Microfluidic device for evaluating toxic effect of pollutants
By designing the concentration gradient generation and detection zone of the microfluidic control device, the high cost and low throughput problems of pollutant toxicity assessment in the prior art are solved, and efficient and integrated pollutant toxicity effect assessment is achieved, which can reflect the interaction between cells and tissues.
Patent Information
- Application Number
- CN202422290578.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-19
- Publication Date
- 2025-08-12
- Estimated Expiration
- 2034-09-19
AI Technical Summary
The existing methods for toxicity assessment of pollutants rely on animal models and large-scale in vitro cell tests, which have high costs, difficult to reproduce the evaluation results, ethical problems and low flux, making it difficult to reflect the interactions between different cells or tissues and organs.
A microfluidic control device is designed, including a cell layer and a pollutant exposure layer, with a concentration gradient generation area, an exposure area and a detection area. The pollutant concentration gradient is generated through the S-shaped curved array, and the cell secreted substances are captured in real time in the detection area to achieve high-throughput, multi-parameter toxic effect evaluation.
A high-throughput, highly integrated pollutant toxicity assessment is achieved, which can reflect the interactions of different cells or tissues and organs, simplify sample preparation and detection processes, and provide multi-parameter biomarker detection.
Smart Images
Figure CN223214098U_ABST
Abstract
Description
Technical Field
[0001] The utility model relates to a microfluidic device for evaluating the toxic effects of pollutants. Background Art
[0002] At present, the toxic effects and mechanisms of many pollutants are discovered through animal models, but 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" principle 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 a solid support. This model provides a simple, economical and reproducible 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 pollutant toxicity assessment. There is an urgent need for a rapid, high-throughput pollutant toxicity effect assessment platform that can comprehensively reflect the interactions between different organisms. Utility Model Content
[0003] The main purpose of the utility model is to provide a microfluidic device for evaluating the toxic effects of pollutants.
[0004] The technical solution adopted by the utility model to solve its technical problems is:
[0005] A microfluidic device for evaluating the toxic effects of pollutants 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, wherein:
[0006] The cell layer has at least two cell culture channels, and the at least two cell culture channels are arranged in a first direction;
[0007] The pollutant exposure layer has a concentration gradient generation area, an exposure area and a detection area connected in sequence, the concentration gradient generation area includes at least two sample 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, 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 intersects with the at least 3 parallel flow channels of the pollutant exposure layer respectively; the detection area includes at least one area modified with a capture element.
[0008] Another technical solution of the present utility model is:
[0009] A microfluidic device for evaluating the toxic effects of pollutants comprises a cell layer provided on a first carrier and a pollutant exposure layer provided on a second carrier, wherein:
[0010] The cell layer has at least two cell culture channels, and the at least two cell culture channels are arranged in a first direction;
[0011] The pollutant exposure layer has a concentration gradient generation area, an exposure area and a detection area connected in sequence, the concentration gradient generation area includes at least two sample 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, 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 intersects with the at least 3 parallel flow channels of the pollutant exposure layer respectively; the detection area includes at least one area modified with a capture element.
[0012] In a preferred embodiment, the angle between the first direction and the second direction is 30 degrees to 90 degrees.
[0013] In a preferred embodiment, there are at least three concentration gradients, and there are also at least three corresponding flow channels in the exposed area.
[0014] In a preferred embodiment, the concentration gradient generating area is composed of a plurality of bends.
[0015] In a preferred embodiment, a fishbone structure is provided in the curve for inducing vortex generation.
[0016] In a preferred embodiment, the detection area comprises a plurality of micro-pillar arrays.
[0017] 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.
[0018] Compared with the background technology, this technical solution has the following advantages:
[0019] Existing methods for assessing the toxicity of pollutants mainly rely on animal models and large-scale in vitro cell testing methods. Animal models have the problems of high cost, difficulty in reproducing assessment results, and animal ethics issues. However, large-scale cell testing methods have low throughput and low integration, and are difficult to reflect the interactions between different cells or tissues and organs. The present invention provides a microfluidic device that can automatically generate a concentration gradient of pollutants; 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, multiple 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 sub-contents of cells, tissues, and organoids after exposure to pollutants, as well as proteins, factors, vesicles and other substances in the effluent, can be simultaneously detected to achieve multi-parameter characterization of biomarkers of the toxic effects of pollutants.
[0020] The chip integrates pollutant concentration gradient generation, pollutant exposure, real-time capture and detection of cell, tissue or organoid secretions, and surface marker and content detection, thus avoiding the tedious sample preparation, sample addition and detection process.
[0021] The utility model can be used to study the interaction between cells, tissues and organs. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The present invention will be further described below with reference to the accompanying drawings and embodiments.
[0023] 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.
[0024] Figure 2 Schematic diagram of the concentration gradient generation area of the microfluidic device in Example 1.
[0025] Figure 3 Schematic diagram of the detection area of the microfluidic device of Example 1 and Example 2.
[0026] 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 utility model, wherein A is the pollutant exposure layer; and B is the cell layer.
[0027] Figure 5 The left picture shows the cell growth status in the chip, where the left picture is 24 hours and the right picture is 48 hours.
[0028] Figure 6 is the fluorescence intensity of the flow channel in the exposed area.
[0029] Figure 7is the fluorescence intensity of the detection area. DETAILED DESCRIPTION
[0030] Example 1
[0031] See also Figures 1 to 3 The microfluidic device for evaluating the toxic effects of pollutants of the present invention includes a common substrate C, a cell layer B fixed on the common substrate C in the first step, and a pollutant exposure layer A fixed on the common substrate C in the second step.
[0032] The common substrate C may be glass, PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate), or the like.
[0033] The cell layer B comprises at least two parallel longitudinal flow channels B2 ( Figure 1 The longitudinal flow channel B2 has grooves along its bottom length (in the vertical 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 channels B2 can be made of glass, PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate), or the like.
[0034] Use a clamp to fix the cell layer to the common substrate C with the groove facing downward, and culture at least one cell in the longitudinal flow channel B2. After the cells adhere to the wall, remove the cell layer B to pre-anchor the cells on the surface of the common substrate C.
[0035] Pollutant exposure layer A in the horizontal direction ( Figure 1 From left to right, the device 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), etc.
[0036] The pollutant exposure layer A is fixed with a clamp to the common substrate C anchored with cells, and can be used for the exposure toxicity study 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) and at least one group of S-shaped bend arrays A22. The fishbone-shaped 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 fed with a pure solvent free of pollutants, and the other injection port A20 is fed with pollutants (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 inlets first enter the upper and lower portions of a vertically oriented pipe, respectively. This pipe is provided with three outlets: upper, middle, and lower. Each outlet passes through an S-shaped bend (three bends in total). Each S-shaped bend has an outlet, for a total of three outlets. These three outlets enter the upper, middle, and lower portions of a second vertically oriented pipe, respectively. This pipe is provided with four outlets, each of which passes through an S-shaped bend (four bends in total). Similarly, five S-shaped outlets A23, A24, A25, A26, and A27 are formed.
[0037] Exposure zone A3 includes at least three parallel transverse flow channels A31. In this embodiment, there are five transverse flow channels A31. The left ends of these five transverse flow channels A31 are connected to one of the five outlets A23, A24, A25, A26, and A27 of concentration gradient generation zone A2. The right ends of these five transverse flow channels A31 are connected to one of the five inlets A33, A34, A35, A36, and A37 of detection zone A4. The bottoms of these transverse flow channels A31 are also grooved along their lengths.
[0038] The longitudinal flow channel B2 of cell layer B and the transverse flow channel A31 of exposure area A3 intersect perpendicularly. As previously described, a clamp is used to clamp the longitudinal flow channel B2 of cell layer B to the shared substrate C. Cell culture medium is introduced into flow channel B2, and the clamp is removed to obtain the shared substrate C with anchored cells. The exposed layer A and the shared substrate C with anchored cells are then clamped together with the clamp. The cells anchored by the longitudinal flow channel B2 on the shared substrate C are perpendicular to the transverse flow channel A31, exposing the cells on the shared substrate C to the varying concentration gradients of contaminants in the transverse flow channel A31 of exposure area A3.
[0039] The detection zone includes at least one area modified with a capture motif (such as an antibody, peptide, or nucleic acid aptamer). After exposure to pollutants, cells can secrete functional biomarkers, cytokines, metabolites, and extracellular vesicles, which are captured in this area. The detection zone contains several microcolumn arrays (circular or polygonal), which can increase the specific surface area of the detection zone, thereby improving the capture efficiency of cell secretions. Combined with the signal output system, the toxic effects of pollutants can be evaluated. 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 toxicity effect assessment system.
[0040] See also Figure 3 In this embodiment, the five inlets A33, A34, A35, A36, and A37 of the detection zone in pollutant exposure layer A each correspond to a row of capture areas. Each row of capture areas has five detection zones. Taking inlet A33 as an example, the corresponding row of capture areas has five detection zones: A411, A412, A413, A414, and A415. These five detection zones are arranged in parallel and each has a detection outlet: A421, A422, A423, A424, and A425.
[0041] Specific detection examples of the microfluidic device used in the utility model for evaluating the toxic effects of pollutants:
[0042] Cell layer assessment
[0043] Mouse neuronal cells HT22 were introduced into the longitudinal channel B2 of the cell layer B3 of the microfluidic chip. After culturing for 24 hours and 48 hours, 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.
[0044] Pollutant exposure assessment
[0045] 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.
[0046] 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 secretions corresponding to the exposed area can flow evenly into the detection area and be captured for subsequent analysis.
[0047] Example 2
[0048] 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 and a pollutant exposure layer A located at the top.
[0049] The cell layer B is placed on a first carrier B1, which can be made of 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 this embodiment, the three longitudinal flow channels B2 are grooves on the first carrier B1. Each channel has an inlet B21 and an outlet B22. A glass slide is placed on the carrier B1 so that it covers the longitudinal flow channels B2. At least one type of cell is cultured in the chip. After the cells adhere, the glass slide is removed to pre-anchor the cells on the chip surface.
[0050] The pollutant exposure layer A is provided on the second carrier A1, which can be made of glass, PDMS, PMMA, etc. The carrier A1 is laterally ( Figure 4 (from left to right) is provided with 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 are all etched on the second carrier A1. The structures of the concentration gradient generating area A2, the exposure area A3 and the detection area A4 are the same as those in Example 1. The pollutant exposure layer A is fixed to the cell layer B with a clamp, and can be used for the exposure toxicity study of pollutants to cells. The concentration gradient generating 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. One of the injection ports A21 introduces a pure solvent without pollutants, and the other injection port A20 introduces a pollutant (pre-dissolved in the solvent in A21). After the two are mixed in the concentration gradient generating 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.
[0051] Exposure zone A3 includes at least three parallel transverse flow channels A31. In this embodiment, there are five transverse flow channels A31. The left ends of these five transverse flow channels A31 are connected to one of the five outlets A23, A24, A25, A26, and A27 of the pollutant exposure layer A. The right ends of these five transverse flow channels A31 are connected to the detection zone A4.
[0052] 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.
[0053] The detection zone includes at least one area modified with a capture motif (such as an antibody, peptide, or nucleic acid aptamer). After exposure to pollutants, cells can secrete functional biomarkers, cytokines, metabolites, and extracellular vesicles, which are captured in this area. The detection zone contains several microcolumn arrays (circular or polygonal), which can increase the specific surface area of the detection zone, thereby improving the capture efficiency of cell secretions. Combined with the signal output system, the toxic effects of pollutants can be evaluated. 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 toxicity effect assessment system.
[0054] See also Figure 3 In this embodiment, the five inlets A33, A34, A35, A36, and A37 of the detection zone in pollutant exposure layer A each correspond to a row of capture areas. Each row of capture areas has five detection zones. Taking inlet A33 as an example, the corresponding row of capture areas has five detection zones: A411, A412, A413, A414, and A415. These five detection zones are arranged in parallel and each has a detection outlet: A421, A422, A423, A424, and A425.
[0055] The above description is merely a preferred embodiment of the present invention and therefore cannot be used to limit the scope of implementation of the present invention. In other words, 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 microfluidic device for evaluating the toxic effects of pollutants, characterized by: The method comprises a common substrate, a cell layer fixed on the common substrate in a first step, and a pollutant-exposed layer fixed on the common substrate in a second step, 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 connected in sequence, the concentration gradient generation area includes at least two sample 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, 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 intersects with the at least 3 parallel flow channels of the pollutant exposure layer respectively; the detection area includes at least one area modified with a capture element.
2. A microfluidic device for evaluating the toxic effects of pollutants, characterized by: The method comprises a cell layer disposed on a first carrier and a pollutant-exposed layer disposed on a second carrier, 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 connected in sequence, the concentration gradient generation area includes at least two sample 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, 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 intersects with the at least 3 parallel flow channels of the pollutant exposure layer respectively; the detection area includes at least one area modified with a capture element.
3. A microfluidic device for evaluating the toxic effects of pollutants according to claim 1 or 2, characterized in that: The angle between the first direction and the second direction is 30 degrees to 90 degrees.
4. A microfluidic device for evaluating the toxic effects of pollutants according to claim 1 or 2, characterized in that: There are at least three concentration gradients, and there are also at least three corresponding flow channels in the exposed area.
5. A microfluidic device for evaluating the toxic effects of pollutants according to claim 1 or 2, characterized in that: The concentration gradient generating area is composed of a plurality of bends.
6. The microfluidic device for evaluating the toxic effects of pollutants according to claim 5, characterized in that: There is a fishbone structure in the curve to induce vortex generation.
7. A microfluidic device for evaluating the toxic effects of pollutants according to claim 1 or 2, characterized in that: The detection area contains multiple micro-pillar arrays.
8. A microfluidic device for evaluating the toxic effects of pollutants according to claim 1 or 2, characterized in that: Each outlet of the pollutant exposure layer corresponds to a column of capture areas, and each column of capture areas includes multiple detection areas.