CN106404885A - Spraying system based on matrix-assisted laser desorption ionization mass spectrometry imaging, and biological sample analysis method - Google Patents
Spraying system based on matrix-assisted laser desorption ionization mass spectrometry imaging, and biological sample analysis method Download PDFInfo
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- CN106404885A CN106404885A CN201510452507.5A CN201510452507A CN106404885A CN 106404885 A CN106404885 A CN 106404885A CN 201510452507 A CN201510452507 A CN 201510452507A CN 106404885 A CN106404885 A CN 106404885A
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Abstract
The present invention discloses a spraying system based on matrix-assisted laser desorption ionization mass spectrometry imaging, and a biological sample analysis method. The spraying system comprises: an injection device for supplying a matrix solution; a capillary tube connected to the injection device and used for supplying the matrix solution to the capillary tube; a power supply device connected to the capillary tube and used for atomizing the matrix solution in the capillary tube so as to obtain matrix solution mist droplets; a sheath gas pipe arranged on the capillary tube in a sleeved manner; a sheath gas supply device connected to the sheath gas pipe and used for performing spray coating on the matrix solution mist droplets by using the sheath gas; a sample table movably arranged on the lower end of the capillary tube; and a sample table control device connected to the sample table and used for controlling the moving of the sample table. According to the present invention, with the system, the uniform spray coating of the matrix on the biological sample surface can be achieved; and the system has advantages of simple structure, cheap and easily available fittings, and strong controllability.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to a kind of be used for Matrix Assisted Laser Desorption ionogen
The spraying system of spectrum imaging and the method for analysis biological specimen.
Background technology
MALDI-TOF-MS imaging (MALDI-MSI) is that directly feasible region from biological specimen is special for one kind
The technology of opposite molecule detection.At present, this technology has been widely used in determining of protein in biological tissue section, lipid etc.
Property, the exploration of quantitation, in-situ study and biomarker, pathogenic mechanism research etc., and in biochemistry, clinical doctor
Etc. field just plays an increasingly important role.
In MALDI-TOF-MS imaging process, the preparation of sample is highly important link, wherein biological
The spraying of sample surface matrix is the most key step, determines the success or failure of mass spectrum imaging.Existing at present multiple commercialization matrix
Spraying instrument occurs, and these instruments are substantially capable of matrix and form uniform crystallizing layer in tissue sample surface, such as Brooker
The ImagePre imaging of tissue matrix atomizer of dalton company, μM atrix Spotter of HST etc..But, these set
Standby purchase is sufficiently expensive, needs 4-10 ten thousand U.S. dollar, and the alternative condition for operation requirement, reagent and matrix is all more severe
Carve it is difficult to meet the needs of routine experimentation.
Therefore, develop the instrument in sample tissue surface simplicity, quick spraying uniform high-quality matrix crystallization coating, very
Meaningful.
Content of the invention
It is contemplated that at least solving one of technical problem in correlation technique to a certain extent.For this reason, the one of the present invention
Purpose is to propose the side of a kind of spraying system for MALDI-TOF-MS imaging and analysis biological specimen
Method, this system can realize the even application of biological specimen surface matrix, and this system architecture is simple, accessory is cheap and easy to get,
Controllability is strong.
In one aspect of the invention, the present invention proposes a kind of spraying for MALDI-TOF-MS imaging
System, according to embodiments of the invention, this system includes：
Injection device, described injection device is suitable to supply matrix solution；
Capillary, described capillary is connected with described injection device, and is suitable to supply described matrix solution to described capillary；
Electric supply installation, described electric supply installation is connected with described capillary, and is suitable to the matrix solution in described capillary is carried out
Atomization, to obtain matrix solution droplet；
Sheath tracheae, described sheath tracheae is set on described capillary；
Sheath air feed system, described sheath air feed system is connected with described sheath tracheae, and it is molten to described matrix to be suitable for use with sheath gas
Liquid mist is dripped and is sprayed；
Sample stage, described sample stage is movably disposed at the lower end of described capillary；And
Sample stage control device, described sample stage control device is connected with described sample stage, and is suitable to control described sample stage
Mobile.
Thus, the spraying system for MALDI-TOF-MS imaging according to embodiments of the present invention passes through to adopt
Electron spray principle, can obtain superfine matrix solution spraying, such that it is able to form the less base of particle diameter on biological specimen surface
Matter crystallizes, and then is conducive to MALDI-TOF-MS imaging for the analysis of biological specimen, and this system knot
Structure is simple, accessory is cheap and easy to get, and controllability is strong.
In addition, the spraying system for MALDI-TOF-MS imaging according to the above embodiment of the present invention also may be used
To have the technical characteristic adding as follows：
In some embodiments of the invention, described sheath tracheae includes the first connected pipeline section and the second pipeline section.
In some embodiments of the invention, described first pipeline section be polyfluortetraethylene pipe, described second pipeline section be plastic tube,
Rubber tube, glass tube or earthenware.Thus, it is possible to significantly improve system run all right.
In some embodiments of the invention, described matrix solution is the mixed solution for matrix and organic solvent.
In some embodiments of the invention, described matrix be selected from DHB, alpha-cyano -4- hydroxycinnamic acid,
Alpha-cyano -4- chloro-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- dihydroxy
At least one in benzoylformaldoxime, N- (5- nitro -2- pyridine radicals) -1,2- ethylenediamine, 3- aminoquinoline, described organic solvent
For acetonitrile solution, in described acetonitrile solution, acetonitrile and the volume ratio of water are 8：2.Thus, not only can be prevented effectively from spray coated
The blocking of pipeline in journey, and the quality of matrix crystallization can be significantly improved.
In some embodiments of the invention, the flow velocity of described sheath gas is 30～70 ls/h.Thus, it is possible to improve further
The quality of matrix crystallization.
In some embodiments of the invention, the flow velocity of described sheath gas is 50 ls/h.Thus, it is possible to improve base further
The quality of matter crystallization.
In some embodiments of the invention, the mass concentration of described matrix solution is 1～50 mg/ml.Thus, it is possible to
Improve the quality of biological specimen surface matrix crystallization further.
In some embodiments of the invention, the mass concentration of described matrix solution is 30 mg/ml.Thus, it is possible to enter
One step improves the quality of biological specimen surface matrix crystallization.
In some embodiments of the invention, the distance between described capillary lower end and described sample stage are 4.5～10.5 centimetres.
Thus, it is possible to improve the quality of biological specimen surface matrix crystallization further.
In some embodiments of the invention, the distance between described capillary lower end and described sample stage are 8.5 centimetres.By
This, can improve the quality of biological specimen surface matrix crystallization further.
In some embodiments of the invention, the voltage of described electric supply installation is 3500～6000 volts.Thus, it is possible to further
Improve the quality of biological specimen surface matrix crystallization.
In some embodiments of the invention, the voltage of described electric supply installation is 4500 volts.Thus, it is possible to improve life further
The quality of thing sample surface matrix crystallization.
In some embodiments of the invention, the flow velocity of described matrix solution is 120～300 micro- ls/h.Thus, it is possible to enter
One step improves the quality of biological specimen surface matrix crystallization.
In some embodiments of the invention, the flow velocity of described matrix solution is 250 micro- ls/h.Thus, it is possible to further
Improve the quality of biological specimen surface matrix crystallization.
In some embodiments of the invention, the internal diameter of described capillary is 50～150 microns, and the external diameter of described capillary is
150～250 microns.Thus, it is possible to improve the quality of biological specimen surface matrix crystallization further.
In some embodiments of the invention, the internal diameter of described capillary is 100 microns, and the external diameter of described capillary is 200
Micron.Thus, it is possible to improve the quality of biological specimen surface matrix crystallization further.
In some embodiments of the invention, described capillary is conductive metal pipe.
In some embodiments of the invention, described sheath gas is at least one in nitrogen and argon gas.
In another aspect of the present invention, the present invention proposes a kind of method of analysis biological specimen, according to the enforcement of the present invention
Example, the method includes：
(1) in biological specimen surface spraying matrix solution, so that matrix crystallization is formed on described biological specimen surface；And
(2) biological specimen that step (1) is obtained is analyzed,
Wherein, described step (1) is using the spraying system for MALDI-TOF-MS imaging described above
System is carried out.
Thus, the method for analysis biological specimen according to embodiments of the present invention is by using above-mentioned for Matrix Assisted Laser Desorption
The spraying system of MALDI-MS imaging carries out to biological sample surface spraying matrix solution, can be formed all on biological specimen surface
Even scattered tiny matrix crystallization, and then be conducive to the analysis further for biological specimen, and the method analysis result essence
Really, experimental result has higher reappearance.
The additional aspect of the present invention and advantage will be set forth in part in the description, and partly will become bright from the following description
Aobvious, or recognized by the practice of the present invention.
Brief description
The above-mentioned and/or additional aspect of the present invention and advantage will be apparent from from reference to the description to embodiment for the accompanying drawings below
With easy to understand, wherein：
Fig. 1 is the knot of the spraying system for MALDI-TOF-MS imaging according to an embodiment of the invention
Fruit schematic diagram；
Fig. 2 is that in experimental example 1, the distance between capillary lower end and sample stage are the host crystal micrograph of gained during 4.5cm；
Fig. 3 is that in experimental example 1, the distance between capillary lower end and sample stage are the host crystal micrograph of gained during 6.5cm；
Fig. 4 is that in experimental example 1, the distance between capillary lower end and sample stage are the host crystal micrograph of gained during 8.5cm；
Fig. 5 is that in experimental example 1, the distance between capillary lower end and sample stage are micro- for the host crystal of gained during 10.5cm
Figure；
Fig. 6 is the host crystal micrograph of experimental example 2 gained；
Fig. 7 is the host crystal micrograph of experimental example 3 gained；
Fig. 8 is the host crystal micrograph of experimental example 4 gained；
Fig. 9 is the host crystal micrograph of experimental example 5 gained；
Figure 10 is the Mice brain tissues sample micrograph of experimental example 6 gained；
The MALDI-MS imaging collection of illustrative plates of the Mice brain tissues sample of Figure 11 experimental example 6 gained.
Specific embodiment
Embodiments of the invention are described below in detail, the example of described embodiment is shown in the drawings, wherein identical from start to finish
Or the element that similar label represents same or similar element or has same or like function.Retouch below with reference to accompanying drawing
The embodiment stated is exemplary it is intended to be used for explaining the present invention, and is not considered as limiting the invention.
In describing the invention it is to be understood that term " " center ", " longitudinal ", " horizontal ", " length ", " width ",
" thickness ", " on ", D score, "front", "rear", "left", "right", " vertical ", " level ", " top ", " bottom ", " interior ", " outward ",
The orientation of instruction such as " clockwise ", " counterclockwise ", " axial ", " radially ", " circumferential " or position relationship are based on shown in the drawings
Orientation or position relationship, be for only for ease of description the present invention and simplify description, rather than instruction or hint indication device or
Element must have specific orientation, with specific azimuth configuration and operation, be therefore not considered as limiting the invention.
Additionally, term " first ", " second " be only used for describe purpose, and it is not intended that instruction or hint relative importance or
Person implies the quantity indicating indicated technical characteristic.Thus, define " first ", the feature of " second " can express or
Implicitly include at least one this feature.In describing the invention, " multiple " are meant that at least two, such as two,
Three etc., unless otherwise expressly limited specifically.
In the present invention, unless otherwise clearly defined and limited, the art such as term " installation ", " being connected ", " connection ", " fixation "
Language should be interpreted broadly, for example, it may be being fixedly connected or being detachably connected or integral；Can be machinery
Connect or electrically connect；Can be to be joined directly together it is also possible to be indirectly connected to by intermediary, can be two units
Connection within part or the interaction relationship of two elements, limit unless otherwise clear and definite.Ordinary skill for this area
For personnel, above-mentioned term concrete meaning in the present invention can be understood as the case may be.
In the present invention, unless otherwise clearly defined and limited, fisrt feature second feature " on " or D score can be
One and second feature directly contact, or the first and second features pass through intermediary mediate contact.And, fisrt feature is
Two features " on ", " top " and " above " but fisrt feature are directly over second feature or oblique upper, or are merely representative of first
Characteristic level height is higher than second feature.Fisrt feature second feature " under ", " lower section " and " below " can be fisrt feature
Immediately below second feature or obliquely downward, or it is merely representative of fisrt feature level height and is less than second feature.
In one aspect of the invention, the present invention proposes a kind of spraying for MALDI-TOF-MS imaging
System.According to embodiments of the invention, this system includes：Injection device, described injection device is suitable to supply matrix solution；
Capillary, described capillary is connected with described injection device, and is suitable to supply described matrix solution to described capillary；For
Electric installation, described electric supply installation is connected with described capillary, and is suitable to the matrix solution in described capillary is atomized,
To obtain matrix solution droplet；Sheath tracheae, described sheath tracheae is set on described capillary；Sheath air feed system, described
Sheath air feed system is connected with described sheath tracheae, and is suitable for use with sheath gas described matrix solution droplet is sprayed；Sample stage,
Described sample stage is movably disposed at the lower end of described capillary；And sample stage control device, described sample stage control dress
Put and be connected with described sample stage, and be suitable to control the movement of described sample stage.Inventor finds, by applying to matrix solution
Voltage, so that matrix solution is disperseed to be formed tiny matrix solution droplet, and even application in the presence of sheath gas
To biological specimen surface, thus forming the tiny matrix crystallization of particle diameter on biological specimen surface, and then Matrix-assisted is conducive to swash
Light parse MALDI-MS imaging for biological specimen analysis, simultaneously the injection rate of this system mesostroma solution, sheath gas,
Applied voltage all can be adjusted, such that it is able to the more flexible preparation realized to the sample that different analyses require, and by
Adjustable in the distance between capillary lower end spout and sample stage, such that it is able to realize the control to matrix spray area, secondly
This system architecture is simple, accessory is cheap and easy to get, and such that it is able to significantly reduce equipment cost, in addition this system can be used for extremely
Under the conditions of for complex matrices solution spraying, the such as nanocrystalline, spraying of the system such as highly basic, chloroform, thus improve this system
Applicability.
Below with reference to Fig. 1, the embodiment of the present invention is carried out for the spraying system that MALDI-TOF-MS is imaged
Describe in detail.According to embodiments of the invention, this system includes：
Injection device 100：According to embodiments of the invention, injection device 100 is suitable to supply matrix solution.According to the present invention
An embodiment, matrix solution can be the mixed solution of matrix and organic solvent.According to still a further embodiment,
The particular type of matrix is not particularly restricted, and those skilled in the art can be selected according to actual needs, according to this
Bright specific embodiment, matrix can be selected from DHB, 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 in N- (5- nitro -2- pyridine radicals) -1,2- ethylenediamine, 3- aminoquinoline.Another enforcement according to the present invention
Example, the particular type of organic solvent is not particularly restricted, and those skilled in the art can be selected according to actual needs,
According to a particular embodiment of the invention, organic solvent can be acetonitrile solution.Inventor finds, the acetonitrile solution of matrix is permissible
Form the crystallization of reduced size in dry run, thus can be in biological sample surface shape in biological sample spraying process
Become tiny matrix crystallization, and then be conducive to the follow-up analysis to biological sample.
According to another embodiment of the invention, in acetonitrile solution, the proportioning of acetonitrile and water is not particularly restricted, this area skill
Art personnel can be selected according to actual needs, according to a particular embodiment of the invention, the body of acetonitrile and water in acetonitrile solution
Long-pending ratio can be 8：2.Inventor finds although the acetonitrile solution of matrix can form the crystallization of fine size, but if second
Nitrile excessive concentration, because acetonitrile has stronger volatility, is easily caused the blocking of system pipeline, thus affecting the matter spraying
Amount and efficiency, and add water in acetonitrile and can slow down the formation of crystallization, prevent pipeline blockage, but the presence of water can affect again
The quality of crystallization, leads to crystal size to become big, and the present inventor is had been surprisingly found that by many experiments, when acetonitrile and water
Volume ratio be 8：When 2, both can ensure that the matrix crystallization forming better quality, can effectively prevent the stifled of system pipeline again
Plug.
According to still another embodiment of the invention, the concentration of matrix solution is not particularly restricted, and those skilled in the art are permissible
Selected according to actual needs, according to a particular embodiment of the invention, the mass concentration of matrix solution can be 1～50 milligram
/ milliliter.Inventor finds, if matrix solution solubility is too high so that the matrix easily forming bulk in biological surface crystallizes,
Thus affecting matrix coating quality, and matrix solution concentration is too low so that the matrix crystallization that biological specimen surface is formed more divides
Dissipate, thus, the matrix solution using concentration range of the present invention can significantly improve matrix coating quality.According to the present invention again
One embodiment, the mass concentration of matrix solution can be 30 mg/ml.Thus, it is possible to improve matrix spraying further
Quality.
According to still another embodiment of the invention, the flow velocity of matrix solution is not particularly restricted, and those skilled in the art are permissible
Selected according to actual needs, according to a particular embodiment of the invention, the flow velocity of matrix solution is 120～300 micro- ls/h.
Inventor finds, if the flow velocity of matrix solution is relatively low so that host crystal is uneven in biological specimen surface distributed, and if base
Matter solution flow rate is higher so that the nebulization efficiency of follow-up matrix solution reduces, thus reducing matrix coating quality.According to this
Another embodiment of invention, the flow velocity of matrix solution can be 250 micro- ls/h.Thus, it is possible to improve biological further
The matrix coating quality of sample surfaces.
According to another specific embodiment of the present invention, injection device can be syringe pump.
Capillary 200：According to embodiments of the invention, capillary 200 is connected with injection device 100, and is suitable to matrix
Solution supplies to capillary.According to one embodiment of present invention, injection device 100 can be by nut and capillary 200
Realize being seamlessly connected.
According to still a further embodiment, capillary can be conductive metal pipe, for example, can be copper pipe or steel pipe.By
This, can significantly improve the nebulization efficiency of follow-up 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 root
Selected according to being actually needed, according to a particular embodiment of the invention, the internal diameter of capillary can be 50～150 microns, external diameter
Can be 150～250 microns.Inventor finds, the capillary of this size not only can significantly improve the atomization effect of matrix solution
Rate, and the matrix crystalline quality of follow-up spraying process can be significantly improved.According to still another embodiment of the invention, capillary
The internal diameter of pipe can be 100 microns, and external diameter can be 200 microns.Thus, it is possible to improve matrix coating quality further.
Electric supply installation 300：According to embodiments of the invention, electric supply installation 300 is connected with capillary 200, and is suitable to hair
Matrix solution in tubule is atomized, such that it is able to obtain matrix solution droplet.Inventor finds, by applying to capillary
Making alive, so that the matrix solution in capillary is disperseed to be formed tiny matrix solution droplet, and in follow-up sheath gas
In the presence of even application to biological specimen surface, thus forming the tiny matrix crystallization of particle diameter on biological specimen surface, and then
Be conducive to MALDI-TOF-MS imaging for the analysis of biological specimen.
According to one embodiment of present invention, the voltage of electric supply installation is not particularly restricted, and those skilled in the art can root
Selected according to being actually needed, according to a particular embodiment of the invention, the voltage of electric supply installation can be 3500～6000 volts.
Inventor finds, can significantly improve the nebulization efficiency of matrix solution, thus being formed on biological specimen surface under this condition of high voltage
Host crystal thinner, and host crystal is uniform in biological sample surface distributed.According to still a further embodiment,
The voltage of electric supply installation can be 4500 volts.Thus, it is possible to improve biological sample surface matrix coating quality further.
According to one embodiment of present invention, electric supply installation can be high-voltage DC power supply.
Sheath tracheae 400：According to embodiments of the invention, sheath tracheae 400 is set on capillary 200.According to the present invention's
One embodiment, sheath tracheae can include the first connected pipeline section and the second pipeline section, and wherein, the first pipeline section is set in capillary
Upper semisection, that is, the first pipeline section be connected with injection device, the second pipeline section is set in the lower semisection of capillary, and the first pipeline section
It is connected with the second pipeline section by nut.According to a particular embodiment of the invention, the first pipeline section and the second pipeline section can be independently
Employing insulation tube, the such as first pipeline section can adopt polyfluortetraethylene pipe, the second pipeline section can using plastic tube, rubber tube,
Glass tube or earthenware.Thus, it is possible to significantly improve system run all right.
Sheath air feed system 500：According to embodiments of the invention, sheath air feed system 500 is connected with sheath tracheae 400, and
It is suitable for use with sheath gas matrix solution droplet is sprayed.According to one embodiment of present invention, sheath gas can adopt indifferent gas
Body, for example, can adopt nitrogen or argon gas.
According to still a further embodiment, the flow velocity of sheath gas is not particularly restricted, and those skilled in the art can basis
It is actually needed and is selected, according to a particular embodiment of the invention, the flow velocity of sheath gas can be 30～70 ls/h.Inventor
Find, if the flow velocity of sheath gas is too low so that the host crystal being formed on biological specimen surface is susceptible to reunite, and if sheath gas
Flow velocity is too high, the host crystal skewness leading to biological specimen surface to be formed, and thus selects the sheath gas of this scope,
The thinner host crystal of particle diameter can be formed on biological specimen surface, and the distribution of this host crystal is more uniform.According to this
Another bright specific embodiment, the flow velocity of sheath gas can be 50 ls/h.Thus, it is possible to improve biological sample further
Surface matrix coating quality.
Sample stage 600：According to embodiments of the invention, sample stage 600 is movably disposed at the lower end of capillary 200,
Such that it is able to realize the control to matrix spray area as needed.Specifically, sample stage ground connection.According to the present invention one
The distance between lower end of embodiment, sample stage and capillary can be 4.5～10.5 centimetres.Inventor finds, works as sample stage
When too low with the distance of the lower end of capillary, the host crystal particle diameter that formed on biological sample surface is larger, and (particle diameter is more than 40 micro-
Rice), and with the more bulk base when the distance of sample stage and the lower end of capillary is too high, being formed on biological sample surface
Matter crystallizes, and host crystal dispersion is uneven.Thus, using the distance range of the present invention, not only can be in biological sample
Surface forms the relatively low host crystal of particle diameter, and host crystal is more uniform in biological sample Dispersion on surface, thus being conducive to
Subsequent process mesostroma Assisted Laser Desorption MALDI-MS is imaged the analysis for biological specimen.Being embodied as according to the present invention
The distance between example, capillary and sample stage can be 8.5 centimetres.Inventor is had been surprisingly found that by many experiments, this distance
Obtained by lower, host crystal size less (particle diameter is 10 microns), host crystal are uniformly dispersed in sample surfaces, and spray
Apply area to be suitable for.
Sample stage control device 700：According to embodiments of the invention, sample stage control device 700 is connected with sample stage 600,
And be suitable to control the movement of sample stage 600.Specifically, sample stage control device can realize the essence in two-dimensional directional for the sample stage
Really at the uniform velocity move, such that it is able to significantly improve the flexibility of system operatio.
Spraying system for MALDI-TOF-MS imaging according to embodiments of the present invention is by adopting electron spray
Principle, can obtain superfine matrix solution spraying, such that it is able to form the less matrix crystallization of particle diameter on biological specimen surface,
And then be conducive to MALDI-TOF-MS imaging for the analysis of biological specimen, and this system architecture simple,
Accessory is cheap and easy to get, and controllability is strong.
Understand for convenience, below to the spray being imaged for MALDI-TOF-MS using the embodiment of the present invention
The technique that mist system is sprayed to biological sample surface is described in detail.
First the ito glass being loaded with biological tissue section sample is placed on sample stage, open sample stage control device so that
Section sample at the uniform velocity can reciprocate in specific two dimension interval, then adjusts the distance between sample stage and capillary lower end,
Open sheath air feed system, and adjust sheath gas as needed, be then turned on electric supply installation, and adjust supply voltage,
Open injection device afterwards so that the matrix solution in capillary is atomized the tiny matrix solution droplet of formation under hyperbaric environment,
And this matrix solution droplet is disperseed by sheath gas during spraying from capillary lower end, along with the movement of sample stage, can
To form homogeneous matrix crystallization coating on histotomy surface.
In the second aspect of the invention, the present invention proposes a kind of method of analysis biological specimen.Enforcement according to the present invention
Example, the method includes：(1) in biological specimen surface spraying matrix solution, so that base is formed on described biological specimen surface
Matter crystallizes；And the biological specimen that (2) obtain to step (1) is analyzed, wherein, described step (1) is using upper
State what the described spraying system for MALDI-TOF-MS imaging was carried out.Inventor finds, by using
The above-mentioned spraying system for MALDI-TOF-MS imaging carries out to biological sample surface spraying matrix solution,
Homodisperse tiny matrix crystallization can be formed on biological specimen surface, and then be conducive to dividing further for biological specimen
Analysis, and the method analysis result is accurately, and experimental result has higher reappearance.Specifically, this analysis method can be
Qualitative, quantitation, the in-situ study of protein, lipid etc. and the analysis of biomarker in biological tissue section.Need
Bright, above-mentioned same for the feature and advantage described by the spraying system being imaged for MALDI-TOF-MS
The method that sample is applied to this analysis biological specimen, here is omitted.
Below with reference to specific embodiment, present invention is described, it should be noted that these embodiments are only descriptive
, and limit the present invention never in any form.
Experimental example 1
Determination of distance between capillary lower end and sample stage：
Experiment condition：Biological tissue samples adopt normal mouse brain tissue slice, and matrix adopts DHB
(DHB), sheath gas (nitrogen) flow velocity is 50L/h, and in acetonitrile solution, acetonitrile and water volume ratio are 8:2, matrix solution dense
Spend for 30mg/mL, matrix solution flow velocity is 250 μ L/h, electric supply installation voltage is 4500V, spray time is 12min, hair
The distance between tubule lower end and sample stage are respectively 4.5cm, 6.5cm, 8.5cm and 10.5cm.
Operating procedure：First the ito glass being loaded with Mice brain tissues section is placed on sample stage, opens sample stage and control dress
Put so that section sample at the uniform velocity can reciprocate in specific two dimension interval, then adjust between sample stage and capillary lower end
Distance, open sheath air feed system, and adjust sheath gas, be then turned on electric supply installation, and adjust supply voltage,
Open injection device afterwards so that the matrix solution in capillary is atomized the tiny matrix solution droplet of formation under hyperbaric environment,
And this matrix solution droplet is disperseed by sheath gas during spraying from capillary lower end, along with the movement of sample stage, can
To form homogeneous matrix crystallization coating in slice surface, gained host crystal micrograph is respectively as Fig. 2 (4.5cm), Fig. 3
(6.5cm), shown in Fig. 4 (8.5cm), Fig. 5 (10.5cm).
Change with distance between capillary lower end and sample stage be can be seen that by Fig. 2-5, the pattern that DHB crystallizes has aobvious
The difference of work property, when distance is for 4.5cm, crystalline size is 40 μm about；When distance is for 6.5cm, crystalline size subtracts
Little to less than 10 μm；When distance continues to increase to 8.5cm, crystalline size is 10 μm about；When distance continues to increase to
During 10.5cm, from figure 5 as can be seen that sample surfaces occur in that more bulk DHB crystallization, and DHB crystal divides
Dissipate not uniform, most of crystal particle diameter is more than 20 μm.Thus, the spray area of Comprehensive Experiment result and reality, confirms
When distance is for 8.5cm, obtained crystalline size is less, be uniformly dispersed, spray area is suitable for, therefore capillary lower end
Distance is the optimal spacing carrying out DHB matrix spraying for 8.5cm and sample stage between.
Experimental example 2
The impact to host crystal quality for the high voltage environment：
Experiment condition：Biological tissue samples adopt normal mouse brain tissue slice, and matrix adopts DHB
(DHB), sheath gas (nitrogen) flow velocity is 50L/h, and in acetonitrile solution, acetonitrile and water volume ratio are 8:2, matrix solution dense
Spend for 30mg/mL, matrix solution flow velocity is 250 μ L/h, and spray time is 12min, between capillary lower end and sample stage
Distance is 8.5cm, and the voltage of electric supply installation is 0V.
Operating procedure：With experimental example 1, gained host crystal micrograph is respectively as shown in Figure 6.
As seen from Figure 6, under other conditions same case, the DHB crystal size closing high pressure gained substantially becomes big,
Increased general 1 times compared to crystal (Fig. 4) size applying high pressure, but do not affect the scattered homogeneity of DHB crystallization.
Thus, the atomization that high pressure can be conducive to matrix solution is applied to matrix solution, be conducive to obtaining thinner drop, and then shape
Become smaller size of host crystal.
Experimental example 3
The impact to host crystal quality for the matrix solution flow velocity：
Experiment condition：Biological tissue samples adopt normal mouse brain tissue slice, and matrix adopts DHB
(DHB), sheath gas (nitrogen) flow velocity is 50L/h, and in acetonitrile solution, acetonitrile and water volume ratio are 8:2, matrix solution dense
Spend for 30mg/mL, the voltage of electric supply installation is 4500V, and spray time is 12min, between capillary lower end and sample stage
Distance be 8.5cm, matrix solution flow velocity is 125 μ L/h, and gained host crystal micrograph is respectively as shown in Figure 7.
Operating procedure：With experimental example 1, gained host crystal micrograph is respectively as shown in Figure 7.
As shown in Figure 7, under the conditions of matrix solution flow velocity is 125 μ L/h, it is 250 μ L/h (figure compared to matrix solution flow velocity
4), DHB crystal size is 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, consider, matrix solution flow velocity is the matrix solution flow velocity that 250 μ L/h are optimal spraying.
Experimental example 4
The impact to host crystal quality for the organic solvent：
Experiment condition：Biological tissue samples adopt normal mouse brain tissue slice, and matrix adopts DHB
(DHB), sheath gas (nitrogen) flow velocity is 50L/h, and organic solvent adopts acetonitrile, and the concentration of matrix solution is 30mg/mL,
Matrix solution flow velocity is 250 μ L/h, and spray time is 12min, and the distance between capillary lower end and sample stage are 8.5cm,
The voltage of electric supply installation is 4500V.
Operating procedure：With experimental example 1, gained host crystal micrograph is as shown in Figure 8.
As seen from Figure 8, it is significantly less than using having using 100% acetonitrile as DHB crystalline size obtained by organic solvent
Machine solvent is that (in acetonitrile solution, acetonitrile and water volume ratio are 8 to acetonitrile solution:2) gained host crystal (Fig. 4), but adopt
While smaller szie crystal, the distribution of DHB crystal is very uneven, or even goes out as obtained by organic solvent for 100% acetonitrile
Show bulk DHB flaky crystal group, with the naked eye i.e. distinguishable, have a strong impact on matrix coating quality, reason is likely due to
So that DHB also there occurs ionization while atomization, the DHB of ionization there occurs reunion again for the presence of high voltage electric field,
In acetonitrile system, this effect is particularly evident, have impact on dispersiveness, or even defines crystallization group.Comprehensive above analysis, finally
System from acetonitrile/water=8/2 (V/V) carries out the spraying of DHB matrix.And all the time, host solvents spray for matrix
The impact applying effect is all a highly important factor.The matrix spraying instrument of any commercialization in use, is required for
Host solvents are explored, to reach the matrix spraying effect of optimum, and the selection of solvent will be followed " because of matrix
And different " principle.
Experimental example 5
Experiment condition：Biological tissue samples adopt normal mouse brain tissue slice, and matrix adopts DHB
(DHB).
Operating procedure：The commercial spray equipment (HST uMatrix Spotter) of laboratory apparatus, using routine operation to normally little
Murine brain section is sprayed, and gained host crystal micrograph is as shown in Figure 9.
As shown in Figure 9, compared with existing commercial apparatus, using the paint finishing gained spraying matrix crystallization (figure of the present invention
4) smaller, more homogeneous and simple to operate, acid and alkali resistance, salt, it is not easily blocked, be easy to safeguard.
Experimental example 6
Experiment condition：Biological tissue samples adopt normal mouse brain tissue slice, and matrix adopts DHB
(DHB), sheath gas (nitrogen) flow velocity is 50L/h, and in acetonitrile solution, acetonitrile and water volume ratio are 8:2, matrix solution dense
Spend for 30mg/mL, matrix solution flow velocity is 250 μ L/h, electric supply installation voltage is 4500V, spray time is 12min, hair
The distance between tubule lower end and sample stage are 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
Carry out MALDI-TOF-MS imaging analysis, result is as shown in figure 11.
Can be seen by Figure 11, very clearly mass spectrum imaging result can be obtained using the present invention, meet the requirement of experiment.
This result is closely bound up with the quality of DHB crystallization and forming process, not only illustrates this patent from the quality of crystal
The validity of invention instrument, more demonstrates the huge actual application value of this patent of invention from final experimental result.
In the description of this specification, reference term " embodiment ", " some embodiments ", " example ", " specific example ",
Or the description of " some examples " etc. means specific features with reference to this embodiment or example description, structure, material or feature bag
It is contained at least one embodiment or the example of the present invention.In this manual, to the schematic representation of above-mentioned term necessarily
It is directed to identical embodiment or example.And, the specific features of description, structure, material or feature can be arbitrary
Combine in an appropriate manner in individual or multiple embodiment or example.Additionally, in the case of not conflicting, the skill of this area
The feature of the different embodiments described in this specification or example and different embodiment or example can be combined by art personnel
And combination.
Although embodiments of the invention have been shown and described above it is to be understood that above-described embodiment is exemplary,
It is not considered as limiting the invention, those of ordinary skill in the art within the scope of the invention can be to above-described embodiment
It is changed, changes, replacing and modification.
Claims (20)
1. a kind of spraying system for MALDI-TOF-MS imaging is it is characterised in that include：
Injection device, described injection device is suitable to supply matrix solution；
Capillary, described capillary is connected with described injection device, and is suitable to supply described matrix solution to described capillary；
Electric supply installation, described electric supply installation is connected with described capillary, and is suitable to the matrix solution in described capillary is carried out
Atomization, to obtain matrix solution droplet；
Sheath tracheae, described sheath tracheae is set on described capillary；
Sheath air feed system, described sheath air feed system is connected with described sheath tracheae, and it is molten to described matrix to be suitable for use with sheath gas
Liquid mist is dripped and is sprayed；
Sample stage, described sample stage is movably disposed at the lower end of described capillary；And
Sample stage control device, described sample stage control device is connected with described sample stage, and is suitable to control described sample stage
Mobile.
2. system according to claim 1 is it is characterised in that described sheath tracheae includes connected the first pipeline section and second
Pipeline section.
3. system according to claim 2 is it is characterised in that described first pipeline section is polyfluortetraethylene pipe, described the
Two pipeline sections are plastic tube, rubber tube, glass tube or earthenware.
4. system according to claim 1 is it is characterised in that described matrix solution is matrix and the mixing of organic solvent
Solution.
5. system according to claim 4 it is characterised in that described matrix be selected from DHB, α-
Cyano group -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- dihydroxy
In benzoylformaldoxime, 2,6- resacetophenone, N- (5- nitro -2- pyridine radicals) -1,2- ethylenediamine and 3- aminoquinoline at least
One kind,
Described organic solvent is acetonitrile solution, and in described acetonitrile solution, acetonitrile and the volume ratio of water are 8：2.
6. system according to claim 4 it is characterised in that described matrix solution mass concentration be 1～50 milligram/
Milliliter.
7. system according to claim 6 it is characterised in that described matrix solution mass concentration be 30 milligrams/in the least
Rise.
8. system according to claim 1 is it is characterised in that the flow velocity of described sheath gas is 30～70 ls/h.
9. system according to claim 8 is it is characterised in that the flow velocity of described sheath gas is 50 ls/h.
10. system according to claim 1 is it is characterised in that between described capillary lower end and described sample stage
Distance is 4.5～10.5 centimetres.
11. systems according to claim 10 are it is characterised in that between described capillary lower end and described sample stage
Distance is 8.5 centimetres.
12. systems according to claim 1 are it is characterised in that the voltage of described electric supply installation is 3500～6000 volts.
13. systems according to claim 12 are it is characterised in that the voltage of described electric supply installation is 4500 volts.
14. systems according to claim 1 it is characterised in that described matrix solution flow velocity be 120～300 microlitres/
Hour.
15. systems according to claim 14 it is characterised in that described matrix solution flow velocity be 250 microlitres/little
When.
16. systems according to claim 1 it is characterised in that described capillary internal diameter be 50～150 microns, institute
The external diameter stating capillary is 150～250 microns.
17. systems according to claim 16 are it is characterised in that the internal diameter of described capillary is 100 microns, described
The external diameter of capillary is 200 microns.
18. systems according to claim 17 are it is characterised in that described capillary is conductive metal pipe.
19. systems according to claim 1 it is characterised in that described sheath gas be in nitrogen and argon gas at least
A kind of.
A kind of 20. methods of analysis biological specimen are it is characterised in that include：
(1) in biological specimen surface spraying matrix solution, so that matrix crystallization is formed on described biological specimen surface；And
(2) biological specimen that step (1) is obtained is analyzed,
Wherein, described step (1) is to ionize for Matrix Assisted Laser Desorption using described in any one of claim 1-19
The spraying system of mass spectrum imaging is carried out.
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