Method for evaluating toxic effect of pollutants

Abstract

The invention discloses a method for evaluating the toxic effect of pollutants, the method takes a microfluidic device as an analysis platform, the microfluidic device comprises a cell layer flow channel fixed on a substrate in the first step and a pollutant exposure layer fixed on the substrate in the second step, the cell layer is provided with at least two cell culture channels, and the pollutant exposure layer is provided with at least two cell culture channels. The gas arrangement direction is a first direction; the pollutant exposure layer is provided with a concentration gradient generation area, an exposure area and a detection area which are communicated in sequence; the exposure area is provided with at least three flow channels which are arranged in parallel, the flow channels have a second direction, and an included angle is formed between the first direction and the second direction, so that each cell culture channel in the at least two cell culture channels of the cell layer is crossed with the at least three flow channels which are arranged in parallel of the pollutant exposure layer; the detection area at least comprises an area modified with a capture element. According to the invention, tedious sample preparation, sample adding and detection processes can be avoided.

Description

Technical Field

[0001] The present invention relates to a method for evaluating the toxic effects of pollutants. Background Art

[0002] At present, the toxic effects and mechanisms of many pollutants have been discovered through animal models. However, due to differences between species, the results of animal model verification are difficult to reproduce in humans. At the same time, a large number of animal experiments do not conform to the "3R" principles of toxicological testing: replacement, reduction, and optimization. In vitro cell culture technology has become a key tool for evaluating the toxic effects of pollutants. It simulates the in vivo cell environment by spreading cells on solid supports. This model provides a simple, economical and repeatable experimental platform and is widely used in toxicity screening and environmental monitoring. However, this method has problems such as low throughput, difficulty in docking with downstream detection devices, and difficulty in reflecting the interactions between different cells, tissues, and organs, which reduces the efficiency of evaluating the toxic effects of pollutants. There is an urgent need for a rapid, high-throughput pollutant toxicity effect assessment platform that can comprehensively reflect the interactions between different organisms. Summary of the invention

[0003] The main purpose of the present invention is to provide a method for evaluating the toxic effects of pollutants.

[0004] The technical solution adopted by the present invention to solve its technical problem is:

[0005] A method for evaluating the toxic effects of pollutants, using a microfluidic device, comprises the following steps:

[0006] Step 1, culturing cells, tissues or organoids in a cell culture channel of the cell layer;

[0007] Step 2: injecting pollutants into the concentration gradient generating area of ​​the pollutant exposure layer to form a pollutant concentration gradient;

[0008] Step 3: The pollutants come into contact with cultured cells, tissues or organoids in the exposure area, causing the cells, tissues or organoids to have physiological reactions and interact with each other;

[0009] Step 4: Detect physiological reaction products in the detection area to evaluate the toxic effects of the pollutants;

[0010] Step 5, collecting the effluent and testing the physiological reaction products in the effluent to evaluate the toxic effects of the pollutants;

[0011] Step 6: Detect the surface and internal components of cells, tissues or organoids to evaluate the toxic effects of pollutants;

[0012] The microfluidic device comprises a cell layer and a pollutant exposure layer, the cell layer has at least two cell culture channels, and the at least two cell culture channels are arranged in a first direction.

[0013] The pollutant exposure layer has a concentration gradient generation area, an exposure area and a detection area which are connected in sequence, and the concentration gradient generation area includes at least two injection ports and at least one group of S-shaped bend arrays to generate a concentration gradient; the exposure area is provided with at least 3 parallel flow channels, and the flow channels have a second direction, and the first direction and the second direction have an angle so that each of the at least 2 cell culture channels of the cell layer respectively intersects with the at least 3 parallel flow channels of the pollutant exposure layer; the detection area includes at least one area modified with a capture element.

[0014] In a preferred embodiment, the physiological reaction products described in step 4 include functional biomarkers, cytokines, metabolites and extracellular vesicles secreted by cells.

[0015] In a preferred embodiment, the physiological reaction products in step five include functional biomarkers, cytokines, metabolites and extracellular vesicles secreted by cells.

[0016] In a preferred embodiment, the surface and content components described in step six include functional biomarkers, microvesicles, and organelles.

[0017] In a preferred embodiment, the microfluidic device includes a common substrate, a cell layer fixed on the common substrate in a first step, and a contaminant-exposed layer fixed on the common substrate in a second step.

[0018] In another preferred embodiment, the microfluidic device includes a cell layer disposed on a first carrier and a contaminant exposure layer disposed on a second carrier.

[0019] In a preferred embodiment, the angle between the first direction and the second direction is 30 degrees to 90 degrees.

[0020] In a preferred embodiment, there are at least three concentration gradients and at least three corresponding flow channels in the exposure area.

[0021] In a preferred embodiment, the concentration gradient generating area is composed of a plurality of bends, and the bends have a fishbone-shaped structure for inducing eddy current generation.

[0022] In a preferred embodiment, the detection zone comprises a plurality of micro-pillar arrays.

[0023] In a preferred embodiment, the capture element includes at least one of an antibody, a polypeptide, and a nucleic acid aptamer.

[0024] In a preferred embodiment, each outlet of the pollutant exposure layer corresponds to a column of capture areas, and each column of capture areas includes a plurality of detection areas.

[0025] Compared with the background technology, this technical solution has the following advantages:

[0026] Existing pollutant toxicity assessment methods mainly rely on animal models and large-scale in vitro cell test methods. Animal models have high costs, difficult to reproduce assessment results, and animal ethics issues. However, large-scale cell test methods have low throughput and low integration, and it is difficult to reflect the interactions between different cells or tissues and organs. The present invention relates to a microfluidic device that can automatically generate a pollutant concentration gradient; a variety of cell culture channels are designed in the chip to expose a variety of different cells, tissues or organoids to gradient pollutants, which can reflect the dose-response relationship of pollutants and the interaction between different entities with high throughput; at the same time, a plurality of capture areas can be designed at the chip outlet to capture and analyze proteins, factors, vesicles and other substances secreted by cells, tissues, and organoids in real time; in addition, the surface and contents of cells, tissues, and organoids after exposure to pollutants, and proteins, factors, vesicles and other substances in the effluent can be detected simultaneously to achieve multi-parameter characterization of biomarkers of pollutant toxic effects.

[0027] The invention has a high integration level. The chip integrates pollutant concentration gradient generation, pollutant exposure, real-time capture and detection of cell, tissue or organoid secretions, surface marker and inclusion detection, avoiding cumbersome sample preparation, sample addition and detection processes.

[0028] The present invention can be used to study the interaction between cells, tissues and organs. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0030] Figure 1 This is a chip design diagram of a microfluidic device for evaluating the toxic effects of pollutants according to Example 1 of the present invention, wherein A is the pollutant exposure layer; and B is the cell layer.

[0031] Figure 2 Schematic diagram of the concentration gradient generating area of ​​the microfluidic device in Example 1.

[0032] Figure 3 Schematic diagram of the detection area of ​​the microfluidic device of Example 1 and Example 2.

[0033] Figure 4 This is a chip design diagram of a microfluidic device for evaluating the toxic effects of pollutants according to Example 2 of the present invention, wherein A is the pollutant exposure layer; and B is the cell layer.

[0034] Figure 5The left picture shows the cell growth status in the chip, where the left picture is 24h and the right picture is 48h.

[0035] Figure 6 is the fluorescence intensity of the flow channel in the exposed area.

[0036] Figure 7 is the fluorescence intensity of the detection area. DETAILED DESCRIPTION

[0037] Example 1

[0038] See also Figures 1 to 3 The microfluidic device for evaluating the toxic effects of pollutants of the present invention comprises a common substrate C, a cell layer B fixed to the common substrate C in the first step, and a pollutant exposure layer A fixed to the common substrate C in the second step.

[0039] The common substrate C may be glass, PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate), or the like.

[0040] The cell layer B comprises at least two parallel longitudinal flow channels B2 ( Figure 1 The longitudinal flow channel B2 has a groove at the bottom along the length direction. In this embodiment, there are three longitudinal flow channels B2. Each flow channel has an inlet B21 and an outlet B22. The longitudinal flow channel B2 can be made of glass, PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate), etc.

[0041] Use a clamp to fix the cell layer and the common substrate C with the groove facing downward, and pass at least one cell into the longitudinal flow channel B2 for culture. After the cells adhere to the wall, remove the cell layer B to obtain the common substrate C anchored with cells.

[0042] Pollutant exposure layer A in the horizontal direction ( Figure 1 From left to right) includes a concentration gradient generating area A2, an exposure area A3 and a detection area A4 which are connected in sequence. The concentration gradient generating area A2, the exposure area A3 and the detection area A4 can be made of glass, PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate) and the like.

[0043] The pollutant exposure layer A is fixed with a clamp to the common substrate C anchored with cells, and can be used to study the toxic effects of pollutants on cells. The concentration gradient generation area A2 of the pollutant exposure layer includes two injection ports (the first injection port A21 and the second injection port A20, respectively) and at least one group of S-shaped bend arrays A22. The fishbone structure A221 is designed in the bend to induce the formation of vortices and improve the mixing efficiency between different fluids. One of the injection ports A21 is introduced into a pure solvent without pollutants, and the other injection port is introduced into the pollutant (pre-dissolved in the solvent in A21). After the two are mixed in the concentration gradient generation area, different pollutant concentration gradients are formed and flow into the exposure area. In this embodiment, five outlets are formed by the S-shaped bend array A22, namely outlets A23, A24, A25, A26 and A27. These five outlets form a pollutant concentration gradient from low to high. Specifically, the two injection ports first enter the upper and lower parts of a vertical pipeline respectively, and the pipeline is provided with three outlets, namely, upper, middle and lower outlets, each of which passes through an S-shaped bend (three bends in total), and each S-shaped bend has an outlet, and there are three outlets in total, and these three outlets enter the upper, middle and lower parts of a second vertical pipeline respectively, and the pipeline is provided with four outlets, and each outlet passes through an S-shaped bend (four bends in total). By analogy, five S-shaped bend outlets A23, A24, A25, A26 and A27 are formed.

[0044] The exposure area A3 comprises at least two parallel transverse flow channels A31. In this embodiment, there are five transverse flow channels A31. The left ends of the five transverse flow channels A31 are connected to one of the five outlets A23, A24, A25, A26 and A27 of the concentration gradient generation area A2. The right ends are connected to one of the five inlets A33, A34, A35, A36 and A37 of the detection area A4. A groove is also provided at the bottom of the transverse flow channel A31 along the length direction.

[0045] The longitudinal flow channel B2 of the cell layer B and the transverse flow channel A31 of the exposure area A3 intersect vertically. As mentioned above, the longitudinal flow channel B2 of the cell layer B is clamped with the common substrate C using a clamp, and the cell culture adherent is introduced into the flow channel B2, and the clamp is removed to anchor the cells to the common substrate C. Then the exposed layer A and the common substrate C anchored with cells are clamped with a clamp, and the cells anchored by the longitudinal flow channel B2 on the common substrate C are perpendicular to the transverse flow channel A31, so that the cells on the common substrate C are exposed to pollutants with different concentration gradients in the transverse flow channel A31 in the exposure area A3.

[0046] The detection area includes at least one area modified with a capture motif (such as an antibody, peptide, nucleic acid aptamer, etc.). After being exposed to pollutants, cells can secrete functional biomarkers, cytokines, metabolites, and extracellular vesicles, etc., which are captured in this area. The detection area contains several microcolumn arrays (circular, polygonal), which can increase the specific surface area of ​​the detection area, thereby improving the capture efficiency of cell secretions, and combining the signal output system to evaluate the toxic effects of pollutants. In addition, the cells exposed to pollutants are combined with cell surface / intracellular markers (proteins, nucleic acids, lipids, etc.) and organelle detection strategies to establish a multi-dimensional pollutant toxic effect evaluation system.

[0047] See also Figure 3 In this embodiment, the five entrances A33, A34, A35, A36 and A37 of the detection area in the pollutant exposure layer A correspond to a column of capture areas, and each column of capture areas has 5 detection areas. Taking the entrance A33 as an example, the corresponding column of capture areas has five detection areas A411, A412, A413, A414 and A415. These five detection areas are arranged in parallel. And each has a detection outlet, which are A421, A422, A423, A424 and A425.

[0048] Specific detection examples of the microfluidic device of the present invention for evaluating the toxic effects of pollutants:

[0049] Cell layer assessment

[0050] Mouse neuron cells HT22 were introduced into the longitudinal channel B2 of the cell layer B of the microfluidic chip. After culturing for 24 h and 48 h, the cell growth was observed under a microscope. Figure 5 It can be seen that the cells are growing well, so cell culture can be carried out in this microfluidic chip.

[0051] Pollutant exposure assessment

[0052] PBS was injected into the first injection port A21 at a rate of 0.1 mL / h, and 10 μg / mL rhodamine B solution was injected into the second injection port A20. The fluorescence intensity of the five flow channels in the exposure area A3 was quantified and linear analysis was performed. The results showed that the five flow channels formed a good concentration gradient (R 2 =0.99, Figure 6 ), so the pollutant exposure layer of the chip can form a better concentration gradient.

[0053] The fluorescence intensity of the five detection areas corresponding to the first transverse flow channel A31 of the exposure area A3 was quantified. The results showed that the intensity of the five detection areas was relatively uniform (CV = 1.46%, Figure 7 ). Therefore, the cell secretion substances corresponding to the exposure area can flow into the detection area evenly and be captured for subsequent analysis.

[0054] Example 2

[0055] See also Figure 4 The microfluidic device for evaluating the toxic effects of pollutants of the present invention includes a cell layer B located at the bottom layer and a pollutant exposure layer A located at the top layer.

[0056] The cell layer B is disposed on a first carrier B1, which may be glass, PDMS (polydimethylsiloxane), PMMA, etc. The upper surface of the carrier B1 is provided with at least two parallel longitudinal flow channels B2 ( Figure 4 In the vertical direction), in this embodiment, there are three longitudinal channels B2. The longitudinal channels B2 are grooves on the first carrier B1, and each channel has an inlet B21 and an outlet B22. A glass slide is placed on the carrier B1 so that the glass slide covers the longitudinal channels B2, at least one cell is cultured in the chip, and the glass slide is removed after the cells adhere to the wall, so that the cells can be pre-anchored on the chip surface.

[0057] The pollutant exposure layer A is disposed on the second carrier A1, and the second carrier A1 can be glass, PDMS, PMMA, etc. The carrier A1 is transversely ( Figure 4 In the embodiment 1, the pollutant exposure layer A is fixed to the cell layer B with a clamp, and can be used for the study of the exposure toxicity of pollutants to cells. The concentration gradient generation area A2 of the pollutant exposure layer includes two injection ports (the first injection port A21 and the second injection port A20, respectively) and at least one group of S-shaped bend arrays A22. A fishbone-shaped structure A221 is designed in the bend to induce the formation of vortices and improve the mixing efficiency between different fluids. A pure solvent without pollutants is introduced into one of the injection ports A21, and a pollutant (pre-dissolved in the solvent in A21) is introduced into the other injection port. After the two are mixed in the concentration gradient generation area, different pollutant concentration gradients are formed and flow into the exposure area. In this embodiment, five outlets are formed by the S-shaped bend array A22, namely outlets A23, A24, A25, A26 and A27. These five outlets form a pollutant concentration gradient from low to high.

[0058] The exposure area A3 comprises at least two parallel transverse flow channels A31. In this embodiment, there are five transverse flow channels A31. The left ends of the five transverse flow channels A31 are respectively connected to one of the five outlets A23, A24, A25, A26 and A27 of the pollutant exposure layer A. The right ends are connected to the detection area A4.

[0059] The longitudinal flow channel B2 of the cell layer B and the transverse flow channel A31 of the exposure area A3 intersect vertically, and the transverse flow channel A31 of the exposure area A3 is located on the upper surface of the cells anchored by the longitudinal flow channel B2, so that the cells in this exposure area A3 are exposed to pollutants with different concentration gradients.

[0060] The detection area includes at least one area modified with a capture motif (such as an antibody, peptide, nucleic acid aptamer, etc.). After being exposed to pollutants, cells can secrete functional biomarkers, cytokines, metabolites, and extracellular vesicles, etc., which are captured in this area. The detection area contains several microcolumn arrays (circular, polygonal), which can increase the specific surface area of ​​the detection area, thereby improving the capture efficiency of cell secretions, and combining the signal output system to evaluate the toxic effects of pollutants. In addition, the cells exposed to pollutants are combined with cell surface / intracellular markers (proteins, nucleic acids, lipids, etc.) and organelle detection strategies to establish a multi-dimensional pollutant toxic effect evaluation system.

[0061] See also Figure 3 In this embodiment, the five entrances A33, A34, A35, A36 and A37 of the detection area in the pollutant exposure layer A correspond to a column of capture areas, and each column of capture areas has 5 detection areas. Taking the entrance A33 as an example, the corresponding column of capture areas has five detection areas A411, A412, A413, A414 and A415. These five detection areas are arranged in parallel. And each has a detection outlet, which are A421, A422, A423, A424 and A425.

[0062] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for evaluating the toxic effects of pollutants, using a microfluidic device, the method comprising the following steps: Step 1, culturing cells, tissues or organoids in a cell culture channel of the cell layer; Step 2: injecting pollutants into the concentration gradient generating area of ​​the pollutant exposure layer to form a pollutant concentration gradient; Step 3: The pollutants come into contact with cultured cells, tissues or organoids in the exposure area, causing the cells, tissues or organoids to have physiological reactions and interact with each other; Step 4: Detect physiological reaction products in the detection area to evaluate the toxic effects of the pollutants; Step 5, collecting the effluent and testing the physiological reaction products in the effluent to evaluate the toxic effects of the pollutants; Step 6: Detect the surface and internal components of cells, tissues or organoids to evaluate the toxic effects of pollutants; The microfluidic device comprises a cell layer and a pollutant exposure layer, wherein the cell layer has at least two cell culture channels, and the at least two cell culture channels are arranged in a first direction; The pollutant exposure layer has a concentration gradient generation area, an exposure area and a detection area which are connected in sequence, and the concentration gradient generation area includes at least two injection ports and at least one group of S-shaped bend arrays to generate a concentration gradient; the exposure area is provided with at least 3 parallel flow channels, and the flow channels have a second direction, and the first direction and the second direction have an angle so that each of the at least 2 cell culture channels of the cell layer respectively intersects with the at least 3 parallel flow channels of the pollutant exposure layer; the detection area includes at least one area modified with a capture element.

2. A method for evaluating the toxic effects of pollutants according to claim 1, It is characterized in that The physiological response products described in step 4 include functional biomarkers, cytokines, metabolites and extracellular vesicles secreted by cells.

3. A method for evaluating the toxic effects of pollutants according to claim 1, It is characterized in that The physiological reaction products described in step five include functional biomarkers, cytokines, metabolites and extracellular vesicles secreted by cells; the surface and inclusion components described in step six include functional biomarkers, microvesicles and organelles.

4. A method for evaluating the toxic effects of pollutants according to claim 1, Features: The microfluidic device comprises a common substrate, a cell layer fixed on the common substrate in the first step, and a pollutant exposure layer fixed on the common substrate in the second step.

5. A method for evaluating the toxic effects of pollutants according to claim 1, Features: The microfluidic device comprises a cell layer arranged on a first carrier and a pollutant exposure layer arranged on a second carrier.

6. A method for evaluating the toxic effects of pollutants according to claim 4 or 5, Features: The angle between the first direction and the second direction of the microfluidic device is 30 degrees to 90 degrees.

7. A method for evaluating the toxic effects of pollutants according to claim 4 or 5, Features: There are at least three concentration gradients, and there are also at least three corresponding flow channels in the exposure area.

8. A method for evaluating the toxic effects of pollutants according to claim 4 or 5, Features: The concentration gradient generating area is composed of a plurality of bends, and the bends have a fishbone-shaped structure for inducing eddy current generation.

9. A method for evaluating the toxic effects of pollutants according to claim 4 or 5, Features: The detection area comprises a plurality of micro-column arrays.

10. A method for evaluating the toxic effects of pollutants according to claim 1, Features: The capture element includes at least one of an antibody, a polypeptide, and a nucleic acid aptamer; each outlet of the pollutant exposure layer corresponds to a column of capture areas, and each column of capture areas includes a plurality of detection areas.

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