CN113655049A - Imaging detection method for nano pollutants in organism - Google Patents
Imaging detection method for nano pollutants in organism Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
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- Health & Medical Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Physics & Mathematics (AREA)
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- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention provides an imaging detection method of nano pollutants in organisms, which combines organic micromolecules with characteristic Raman fingerprints with the nano pollutants and is matched with a proper biological fixation method, so that the nano pollutants in the organisms can be well detected, and the spatial distribution of the pollutants can be observed. The method can realize the rapid, in-situ and quantitative analysis of the nano pollutants in the organisms, truly reflect the distribution of the nano pollutants in the organisms in the environment, and reveal the migration and transformation process of the nano pollutants in the actual environment, thereby evaluating the biological safety risk of the nano pollutants.
Description
Technical Field
The invention relates to the field of nano pollutant analysis and detection, in particular to an imaging detection method for nano pollutants in a living body.
Background
The nano material has excellent optical, electric, thermal, magnetic and other properties, and is widely applied to the industries of biological medical treatment, electronic devices, environmental remediation, food packaging and the like. However, a large amount of nano materials enter the environment after being used, potential toxicity is caused to environmental water bodies and organisms, and the concentration in the environment can reach ppb-ppm level. The current method for detecting the nanometer material in the organism mainly comprises the following steps: scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM), nanoscale secondary ion mass spectrometry (nanoSIMS), laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), fluorescence microscope, and the like. However, these methods require complex pre-treatment (such as fluorescent modification) on the sample, and cannot perform in-situ detection, and have certain requirements on the size of the sample, cannot detect various nano-pollutants, and cannot realize convenient and fast imaging detection, and the fluorescent modification can change the surface properties of the nano-materials, thereby affecting the real distribution of the nano-materials in the living body.
Surface Enhanced Raman Spectroscopy (SERS) is a detection method that utilizes the surface plasmon resonance effect of noble metals (Au, Ag, Pt, etc.) to enhance the raman signal of its surface species. SERS has the advantages of in-situ, rapid detection, good biocompatibility, simple sample treatment and the like. The method is widely applied to the fields of medical cancer treatment, biosensing, food safety detection and the like.
The SERS technology is applied to the field of environment, the distribution and migration process of nano pollutants in an environmental water body in an organism can be detected, the biotoxicity of the nano pollutants is researched, and the biological safety risk of the nano pollutants is evaluated. However, the types of nanoparticles in the environment are complex, the shapes and the sizes are various, and a corresponding SERS system is difficult to construct for detection. Therefore, a method for detecting nano-pollutants in organisms by using SERS (surface enhanced Raman Scattering) is not developed at present.
Disclosure of Invention
In view of the problems in the prior art, the invention provides an imaging detection method for nano-pollutants in a living body, which can rapidly detect a plurality of nano-pollutants in the living body in situ and observe the spatial distribution of the nano-pollutants in the living body. The invention can truly reflect the distribution of the nano-pollutants in the organism in the environment, realize the rapid, in-situ and quantitative analysis of the nano-pollutants in the organism and evaluate the biological safety risk of the nano-pollutants.
In order to solve the technical problem, the invention adopts the following technical scheme:
an imaging detection method of nano-pollutants in organisms comprises the following steps:
(1) coating Raman active molecules on the surfaces of the nano particles, and then coating nano pollutants to be detected to obtain core-shell structure nano pollutants;
adding the core-shell structure nano pollutant into a biological culture solution, and culturing to ensure that the core-shell structure nano pollutant is taken in a living body; after the culture is finished, taking out the polluted organisms and fixing the polluted organisms on a glass slide to obtain a pretreated sample;
(2) performing Raman detection and imaging on the pretreated sample by using a laser micro-Raman spectrometer to obtain a Raman spectrum of the pretreated sample; and then processing the Raman spectrum through imaging software according to the characteristic peak of the Raman active molecule to obtain a Raman imaging graph of the nano-pollutants, namely obtaining the spatial distribution of the nano-pollutants in the organism. In the Raman imaging image, different colors represent the abundance of the nano-pollutants at the point, and the obtained Raman image is superposed with the biological optical photograph to obtain the spatial distribution of the nano-pollutants in the organism.
Further, the nanoparticles are gold nanoparticles or silver nanoparticles.
Further, the raman-active molecule is at least one of crystal violet, rhodamine 6G, DTTC, DTDC, IR-775 chloride, p-nitrobenzophenol, p-aminophenol and p-dithiol. The choice of raman-active molecules is very important: firstly, Raman molecules can be well combined on the surface of the nano particles and combined with nano pollutants; in addition, a specific raman molecule needs to be selected so that the nano-pollutants can uniformly grow on the surface of the nano-particles, and the stability of the raman molecule and the uniform appearance of the nano-particles are ensured. The invention utilizes the combination of the organic micromolecules with characteristic Raman fingerprints and the nano pollutants, can well detect the nano pollutants in organisms and observe the spatial distribution of the pollutants.
Further, the nano pollutants are Ag and SiO2、TiO2、MnO2And a nano plastic.
Further, the organism is a plant (such as duckweed, etc.), a microorganism (such as Escherichia coli, etc.) or an animal (such as a nematode, etc.).
Further, the preparation method of the core-shell structure nano pollutant comprises the following steps:
step 2, dispersing the nano particles in a solvent containing a surface stabilizer to obtain a nano particle dispersion liquid; then adding the Raman active molecule dispersion liquid, stirring for 20min-2h at 25-50 ℃, centrifuging, washing, adding a solvent containing a surface stabilizer, and re-suspending to the original volume to obtain the nano-particle/Raman active molecule dispersion liquid;
and 3, adding the precursor solution into the nanoparticle/Raman active molecule dispersion solution, performing hydrothermal reaction, centrifuging, washing, and dispersing with water to obtain the core-shell structure nano pollutant.
Further, the conditions of the hydrothermal reaction and the types of the surface stabilizer, the solvent and the precursor are determined according to the type of the nano-pollutants to be detected, such as: when the nano pollutant is Ag, the hydrothermal reaction temperature is 70 ℃, the time is 3 hours, the surface stabilizer is hexadecyl trimethyl ammonium chloride, the solvent is water, and the precursor is silver nitrate and ascorbic acid; when the nano pollutant is SiO2The hydrothermal reaction temperature is 25 ℃, the time is 36 hours, the surface stabilizer is 3-aminopropyl triethoxysilane or 3-mercaptopropyl trimethoxysilane, the solvent is ethanol, and the precursor is tetraethoxysilane; when the nano-pollutant is TiO2The hydrothermal reaction temperature is 25 ℃, the time is 1h, the surface stabilizer is hexadecyl trimethyl ammonium chloride, the solvent is water, and the precursor is butyl titanate; when the nano pollutant is MnO2The hydrothermal reaction temperature is 25 ℃, the time is 0.5h, the surface stabilizer is polyvinylpyrrolidone, the solvent is water, and the precursor is potassium permanganate; when the nano-pollutants are nano-plastics, the hydrothermal reaction temperature is 70 ℃, the time is 1.5h, the surface stabilizer is sodium dodecyl sulfate, the solvent is water, and the precursor is divinylbenzene and styrene.
Further: the concentration of the precursor solution is 5-50 mM; the concentration of the surface stabilizer in the solvent containing the surface stabilizer is 25-50 mM; the concentration of the nanoparticles in the nanoparticle dispersion liquid is 1-10 nM; the concentration of the Raman active molecule dispersion liquid is 0.1-1mM, and the used solvent is at least one of ethanol, DMSO and DMF; the volume ratio of the nanoparticle dispersion liquid to the Raman active molecule dispersion liquid is 10-100: 1; the volume ratio of the nanoparticle/Raman active molecule dispersion liquid to the precursor solution is 10-100: 1.
Further, the fixing method comprises the following steps: taking out the polluted organisms, transferring the polluted organisms onto a glass slide, dripping a fixing solution, and fixing for 10-60 min at 4-25 ℃; the fixing liquid is formed by mixing glycerol or 1, 2-propylene glycol and water according to the mass ratio of 1: 0-4. The choice of fixative is critical to the processing of the sample: the sample needs to be kept moisture in the fixing process, so that biological shrinkage and morphological change which affect subsequent detection are avoided; meanwhile, the addition of the fixing liquid does not influence the biological Raman signal, and the accuracy of biological imaging is ensured.
Further, the conditions of the raman detection in step 2) are as follows:
adopting 633nm or 785nm laser with laser energy of 10-100%;
the grating is 600lines/mm or 1800 lines/mm;
the spatial resolution is 1-50 mu m/pixel, and the single-pixel acquisition time is 0.001-1 s;
the spectrum collection range is 500-2000 cm-1；
Other parameters are system default values.
Compared with the prior art, the invention has the beneficial effects that:
1. the method can realize the rapid, in-situ and quantitative analysis of the nano pollutants in the organisms, truly reflect the distribution of the nano pollutants in the organisms in the environment, and reveal the migration and transformation process of the nano pollutants in the actual environment, thereby evaluating the biological safety risk of the nano pollutants.
2. The method of the invention has no limit on the size of the living beings, can detect living beings with different sizes and types, and can detect nano pollutants with various types, different shapes and different charges at the same time.
3. The invention simplifies the complicated sample pretreatment steps of the traditional method, and the nano pollutants do not need to be marked by external sources and can reflect the real distribution of the nano pollutants in organisms.
Drawings
Fig. 1 is a TEM image of core-shell structured nano Ag contaminants prepared in example 1 of the present invention.
Fig. 2 is a raman spectrum of the core-shell structured nano Ag contaminant prepared in example 1 of the present invention.
FIG. 3 is a Raman image of Escherichia coli contaminated with Ag nanoparticles in example 1 of the present invention, wherein the size of the Raman image is 2 μm.
FIG. 4 shows core-shell structure nano SiO prepared in example 2 of the present invention2TEM images of contaminants.
FIG. 5 shows the core-shell structure of nano SiO prepared in example 2 of the present invention2Raman spectra of contaminants.
FIG. 6 shows a nano SiO solid in example 2 of the present invention2Raman images of contaminated Spirodela delavayi, all with scale of 1 mm.
Fig. 7 is a TEM image of the core-shell structured nano Ag contaminant prepared in example 3 of the present invention.
Fig. 8 is a raman spectrum of the core-shell structured nano Ag contaminant prepared in example 3 of the present invention.
FIG. 9 is a Raman image of nematodes contaminated with Ag nanoparticles of example 3 of the present invention, each at 100 μm scale.
FIG. 10 shows the core-shell structure of nano SiO prepared in example 4 of the present invention2TEM image of (+) contaminants.
FIG. 11 shows the core-shell structure of nano SiO prepared in example 4 of the present invention2(-) - (upper diagram) and SiO2Raman spectra of (+) contaminants (lower panel).
FIG. 12 shows simultaneous exposure to nano SiO in example 4 of the present invention2(-) and nano SiO2Raman images of (+) contaminated Spirodela polyrhiza, all 100 μm on scale.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The source of the raw materials used in the following examples is not particularly limited, and may be generally commercially available.
The biological culture methods in the following examples are all standard methods.
The Raman spectrometer used in the following examples was a laser confocal micro-Raman spectrometer (HORIBA Co., Japan).
Example 1 distribution of Nano Ag in E.coli
50mM silver nitrate aqueous solution and 50mM ascorbic acid aqueous solution were prepared, respectively.
Dispersing gold nanoparticles in 10mL of water containing 50mM hexadecyl trimethyl ammonium chloride at the concentration of 1nM to obtain a gold nanoparticle dispersion liquid; then, 100 mu L of 0.1mM p-aminophenol ethanol solution is added dropwise, the mixture is stirred for 20min at 50 ℃, centrifuged for 10min at 9000rpm, washed by water containing 50mM hexadecyl trimethyl ammonium chloride, and finally added with the water containing 50mM hexadecyl trimethyl ammonium chloride to be resuspended to 10mL, so that gold nano-particles/p-aminophenol dispersion liquid is obtained;
adding 75 mu L of 50mM silver nitrate aqueous solution and 300 mu L of 50mM ascorbic acid aqueous solution into the gold nanoparticle/p-aminophenol dispersion liquid, heating in 70 ℃ water bath for 3h, centrifuging at 8000rpm for 5min, and dispersing with water to prepare the core-shell structure nano Ag pollutant.
Step 2, contamination with Escherichia coli
Culturing Escherichia coli in LB culture solution until OD is 0.8, washing with PBS buffer solution for 3 times, dispersing in PBS buffer solution, adding the core-shell structure nanometer Ag pollutant with final concentration of 10mg/L, and culturing for 24h to obtain Escherichia coli polluted by nanometer Ag.
Step 3, pretreatment
The Escherichia coli polluted by the nano Ag is centrifuged at 6000rpm for 5min, 5 mul of the Escherichia coli is absorbed and dropped on a glass slide, 10 mul of fixing solution (formed by mixing glycerol and water according to the mass ratio of 3: 2) is added, the Escherichia coli is fixed for 20min at 20 ℃, and then a cover glass is covered to lightly press the Escherichia coli.
Performing Raman detection and imaging on the pretreated sample by using a laser Raman spectrometer to obtain a Raman spectrum of the pretreated sample, wherein the detection conditions are as follows:
785nm laser is adopted, and the laser energy is 100%;
the grating is 600 lines/mm;
the spatial resolution is 2 mu m/pixel, and the single-pixel acquisition time is 0.1 s;
the spectrum collection range is 1000-1600cm-1；
other parameters are system default values.
Fig. 1 is a TEM image of the core-shell nano Ag contaminant prepared in this example, which shows that the nano contaminant has uniform and regular morphology, no modification on the outer surface, and a particle size of about 90 nm.
FIG. 2 is a Raman spectrum of the core-shell nano Ag contaminant prepared in this example, wherein the nano contaminant is 1078cm-1、1143cm-1、1390cm-1And 1437cm-1Has stronger Raman characteristic peak, and can be used for rapidly detecting the spatial distribution of the nano pollutants in the organism.
Based on the characteristic peak of the Raman active molecule p-aminophenol, 1078cm was subjected to image processing using Labspec 6 (HORIBA Co., Japan)-1The raman characteristic peak intensity of the nano-pollutants is plotted to obtain a raman imaging graph of the nano-pollutants, and then the spatial distribution of the nano-pollutants in the organism can be obtained, as shown in fig. 3. As can be seen from the figure, the concentration of the nano-pollutants is lower at the edge of the Escherichia coli, and the concentration of the nano-pollutants is higher at the center, which indicates that the Escherichia coli continuously absorbs or intakes the nano-pollutants through endocytosis, and the nano-pollutants can be transferred to other organisms through food chains.
From the above, the method of the invention is applicable to detection of microorganisms, has the advantages of high resolution and high detection speed, and can be used for researching the transmission mechanism of nano pollutants in the food chain and the toxicity of the nano pollutants to the microorganisms.
Example 2 Nano SiO2Distribution in duckweed
100 mu M ethyl orthosilicate ethanol solution is prepared.
Dispersing gold nanoparticles in 10mL of ethanol containing 25mM 3-mercaptopropyl trimethoxy silane at the concentration of 1nM to obtain a gold nanoparticle dispersion liquid; then 200. mu.L of 0.15mM ethanol solution of DTTC was added dropwise, stirred at 25 ℃ for 30min, centrifuged at 8000rpm for 10min, washed with 25mM 3-mercaptopropyltrimethoxysilane in ethanol, and finally added with 25mM 3-mercaptopropyltrimethoxysilane in ethanol and resuspended to 10mL to obtain gold nanoparticle/DTTC dispersion.
Adding 400 mu L of 100 mu M ethyl orthosilicate ethanol solution into the gold nanoparticle/DTTC dispersion liquid under stirring, heating in water bath at 25 ℃ for 36h, centrifuging at 8000rpm for 5min, and dispersing with water to obtain the core-shell structure nano SiO2A contaminant.
Step 2, contamination of duckweed
Culturing duckweed seed in 1% Hoagland nutrient solution for 7 days, and adding the core-shell structure nano SiO with final concentration of 10mg/L2Culturing for 8h to obtain the nano SiO2Contaminated duckweed.
Step 3, pretreatment
Selecting a strain of nano SiO2Shearing the contaminated duckweed, placing on a glass slide, adding 20 μ L of fixative (prepared by mixing glycerol and water at a mass ratio of 2: 3), fixing at 20 deg.C for 10min, and slightly pressing with a cover glass.
Performing Raman detection and imaging on the pretreated sample by using a laser Raman spectrometer to obtain a Raman spectrum of the pretreated sample, wherein the detection conditions are as follows:
785nm laser is adopted, and the laser energy is 100%;
the grating is 600 lines/mm;
the spatial resolution is 20 mu m/pixel, and the single-pixel acquisition time is 0.2 s;
the spectrum collection range is 700-1300cm-1；
Lens 10 ×;
other parameters are system default values.
FIG. 4 shows the core-shell structure of nano SiO prepared in this example2The TEM image of the pollutant shows that the nanometer pollutant has uniform and regular appearance, no modification on the outer surface and particle size of about 125.5 nm.
FIG. 5 shows the core-shell structure nano SiO prepared in this example2Raman spectrum of the contaminant, from which it can be seen that the nano-contaminant is 780cm-1、845cm-1And 1125cm-1Has stronger Raman characteristic peak. Can be used for rapidly detecting the spatial distribution of the nano-pollutants in the organism.
Based on the characteristic peak of the Raman active molecule DTTC, 845cm of Raman active molecule was imaged using Labspec 6 (HORIBA Co., Japan) image processing software-1The raman characteristic peak intensities of (a) were plotted to obtain the distribution of the nano-contaminants in the plant, as shown in fig. 6. As can be seen from the figure, the nano-pollutants are mainly distributed in the root tips and the leaf edges, and the leaf center is small, which is consistent with the current research result, the nano-pollutants are taken in by the plants mainly through the root system, and then are transported upwards to the leaves.
Therefore, the method has high resolution and signal-to-noise ratio, is not influenced by the self background signal of the plant, can analyze the spatial distribution of the pollutants in each organ of the plant, and can be used for researching the migration and transformation process of the nano pollutants in the plant.
Example 3 distribution of Nano Ag in nematode bodies
50mM silver nitrate aqueous solution and 50mM ascorbic acid aqueous solution were prepared, respectively.
Dispersing gold nanoparticles in 10mL of water containing 50mM hexadecyl trimethyl ammonium chloride at the concentration of 1nM to obtain a gold nanoparticle dispersion liquid; then 1mL of 0.16mM IR-775 chloride in DMF was added dropwise, stirred at 30 ℃ for 1.5h, centrifuged at 9000rpm for 10min, washed with 50mM cetyltrimethylammonium chloride in water, and finally resuspended to 10mL with 50mM cetyltrimethylammonium chloride in water to obtain gold nanoparticle/IR-775 chloride dispersion.
Adding 25 mu L of 50mM silver nitrate aqueous solution and 100 mu L of 50mM ascorbic acid aqueous solution into the gold nanoparticle/IR-775 chloride dispersion liquid under stirring, heating in 70 ℃ water bath for 3h, centrifuging at 8000rpm for 5min, and dispersing with water to obtain the core-shell structure nano Ag pollutant.
Step 2, contamination by nematodes
And (3) culturing the nematode oosperm in medium-hardness regenerated water for 60h, adding the core-shell structure nano Ag pollutant with the final concentration of 15mg/L, and culturing for 48h to obtain the nematode polluted by the nano Ag.
Step 3, pretreatment
The nematodes contaminated by the nano Ag are centrifuged for 2min at 400rpm, 5 mul of the nematodes are absorbed and dropped on a glass slide, 15 mul of a fixative (prepared by mixing glycerol and water according to a mass ratio of 1: 1) is added, the mixture is fixed for 30min at 20 ℃, and then a cover glass is covered to lightly press the mixture.
Performing Raman detection and imaging on the pretreated sample by using a laser Raman spectrometer to obtain a Raman spectrum of the pretreated sample, wherein the detection conditions are as follows:
785nm laser is adopted, and the laser energy is 100%;
the grating is 600 lines/mm;
the spatial resolution is 3 mu m/pixel, and the single-pixel acquisition time is 0.01 s;
the spectrum collection range is 450-1100cm-1；
other parameters are system default values.
Fig. 7 is a TEM image of the core-shell nano-Ag contaminant prepared in this example, which shows that the nano-Ag contaminant has uniform and regular morphology, no modification on the outer surface, and a particle size of about 77.5 nm.
FIG. 8 shows the present embodimentThe Raman spectrogram of the prepared core-shell structure nano Ag pollutant shows that the nano Ag pollutant is 516cm-1、563cm-1And 931cm-1Has stronger Raman characteristic peak, and can be used for rapidly detecting the spatial distribution of the nano pollutants in the organism.
From the characteristic peak of Raman active molecule IR 775-chloride, 563cm was paired with Labspec 6 (HORIBA, Japan) image processing software-1The raman characteristic peak intensity of the nano-pollutants is plotted to obtain a raman imaging graph of the nano-pollutants, and the spatial distribution of the nano-pollutants in the organism can be obtained, as shown in fig. 9. As can be seen from the figure, the nano-pollutants are distributed in a nematode digestive system, including a pharyngeal pump, the middle part of an intestinal tract and a rectum area, the pollutant distribution area is obvious, the signal is strong, the resolution ratio is high, and the influence of the background signal of an animal is avoided.
Example 4 SiO with different charges2Distribution in plant root tips
100 mu M ethyl orthosilicate ethanol solution is prepared.
Dispersing gold nanoparticles in 10mL of ethanol containing 25mM 3-mercaptopropyl trimethoxy silane at the concentration of 1nM to obtain a gold nanoparticle dispersion liquid; then 200. mu.L of 0.15mM ethanol solution of DTTC was added dropwise, stirred at 25 ℃ for 30min, centrifuged at 8000rpm for 10min, washed with 25mM 3-mercaptopropyltrimethoxysilane in ethanol, and finally added with 25mM 3-mercaptopropyltrimethoxysilane in ethanol and resuspended to 10mL to obtain gold nanoparticle/DTTC dispersion. Adding 400 mu L of 100 mu M ethyl orthosilicate ethanol solution into the gold nanoparticle/DTTC dispersion liquid under stirring, heating in water bath at 25 ℃ for 36h, centrifuging at 8000rpm for 5min, and dispersing with water to obtain the core-shell structure nano SiO2Contaminants, the nano-contaminants are negatively charged and are denoted as SiO2(-)。
Dispersing gold nanoparticles in 10mL of ethanol containing 25mM 3-aminopropyltriethoxysilane at a concentration of 1nM to obtain a gold nanoparticle dispersion liquid; then 200. mu.L of 0.15mM DTDC ethanol solution was added dropwise at 25 deg.CStirring for 30min, centrifuging at 8000rpm for 10min, washing with ethanol containing 25mM 3-aminopropyltriethoxysilane, and adding ethanol containing 25mM 3-aminopropyltriethoxysilane to resuspend to 10mL to obtain gold nanoparticle/DTDC dispersion. Adding 400 mu L of 100 mu M ethyl orthosilicate and ethanol solution into the gold nanoparticle/DTDC dispersion liquid under stirring, heating in water bath at 25 ℃ for 36h, centrifuging at 8000rpm for 5min, and dispersing with water to obtain the core-shell structure nano SiO2Contaminants, the nano-contaminants being positively charged, SiO2(+)。
Step 2, contamination of duckweed
Culturing duckweed seeds in 1% Hoagland nutrient solution for 7 days, and adding the core-shell structure nano SiO with final concentration of 5mg/L2(-) and core-shell structure nano SiO2Culturing for 8h to obtain synchronously-received core-shell structure nano SiO2(-) and core-shell structure nano SiO2(+) contaminated Spirodela polyrhiza.
Step 3, pretreatment
Selecting a polluted duckweed, cutting off roots, stems and leaves, placing on a glass slide, adding 20 mu L of fixing solution (prepared by mixing glycerol and water according to the mass ratio of 2: 3), fixing at 20 ℃ for 10min, and then covering a cover glass to lightly press.
Performing Raman detection and imaging on the pretreated sample by using a laser Raman spectrometer to obtain a Raman spectrum of the pretreated sample, wherein the detection conditions are as follows:
785nm laser is adopted, and the laser energy is 100%;
the grating is 600 lines/mm;
the spatial resolution is 20 mu m/pixel, and the single-pixel acquisition time is 0.2 s;
the spectrum collection range is 450-650cm-1；
Lens 10 ×;
other parameters are system default values.
FIG. 10 shows the core-shell structure nano SiO prepared in this example2TEM image of (+) contaminant, from which it can be seen that the nano contaminant morphologyUniform and regular core-shell structure nano SiO prepared in figure 42The (-) contaminant particle size is similar.
FIG. 11 shows the core-shell structure nano SiO prepared in this example2(-) - (upper diagram) and SiO2The Raman spectrum of the (+) contaminant (lower panel) is shown in the following figure: SiO 22(+) at 508cm-1、580cm-1Has a strong Raman characteristic peak and SiO2(-) at 508cm-1Has stronger Raman characteristic peak. Therefore, 580cm can be used-1Represents SiO2(+) distribution in the plant and 508cm-1SiO representing two charges2The distribution in the plant can be deduced according to the relation of peak areas2The distribution of (-) in the plant can be used for rapidly detecting the spatial distribution of the nano-pollutant in the organism.
According to two kinds of SiO2Characteristic peaks of Raman-active molecules DTTC and DTDC, SiO was subjected to Labspec 6 (HORIBA, Japan) image processing software2Unique 580cm in (+)-1The Raman characteristic peak intensity of the SiO is obtained by mapping2Distribution of (+) nano-contaminants in plants; according to 580cm in DTDC Raman molecule-1The peak area of (A) was calculated to be 508cm-1Peak area of (2), will be 508cm-1Subtracting the calculated peak area from the total peak area to obtain the peak area of DTTC, at which 508cm-1The Raman characteristic peak intensity is plotted to obtain the SiO2Distribution of (-) nano-pollutants in plants, as shown in fig. 12. It can be seen from the figure that the positively charged nano-contaminants are distributed in the roots significantly more than the negatively charged nano-contaminants, since the positively charged nano-contaminants are more easily adsorbed on the surface of the plant.
From the above, the method of the invention has high resolution and signal-to-noise ratio, is not influenced by the self background signal of the plant, can analyze the spatial distribution of the pollutants in each organ of the plant, can analyze the distribution of various pollutants in the plant, and can be used for researching the migration and transformation process of nano pollutants in the plant.
The present invention is not limited to the above exemplary embodiments, and any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. An imaging detection method of nano pollutants in a living body is characterized by comprising the following steps:
(1) coating Raman active molecules on the surfaces of the nano particles, and then coating nano pollutants to be detected to obtain core-shell structure nano pollutants;
adding the core-shell structure nano pollutant into a biological culture solution, and culturing to ensure that the core-shell structure nano pollutant is taken in a living body; after the culture is finished, taking out the polluted organisms and fixing the polluted organisms on a glass slide to obtain a pretreated sample;
(2) performing Raman detection and imaging on the pretreated sample by using a laser micro-Raman spectrometer to obtain a Raman spectrum of the pretreated sample; and then processing the Raman spectrum through imaging software according to the characteristic peak of the Raman active molecule to obtain a Raman imaging graph of the nano-pollutants, namely obtaining the spatial distribution of the nano-pollutants in the organism.
2. The imaging detection method according to claim 1, characterized in that: the nano-particles are gold nano-particles or silver nano-particles.
3. The imaging detection method according to claim 1, characterized in that: the Raman active molecule is at least one of crystal violet, rhodamine 6G, DTTC, DTDC, IR-775 chloride, p-nitrophenol, p-aminophenol and p-dithiol.
4. The imaging detection method according to claim 1, characterized in that: the nano pollutants are Ag and SiO2、TiO2、MnO2And a nano plastic.
5. The imaging detection method according to claim 1, characterized in that: the organism is a plant, microorganism or animal.
6. The imaging detection method according to claim 1, characterized in that: the preparation method of the core-shell structure nano pollutant comprises the following steps:
step 1, dissolving a precursor of a nano pollutant to be detected in a solvent to obtain a precursor solution;
step 2, dispersing the nano particles in a solvent containing a surface stabilizer to obtain a nano particle dispersion liquid; then adding the Raman active molecule dispersion liquid, stirring for 20min-2h at 25-50 ℃, centrifuging, washing, adding a solvent containing a surface stabilizer, and re-suspending to the original volume to obtain the nano-particle/Raman active molecule dispersion liquid;
and 3, adding the precursor solution into the nanoparticle/Raman active molecule dispersion solution, performing hydrothermal reaction, centrifuging, washing, and dispersing with water to obtain the core-shell structure nano pollutant.
7. The imaging detection method of claim 6, wherein:
the concentration of the precursor solution is 5-50 mM;
the solvent is at least one of ethanol and water;
the surface stabilizer is at least one of hexadecyl trimethyl ammonium chloride, polyvinylpyrrolidone, sodium dodecyl sulfate, 3-aminopropyl triethoxysilane and 3-mercaptopropyl trimethoxysilane;
the concentration of the surface stabilizer in the solvent containing the surface stabilizer is 25-50 mM;
the concentration of the nanoparticles in the nanoparticle dispersion liquid is 1-10 nM;
the concentration of the Raman active molecule dispersion liquid is 0.1-1mM, and the used solvent is at least one of ethanol, DMSO and DMF;
the volume ratio of the nanoparticle dispersion liquid to the Raman active molecule dispersion liquid is 10-100: 1;
the volume ratio of the nanoparticle/Raman active molecule dispersion liquid to the precursor solution is 10-100: 1.
8. The imaging detection method according to claim 1, characterized in that the fixing method is: taking out the polluted organisms, transferring the polluted organisms onto a glass slide, dripping a fixing solution, and fixing for 10-60 min at 4-25 ℃;
the fixing liquid is formed by mixing glycerol or 1, 2-propylene glycol and water according to the mass ratio of 1: 0-4.
9. The detection method according to claim 1, wherein the conditions of the raman detection in step 2) are:
adopting 633nm or 785nm laser with laser energy of 10-100%;
the grating is 600lines/mm or 1800 lines/mm;
the spatial resolution is 1-50 mu m/pixel, and the single-pixel acquisition time is 0.001-1 s;
the spectrum collection range is 500-2000 cm-1。
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