阅读说明：本技术 制备maldi-tof ms成像基质的超声喷雾装置和方法及应用 (Ultrasonic spraying device and method for preparing MALDI-TOF MS imaging matrix and application ) 是由 聂宗秀 黄熹 占铃鹏 孙洁 刘会会 于 2018-06-08 设计创作，主要内容包括：本发明涉及基质喷涂领域,公开了制备MALDI-TOF MS成像基质的超声喷雾装置和方法及应用。一种制备MALDI-TOF MS成像基质的超声喷雾装置,该装置包括：溶液池(1)、置于溶液池(1)的底座凹槽内的雾化片(2),以及与所述雾化片(2)通过电线连接的控制器(3)；其中,所述溶液池(1)用于盛放含有基质的喷涂液,所述控制器(3)发生电子高频震荡使所述雾化片(2)产生高频谐振,将所述喷涂液打散形成雾滴。采用本发明的超声喷雾装置进行基质喷涂具有成本低廉,制备的MALDI-TOF MS成像基质的晶粒致密细小的优点,能够得到高分辨率的MALDI-TOF MS成像效果。(The invention relates to the field of matrix spraying, and discloses an ultrasonic spraying device and method for preparing a MALDI-TOF MS imaging matrix and application of the ultrasonic spraying device and method. An ultrasonic spray apparatus for preparing a MALDI-TOF MS imaging matrix, the apparatus comprising: the device comprises a solution pool (1), an atomizing sheet (2) arranged in a groove of a base of the solution pool (1), and a controller (3) connected with the atomizing sheet (2) through a wire; the solution pool (1) is used for containing spraying liquid containing a matrix, and the controller (3) generates electronic high-frequency oscillation to enable the atomizing plate (2) to generate high-frequency resonance and break up the spraying liquid to form fog drops. The ultrasonic spraying device for matrix spraying has the advantages of low cost and compact and fine crystal grains of the prepared MALDI-TOF MS imaging matrix, and can obtain the MALDI-TOF MS imaging effect with high resolution.)

1. An ultrasonic spray apparatus for preparing a matrix for MALDI-TOF MS imaging, the apparatus comprising: the device comprises a solution pool (1), an atomizing sheet (2) arranged in a groove of a base of the solution pool (1), and a controller (3) connected with the atomizing sheet (2) through a wire;

The solution pool (1) is used for containing spraying liquid containing a matrix, and the controller (3) generates electronic high-frequency oscillation to enable the atomizing plate (2) to generate high-frequency resonance and break up the spraying liquid to form fog drops.

2. The ultrasonic spraying device according to claim 1, wherein the diameter of the atomizing plate (2) is 10-25mm, and the diameter of the micropores of the atomizing plate is 0.5-5 μm.

3. The ultrasonic spraying device according to claim 1, wherein the atomizing plate (2) is fixed with the solution tank (1) by a nozzle ferrule (4).

4. A method for preparing a MALDI-TOF MS imaging matrix from an ultrasonic spray device according to any one of claims 1 to 3, comprising the steps of: and (3) connecting the atomizing sheet (2) with the controller (3) through an electric wire, starting the controller (3), spraying the spraying liquid containing the matrix in the solution pool (1) onto the surface of the sample slice to be detected through the atomizing sheet (2), and forming the MALDI-TOF MS imaging matrix on the surface of the sample slice to be detected.

5. The method of claim 4, wherein the spray coating liquid further comprises a solvent; preferably, the solvent is one or more of methanol, ethanol, water and acetonitrile.

6. a method according to claim 4 or 5, wherein the concentration of matrix in the spray solution is 4-15 mg/ml;

Preferably, the substrate is 1, 5-dihydroxybenzoic acid, 1, 5-naphthalenediamine hydrochloride or naphthylethylenediamine hydrochloride.

7. The method of claim 4, wherein the sample slice to be tested is an animal sample slice, a plant sample slice, a bacterial sample slice or a fingerprint sample slice.

8. The method of claim 7, wherein the sample slice to be tested contains a biomolecule; preferably, the biomolecule has a molecular weight of less than 1000 Da;

preferably, the biomolecule is one or more of a polypeptide, an oligotitanium, an organic acid, adenosine phosphate, glucose, a fatty acid, a phosphate, and a sulfatide;

preferably, the organic acid is taurine and/or a fatty acid;

Preferably, the polypeptide is glutathione.

9. The method according to claim 4, wherein the distance between the sample section to be tested and the nebulization plate (2) is greater than 5cm, preferably 5-20 cm.

10. Use of an ultrasonic spray device for the preparation of a MALDI-TOF MS imaging matrix according to any one of claims 1 to 3 or a method according to any one of claims 4 to 9 in metabolomics, oncology and botany.

Technical Field

The invention relates to the field of matrix spraying, in particular to an ultrasonic spraying device and method for preparing a MALDI-TOF MS imaging matrix and application of the ultrasonic spraying device and method.

background

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is a soft ionization biological mass spectrum developed in recent years (reported by Karas et al and Tanaka et al 1988), and the principle of MALDI-TOF MS is that laser is used for irradiating cocrystal formed by matrix and sample molecules, the matrix absorbs energy from the laser and transfers the energy to the biomolecules, so that the biomolecules are desorbed and ionized, and the ionization process is realized. A data acquisition process of MALDI-TOF MS includes firstly dropping a mixed solution of a sample and a matrix on a sample plate, drying and crystallizing in air (or vacuum), then sending the sample plate into a vacuum sample bin, ionizing the matrix and the sample by pulse laser, starting acceleration of generated molecular ions under the action of acceleration voltage, accelerating ions with different mass-to-charge ratios to different speeds, then entering a flight tube, determining the mass-to-charge ratio of the molecular ions according to the relation between the flight time and the mass-to-charge ratio because the flight tube type high vacuum non-field area is provided and the ions move at a constant speed, so that the distance is constant, the time (flight time) for the ions with different mass-to-charge ratios to reach a detector is different, and finally outputting signals by a digital control system to obtain a mass spectrum of the mass spectrum. When the laser of the MALDI ion source is used for carrying out two-dimensional point-by-point scanning on the biological section, different molecular weight molecules of each scanning point are detected by a mass spectrometer, and a two-dimensional heat map is carried out on the biological molecules with a certain molecular weight according to the detected signal intensity, so that a MALDI-TOFMS imaging graph with space distribution characteristics can be obtained.

To obtain high quality MALDI-TOF MS imaging results, it is necessary to spray the matrix uniformly onto the biological sections. Both the crystal size of the matrix and the crystallization process can have a decisive influence on the imaging quality. Generally, the denser and finer the grains of the matrix, the stronger the mass spectral peak signal intensity, and the higher the imaging resolution that can be supported. In addition, the more the solvent in which the substrate is dissolved is in contact with the chips, the better the extraction effect on the biomolecules, and the higher the signal intensity can be. At present, the main methods for spraying the substrate include sublimation method, art spray gun method, pulse type vibration spray method, electrospray method, and the like. Substrate grains sprayed by the art spray gun method and pulsed vibro-electrospray tend to be large (greater than 50 microns in size) and are not suitable if imaging experiments are to be conducted with a resolution better than 50 microns. The electrospraying method can obtain slices with the particle size of about 10 micrometers, but the instrument based on the principle needs to use a high-voltage power supply and high-voltage sheath gas, and the price is very high. Therefore, it is desirable to develop a cheap and efficient matrix spray method that yields high quality MALDI-TOF MS imaging results.

Disclosure of Invention

The invention aims to overcome the problems of large matrix crystal grain, low signal intensity, high cost and poor MALDI-TOF MS imaging effect in the prior art, and provides an ultrasonic spraying device and a method for preparing the MALDI-TOF MS imaging matrix and application.

In order to achieve the above object, the present invention provides in a first aspect an ultrasonic spray device for preparing a matrix for MALDI-TOF MS imaging, wherein the device comprises: the device comprises a solution pool 1, an atomizing sheet 2 arranged in a groove of a base of the solution pool 1, and a controller 3 connected with the atomizing sheet 2 through a wire;

The solution tank 1 is used for containing spraying liquid containing a matrix, and the controller 3 generates electronic high-frequency oscillation to enable the atomizing plate 2 to generate high-frequency resonance and break up the spraying liquid to form fog drops.

In a second aspect, the present invention provides a method for preparing a MALDI-TOF MS imaging matrix from the ultrasonic spray device described above, comprising the steps of: and connecting the atomizing plate 2 with the controller 3 through an electric wire, starting the controller 3, spraying the spraying liquid containing the matrix in the solution pool 1 onto the surface of the sample slice to be detected through the atomizing plate 2, and forming the MALDI-TOF MS imaging matrix on the surface of the sample slice to be detected.

In a third aspect, the invention provides the use of an ultrasonic spray device as described above for the preparation of a MALDI-TOF MS imaging matrix or a method as described above in metabolomics, oncology and botany.

According to the technical scheme, a high-voltage power supply and a sheath gas are not needed, equipment and material consumption cost is reduced, the prepared MALDI-TOF MS imaging matrix is compact and fine in crystalline grain, the defects that the crystalline grain is large and the signal intensity is not high in the traditional spray coating process are overcome, and the MALDI-TOF MS imaging effect with high resolution can be obtained.

drawings

FIG. 1 is a schematic diagram of an ultrasonic spray apparatus for preparing a MALDI-TOF MS imaging matrix according to the present invention;

FIG. 2a is a photograph of a co-crystal of the MALDI-TOF MS imaging matrix of example 1 with a sample;

FIG. 2b is a photograph of a co-crystal of the MALDI-TOF MS imaging matrix of example 2 with a sample;

FIG. 2c is a photograph of a co-crystal of the MALDI-TOF MS imaging matrix of comparative example 1 with a sample;

FIG. 2d is a photograph of a co-crystal of the MALDI-TOF MS imaging matrix of comparative example 2 with a sample;

FIG. 3a is a graph of positive ion whole brain HE staining and cerebellum HE staining;

FIG. 3b is a picture of positive ion MALDI-TOF MS imaging of example 1;

FIG. 3c is a picture of positive ion MALDI-TOF MS imaging of comparative example 1;

FIG. 3d is a plot of the mass spectrum signal of positive ion MALDI-TOF MS;

FIG. 4a is a graph of negative ion whole brain HE staining and cerebellum HE staining;

FIG. 4b is a picture of negative ion MALDI-TOF MS imaging of example 2;

FIG. 4c is a picture of negative ion MALDI-TOF MS imaging of comparative example 2;

FIG. 4d is a graph of the mass spectrum signal of negative ion MALDI-TOF MS.

description of the reference numerals

1. solution pool 2, atomizing piece

3. controller 4, shower nozzle cutting ferrule

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

in a first aspect, the present invention provides an ultrasonic spray apparatus for preparing a matrix for MALDI-TOF MS imaging, as shown in figure 1, wherein the apparatus comprises: the device comprises a solution pool 1, an atomizing sheet 2 arranged in a groove of a base of the solution pool 1, and a controller 3 connected with the atomizing sheet 2 through a wire;

The solution tank 1 is used for containing spraying liquid containing a matrix, and the controller 3 generates electronic high-frequency oscillation to enable the atomizing plate 2 to generate high-frequency resonance and break up the spraying liquid to form fog drops.

In the invention, the solution tank 1 is provided with a base, a groove is arranged in the base, the atomizing plate 2 is arranged in the groove, and the spraying liquid in the solution tank 1 can flow into the atomizing plate 2.

In the invention, the controller 3 generates electronic high-frequency oscillation, and the atomizing plate 2 breaks up the spraying liquid to form fog drops by utilizing the electronic high-frequency oscillation generated by the controller 3.

In the present invention, the diameter of the atomizing plate 2 is determined as required, but is not limited to 10 to 25mm, for example, 10mm, 12mm, 15mm, 17mm, 20mm, 22mm, 25mm, and any value in the range of any two of these values. The diameter of the pores of the atomizing sheet may also be as desired, and may be from 0.5 to 5 μm, for example, 0.5 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, and any value within the range of any two of these values.

In the present invention, the atomizing plate 2 and the solution tank 1 can be fixed by a nozzle sleeve 4, but the solid manner is not limited thereto.

In the invention, time-of-flight mass spectrometry (TOF MS) is used as an analysis means, but the ultrasonic spraying device of the MALDI-TOF MS imaging matrix can also be used together with other mass analyzers.

In a second aspect, the present invention provides a method for preparing a MALDI-TOF MS imaging matrix from the ultrasonic spray device described above, comprising the steps of: and connecting the atomizing plate 2 with the controller 3 through an electric wire, starting the controller 3, spraying the spraying liquid containing the matrix in the solution pool 1 onto the surface of the sample slice to be detected through the atomizing plate 2, and forming the MALDI-TOF MS imaging matrix on the surface of the sample slice to be detected.

according to the method of the present invention, the spraying liquid further contains a solvent, wherein the solvent is used for compatibility with the sample to be tested, and may be one or more of methanol, ethanol, water and acetonitrile, but is not limited thereto.

According to the method of the invention, the concentration of the matrix in the spray liquid may be, but is not limited to: 4-15 mg/ml. Preferably, the substrate is 1, 5-dihydroxybenzoic acid, 1, 5-naphthalenediamine hydrochloride or naphthylethylenediamine hydrochloride. However, the matrix is not limited thereto, and the purpose is to form a matrix for MALDI-TOF MS imaging.

According to the method of the present invention, the sample slice to be tested may be, but is not limited to: animal sample slices, plant sample slices, bacterial sample slices, or fingerprint sample slices.

According to the method of the invention, the sample section to be tested contains a biomolecule, preferably having a molecular weight of less than 1000 Da. In particular, the biomolecule may be one or more of a polypeptide, an oligotitanium, an organic acid, adenosine phosphate, glucose, a fatty acid, a phosphate, and a sulfatide. Wherein, the organic acid can be taurine and/or fatty acid; the polypeptide may be glutathione.

According to the method of the invention, when the spraying liquid containing the matrix in the solution pool 1 is sprayed to the surface of the sample slice to be tested through the atomizing plate 2, the distance between the sample slice to be tested and the atomizing plate 2 is more than 5cm, preferably 5-20 cm. I.e. the spraying distance is greater than 5cm, preferably 5-20 cm.

According to the method of the invention, the spraying amount of the spraying liquid is such that the coating amount for forming the MALDI-TOF MS imaging matrix on the surface of the sample slice to be tested is 1-10 mg/glass plate (i.e., 1-10mg of matrix is coated on each glass plate, wherein the size of the glass plate can be, for example, 76mm long, 26mm wide and 1mm thick). For different sample slices to be detected, namely different biomolecules, when the coating amount of the matrix is different, the obtained MALDI-TOF MS imaging effect is also different, so that the aim of obtaining the matrix spraying amount with the best imaging effect is fulfilled.

For example, the images of the co-crystallization of the MALDI-TOF MS imaging matrix and the sample of the invention are shown in FIG. 2a and FIG. 2b, while the images of the co-crystallization of the MALDI-TOF MS imaging matrix and the sample of the prior art are shown in FIG. 2c and FIG. 2d, and the comparison shows that the co-crystallization prepared by the ultrasonic spraying device of the invention has better imaging effect.

In a third aspect, the invention provides the use of an ultrasonic spray device as described above for the preparation of a MALDI-TOF MS imaging matrix or a method as described above in metabolomics, oncology and botany.

The present invention will be described in detail below by way of examples.

in the following examples, the atomizing sheet was purchased from the Dow electronics Co., Ltd, and the diameter of the atomizing sheet was 16mm, and the diameter of the fine pores of the atomizing sheet was 3 μm;

The controller is purchased from British electronic Co., Ltd, the oscillation frequency is 110kHz, and the driving voltage is 5V;

Optical microscopes available from olympus under model number IX 73;

MALDI-TOF MS was purchased from Bruk corporation under the model UltraFlextreme;

A commercially available pulsed matrix nebulizer was purchased from Bruker under the model Imageprep.