CN112816477B - Method for researching interaction between solid powder and biological sample by using microfluid chip - Google Patents
Method for researching interaction between solid powder and biological sample by using microfluid chip Download PDFInfo
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- CN112816477B CN112816477B CN202110085151.1A CN202110085151A CN112816477B CN 112816477 B CN112816477 B CN 112816477B CN 202110085151 A CN202110085151 A CN 202110085151A CN 112816477 B CN112816477 B CN 112816477B
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Abstract
The invention belongs to the field of life science, relates to a method for researching interaction between solid powder and a biological sample by utilizing a microfluid chip, and can provide a reaction environment for interaction between biological cells or tissues and the solid powder sample; the device mainly comprises a liquid outlet 1, a liquid outlet channel 2, a solid powder sample filling area 3, a biological sample channel 4, a biological sample inlet 5, a liquid inlet channel 6 and a liquid inlet 7; wherein, the solid powder sample filling area 3 can be filled with a trace amount of solid powder sample; liquid enters from the liquid inlet 7, flows out from the liquid outlet 1 through the liquid inlet channel 6, the biological sample channel 4, the solid powder sample filling area 3 and the liquid outlet channel 2, and a biological sample can enter from the biological sample inlet 5 and reach the solid powder sample filling area 3 through the biological sample channel 4 to interact with the solid powder sample; the invention has simple structure and is suitable for researching the interaction between the precious solid powder sample and biological cells or tissues.
Description
Technical Field
The invention provides a micro-fluidic chip capable of realizing interaction between a solid powder sample and biological cells or tissues, and the chip can realize interaction experiments and real-time observation of milligram-level solid powder samples and biological cells or tissues. The invention has definite structure and simple operation, can be applied to the crossed fields of materials science and biology, researches the interaction relation between biological cells or tissues and solid powder materials, and the like.
Background
Since the invention, the microfluidic chip has the advantages of low manufacturing cost, controllable reaction conditions, small material consumption and the like, and is widely applied as a carrier of biological/chemical experiments.
However, the prior art has the following problems:
on one hand, the microfluidic chip itself is used as a reaction carrier of a micro volume, and the reaction object of the microfluidic chip is often a micro organism such as a cell, a bacterium and the like with a diameter of micron order or even nanometer order, and is basically a liquid phase reaction. The interaction between solids, in particular powders having a diameter of tens of microns or even hundreds of microns, and organisms has been studied less frequently and is worth making intensive studies by means of microfluidic chip technology. The acquisition or preparation of some solid powder samples is difficult and extremely costly, and therefore the quantities obtained are extremely limited; however, the potential effects of these solid powder samples on biological cells or tissues, and even possible damage, require more detailed studies.
On the other hand, the existing microfluidic chip technology is mostly liquid phase reaction due to the design principle, that is, the observable reaction result is generated by the mutual mixing of solution or suspension; a small part of the microfluidic chip can realize cell culture and even tissue culture. However, when the above microfluidic chips are used for studying chemical or biological effects of solid powders, the methods adopted by the microfluidic chips are to prepare suspensions or prepare nanoparticles, the substance of the microfluidic chips is still a liquid phase reaction, and the surface reaction of the solid powders cannot be studied, and the influence of some physical properties (such as surface roughness and porosity) of the solid powders on biological cells or tissues cannot be judged.
Disclosure of Invention
Aiming at the problems in the prior art, the invention designs the microfluidic chip to overcome the defect that the potential influence of a solid powder sample on biological cells or tissues is not feasible to research in the prior art; on the other hand, the potential influence of the solid powder surface on biological cells or tissues is researched through the specially designed microfluidic chip.
The invention aims to construct a method for researching the interaction between solid powder and cells and biological tissues by utilizing a microfluidic chip, which is realized by the microfluidic chip capable of realizing the interaction between a solid powder sample and the biological cells or tissues and is used for researching the interaction between the solid powder with the diameter of 20-500 mu m and the cells or even the biological tissues.
The microfluidic chip comprises a liquid outlet 1, a liquid outlet channel 2, a solid powder sample filling area 3, a biological sample channel 4, a biological sample inlet 5, a liquid inlet channel 6 and a liquid inlet 7 from left to right. Wherein, the liquid can be filled from the liquid inlet 7 and flows through the liquid inlet channel 6, the biological sample channel 4, the solid powder sample filling area 3 and the liquid outlet channel 2 until the liquid outlet 1 flows out; a solid powder sample filling area 3 is arranged between the biological sample channel 4 and the liquid outlet channel 2, and the solid powder sample filling area 3 can be filled and packaged with micro solid powder with the diameter of 20-500 mu m; a biological sample inlet 5 is arranged between the biological sample channel 4 and the liquid inlet channel 6, and biological samples such as cells or biological tissues can be injected or grown from the biological sample inlet 5, enter the solid powder sample filling area 3 through the biological sample channel 4 and interact with the filled and packaged solid powder sample. Biological tissue includes raw or treated samples of roots, stems, leaves of plants.
Wherein, the biological sample channel 4 is designed with a convergent section towards the direction of the solid powder sample filling area 3, and the width of the convergent section is gradually reduced; the depth of the liquid outlet channel 2 is less than the depth D of the solid powder sample filled in the solid powder sample filling area 3 1 Particle size to avoid as much as possible the solid powder sample flowing out with the outlet channel 2 during the whole operation step, wherein D 1 The particle size distribution curve shows a particle size of 1% by volume from the small particle size side. The liquid outlet 1, the biological sample inlet 5 and the liquid inlet 7 are all holes with the diameter of 200-2000 mu m; the liquid outlet channel 2 and the liquid inlet channel 6 are pipelines with the width of 200-1000 μm, the length of 5-20 mm and the depth of 20-100 μm.
The solid powder sample filling area 3 is a quadrilateral pit with the side length of 1mm to 5mm and the depth of 50 mu m to 500 mu m, after the micro-fluidic chip is cured for the first time, the solid powder sample with the grain size of not more than 500 mu m can be filled into the solid powder sample filling area 3, and then secondary curing is carried out on a cover glass, and the solid powder sample is packaged between the curing chip and the cover glass; the solid powder sample loading area can be used as a microscopic observation chamber at the same time as the reaction chamber, so as to observe the interaction between the solid powder sample and the organism under a microscope.
The biological sample channel 4 is a special pipeline with a convergent section, the total length of the biological sample channel 4 is 5mm to 20mm, the depth of the pipeline is 50 mu m to 500 mu m, the length of the convergent section is 2mm to 10mm, the width of the narrowest part of the convergent section is 50 mu m to 500 mu m, the biological sample channel is communicated with the solid powder sample filling area 3, and the width of the widest part of the convergent section is 200 mu m to 1000 mu m and is communicated with the biological sample inlet 5 and the liquid inlet channel 6.
The microfluidic chip can be used for researching the interaction between solid powder with the diameter of 20-500 mu m and cells and even biological tissues. Because the dosage of the solid powder is very small, the invention is suitable for the biological interaction experiment of the precious solid powder sample. Meanwhile, the invention can also realize the real-time microscopic observation of the interaction experiment.
The invention has the advantages that:
1. the potential influence of the solid powder sample on biological cells or tissues is researched by adopting the microfluidic chip, a plurality of experiments can be carried out by using a very small amount of samples, reliable experimental data can be obtained, and the method has remarkable advantages when being applied to biological interaction experiments of solid powder samples which are expensive and difficult to obtain;
2. according to the invention, the space between the cover glass and the chip substrate is constructed, so that the solid powder is directly packaged, the defect that the traditional liquid phase reaction cannot be used for researching the surface reaction of the solid powder can be avoided, and a solution is provided for researching the potential influence of the surface of the solid powder on biological cells or tissues;
3. the convergence section designed on the biological sample channel 4 can ensure that some cells, microorganisms and even biological tissues can pass through the biological sample channel in one direction and do not return, enter the solid powder sample filling area 3, and interact with the packaged particles, so that the movement direction of the biological cells or tissues in the chip is controlled;
4. the invention takes the cover glass as the glass sealing material, and can clearly observe the interaction between the solid powder sample and the organism in the micro-fluidic chip in real time;
5. the depth of the liquid outlet channel 2 is less than the depth D of the solid powder sample filled in the solid powder sample filling area 3 1 The particle size can avoid the solid powder sample from flowing out along with the liquid outlet channel 2 in the whole operation step, and the accuracy of subsequent detection is improved.
Drawings
FIG. 1 is an overall block diagram of the present invention;
FIG. 2 is a schematic diagram of the operation of the present invention
In the figure:
1-liquid outlet 2-liquid outlet channel 3-solid powder sample filling area 4-biological sample channel 5-biological sample inlet 6-liquid inlet channel 7-liquid inlet
Detailed Description
The present invention will be described in further detail below with reference to the accompanying drawings.
As shown in fig. 2, the operation steps of the present invention can be divided into 4 operations: filling a solid powder sample, packaging the solid powder sample, injecting a biological sample for reaction, and detecting the sample.
Due to the special design of the present invention, it is necessary to perform the operation of "filling solid powder sample" to fill a minute amount of solid powder into the solid powder sample filling area 3, as shown in fig. 2 (a).
After the operation of "filling the solid powder sample" is performed, the operation of "encapsulating the solid powder sample" can be performed, specifically, the solid powder overflowing the solid powder sample filling area 3 is removed slightly, and the chip is encapsulated by the cover fragment, at which time the sample in the solid powder sample filling area 3 is fixed, as shown in fig. 2 (b).
After the operation of packaging the solid powder sample is realized, the operation of injecting biological sample reaction can be carried out, specifically, biological cells or tissues are injected from a biological sample inlet 5, and the biological cells or tissues finally flow to the solid powder sample filling area 3 through a biological sample channel 4 due to the directional flow of liquid; FIG. 2 (c) shows the flow of biological cells in the biological sample channel 4, and FIG. 2 (d) shows the growth of biological tissue (for example, roots of some kinds of plants) in the biological sample channel 4; because the depth of the liquid outlet channel 2 is smaller than the grain diameter of the solid powder sample, the solid powder sample cannot flow out along with the liquid outlet channel 2 in the whole operation step; after entering the solid powder sample loading area 3, the biological sample interacts with the solid powder therein.
After the operation of injecting biological sample reaction is realized, the operation of sample detection can be carried out; because the transparent cover glass is adopted, the surface states of the solid powder and the biological sample can be observed by adopting an optical microscope; in addition, the micro-fluid chip is opened, the solid powder and the biological sample are taken out, the surface property of the solid powder is detected, and the subsequent separation culture and growth index detection of the biological sample are carried out.
Claims (7)
1. A method for studying the interaction between a solid powder and a biological sample by using a microfluidic chip, characterized in that the method is carried out by a microfluidic chip capable of causing the interaction between the solid powder sample and the biological sample; the biological sample comprises biological cells or tissues; the microfluidic chip integrally comprises a bottom substrate material and a top cover glass, and the substrate material and the cover glass are combined to form a space between the substrate material and the cover glass; the microfluid chip comprises a liquid outlet (1), a liquid outlet channel (2), a solid powder sample filling area (3), a biological sample channel (4), a biological sample inlet (5), a liquid inlet channel (6) and a liquid inlet (7) from left to right; one end of the liquid outlet channel (2) is connected with the liquid outlet (1), and the other end of the liquid outlet channel is connected with the solid powder sample filling area (3); one end of the solid powder sample filling area (3) is connected with the liquid outlet channel (2), and the other end is connected with the biological sample channel (4); one end of the biological sample passage (4) is connected with the solid powder sample filling area (3), and the other end is connected with the liquid inlet passage (6); one end of the liquid inlet channel (6) is connected with the biological sample channel (4), and the other end is connected with the liquid inlet (7); the biological sample inlet (5) is positioned at the junction position of the liquid inlet channel (6) and the biological sample channel (4), so that liquid can be filled from the liquid inlet (7) and flows out of the liquid outlet (1) through the liquid inlet channel (6), the biological sample channel (4), the solid powder sample filling area (3) and the liquid outlet channel (2);
the method specifically comprises the following steps:
(1) Filling solid powder samples: filling a trace amount of solid powder into a solid powder sample filling area (3);
(2) Packaging solid powder sample: gently removing the solid powder overflowing the solid powder sample filling area (3) after the filling in the step (1), and packaging the chip by using a cover fragment, wherein the sample in the solid powder sample filling area (3) is fixed, and then performing the step (3);
(3) Injecting a biological sample for reaction: injecting biological cells or tissues from a biological sample inlet (5), wherein the biological cells or tissues finally flow to the solid powder sample filling area (3) through the biological sample channel (4) due to the directional flow of the liquid, and then performing the step (4);
(4) Sample detection: directly placing the microfluid chip under an optical microscope to observe the surface states of solid powder and a biological sample; or opening the microfluidic chip, taking out the solid powder and the biological sample, detecting the surface property of the solid powder, and detecting the growth index of the biological sample after the separation culture of the biological sample.
2. The method of claim 1, wherein the particle size of the solid powder sample is from 50 μ ι η to 500 μ ι η.
3. The method according to any of claims 1-2, wherein the outlet (1), the biological sample inlet (5) and the inlet (7) are each a hole having a diameter of 200 μm to 2000 μm; the liquid outlet channel (2) and the liquid inlet channel (6) are pipelines with the width of 200-1000 μm, the length of 5-20 mm and the depth of 20-100 μm.
4. A method according to any one of claims 1 to 3, wherein the solid powder sample loading area (3) is a quadrangular pit having a side of 1mm to 5mm and a depth of 50 μm to 500 μm, and after the microfluidic chip is once cured, a solid powder sample having a particle size of not more than 500 μm can be loaded into the solid powder sample loading area (3), and then a secondary curing is performed on a cover glass to enclose the solid powder sample between the cured chip and the cover glass; the solid powder sample loading area can be used as a microscopic observation chamber at the same time as the reaction chamber, so as to observe the interaction between the solid powder sample and the organism under a microscope.
5. The method according to any one of claims 1 to 4, wherein the biological sample channel (4) is a special tube having a convergent section, the biological sample channel (4) has a total length of 5mm to 20mm, a depth of 50 μm to 500 μm, a length of 2mm to 10mm, a width of 50 μm to 500 μm at the narrowest part of the convergent section and communicates with the solid powder sample loading region (3), and a width of 200 μm to 1000 μm at the widest part of the convergent section and communicates with the biological sample inlet (5) and the liquid inlet channel (6).
6. The method according to any of claims 1 to 5, wherein the depth of the liquid outlet channel (2) is smaller than the D of the solid powder sample filled in the solid powder sample filling area (3) 1 Particle size of wherein D 1 The particle size represents a particle size of 1% by volume from the small particle size side in the particle size distribution curve of the solid powder sample.
7. The method of any one of claims 1 to 6, wherein the tissue comprises a raw or physically or chemically processed sample of a root, stem, leaf of the plant.
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