CN111982622A - Method for tissue sample mass spectrum imaging - Google Patents
Method for tissue sample mass spectrum imaging Download PDFInfo
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- CN111982622A CN111982622A CN202010775751.6A CN202010775751A CN111982622A CN 111982622 A CN111982622 A CN 111982622A CN 202010775751 A CN202010775751 A CN 202010775751A CN 111982622 A CN111982622 A CN 111982622A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
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- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
- G01N27/626—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using heat to ionise a gas
- G01N27/628—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using heat to ionise a gas and a beam of energy, e.g. laser enhanced ionisation
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Abstract
The invention relates to a method for tissue sample mass spectrum imaging, which comprises the following steps: (1) uniformly dispersing the monolayer graphene oxide in water by using ultrasound to form a dispersion liquid; (2) dripping the dispersion liquid on the surface of the conductive glass; (3) placing the conductive glass on a horizontal plane, and naturally drying to obtain an imaging film; (4) the tissue sample is placed on top of the imaging film, subjected to mass spectrometric detection and imaged. Compared with the prior art, the invention has the advantages of good film uniformity, good coating repeatability, simple manufacture, long storage time and the like.
Description
Technical Field
The invention relates to the field of mass spectrometry imaging, in particular to a method for mass spectrometry imaging of a tissue sample.
Background
Mass Spectrometry Imaging (MSI) is a powerful analytical tool that allows for the non-targeted study of the type of molecules in a sample and the spatial distribution of the sample. It has the ability to image thousands of molecules (e.g., metabolites, lipids, peptides, proteins and glycans) in a single experiment without labeling. Combining the information obtained from mass spectrometry with visualization of the spatial distribution of sample regions makes it a valuable chemical analysis tool for biological sample characterization.
The mass spectrum imaging method based on Matrix Assisted Laser Desorption Ionization (MALDI) mainly comprises the steps of sample preparation, matrix screening and deposition and imaging. Wherein the substrate is deposited by spraying the screened substrate on the surface of the sample mainly by a special spraying device, and the substrate should form particles with uniform size on the surface of the sample and be uniformly distributed on the surface of the sample. Since the uniformity of the substrate deposition is affected by the spray device, the uniformity of the substrate deposition will also directly affect the quality of the mass spectrometry imaging. To reduce the effect of the spray device on the uniformity of the deposition of the substrate, there is an increasing search for pre-coated or substrate substrates.
At present, a compressed matrix film (CMTF) method has been reported as a precoated matrix, and a sample analysis is performed by compressing a solid matrix into a film. Because no solvent is required, the crystallization process is bypassed, thereby reducing sample-to-sample variation due to crystallization. In addition, a porous silicon method is adopted, a sample to be detected is placed on the surface of the porous silicon for imaging analysis, and the sample is analyzed and ionized on the surface of the porous silicon, so that the matrix deposition step is avoided. There are also reports in the literature of the use of small-molecule organic substrates such as 2, 5-dihydroxybenzoic acid, α -cyano-4-hydroxycinnamic acid, etc. as precoated substrates, but special techniques are required to reduce the particle size of the crystals.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the method for tissue sample mass spectrometry imaging, which has the advantages of good film uniformity, good coating repeatability, simple manufacture and long storage time.
The purpose of the invention can be realized by the following technical scheme:
a method for using graphene oxide as a precoating substrate has not been reported. According to the invention, the adsorption effect of the matrix-single-layer graphene oxide dispersion liquid on the surface of the conductive glass of the tin oxide is utilized, so that the single-layer graphene oxide dispersion liquid forms a uniform film on the surface of the conductive glass, and the film has good uniformity, good repeatability, simple manufacture and long storage time, and can be well applied to the mass spectrometry imaging process, and the specific scheme is as follows:
a method of tissue sample mass spectral imaging, the method comprising the steps of:
(1) uniformly dispersing the monolayer graphene oxide in water by using ultrasound to form a dispersion liquid;
(2) dripping the dispersion liquid on the surface of the conductive glass;
(3) placing the conductive glass on a horizontal plane, and naturally drying to obtain an imaging film;
(4) the tissue sample is placed on top of the imaging film, subjected to mass spectrometric detection and imaged.
Further, the concentration of the single-layer graphene oxide in the dispersion liquid is 0.5-3 mg/mL.
Further, the concentration of the single-layer graphene oxide in the dispersion liquid is 2.5 mg/mL.
Furthermore, the conductive glass is indium tin oxide conductive glass.
Further, the ultrasonic power is 100-600W, and the time is 0.5-6 h.
Further, the specific steps of the step (2) are as follows:
(2-1) centrifuging the dispersion; cleaning the conductive glass by removing the large single-layer graphene oxide particles (2-2) with unstable dispersion; make the dispersion lay flat on the surface
And (2-3) dropwise applying the centrifuged dispersion liquid on the surface of the cleaned conductive glass.
Further, the rotation speed of the centrifugation is 10000-20000rpm, the times are 3-5 times, and the time of each centrifugation is 2.5-4 min.
Further, the cleaning is carried out by adopting a sodium hydroxide alcoholic solution.
Further, the drop volume is 0.1-0.9 ml.
Further, the drop volume was 0.3 ml.
The present invention uses graphene oxide as a pre-coating matrix. The pre-coated substrate is used here on the underside of the tissue section, i.e. on the top of the slide. At present, a spraying method of spraying graphene oxide on a tissue section has been reported, and a method of using a small molecular organic matrix, for example, 2, 5-dihydroxybenzoic acid, α -cyano-4-hydroxycinnamic acid, etc., as a precoated matrix has also been reported. However, a method of using graphene oxide as a precoating substrate has not been reported.
Compared with the prior art, the invention has the following advantages:
(1) the film uniformity is good. Unlike the small molecular matrix which is easy to crystallize into large particles, the graphene oxide is used as a nano material and has better surrounding uniformity;
(2) the coating repeatability is good. The very similar precoated glass slide can be obtained after multiple times of manufacture;
(3) the preparation is simple, the operation is easy, the storage time is long, and the graphene oxide is not easy to change.
Drawings
FIG. 1 is an image under an optical microscope of a pre-coated substrate film of the present invention;
FIG. 2 is an image under an electron microscope of a pre-coated substrate film of the present invention;
FIG. 3 is a graph showing the effect of absolute amount of matrix used on the signal intensity of mass spectra in the present invention;
FIG. 4 is a graph of the effect of the number of post-scan scans on the signal response of mass spectrometry for tissue samples covered in example 1;
FIG. 5 is an ion image of different lipids in rat brain tissue sections and an optical picture of Nissl staining in example 1;
FIG. 6 shows the imaging results of the same tissue in example 1 at different times and the imaging results of adjacent tissues;
FIG. 7 is a comparison of signal intensity of two experiments in which the SLGO film of example 1 is separated by one month;
FIG. 8 shows the MALDI-MS imaging results of one month storage of the tissue-coated SLGO film of example 1.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments.
A method of tissue sample mass spectral imaging, the method comprising the steps of:
(1) uniformly dispersing the monolayer graphene oxide in water by using ultrasound to form a dispersion liquid; the concentration of the single-layer graphene oxide in the dispersion liquid is 0.5-3mg/mL, the ultrasonic power is 100-600W, and the time is 0.5-6h
(2) Dripping the dispersion liquid on the surface of the conductive glass; the conductive glass is indium tin oxide conductive glass
(2-1) centrifuging the dispersion; so as to remove the large single-layer graphene oxide particles with unstable dispersion, the rotation speed of centrifugation is 10000-20000rpm, the times are 3-5 times, and the time of each centrifugation is 2.5-4 min.
(2-2) washing the conductive glass; make the dispersion lay flat on the surface
(2-3) dropwise coating the centrifuged dispersion liquid on the surface of the conductive glass cleaned by the sodium hydroxide alcoholic solution, wherein the dropwise coating volume is 0.1-0.9 ml.
(3) Placing the conductive glass on a horizontal plane, and naturally drying to obtain an imaging film;
(4) the tissue sample is placed on top of the imaging film, subjected to mass spectrometric detection and imaged.
The invention comprises the following raw materials: single-layer graphene oxide, as shown in FIGS. 1-2, (GO, 99.9 wt% purity, 0.335-1.2nm thickness, 300-500nm lamella diameter) was purchased from Suzhou Hengqiu NanoH. Indium tin oxide conductive glass slides were purchased from luoyang tengchang xu kun biotechnology limited (25 × 75 × 1.1mm specification). The tissue samples were from Sprague-Dawley rats (Experimental animals center of Chinese academy of sciences, Shanghai, China) weighing 200-.
The equipment used in the invention: SolariX model 7.0 Fourier transform ion cyclotron resonance Mass Spectrometry (FT-ICR-MS, Bruker, Switzerland). Scanning electron microscope (SEM, S-3400N type, Hitachi). PS-100A ultrasonic cleaner (Guandong Dongguan Jiekang Co.). Cryomicrotomes (Leica, Leider Lane Buffalo Grove, IL, USA). The magnification of the optical image is 500 times, and the magnification of the scanning electron microscope image is 500 times.
Different volumes (0.1mL, 0.3mL, 0.5mL, 0.7mL and 0.9mL) of uniformly dispersed single-layer graphene oxide solutions (0.5 mg/mL, 1mg/mL, 1.5mg/mL, 2mg/mL, 2.5mg/mL and 3mg/mL) with different concentrations are respectively absorbed and deposited on the surface of the treated indium tin oxide conductive glass slide (the effective area of deposition is 25mm multiplied by 55 mm). Four different chemicals (AMP, CAMP, Inosine, L-Phenylalanine) were spotted on the surface of the film in an amount of 2. mu.l each time, and dried in a desiccator and spotted twice. By mass spectrometry, the signal intensity response at each concentration is multiplied by its corresponding volume, and the resulting value is plotted as the abscissa and the signal intensity as the ordinate, to form a histogram, as shown in FIG. 3, which is a graph showing the effect of the absolute amount (mg) of matrix used on the signal intensity of the mass spectrum. It can be seen that the best mass spectral signal response can be obtained at absolute coating levels of 0.6-0.75 mg.
Example 1
The concentration of the single-layer graphene oxide is 2.5mg/mL, and the ultrasonic power is 600W for 4 h. Centrifugation was carried out at 10000rpm, 13000rpm, 10000rpm for 3min, respectively. The drop volume was 0.3 ml. The indium tin oxide conductive glass is cleaned by sodium hydroxide alcoholic solution to achieve the effect of spreading liquid drops. And (3) dripping 0.3mL of single-layer graphene oxide with the concentration of 2.5mg/mL on the surface of the cleaned indium tin oxide conductive glass, placing the indium tin oxide conductive glass on a horizontal desktop, and naturally drying the indium tin oxide conductive glass. Rat brain tissue was placed on top of the membrane directly into mass spectrometry detection and imaged.
The same position of the sample was chosen and the effect of different scan times (5, 10, 15, 20, 25, 30 respectively) on the mass spectrum signal intensity was examined, as shown in fig. 4, which fig. 4 shows the effect of scan times on the mass spectrum signal response. It can be seen that 25 scans gave a better mass spectral signal response.
Adjacent sections were taken for Nissl staining. The same rat brain tissue was imaged on the first and sixth days, respectively, and adjacent tissue to the above tissue was selected for imaging, as shown in fig. 5-6. Fig. 5 shows ion images of different lipids in rat brain tissue sections obtained with 100 μm spatial resolution and optical images of Nissl staining using a monolayer graphene oxide film as a pre-coated substrate matrix. Except for the lower right image which is an optical image for comparison, the rest are mass spectrum imaging images of different lipid components. In FIG. 6, A and B are the results of mass spectrometry imaging of the same slice at different times A) day one, B) day six, and C) the mass spectrometry imaging of adjacent slices.
Both figures show that the graphene oxide pre-coated matrix proposed using the present method can be used for mass spectrometric imaging of different components in tissue sections.
Four chemical samples, namely cholesterol, (1-hexadecanoyl-2) -octadecenoyl-sn-glycerol-3-phosphoric acid (sodium salt) (PA), 1, 2-dipalmitoyl-sn-glycerol-3-Phosphorylcholine (PC), 1, 2-dicinnamoyl-sn-glycerol-3-phosphatidylcholine (DMPC) and DPMC, were selected to evaluate the storage timeliness of the SLGO film (single-layer graphene oxide film) prepared in the same batch. The four chemical samples were tested twice, separated by one month. The tissue was placed on top of the single graphene oxide film and stored for one month for imaging analysis, as shown in fig. 7-8, fig. 7 is an evaluation of the aging of the SLGO film: comparison of signal intensity of two experiments separated by one month. FIG. 8 shows the MALDI-MS imaging results after one month storage of the organized SLGO film. Fig. 7-8 all show that the graphene oxide pre-coated matrix provided by the invention has long storage time, and can still be used for mass spectrometry imaging of different components in tissue slices after being stored for one month.
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.
Claims (10)
1. A method of tissue sample mass spectral imaging, the method comprising the steps of:
(1) uniformly dispersing the monolayer graphene oxide in water by using ultrasound to form a dispersion liquid;
(2) dripping the dispersion liquid on the surface of the conductive glass;
(3) placing the conductive glass on a horizontal plane, and naturally drying to obtain an imaging film;
(4) the tissue sample is placed on top of the imaging film, subjected to mass spectrometric detection and imaged.
2. The method according to claim 1, wherein the concentration of graphene oxide in the dispersion is 0.5-3 mg/mL.
3. The method according to claim 2, wherein the concentration of graphene oxide in the dispersion is 2.5 mg/mL.
4. The method according to claim 1, wherein the conductive glass is indium tin oxide conductive glass.
5. The method as claimed in claim 1, wherein the ultrasonic power is 100-600W and the time is 0.5-6 h.
6. The method for tissue sample mass spectrometry imaging according to claim 1, wherein the step (2) comprises the following specific steps:
(2-1) centrifuging the dispersion;
(2-2) washing the conductive glass;
and (2-3) dropwise applying the centrifuged dispersion liquid on the surface of the cleaned conductive glass.
7. The method as claimed in claim 6, wherein the rotation speed of centrifugation is 10000-20000rpm for 3-5 times, and the time of each centrifugation is 2.5-4 min.
8. The method of claim 6, wherein the washing is performed with an alcoholic solution of sodium hydroxide.
9. The method of claim 6, wherein the drop volume is 0.1-0.9 ml.
10. The method of claim 9, wherein the drop volume is 0.3 ml.
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