A method for assessing the toxic effects of pollutants

Abstract

The application discloses a method for evaluating the toxic effect of pollutants, which takes a microfluidic device as an analysis platform, and the microfluidic device comprises a cell layer channel fixed on a substrate in a first step and a pollutant exposure layer fixed on the substrate in a second step, wherein the cell layer has at least two cell culture channels, and the gas arrangement direction is the first direction; the pollutant exposure layer has a concentration gradient generation area, an exposure area and a detection area which are sequentially connected; the exposure area is provided with at least three parallel flow channels, the flow channels have a second direction, and the first direction and the second direction have an included angle, so that each cell culture channel of the at least two cell culture channels of the cell layer and the at least three parallel flow channels of the pollutant exposure layer are crossed respectively; and the detection area at least comprises a region modified with a capture element. The application can avoid complicated sample preparation, sample adding and detection processes.

Description

Technical Field

[0001] This invention relates to a method for assessing the toxic effects of pollutants. Background Technology

[0002] Currently, the toxic effects and mechanisms of many pollutants are discovered through animal models. However, due to interspecies differences, the results validated in animal models are difficult to reproduce in humans. Furthermore, many 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 assessing the toxic effects of pollutants. It simulates the in vivo cellular environment by growing cells spread 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 suffers from low throughput, difficulty in interfacing with downstream detection devices, and difficulty in reflecting the interactions between different cells, tissues, and organs, thus reducing the efficiency of pollutant toxicity assessment. There is an urgent need for a rapid, high-throughput platform for assessing pollutant toxicity effects that can comprehensively reflect the interactions between different organisms. Summary of the Invention

[0003] The main objective of this invention is to provide a method for assessing the toxic effects of pollutants.

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

[0005] A method for assessing the toxic effects of pollutants, employing a microfluidic device, includes the following steps:

[0006] Step 1: Culture cells, tissues, or organoids in the cell culture channels of the cell layer;

[0007] Step 2: Inject pollutants into the concentration gradient generation zone of the pollutant exposure layer to form a pollutant concentration gradient;

[0008] Step 3: The contaminants come into contact with the cultured cells, tissues, or organoids in the exposure area, causing physiological reactions and interactions in the cells, tissues, or organoids.

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

[0010] Step 5: Collect the effluent and test the physiological reaction products in the effluent to assess the toxic effects of the pollutants;

[0011] Step six: Detect the components on the surface and inside of cells, tissues, or organoids to assess the toxic effects of pollutants;

[0012] The microfluidic device includes a cell layer and a contaminant 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 contaminant exposure layer has a concentration gradient generation zone, an exposure zone, and a detection zone that are connected in sequence. The concentration gradient generation zone includes at least two inlets and at least one set of S-shaped bend arrays to generate a concentration gradient. The exposure zone has at least three parallel flow channels with a second direction. The first direction and the second direction have an angle such that each of the at least two cell culture channels in the cell layer intersects with at least three parallel flow channels in the contaminant exposure layer. The detection zone includes at least one region modified with a capture unit.

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

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

[0016] In a preferred embodiment, the surface and inclusion 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 first fixed on the common substrate, and a contaminant exposure layer second fixed on the common substrate.

[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 flow channels in the corresponding exposure area.

[0021] In a preferred embodiment, the concentration gradient generation region is composed of multiple bends, each bend having a fishbone-shaped structure for inducing eddy current generation.

[0022] In a preferred embodiment, the detection region comprises an array of multiple micropillars.

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

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

[0025] Compared with the prior art, this technical solution has the following advantages:

[0026] Existing methods for assessing pollutant toxicity primarily rely on animal models and large-scale in vitro cell experiments. Animal models suffer from high costs, difficulty in reproducing assessment results, and ethical concerns. Large-scale cell experiments, on the other hand, have low throughput, low integration, and struggle to reflect interactions between different cells, tissues, or organs. This invention relates to a microfluidic device that automatically generates a pollutant concentration gradient. The chip incorporates multiple cell culture channels, exposing various cells, tissues, or organoids to the gradient pollutant. This allows for high-throughput reflection of the dose-response relationship of the pollutant and interactions between different organisms. Simultaneously, the chip outlet can be designed with multiple capture zones to capture and analyze proteins, factors, vesicles, and other substances secreted by cells, tissues, and organoids in real time. Furthermore, it can simultaneously detect the surface and contents of cells, tissues, and organoids after pollutant exposure, as well as proteins, factors, vesicles, and other substances in the effluent, achieving multi-parameter characterization of biomarkers for pollutant toxicity effects.

[0027] This invention features high integration. The chip integrates pollutant concentration gradient generation, pollutant exposure, real-time capture and detection of secretions from cells, tissues, or organoids, and detection of surface markers and contents, avoiding cumbersome sample preparation, addition, and detection processes.

[0028] This invention enables the study of interactions between cells, tissues, and organs. Attached Figure Description

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

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

[0031] Figure 2 This is a schematic diagram of the concentration gradient generation region of the microfluidic device in Example 1.

[0032] Figure 3 This is a schematic diagram of the detection area of ​​the microfluidic device in Examples 1 and 2.

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

[0034] Figure 5The images show the cell growth status in the chip, with the left image showing 24 hours and the right image showing 48 hours.

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

[0036] Figure 7 The fluorescence intensity in the detection area. Detailed Implementation

[0037] Example 1

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

[0039] The common substrate C can be glass, PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate), etc.

[0040] The cell layer B includes at least two parallel longitudinal flow channels B2. Figure 1 The longitudinal flow channels B2 have grooves along their length at the bottom. 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), etc.

[0041] Use clamps to fix the cell layer to the common substrate C with the groove facing down. Introduce at least one type of cell into the longitudinal channel B2 for culture. After the cells adhere to the wall, remove the cell layer B to obtain the common substrate C with the cells anchored.

[0042] Laterally on the pollutant exposure layer A ( Figure 1 From left to right, the zone includes a concentration gradient generation zone A2, an exposure zone A3, and a detection zone A4, which are connected sequentially. The concentration gradient generation zone A2, exposure zone A3, and detection zone A4 can be made of glass, PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate), etc.

[0043] The contaminant exposure layer A is fixed to a shared substrate C anchored with cells using clamps, allowing it to be used for studying the toxic effects of contaminants on cells. The concentration gradient generation zone A2 of the contaminant exposure layer includes two inlets (first inlet A21 and second inlet A20) and at least one set of S-shaped bend arrays A22. A herringbone structure A221 is designed within the bends to induce vortices and improve the mixing efficiency between different fluids. One inlet A21 is supplied with a pure solvent free of contaminants, while the other inlet is supplied with contaminants (pre-dissolved in the solvent in A21). The two mix in the concentration gradient generation zone, forming different contaminant concentration gradients that flow into the exposure zone. In this embodiment, five outlets are formed by the S-shaped bend array A22: outlets A23, A24, A25, A26, and A27. These five outlets form a contaminant concentration gradient from low to high. Specifically, the two inlets first enter the upper and lower parts of a vertically oriented pipe, respectively. This pipe has three outlets: upper, middle, and lower. Each outlet passes through an S-shaped bend (a total of three bends), and each S-shaped bend has an outlet, for a total of three outlets. These three outlets then enter the upper, middle, and lower parts of a second vertically oriented pipe, respectively. This pipe has four outlets, each passing through an S-shaped bend (a total of four bends). This process continues, forming outlets A23, A24, A25, A26, and A27 with five S-shaped bends.

[0044] The exposure zone A3 includes at least two parallel transverse channels A31. In this embodiment, there are five transverse channels A31. The left ends of these five transverse channels A31 are connected to one of the five outlets A23, A24, A25, A26, and A27 of the concentration gradient generation zone A2, respectively. The right ends are connected to one of the five inlets A33, A34, A35, A36, and A37 of the detection zone A4. The bottom of each transverse channel A31 also has grooves along its length.

[0045] The longitudinal flow channel B2 of cell layer B and the transverse flow channel A31 of exposure zone A3 intersect perpendicularly. As previously described, clamps are used to secure the longitudinal flow channel B2 of cell layer B to the common substrate C, and cell culture is introduced into flow channel B2 for adhesion. The clamps are then removed, anchoring the cells to the common substrate C. Next, the exposure layer A is clamped to the common substrate C with the anchored cells. The cells on the common substrate C anchored by the longitudinal flow channel B2 are perpendicular to the transverse flow channel A31, thus exposing the cells on the common substrate C to contaminants of different concentration gradients in the transverse flow channel A31 of exposure zone A3.

[0046] The detection zone includes at least one area modified with capture motifs (such as antibodies, peptides, nucleic acid aptamers, etc.). After exposure to contaminants, cells can secrete functional biomarkers, cytokines, metabolites, and extracellular vesicles, which are captured in this zone. The detection zone contains several micropillar arrays (circular, polygonal), which can increase the specific surface area of ​​the detection zone, thereby improving the capture efficiency of cell secretions. Combined with a signal output system, the toxic effects of contaminants can be assessed. Furthermore, by combining contaminant-exposed cells with cell surface / intracellular markers (proteins, nucleic acids, lipids, etc.) and organelle detection strategies, a multi-dimensional contaminant toxicity effect assessment system can be established.

[0047] See Figure 3 In this embodiment, the five inlets A33, A34, A35, A36, and A37 in the detection area of ​​the pollutant exposure layer A correspond to a column of capture areas. Each column of capture areas contains five detection areas. Taking inlet A33 as an example, its corresponding column of capture areas has five detection areas A411, A412, A413, A414, and A415. These five detection areas are arranged side by side and each has a detection outlet, namely A421, A422, A423, A424, and A425.

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

[0049] Cell layer assessment

[0050] Mouse neuronal cells (HT22) were introduced into the longitudinal channel B2 of cell layer B in a microfluidic chip. Cell growth was observed under a microscope after 24 and 48 hours of culture. Figure 5 As can be seen, the cells are growing well, therefore, cell culture can be performed in this microfluidic chip.

[0051] Pollutant exposure layer assessment

[0052] PBS was injected at a rate of 0.1 mL / h into the first injection port A21, and 10 μg / mL Rhodamine B solution was injected into the second injection port A20. The fluorescence intensity of the five channels in the exposure area A3 was quantified and linear analysis was performed. The results showed that a good concentration gradient (R0) was formed in the five channels. 2 =0.99, Figure 6 Therefore, the contaminant exposure layer of this chip can form a good concentration gradient.

[0053] Fluorescence intensity was quantified in five detection areas corresponding to the first transverse flow channel A31 in exposed area A3. 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.

[0054] Example 2

[0055] See Figure 4 The microfluidic device of the present invention for assessing the toxic effects of pollutants includes a cell layer B located in the lower layer and a pollutant exposure layer A located in the upper layer.

[0056] The cell layer B is disposed on a first carrier B1, which can be glass, PDMS (polydimethylsiloxane), PMMA, etc. The upper surface of the carrier B1 has at least two parallel longitudinal flow channels B2. Figure 4 In this embodiment, the vertical flow channels (the vertical direction) are three longitudinal channels B2. Each longitudinal channel B2 is a groove on the first carrier B1, with an inlet B21 and an outlet B22. A glass slide is placed on the carrier B1 to cover the longitudinal channels B2. At least one type of cell is introduced into the chip for culture. After the cells adhere to the chip, the glass slide is removed, thus pre-anchoring the cells on the chip surface.

[0057] A pollutant exposure layer A is disposed on a second carrier A1, which may be made of glass, PDMS, PMMA, etc. The carrier A1 is positioned laterally (…). Figure 4 From left to right, a concentration gradient generation zone A2, an exposure zone A3, and a detection zone A4 are sequentially connected. The concentration gradient generation zone A2, exposure zone A3, and detection zone A4 are all etched onto the second carrier A1. The structures of the concentration gradient generation zone A2, exposure zone A3, and detection zone A4 are the same as in Example 1. The pollutant exposure layer A is fixed to the cell layer B using a clamp, allowing it to be used for pollutant exposure toxicity studies on cells. The concentration gradient generation zone A2 of the pollutant exposure layer includes two inlets (a first inlet A21 and a second inlet A20) and at least one set of S-shaped bend arrays A22. A fishbone-shaped structure A221 is designed within the bends to induce vortices and improve the mixing efficiency between different fluids. One inlet A21 is filled with a pure solvent free of pollutants, while the other inlet is filled with pollutants (pre-dissolved in the solvent in A21). After mixing in the concentration gradient generation zone, different pollutant concentration gradients are formed and flow into the exposure zone. 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 zone A3 includes at least two 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, respectively. The right ends are connected to the detection zone A4.

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

[0060] The detection zone includes at least one area modified with capture motifs (such as antibodies, peptides, nucleic acid aptamers, etc.). After exposure to contaminants, cells can secrete functional biomarkers, cytokines, metabolites, and extracellular vesicles, which are captured in this zone. The detection zone contains several micropillar arrays (circular, polygonal), which can increase the specific surface area of ​​the detection zone, thereby improving the capture efficiency of cell secretions. Combined with a signal output system, the toxic effects of contaminants can be assessed. Furthermore, by combining contaminant-exposed cells with cell surface / intracellular markers (proteins, nucleic acids, lipids, etc.) and organelle detection strategies, a multi-dimensional contaminant toxicity effect assessment system can be established.

[0061] See Figure 3 In this embodiment, the five inlets A33, A34, A35, A36, and A37 in the detection area of ​​the pollutant exposure layer A correspond to a column of capture areas. Each column of capture areas contains five detection areas. Taking inlet A33 as an example, its corresponding column of capture areas has five detection areas A411, A412, A413, A414, and A415. These five detection areas are arranged side by side and each has a detection outlet, namely A421, A422, A423, A424, and A425.

[0062] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for assessing the toxic effects of pollutants, employing a microfluidic device, the microfluidic device comprising a cell layer and a pollutant exposure layer, the cell layer having at least two cell culture channels arranged in a first direction; The contaminant exposure layer has a concentration gradient generation zone, an exposure zone, and a detection zone connected in sequence. The concentration gradient generation zone includes at least two inlets and at least one set of S-shaped bend arrays to generate a concentration gradient. The exposure zone has at least three parallel flow channels with a second direction. The first direction and the second direction form an angle such that each of the at least two cell culture channels in the cell layer intersects with at least three parallel flow channels in the contaminant exposure layer, thereby simultaneously exposing cells in the same cell culture channel to contaminants at all concentration gradients. The detection zone includes at least one region modified with a capture unit. Each outlet of the contaminant exposure layer corresponds to a column of capture regions, and each column of capture regions includes multiple detection zones. The method includes the following steps: Step 1: Culture cells, tissues, or organoids in the cell culture channels of the cell layer; Step 2: Inject pollutants into the concentration gradient generation zone of the pollutant exposure layer to form a pollutant concentration gradient; Step 3: The contaminants come into contact with the cultured cells, tissues, or organoids in the exposure area, causing physiological reactions and interactions in the cells, tissues, or organoids. Step four: Detect physiological reaction products in the detection area to assess the toxic effects of pollutants; Step 5: Collect the effluent and test the physiological reaction products in the effluent to assess the toxic effects of the pollutants; Step six involves detecting the components on the surface and inside of cells, tissues, or organoids to assess the toxic effects of pollutants.

2. The method for assessing the toxic effects of pollutants according to claim 1, characterized in that, The physiological reaction products described in step four include functional biomarkers secreted by cells, cytokines, metabolites, and extracellular vesicles.

3. The method for assessing the toxic effects of pollutants according to claim 1, 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. The method for assessing the toxic effects of pollutants according to claim 1, characterized in that: The microfluidic device includes a common substrate, a cell layer first fixed on the common substrate, and a contaminant exposure layer second fixed on the common substrate.

5. The method for assessing the toxic effects of pollutants according to claim 1, characterized in that: The microfluidic device includes a cell layer disposed on a first carrier and a pollutant exposure layer disposed on a second carrier.

6. A method for assessing the toxic effects of pollutants according to claim 4 or 5, characterized in that: The angle between the first and second directions of the microfluidic device is 30 degrees to 90 degrees.

7. A method for assessing the toxic effects of pollutants according to claim 4 or 5, characterized in that: The concentration gradient generation region consists of multiple bends, each containing a fishbone-shaped structure designed to induce eddy currents.

8. A method for assessing the toxic effects of pollutants according to claim 4 or 5, characterized in that: The detection area contains multiple micropillar arrays.

9. The method for assessing the toxic effects of pollutants according to claim 1, characterized in that: The capture motif includes at least one of antibodies, peptides, and nucleic acid aptamers.

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