating procedure: with experimental example 1, the Mice brain tissues sample micrograph after spraying is as shown in Figure 10, and to gained sample
Product carry out matrix assisted laser desorption ionization mass spectrometry imaging analysis, as a result as shown in figure 11.
By Figure 11 it can be seen that, using the present invention can obtain very clearly mass spectrum imaging as a result, meet experiment want
It asks.This result is that with DHB crystallization quality and forming process it is closely bound up, not only illustrated from the quality of crystal this specially
The validity of sharp invention instrument more demonstrates the huge practical application value of the patent of invention from final experimental result.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field
Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples
It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, modifies, replacement and variant.
Claims (18)
1. a kind of spraying system characterized by comprising
Injection device, the injection device are suitable for supply matrix solution；
Capillary, the capillary are connected with the injection device, and are suitable for supplying the matrix solution to the capillary；
Power supply unit, the power supply unit are connected with the capillary, and are suitable for carrying out the matrix solution in the capillary
Atomization, to obtain matrix solution droplet；
Sheath tracheae, the sheath tracheae are set on the capillary；
Sheath air feed system, the sheath air feed system are connected with the sheath tracheae, and are suitable for molten to the matrix using sheath gas
Liquid mist drop is sprayed；
Sample stage, the sample stage are movably disposed at the lower end of the capillary；And
Sample stage control device, the sample stage control device are connected with the sample stage, and are suitable for controlling the sample stage
It is mobile,
The voltage of the power supply unit is 4500 volts；
The spraying system is imaged for matrix assisted laser desorption ionization mass spectrometry.
2. system according to claim 1, which is characterized in that the sheath tracheae includes connected the first pipeline section and the second pipe
Section.
3. system according to claim 2, which is characterized in that first pipeline section be polyfluortetraethylene pipe, described second
Pipeline section is plastic tube, rubber tube, glass tube or ceramic tube.
4. system according to claim 1, which is characterized in that the matrix solution is molten for the mixing of matrix and organic solvent
Liquid.
5. system according to claim 4, which is characterized in that the matrix is selected from 2,5-dihydroxybenzoic acid, α-cyanogen
Base -4- hydroxycinnamic acid, alpha-cyano -4- chloro-cinnamic acid, 4- hydroxyl -3,5- dimethoxy-cinnamic acid, 9-aminoacridine, indoles -
3- acetic acid, paranitroanilinum, 3- hydroxyl -2- pyridine carboxylic acid, 2-mercaptobenzothiazole, 2,4,6- trihydroxy-acetophenone, 2,5- bis-
In hydroxy acetophenone, 2,6- resacetophenone, N- (5- nitro -2- pyridyl group) -1,2- ethylenediamine and 3- aminoquinoline extremely
Few one kind,
The organic solvent is acetonitrile solution, and the volume ratio of acetonitrile and water is 8:2 in the acetonitrile solution.
6. system according to claim 4, which is characterized in that the mass concentration of the matrix solution is 1~50 milligram/milli
It rises.
7. system according to claim 6, which is characterized in that the mass concentration of the matrix solution is 30 mg/mls.
8. system according to claim 1, which is characterized in that the flow velocity of the sheath gas is 30~70 ls/h.
9. system according to claim 8, which is characterized in that the flow velocity of the sheath gas is 50 ls/h.
10. system according to claim 1, which is characterized in that between the capillary lower end and the sample stage away from
From being 4.5~10.5 centimetres.
11. system according to claim 10, which is characterized in that between the capillary lower end and the sample stage away from
From being 8.5 centimetres.
12. system according to claim 1, which is characterized in that the flow velocity of the matrix solution be 120~300 microlitres/it is small
When.
13. system according to claim 12, which is characterized in that the flow velocity of the matrix solution is 250 micro- ls/h.
14. system according to claim 1, which is characterized in that the internal diameter of the capillary is 50~150 microns, described
The outer diameter of capillary is 150~250 microns.
15. system according to claim 14, which is characterized in that the internal diameter of the capillary is 100 microns, the capillary
The outer diameter of pipe is 200 microns.
16. system according to claim 15, which is characterized in that the capillary is conductive metal pipe.
17. system according to claim 1, which is characterized in that the sheath gas is at least one in nitrogen and argon gas
Kind.
18. a kind of method for analyzing biological sample characterized by comprising
(1) matrix solution is sprayed on biological sample surface, to form matrix crystallization on the biological sample surface；And
(2) biological sample obtained to step (1) is analyzed,
Wherein, the step (1) is carried out using the described in any item spraying systems of claim 1-17.
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| CN110400740A (en) * | 2019-07-23 | 2019-11-01 | 中国科学院上海有机化学研究所 | A kind of method and apparatus using solvent and gas double auxiliary flame direct ion sample |
| JP7306180B2 (en) * | 2019-09-12 | 2023-07-11 | 株式会社島津製作所 | Sample pretreatment device |
| CN112858450A (en) * | 2021-01-18 | 2021-05-28 | 中国烟草总公司郑州烟草研究院 | Cigarette smoke online collecting and ionizing device and method |
| CN113447561B (en) * | 2021-08-06 | 2022-10-14 | 河北省食品检验研究院 | MALDI-TOF detection method for sheep-derived components in milk powder |
| CN114724921A (en) * | 2022-04-08 | 2022-07-08 | 中国科学院深圳先进技术研究院 | Automatic electrospray spraying device and method for mass spectrometry imaging sample preparation |
| CN116046877B (en) * | 2023-04-03 | 2024-03-15 | 深圳先进技术研究院 | Metabolic analysis method for disease model, embedding mould and spraying device |
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| WO2009124298A2 (en) * | 2008-04-04 | 2009-10-08 | Agilent Technologies, Inc. | Ion sources for improved ionization |
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