CN113654953A - Method for detecting environmental behaviors and biological effects of nano-particle pollutants - Google Patents
Method for detecting environmental behaviors and biological effects of nano-particle pollutants Download PDFInfo
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Abstract
The invention discloses a method for detecting environmental behaviors and biological effects of nano-particle pollutants, which comprises the following steps: firstly, preparing a vesicle suspension dyed by a micro model and a fluorescent dye; secondly, injecting the vesicle suspension into the micro-model from an injection port; then, introducing a background solution into the micro model from the sample inlet, and washing the unloaded vesicles; and finally, introducing a suspension sample of the nano-particle pollutants into the micro-model from the sample inlet, connecting the micro-model with a CCD (charge coupled device) through a microscope to take a picture at regular time, observing the internal fluorescence intensity of the micro-model and the distribution change of the nano-particle pollutants, simultaneously collecting effluent from the sample outlet, measuring the fluorescence intensity by using a fluorescence spectrophotometer at regular time, and judging whether the nano-particle pollutants interact with the phospholipid layer. The method disclosed by the invention can monitor the environmental behaviors of the flow, migration and the like of the nano-particle pollutants in the environmental medium when the biological interface exists; and the interaction between the nano-particle pollutants and the cell membrane can be detected, and the biological effect of the nano-particle pollutants can be evaluated.
Description
Technical Field
The invention relates to the technical field of nano particle pollutant detection, in particular to a method for detecting environmental behaviors and biological effects of nano particle pollutants.
Background
With the continuous progress and development of nanotechnology, various novel nanometer materials emerge endlessly, and enter the production and life of people, so that the nanometer material is widely applied to the fields of military affairs, aerospace, biology, medicine, environment and the like. Therefore, the method has great significance in exploring the environmental behaviors and biological effects of various nano materials.
In the existing environmental quality monitoring, a sample is taken at a sampling point, then a sample to be detected is taken back to a laboratory, and the type, concentration and variation trend of pollutants in the sample to be detected are determined through a series of detection means, so that the environmental pollution condition is evaluated. But for various nano-particle pollutants in the current environment, no good universal detection index and method are available to reflect the environmental behaviors and biological effects of the nano-particle pollutants.
There are many biological interfaces in the environment, including the biological membrane interface formed on the surface of mineral, the animal and plant cell membrane interface, etc. The interaction between the nano-particle pollutants and the environmental biological interface directly determines the migration transformation and fate trend of the nano-particle pollutants and also determines the biological toxicity of the nano-particle pollutants to animal and plant cells. The cell membrane is the first barrier of the cell against external contaminants, and its interaction with the nanoparticle contaminants is of great significance for predicting the environmental risk of the nanoparticle contaminants.
The phospholipid bilayer constitutes the basic skeleton of the cell membrane, and therefore, cell membrane models are common laboratory tools for simulating cell membranes. Compared with living cells, the cell membrane model can simplify experimental conditions, so that the experiment is not interfered by the physiological process of the cells, the variables are controlled one by one, and the interaction between the particles and the cell membrane is better researched. The lipid layer is a common cell membrane model and is obtained by crushing small unilamellar vesicles with the diameter of about 50 nanometers on a certain plane, and the synthesis method of the small unilamellar vesicles comprises a solvent-free method and a solvent replacement method, wherein a mild hydration method in the solvent-free method is simple and is widely applied.
The two-dimensional flow micromodel is a good novel tool for exploring environmental behaviors such as migration, deposition and the like of trace nano-particle pollutants in an environmental medium, and is often used for detecting physical, chemical and biological processes of micron-sized and submicron-sized particles. The two-dimensional or three-dimensional geometric channel on the surface of the micro model is manufactured by adopting laser or plasma etching to carry out ion milling on the surfaces of glass, a silicon plate or a high molecular polymer and the like. The pore structure, the surface material and the like of the micro model can be regulated and controlled according to requirements, and the change process in the pore microenvironment can be directly observed and quantified through a microscope.
At present, no research report for detecting the environmental behaviors and biological effects of the nanoparticle pollutants by using a micromodel and phospholipid layer vesicles exists.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for detecting the environmental behaviors and biological effects of the nano-particle pollutants, which can monitor the environmental behaviors such as the flowing and the migration of the nano-particle pollutants in an environmental medium when a biological interface exists; and the interaction between the nano-particle pollutants and the cell membrane can be detected, and the biological effect of the nano-particle pollutants can be evaluated.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for detecting environmental behavior and biological effects of nanoparticle pollutants, comprising the steps of:
firstly, preparing a micro model required for detection and a vesicle suspension dyed by a fluorescent dye;
the micro-model comprises a glass layer and a PDMS layer which are bonded together, the PDMS layer is prepared by a soft lithography method, a sample flow channel is carved on one side of the PDMS layer facing the glass layer, the sample flow channel comprises a sample inlet area, an observation area and a sample outlet area, the sample inlet area is provided with a sample inlet, the sample outlet area is provided with a sample outlet, a plurality of support columns bonded with the glass layer are arranged in the sample inlet area and the sample outlet area, and a plurality of collecting columns bonded with the glass layer are arranged in the observation area; the vesicles in the vesicle suspension are composed of phospholipid and fluorescent dye loaded on the phospholipid;
secondly, injecting the vesicle suspension into the micro-model from an injection port to enable the vesicles to be uniformly filled in the micro-model, and crushing the vesicles due to hydrophobic acting force after the vesicles are contacted with the plane of the micro-model to form a phospholipid layer which is loaded on the micro-model;
then, introducing a background solution into the micro-model from the sample inlet, flushing the unloaded vesicles, and periodically measuring the fluorescence intensity of the effluent liquid from the sample outlet by using a fluorescence spectrophotometer until the effluent liquid has no fluorescence; simultaneously observing the micro model by using a fluorescence microscope, and determining that a phospholipid layer dyed by a fluorescent dye exists in the micro model;
and finally, introducing a suspension sample of the nano-particle pollutants to be detected into the micro-model from the sample inlet, connecting the micro-model with a CCD (charge coupled device) through a microscope to take a picture at regular time, observing the fluorescence intensity inside the micro-model and the distribution change of the nano-particle pollutants, simultaneously collecting effluent from the sample outlet, measuring the fluorescence intensity by using a fluorescence spectrophotometer at regular time, and judging whether the nano-particle pollutants interact with the phospholipid layer according to the fluorescence intensity in the effluent to cause the phospholipid layer loaded on the micro-model to be broken and flow out along with the sample.
In the above scheme, the depth of the sample flow channel is 26.7 μm.
In the above scheme, the observation area comprises 1875 collecting columns, the diameter of each collecting column is 190 μm, and pores with a porosity of 0.44 are formed between adjacent collecting columns.
In the scheme, the extrusion membrane-passing method is adopted to prepare the suspension of the unilamellar vesicles dyed by the RhB-PE fluorescent dye.
In the above scheme, the preparation method of the unilamellar vesicle suspension is as follows:
dissolving phospholipid in a volume ratio of 2: 1, taking a proper amount of the solution to be uniformly mixed in a glass bottle and drying the solution by blowing nitrogen to form a uniform lipid film on the inner wall of the glass bottle; then 2ml of ultrapure water is added into the glass bottle, and the mixture is hydrated in an oven at 40 ℃ for 30min to obtain phospholipid suspension; finally, the phospholipid suspension was repeatedly extruded 31 times through a filter membrane with a pore size of 50nm using an extruder to produce a unilamellar vesicle suspension with a diameter of 50 nm.
Through the technical scheme, the method for detecting the environmental behaviors and the biological effects of the nano-particle pollutants provided by the invention has the following beneficial effects:
the detection method disclosed by the invention is simple to operate, is intuitive to observe, and can simultaneously monitor the interaction between the nanoparticle pollutants and a cell membrane and the migration and distribution rule of the nanoparticle pollutants in a medium when a biological interface exists. The technical scheme of the invention is beneficial to researching the biological toxicity of the nano-particle pollutants on animal and plant cells and the migration and transformation of the nano-particle pollutants, and has good guiding significance for environmental protection.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a side view of a disclosed micromodel of an embodiment of the present invention;
FIG. 2 is a top view of a PDMS layer of the micro-model according to an embodiment of the present invention;
FIG. 3 is a cross-sectional view of a micro-model viewing area according to an embodiment of the present invention;
FIG. 4 is an enlarged top view of a collection column according to an embodiment of the present invention;
FIG. 5 is a schematic view of vesicles disclosed in the examples of the present invention;
FIG. 6 is a diagram illustrating a process of introducing vesicles into a micromodel according to an embodiment of the present invention;
FIG. 7 is a diagram of a process after sample introduction as disclosed in the embodiments of the present invention.
In the figure, 1, a glass layer; 2. a PDMS layer; 3. a sample introduction area; 4. an observation area; 5. a sample outlet area; 6. a sample inlet; 7. a sample outlet; 8. collecting the column; 9. a phospholipid layer; 10. a fluorescent dye; 11. a vesicle; 12. a support pillar; 13. a pore; 14. a nanoparticle contaminant.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.
The invention provides a method for detecting environmental behaviors and biological effects of nano-particle pollutants, which comprises the following steps:
in the first step, a suspension of vesicles stained with a micromodel and a fluorescent dye is prepared for detection.
The micro model is designed and manufactured according to the requirement to simulate the water environment and the soil environment, and the size, the internal structure, the material and the manufacturing method of the micro model can be customized according to the requirement of an experiment. The examples of the micro-molds mentioned below are those prepared by soft lithography using Polydimethylsiloxane (PDMS) as a raw material used in our experiments. PDMS has a similar potential to silica and the surface is easily loaded with biomolecules. The preparation steps mainly comprise: preparing a silanization mould, pouring PDMS on the mould, curing PDMS, stripping a PDMS model, and bonding the model and glass.
As shown in fig. 1, the prepared micro-model comprises a glass layer 1 and a PDMS layer 2 bonded together, the PDMS layer 2 is prepared by soft lithography, a sample flow channel is engraved on one side of the PDMS layer 2 facing the glass layer 1, as shown in fig. 2, the sample flow channel comprises a sample inlet area 3, an observation area 4 and a sample outlet area 5, the sample inlet area 3 is provided with a sample inlet 6, the sample outlet area 5 is provided with a sample outlet 7, a plurality of support columns 12 bonded with the glass layer 1 are arranged in the sample inlet area 3 and the sample outlet area 5, and a plurality of collection columns 8 bonded with the glass layer 1 are arranged in the observation area 4; the depth of the sample flow channel was 26.7 μm. As shown in fig. 3 and 4, the observation region 4 includes 1875 collection pillars 8, the diameter of each collection pillar 8 is 190 μm, and pores 13 having a porosity of 0.44 are formed between adjacent collection pillars 8. The number of support columns 12 is much smaller than the number of collection columns 8.
As shown in fig. 5, the vesicles 11 in the vesicle suspension are composed of phospholipids and fluorescent dyes 10 supported on the phospholipids. In this example, a suspension of unilamellar vesicles stained with RhB-PE fluorescent dye 10 was prepared using an extrusion coating method. The preparation method comprises the following steps:
dissolving phospholipid in a volume ratio of 2: 1, taking a proper amount of the solution to be uniformly mixed in a glass bottle and drying the solution by blowing nitrogen to form a uniform lipid film on the inner wall of the glass bottle; then 2ml of ultrapure water is added into the glass bottle, and the mixture is hydrated in an oven at 40 ℃ for 30min to obtain phospholipid suspension; finally, the phospholipid suspension was repeatedly extruded 31 times through a filter membrane with a pore size of 50nm using an extruder to produce a unilamellar vesicle suspension with a diameter of 50 nm.
And secondly, injecting the vesicle suspension into the micromodel from the injection port 6 to enable the vesicles 11 to be uniformly filled in the micromodel, wherein as shown in figure 6, the vesicles 11 are broken due to hydrophobic acting force after contacting with the plane of the micromodel to form a phospholipid layer 9 which is loaded on the micromodel.
Thirdly, introducing a background solution into the micro-model from the sample inlet 6, flushing the unloaded vesicle 11, and periodically measuring the fluorescence intensity of the effluent liquid from the sample outlet 7 by using a fluorescence spectrophotometer until the effluent liquid has no fluorescence; and observing the micro model by using a fluorescence microscope, and determining that the phospholipid layer 9 dyed by the fluorescent dye 10 exists in the micro model.
And fourthly, introducing a suspension sample of the nano-particle pollutants 14 to be detected into the micro-model from the sample inlet 6, connecting a microscope with the CCD (charge coupled device) for timed photographing, observing the internal fluorescence intensity of the micro-model and the distribution change of the nano-particle pollutants 14 (which can be compared with an empty micro-model without the vesicle 11), simultaneously collecting effluent from the sample outlet 7, regularly measuring the fluorescence intensity by using a fluorescence spectrophotometer, and judging whether the nano-particle pollutants 14 interact with the phospholipid layer 9 according to the fluorescence intensity in the effluent, wherein as shown in fig. 7, the phospholipid layer 9 loaded on the micro-model is broken and flows out along with the sample.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (5)
1. A method for detecting environmental behavior and biological effects of nanoparticle pollutants, comprising the steps of:
firstly, preparing a micro model required for detection and a vesicle suspension dyed by a fluorescent dye;
the micro-model comprises a glass layer and a PDMS layer which are bonded together, the PDMS layer is prepared by a soft lithography method, a sample flow channel is carved on one side of the PDMS layer facing the glass layer, the sample flow channel comprises a sample inlet area, an observation area and a sample outlet area, the sample inlet area is provided with a sample inlet, the sample outlet area is provided with a sample outlet, a plurality of support columns bonded with the glass layer are arranged in the sample inlet area and the sample outlet area, and a plurality of collecting columns bonded with the glass layer are arranged in the observation area; the vesicles in the vesicle suspension are composed of phospholipid and fluorescent dye loaded on the phospholipid;
secondly, injecting the vesicle suspension into the micro-model from an injection port to enable the vesicles to be uniformly filled in the micro-model, and crushing the vesicles due to hydrophobic acting force after the vesicles are contacted with the plane of the micro-model to form a phospholipid layer which is loaded on the micro-model;
then, introducing a background solution into the micro-model from the sample inlet, flushing the unloaded vesicles, and periodically measuring the fluorescence intensity of the effluent liquid from the sample outlet by using a fluorescence spectrophotometer until the effluent liquid has no fluorescence; simultaneously observing the micro model by using a fluorescence microscope, and determining that a phospholipid layer dyed by a fluorescent dye exists in the micro model;
and finally, introducing a suspension sample of the nano-particle pollutants to be detected into the micro-model from the sample inlet, connecting the micro-model with a CCD (charge coupled device) through a microscope to take a picture at regular time, observing the fluorescence intensity inside the micro-model and the distribution change of the nano-particle pollutants, simultaneously collecting effluent from the sample outlet, measuring the fluorescence intensity by using a fluorescence spectrophotometer at regular time, and judging whether the nano-particle pollutants interact with the phospholipid layer according to the fluorescence intensity in the effluent to cause the phospholipid layer loaded on the micro-model to be broken and flow out along with the sample.
2. The method of claim 1, wherein the sample flow channel has a depth of 26.7 μm.
3. The method of claim 1, wherein the observation area comprises 1875 collection pillars with a diameter of 190 μm, and pores are formed between adjacent collection pillars with a porosity of 0.44.
4. The method for detecting the environmental behaviors and the biological effects of the nanoparticle pollutants as claimed in claim 1, wherein the suspension of the monolayer vesicles dyed by the RhB-PE fluorescent dye is prepared by an extrusion membrane-passing method.
5. The method for detecting the environmental behaviors and the biological effects of the nanoparticle pollutants according to claim 4, wherein the suspension of unilamellar vesicles is prepared by the following steps:
dissolving phospholipid in a volume ratio of 2: 1, taking a proper amount of the solution to be uniformly mixed in a glass bottle and drying the solution by blowing nitrogen to form a uniform lipid film on the inner wall of the glass bottle; then 2ml of ultrapure water is added into the glass bottle, and the mixture is hydrated in an oven at 40 ℃ for 30min to obtain phospholipid suspension; finally, the phospholipid suspension was repeatedly extruded 31 times through a filter membrane with a pore size of 50nm using an extruder to produce a unilamellar vesicle suspension with a diameter of 50 nm.
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