CN111239238A - Rapid mass spectrometry imaging method for tissue sample - Google Patents
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Abstract
The invention discloses a tissue sample rapid mass spectrometry imaging method. The method comprises the steps of S1, preparing a nano imprinting substrate; s2, sample imprinting: imprinting a sample on the nanoimprint substrate such that a tissue sample imprint is obtained on the nanoimprint substrate; s3, mass spectrum imaging: and irradiating the tissue sample blot by using a light source, and detecting by using a mass analyzer to obtain a two-dimensional mass spectrum imaging image with all chemical information on the surface of the tissue sample blot. According to the invention, the nano imprinting substrate is used for replacing the traditional imprinting substrate, and the substrate does not need to be sprayed after imprinting, so that the imaging result is real and reliable, and a false positive result is avoided; the preparation method of the nano imprinting substrate is simple, and the effects of in-situ imprinting and ionic signal enhancement can be realized; compared with the traditional imprinted PTFE material, the nano imprinted substrate has wide universality and application prospect, and can be widely used in a commercial mass spectrometer based on laser desorption/ionization.
Description
Technical Field
The invention relates to the field of mass spectrometry imaging, in particular to a rapid mass spectrometry imaging method for a tissue sample.
Background
Agriculture has had profound effects on human society and the natural environment for thousands of years, and the negative effects of increasing pesticide use levels on living environment, human health, bee survival, etc. have attracted worldwide attention (Larsen, a.e., gains, s.d. & Desch e nes, o.nat. commun.2017,8,302; Woodcock, b.a.et al.science2017,356, 1393-1395; Tsvetkov, n.et al.science2017,356, 1395-. Therefore, an in-depth understanding of absorption, distribution, metabolism and excretion (ADME) of pesticides is crucial to reducing the amount of pesticides and improving the efficiency of pesticides. Due to the lack of visual observation of the delivery and distribution of pesticide molecules in plant tissues by imaging technology, the study of pesticide absorption and long-distance transportation by different plant tissues has been a difficult point of research, and also has hindered the deep understanding of natural plant loading and transportation channels by scientists (Clark, R.D.Pest Man. Sci.2018,74, 1992-. Therefore, the development of a visual imaging technology capable of rapidly tracking the conductance trace of pesticide molecules and analyzing the conductance mechanism of pesticides is the center of research at present.
At present, the method for analyzing the plant tissue endogenous plant and pesticide mainly adopts methods such as chromatography, fluorescence spectrum, isotope labeling, mass spectrum imaging and the like. The traditional chromatographic analysis method is to lose the spatio-temporal distribution information of pesticides and endogenesis by extracting a large amount of plant tissues and determining the average value (Wu, h.et al.j.agric.food.chem.2012,60, 6088-. Fluorescence spectroscopy and isotope labeling imaging techniques can be used for monitoring the distribution of pesticide molecules in plant tissues, but the introduction of a fluorescent label may change the distribution of molecules to be detected, thereby affecting the accuracy of results (Li, d., Li, z., Chen, W. & Yang, x.j.agric.food.chem.2017,65, 4209-; isotopically labeled imaging techniques often require tedious, time-consuming sample pre-treatment procedures (Wang, j.et al.j.agric.food.chem.2014,62, 8791-8798). As a label-free and non-specific detection method, Mass Spectrometry (MSI) has been proven to be a highly sensitive and universal analysis tool that can provide visual distribution information of pesticide molecules and endogenous substances in plant tissues.
Sample preparation methods are a central problem for mass spectrometric analysis of plant or animal tissues (Dong, y.et.front.plant sci.2016,7, 60). At present, the tissue section method is alreadyIs widely used for sample treatment of tissues such as roots, stems and the like of plants, but because plant leaves have protection of a waxy layer and the thicknesses of leaves and petals are thin, samples cannot be prepared by a tissue slicing method, mass spectrometry analysis for plant petals and leaves is slow to develop (Bhandari, D.R. et al. analysis 2015,140,7696-,
A.&spengler, B.plant J.2014,80,161 and 171). Although the pesticide and endogenous molecules of leaves/petals can be directly detected using Desorption electrospray ionization (DESI), the surface relief and protection of the waxy layer of the sample often show "false positive" imaging results. To this end, the c.janfelt and t.pradeep subjects proposed blot imaging strategies based on porous Polytetrafluoroethylene (PTFE) and Thin Layer Chromatography (TLC) materials, respectively (Thunig, j., Hansen, S.H.&Janfelt,Anal.Chem.2011,83,3256-3259；Hemalatha,R.G.&Pradeep, t.j.agric.food.chem.2013,61, 7477-. MSI methods based on laser sampling techniques are widely used for their versatility and reliability, but imaging artifacts due to the low absorbance and surface charge accumulation effects of PTFE and TLC materials on lasers are not negligible.
Besides the preservation of the profile and distribution of endogenesis in tissue samples, imprinted imaging materials are also required to be able to detect endogenesis molecules with high sensitivity. The PTFE and TLC based imprinting materials described above can also be imprinted with additional matrix molecules to improve ionization efficiency, but the introduction of matrix molecules often results in a large number of interfering peaks in the spectra in low mass quantities, and non-uniform deposition of matrix can also result in false positive imaging results. Recently, materials with nanostructures can also be used to improve the ionization efficiency of molecules to be detected, but because such materials cannot be reused after imprinting and are expensive, they have very few applications in mass spectrometry imaging (plant/animal tissue imaging). The above disadvantages limit the existing methods to obtain the imprinting and mass spectrometry imaging information of plant/animal tissue samples in situ, rapidly and with high sensitivity.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a tissue sample rapid mass spectrometry imaging method. The method does not need to spray matrix molecules on the imprinting material, so that the efficiency is higher, and false positive results caused by uneven deposition of the matrix are avoided.
The above object of the present invention is achieved by the following technical solutions:
a method of rapid mass spectrometry imaging of a tissue sample comprising the steps of:
s1, preparing a nano imprinting substrate: soaking a material with liquid phase adsorption capacity in a nanoparticle dispersed phase to obtain the nano imprinting substrate;
s2, sample imprinting: imprinting a sample on the nano-imprinting substrate so as to obtain a tissue sample imprinting on the nano-imprinting substrate, wherein the tissue sample imprinting at least comprises a sample outline and chemical information of distribution of endogenous substances in the sample;
s3, mass spectrum imaging: and irradiating the tissue sample blot by using a light source, and detecting by using a mass analyzer to obtain a two-dimensional mass spectrum imaging image with all chemical information on the surface of the tissue sample blot.
According to the invention, the nano imprinting base material prepared by the method of S1 replaces the traditional imprinting base material, and the nano particles contained in the nano imprinting base material are uniformly embedded into the microporous structure of the imprinting base material, so that the nano particles have the effect of signal enhancement, the imaging result is real and reliable, and the ion signals are improved in an auxiliary manner without spraying matrix molecules on the surface of the imprinting base material, thereby overcoming the defect of imaging false image caused by poor uniformity of the traditional matrix spraying.
The nanoparticles can adopt any metal and/or non-metal nanoparticles having the effect of enhancing mass spectrum signals, and preferably, in the step S1, the nanoparticles are any one of Au, Ag, Cu, C, Ge or Si. Most preferably, in step s1, the nanoparticles are most preferably Au. The nanoparticles can be prepared by any of the known methods known to those skilled in the art. The most common dispersed phase of nanoparticles is a suspension of nanoparticles. As the liquid phase of the suspension, water is most common.
The shape of the nanoparticle can be any shape or a combination of multiple shapes, and the common shape of the nanoparticle is spherical, rod-shaped, star-shaped, nanowire-shaped, nanopore or nanowall. The morphology of the nanoparticles of the invention may be any one of the above or a combination of several of the above.
Preferably, in the step S1, the diameter of the nanoparticles in the nanoparticle dispersed phase is preferably 1-200 nm.
More preferably, in the step S1, the average particle diameter of the nanoparticles in the nanoparticle dispersed phase is preferably 20-30 nm.
Preferably, in the step S1, the nanoparticles preferably have a larger absorption wavelength in a light source with a wavelength of 125-1064 nm.
Preferably, in step s1, the material having the ability to adsorb liquid phase is most commonly available, namely paper. The paper can be common paper, filter paper or rice paper, and can be single-sided or double-sided paper with liquid phase adsorption capacity. As an easily realized way, the material having the ability to adsorb liquid phase is more preferably a filter paper, and the filter paper may be a qualitative or quantitative filter paper. Further, the material having the ability to adsorb a liquid phase is most preferably a quantitative slow filter paper. Preferably, in the step S1, the concentration of the nanoparticle dispersed phase is preferably 0.01-10 g/L, and the soaking time is preferably 5-360 min. More preferably, in the step S1, the concentration of the nanoparticle dispersed phase is more preferably 0.1-1 g/L, and the soaking time is more preferably 5-30 min.
Most preferably, in step S1, the concentration of the nanoparticle dispersed phase is most preferably 0.2g/L, and the soaking time is most preferably 20 min. Under the condition, the concentration signal of the molecule to be detected is stronger, and the signal intensity of the interference peak is lower.
As a specific preparation method, the nano-imprinting substrate can be prepared as follows: preparing a suspension with the Au nano particle concentration of 0.2g/L, putting paper into the suspension for soaking for 20min, taking out the paper, and drying at normal temperature to obtain the nano imprinting base material.
In step s2, the sample may be a liquid residue, a sediment, a colloid, an ionic liquid, etc., on a microorganism tissue, a plant tissue (such as a leaf, a bulb, a petal, etc.), an animal tissue (brain, muscle, kidney, etc.), etc., or a solid surface.
The principle of the mass spectrometry imaging method is similar to that of the conventional mass spectrometry imaging method, after the tissue sample print is prepared, light beams (including light beams generated by laser or other light sources) emitted by a light source are irradiated on the surface of the tissue sample print, and because the light beams and nano particles have a coupling enhancement effect, the desorption and ionization efficiency can be improved, so that ions can be generated under the condition of lower light beam energy. When the beam power density after the nanoparticle enhancement is greater than or equal to the desorption (or atomization) threshold of the sample, the sample molecules (or atoms) in the irradiation range are desorbed (or atomized) and ionized to generate ions due to the energy provided by the output light source. The ions are transmitted into a mass analyzer to be separated and detected, and a mass spectrogram containing molecular and atomic information at a sample sampling point is obtained.
Preferably, in step s3, the mass analyzer is a magnetic field analyzer, a quadrupole analyzer, an ion trap analyzer (including a linear ion trap, an orbital ion trap, a rectangular ion trap, etc.), a time-of-flight analyzer, a fourier transform analyzer.
Preferably, in step s3, the mass spectrometry obtains a two-dimensional mass spectrometry image with all chemical information of the surface of the tissue sample print by means of point-by-point scanning. The chemical information (molecular or element information) of the micro-area on the surface of the sample can be recorded better in a point-by-point scanning mode. The spatial resolution obtainable in this way can be on the micrometer or even nanometer scale and at the same time enables deep analysis of thin layers of molecules and elements.
For better spot-by-spot scanning, the tissue sample blot can be placed on an XY two-dimensional moving platform. The XY two-dimensional moving platform can be a platform adopting any driving mode (a manual mode, a motor driving mode, a piezoelectric ceramic mode, a piezoelectric motor mode and the like), and the control system can adopt a non-feedback or any feedback mode (a direct reading method, a grating ruler, a grating containing ruler and the like) to carry out position control.
In addition, the environment of the mass spectrometry imaging method of the invention can be high pressure, normal pressure, low vacuum or high vacuum, and other gases (such as air, nitrogen, argon, helium, hydrogen or the mixture of the above gases) can be used as auxiliary gases, and the pressure range is 1 × 10-7～1×108Pa。
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the nano imprinting substrate is used for replacing the traditional imprinting substrate, and the substrate does not need to be sprayed after imprinting, so that the imaging result is real and reliable, and a false positive result is avoided; the preparation method of the nano imprinting substrate is simple, and the effects of in-situ imprinting and ionic signal enhancement can be realized; compared with the traditional imprinted PTFE material, the nano imprinted substrate has wide universality and application prospect, and can be widely used in a commercial mass spectrometer based on laser desorption/ionization.
Drawings
Fig. 1 is a schematic diagram of an apparatus for implementing the mass spectrometry imaging method of the present invention, each labeled as: 1-nano imprinting substrate, 2-tissue sample imprinting, 3-light source, 4-ion, 5-mass analyzer and 6-XY two-dimensional moving platform;
FIG. 2 is a diagram showing the particle size distribution of gold nanoparticles in the nano-dispersed phase prepared in the example;
FIG. 3 is a comparison of mass spectra for molecular signal enhancement after soaking a nanoimprinted substrate (filter paper) in a nano-solution for different times;
FIG. 4 is a surface electron microscope image of a) a nano-imprinted substrate (filter paper) before soaking, for uniformity examination of nanoparticles on the filter paper after soaking; b) surface electron microscope images of the nano-imprinting substrate (filter paper) after soaking the nano-particle dispersion phase;
FIG. 5 is a comparison of mass spectra of different types of filter paper under the same conditions for molecular signal enhancement;
FIG. 6 is the result of mass spectrometry imaging of a leaf after imprinting on a nanoimprint substrate, a) leaf optical diagram; b) the imprinting optical diagram of the leaf blades on the nanopaper; c) mass spectrum imaging of the leaf source;
fig. 7 is the result of mass spectrometry imaging of cucumber petals after imprinting on nanoimprint substrate paper, a) petal optical map; b) imprinting the imprinting image on the nanometer paper; c-d) mass spectrum imaging of petal endogenous substances;
FIG. 8 is the result of mass spectrometry imaging of banana bulbs after imprinting on nanoimprint substrate paper, a) optical image of banana bulb profile; b) imprinting the imprinting image on the nanometer paper; c-f) imaging the mass spectrum of various endogenous substances of the banana corms.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It is to be understood that these examples are provided for illustrative purposes only and are not meant to limit the present invention. The following examples are examples of test methods in which specific conditions are not specified, and the apparatus is generally commercially available according to the conventional conditions. Unless otherwise indicated, percentages and parts are by weight.
The apparatus used in the mass spectrometry imaging method of the present invention can be referred to fig. 1.
After the nanoimprint substrate is prepared, plant tissue, such as leaves (fig. 6), is imprinted on the nanoimprint substrate to obtain a tissue sample imprint including profile and endogenesis distribution information, and then a light beam 3 (including a light beam generated by a laser or other light source) is irradiated on a micro-area of the tissue sample imprint surface on the nanoimprint substrate to generate ions 4, so as to realize micro-area sampling analysis. The ions are transmitted into a mass analyzer 5 to be separated and detected, and a mass spectrogram of molecular and atomic information contained in the sampling point of the tissue sample blot 2 to be detected is obtained. And then, all chemical information (molecular or element information) of the micro-area on the surface of the tissue sample print 2 can be recorded in situ in a point-by-point scanning mode through the XY two-dimensional moving platform 6, so that an XY two-dimensional mass spectrum imaging graph of all the chemical information on the surface of the tissue sample print is obtained. The spatial resolution which can be obtained by the method can reach the micrometer or even nanometer scale, and meanwhile, the deep analysis of the molecule and element thin layer can be realized.
In an embodiment, the preparation method of the nano-imprinting substrate specifically comprises the following steps:
and soaking the filter paper in the nanoparticle dispersed phase for 5-360 min, taking out and drying at room temperature to obtain the nanopaper.
The preparation method of the nanoparticle dispersed phase comprises the following steps: preparation of 0.01% by volume HAuCl4The aqueous solution was then heated to boiling and 2mL of a 1% by mass sodium citrate solution was added rapidly with high speed stirring, stirred for 10 minutes and then cooled to room temperature for further use. FIG. 2 is a diagram showing the particle size distribution of gold nanoparticles in the nanoparticle dispersed phase prepared in the present embodiment.
FIG. 3 reflects the effect of different soaking times of filter paper in the nanoparticle dispersed phase on the signal peak in chlorantraniliprole pesticide samples. The pesticide signal is not very obvious at 5min soaking time, such as molecular ion peak ([ M + Na ]]+) Pesticide debris peak ([ M-C)9H9N2OCl]+) Also weaker, the signal of the pesticide molecule gradually increases with time. However, when the soaking time is longer than 20min, a relatively obvious impurity interference peak appears in a spectrogram, and the spectrogram background is increased. Comprehensively considering, the soaking time is 20min as the optimal soaking time.
Fig. 5 reflects the influence of selecting different filter papers (in the figure, 1 is qualitative fast filter paper, 2 is qualitative medium speed filter paper, 3 is qualitative slow filter paper, 4 is quantitative fast filter paper, 5 is quantitative medium speed filter paper, and 6 is quantitative slow filter paper) on the signal in the chlorantraniliprole pesticide sample. The results show that the signal enhancement is best when using quantitative slow filter paper (6 in FIG. 5) as the nanopaper material, which can be selected from [ M + Na [ ]]+And [ M-C9H9N2OCl]+Mass spectrum signal intensity. This is probably because the pores inside the slow filter paper are smaller, and thus the gold nanoparticles can be better embedded, thereby enhancing the reinforcing effect. The impurities contained in the quantitative filter paper interfere less than the qualitative filter paper.
Fig. 6 reflects the mass spectrometry imaging results of the leaves after imprinting on the nanoimprint substrate paper. It can be seen from the figure that the leaf contour and even the vein lines can be completely marked on the nanometer paper, and the time-space distribution information of corresponding endogenous substances and pesticide molecules can be obtained.
Fig. 7 reflects the mass spectrometry imaging results of cucumber petals after imprinting on the nanoimprint substrate paper. The petal outlines can be completely imprinted on the nanopaper respectively, and mass spectrum imaging images of corresponding endogenous substances are obtained, and for sample systems which cannot be sliced to carry out mass spectrum, such as the leaves and the petals, the embodiment shows the wide application prospect of the method for mass spectrum imaging analysis of tissue samples, such as the leaves and the petals.
Fig. 8 reflects the mass spectrometry imaging results of banana bulbs after imprinting on the nanoimprint substrate paper. In the figure, the method can be respectively used for imprinting and imaging analysis of banana corms besides leaves and petals, so that the method covers the field of plant tissue analysis such as stems, leaves and petals, and the broad spectrum and universality of the method are proved. In combination with the advantages of mass spectrometry imaging technology, the method can provide multi-component information in a single analysis, greatly shortens the time for preparing and analyzing the sample, and is an important supplement to the existing analysis method.
The above embodiments are only one of the embodiments of the present invention, and any other changes, modifications, substitutions, combinations, and simplifications which are made without departing from the principles and spirit of the present invention are all equivalent replacements within the protection scope of the present invention.
Claims (10)
1. A method of rapid mass spectrometry imaging of a tissue sample, comprising the steps of:
s1, preparing a nano imprinting substrate: soaking a material with liquid phase adsorption capacity in a nanoparticle dispersed phase to obtain the nano imprinting substrate;
s2, sample imprinting: imprinting a tissue sample on the nanoimprint substrate such that a tissue sample imprint is obtained on the nanoimprint substrate, the tissue sample imprint comprising at least chemical information of sample profile, sample endogenous substance distribution.
S3, mass spectrum imaging: and irradiating the tissue sample blot by using a light source, and detecting by using a mass analyzer to obtain a two-dimensional mass spectrum imaging image with all chemical information on the surface of the tissue sample blot.
2. The method for rapid mass spectrometry imaging of a tissue sample according to claim 1, wherein in step s1, the nanoparticles are any one of Au, Ag, Cu, C, Ge or Si.
3. The method for rapid mass spectrometry imaging of a tissue sample according to claim 1, wherein in step S1, the concentration of the nanoparticle dispersed phase is 0.01-10 g/L, and the soaking time is 5-360 min.
4. The method for rapid mass spectrometry imaging of tissue samples according to claim 1, wherein in step s1, the concentration of the nanoparticle dispersed phase is 0.2g/L, and the soaking time is 20 min.
5. The method for rapid mass spectrometry imaging of tissue samples according to claim 1, wherein in step S1, the diameter distribution of the nanoparticles in the nanoparticle dispersed phase is 1-200 nm.
6. The method for rapid mass spectrometry imaging of tissue samples according to claim 1, wherein in step s1. the material having the ability to adsorb liquid phase is paper.
7. The method for rapid mass spectrometry imaging of tissue samples according to claim 1, wherein in step s1, the morphology of the nanoparticles is spherical, rod-like, star-like, nanowire-like, nanopore and/or nanowall.
8. The method for rapid mass spectrometry imaging of tissue samples according to claim 1, wherein in step S1. the nanoparticles have a larger absorption wavelength at a light source with a wavelength between 125 nm and 1064 nm.
9. The method of rapid mass spectrometry imaging of tissue samples according to claim 1, wherein in step s3. the mass analyzer is a magnetic field analyzer, a quadrupole analyzer, an ion trap analyzer, a time-of-flight analyzer, a fourier transform analyzer.
10. The method for rapid mass spectrometry imaging of tissue samples according to claim 1, wherein in step s3, the mass spectrometry imaging obtains a two-dimensional mass spectrometry imaging map with all chemical information of the imprinted surface of the tissue sample by means of point-by-point scanning.
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