CN217922153U - Microchip device capable of realizing two-dimensional exposure of aerosol and liquid - Google Patents
Microchip device capable of realizing two-dimensional exposure of aerosol and liquid Download PDFInfo
- Publication number
- CN217922153U CN217922153U CN202220927432.7U CN202220927432U CN217922153U CN 217922153 U CN217922153 U CN 217922153U CN 202220927432 U CN202220927432 U CN 202220927432U CN 217922153 U CN217922153 U CN 217922153U
- Authority
- CN
- China
- Prior art keywords
- liquid
- chip
- channel
- aerosol
- exposure
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
A micro chip device capable of realizing two-dimensional exposure of aerosol and liquid comprises a chip layer and a bottom substrate, wherein the chip layer comprises an upper chip provided with an air flow channel, a middle chip and a lower chip provided with a liquid channel; the channel in the lower chip close to the liquid inlet end is zigzag, and the liquid channel at the rear part is an alternate elliptic structure and is used as a cell culture area; the middle chip is a porous film with the aperture of 0.2-12 μm and is used as a cell inoculation culture growth support; strip-shaped openings are formed in the bottom of the airflow channel of the upper chip and the top of the liquid channel of the oval structure of the lower chip and used for being embedded with the middle chip. The device has the advantages that: the porous membrane structure can enable components of aerosol in the gas layer and components of liquid in the liquid layer to be in direct contact with cells, actual exposure concentration and component composition characteristics of tested pollutants are truly reflected, cell exposure conditions are uniform and stable, and accurate and reliable evaluation results can be obtained.
Description
Technical Field
The utility model relates to an in vitro toxicology evaluation research field that environmental contaminant exposes specifically says so a can realize the microchip device that aerosol and liquid multiple concentration gradient two dimensions exposed.
Background
The preparation of concentration gradients is one of the most basic and commonly used procedures in scientific experiments in biology, medicine, analytical chemistry, and the like. The traditional concentration gradient preparation method needs accurate calculation, weighing, repeated solution mixing and constant volume, needs a plurality of chemical containers, has complicated steps, is easy to cause experimental errors and even errors, and is very easy to cause the increase of experimental errors and the reduction of reproducibility due to the difference of operation habits of different experimenters.
The microfluidic chip technology is a technology for highly miniaturizing and integrating different fluid operation steps, and integrates basic operation units such as sample preparation, reaction, separation, detection and the like related to the fields of biology, medicine, chemistry and the like on a chip with a micron-sized channel structure with the size of several square centimeters. The micro-fluidic chip has the characteristic of micro-scale, so that the micro-fluidic chip has the unique advantage of high flux in application.
The microfluidic chip system can be used for toxicological prediction and mechanism research of pollutants in environmental toxicology, and at present, traditional cell experiments are mostly adopted for in vitro toxicity evaluation of environmental pollutant exposure at home and abroad, so that the operation is time-consuming and labor-consuming, and the microenvironment cannot be simulated. A new exposure evaluation model is developed based on the microfluidic chip technology, and micro-environment simulation and multi-dimensional exposure analysis of environmental pollutant research on micro-nano scale can be realized. The concentration gradient generating device based on the microfluidic technology is applied to the fields of drug activity screening, cell and microorganism culture, tissue and organ bionics, trace substance detection and the like. Compared with the traditional concentration gradient prepared manually, the concentration gradient generating device based on the microfluidic technology has great advantages. The microfluidic concentration gradient generation device is designed in advance according to application requirements, can realize accurate control on fluids (gas and liquid), and has the principle that the fluids with different concentrations or different components meet, mix and separate for many times in an internal channel network of the concentration gradient generation device, the concentration gradient is finally formed through a diffusion effect, an equal ratio, an equal difference, an index ratio or any expected concentration gradient can be formed, the generated time and space instantaneous gradient is accurate, controllable, easy to quantify and good in reproducibility, and researchers can be helped to better develop scientific research experiments. Single gas or liquid concentration gradients are easier to implement, but there is currently little concern with aerosol and liquid multi-gradient two-dimensional exposure designs.
In the prior art, there is an exposure device for gas and liquid, and the specific structure is shown in fig. 1, which is composed of an upper chip, a lower chip and a middle chip located therebetween, wherein the middle chip uses a laser engraving machine to engrave a 4x 4 array of porous structures on a 0.5 mm thick organic plastic plate, the pore diameter is 1 mm,0.7 μ L of gel is injected into micropores, the middle chip is used for loading gel, the cells are seeded on the gel, and the cells in the middle gel are indirectly contacted with the upper gas and the lower liquid. However, after practical application, the gel layer can retain components in aerosol and liquid, reduce the types of components contacted by cells, and reduce the concentration of the contacted components, so that the harmful effect of the tested pollutants is underestimated in the application of practical pollutant exposure evaluation, and the stability and the accuracy of a test result are further influenced.
SUMMERY OF THE UTILITY MODEL
The utility model discloses an it is based on above-mentioned prior art situation and a microchip device that can realize that aerosol and the two dimensions of the many concentration gradients of liquid expose that provide, can truly reflect the actual concentration and the composition feature of exposing of the pollutant tested, the cell exposure condition homogeneous is stable, and then obtains accurate reliable assessment result.
The purpose of the utility model is realized through the following technical scheme:
a microchip device capable of realizing two-dimensional exposure of aerosol and liquid with multiple concentration gradients comprises a chip layer and a bottom substrate, wherein the chip layer comprises an upper chip provided with an air flow channel, a middle chip and a lower chip provided with a liquid channel; the channel in the lower chip close to the liquid inlet end is zigzag, and the liquid channel at the rear part is an alternate elliptic structure and is used as a cell culture area; the middle chip layer takes a porous film with the aperture of 0.2-12 mu m as a cell inoculation culture growth support; strip-shaped openings are formed in the bottoms of the airflow channels of the upper chip and the tops of the airflow channels of the oval structures of the lower chip and used for being embedded/attached (also called bonding) with the middle chip. After the upper layer, the middle layer and the lower layer of the chip are bonded, after ethanol cleaning and disinfection, cell suspension is injected through a gas outlet on the upper layer chip and flows into the porous film through a strip-shaped opening at the bottom of an air flow channel of the upper layer chip, so that cells are uniformly inoculated on the porous film in the middle layer of the chip.
The porous film can be made of polycarbonate, polyester, polyethylene terephthalate, polydimethylsiloxane and polytetrafluoroethylene.
The four airflow channels of the upper chip and the four liquid channels of the lower chip can form four concentration gradients，Can realize two-dimensional high-flux toxicology exposure research of aerosol and liquid pollutants. The direction of the air flow channel in the upper chip is vertical to that of the liquid channel in the lower chip.
The height of the airflow channel is 0.8 mm, and the width of the airflow channel is 2 mm; the height of the liquid channel is 0.15 mm, the width of the liquid channel is 0.6 mm, the oval major diameter of the liquid channel cell culture area is 3 mm, and the minor diameter of the liquid channel cell culture area is 2 mm. The reason that the gas channel is higher than the liquid channel is to ensure that the formation of gas concentration gradient in the gas channel can be realized, the gas flow rate is reduced, and the damage of gas flow to cells is reduced.
Two gas inlets and one gas outlet are arranged in the upper chip; two liquid inlets and a liquid outlet are arranged in the lower chip.
The utility model discloses compare prior art's advantage and lie in: the middle layer adopts a porous film structure, cells grow on the microporous film, the upper layer of the chip and the lower layer of the chip are directly communicated through the porous film, and the cells are directly contacted with the upper layer gas and the lower layer liquid. The porous membrane material has good biocompatibility, can be used as a support medium for cell growth, has no toxic influence on cells, is suitable for cell inoculation and long-time continuous culture, can ensure that components of aerosol in a gas layer and components of liquid in a liquid layer are directly contacted with the cells, and the types and the concentrations of the components directly exposed to the cells are consistent with the types and the concentrations of the components in the upper layer aerosol and/or the lower layer liquid actually. Compared with the gel layer structure adopted in the prior art, the gel layer obstructs the communication between the upper layer and the lower layer of the chip, the upper layer and the lower layer become obstructed spaces, cells in the gel layer are in indirect contact with upper layer gas and lower layer liquid, the gel layer can intercept components in aerosol and liquid, the types of the components contacted by the cells are reduced, and the concentration of the contacted components is reduced, so that the hazard effect of the tested pollutants is underestimated in the application of actual pollutant exposure evaluation. In addition, components in aerosol and liquid can generate diffusion phenomena in the gel layer, and the diffusion coefficients of different components are different, so that the components are unevenly distributed in the gel, large errors exist in cell exposure test results at different positions, and the stability and accuracy of the test are influenced. Therefore, the utility model discloses a structural feature can truly reflect the actual concentration and the composition characteristic of exposing of pollutant tested, and the cell exposure condition is homogeneous stable, can obtain accurate reliable assessment result.
Drawings
FIG. 1 is a cross-sectional view of a prior art chip structure;
in fig. 1: 1 'is a gel and 2' is a cell.
FIG. 2 is a cross-sectional view of the chip layer structure of the present invention;
FIG. 3 is a structural view of the upper chip of the present invention;
FIG. 4 is a view of the lower chip structure of the present invention;
FIG. 5 is a structural view of the middle chip layer of the present invention;
FIG. 6 is the overall structure design of the present invention, wherein A is a schematic view of bonding, B: a schematic view of the layers to be bonded;
in FIGS. 2-6: 1: upper chip, 1.1: upper chip gas channel, 1.2: gas a inlet, 1.3: gas B inlet, 1.4: a gas outlet; 2: an interlayer chip (porous film); 3: lower chip, 3.1: lower chip liquid channel, 3.2: liquid a inlet, 3.3: liquid B inlet, 3.4: a liquid outlet; 4: a base substrate.
Fig. 7 is a diagram of the utility model discloses the material object that the preparation obtained, A: top view, B: side view.
Fig. 8 is a top gas channel gas concentration gradient characterization, a: introducing CO 2 Color change map in last 0, 4, 6 min channel, B: the linear plot was analyzed colorimetrically.
Fig. 9 is a lower layer liquid channel liquid concentration gradient characterization, a: the liquid concentration gradient forms a color change diagram (the uppermost channel (1) is a colorless channel for introducing sterile water); b: the color metric analysis is linear.
Detailed Description
The structure of the utility model is further described with the attached drawings as follows:
as shown in fig. 2-7: a can realize the aerosol and liquid multi-concentration gradient two-dimensional microchip device exposed, including chip layer and bottom base plate 4 form, the chip layer includes the upper chip 1 equipped with air current channel 1.1, middle floor chip 2, lower floor chip 3 equipped with liquid channel 3.1; the channel in the lower chip near the liquid inlet end is zigzag, and the liquid channel at the rear part is an alternate elliptic structure as a cell culture area; the middle chip 2 takes a porous film with the aperture of 0.2-12 mu m as a cell inoculation culture growth support; strip-shaped openings are formed in the bottoms of the airflow channels 1.1 of the upper chip 1 and the tops of the liquid channels 3.1 of the oval structures of the lower chip 3 and used for being embedded/attached (also called bonding) with the middle chip 2.
The four airflow channels 1.1 of the upper chip 1 and the four liquid channels 3.1 of the lower chip 3 can form four concentration gradients，Can realize two-dimensional high-flux toxicology exposure research of aerosol and liquid pollutants. The air flow channels 1.1 in the upper chip 1 and the liquid channels 3.1 in the lower chip 3 are oriented perpendicular to each other.
The following manufacturing process of the present invention is further described with reference to the following embodiments:
1. an upper chip:
The method comprises the steps of manufacturing a die of an upper chip by adopting a machining method, wherein the height of a channel is 0.8 mm, mixing Polydimethylsiloxane (PDMS) prepolymer and a curing agent according to a ratio of 10. And placing the upper chip after punching in isopropanol, cleaning by an ultrasonic cleaning instrument, and drying for later use.
2. An intermediate layer chip:
the middle chip 2 is a porous film with a pore size of 0.2-12 μm, as shown in fig. 5, the porous film can be made of polycarbonate, polyester, polyethylene terephthalate, polydimethylsiloxane, polytetrafluoroethylene and other materials, the porous film has good biocompatibility, can be used as a support medium for cell growth, has no toxic effect on cells, is suitable for cell inoculation and long-time continuous culture, and has the porous characteristic of exchanging substances up and down the film, a liquid culture medium below the film can exchange substances directly through micropores on the porous film to provide nutritional support for cells on the film, and cytokines and metabolic molecules secreted by the cells can directly enter a lower culture solution through the micropores of the film, so that online collection of biomarker indexes in a toxicity evaluation experiment is facilitated, and in addition, the characteristics of thin thickness and good transparency of the porous film can also facilitate in-situ detection and analysis of the cells inoculated on the film.
The polycarbonate membrane with the pore diameter of 5 mu m is preferably cut by a laser engraving machine according to the designed size for standby.
3. A lower chip:
the lower chip is a liquid channel layer, the design pattern is shown in figure 4, two liquid inlets 3.2, 3.3 and a liquid outlet 3.4 are arranged on the lower chip, liquid A is introduced into one side of the chip, liquid B is introduced into the other side of the chip, the two paths of liquid are fully mixed and diluted in a zigzag structure at the front end to form four mixed liquids with different concentrations, an oval cell culture area at the rear end also contributes to reducing the fluid shearing force on cells in the fluid flow process, the four channels at the rear end are mutually independent, and the tail ends of the channels are converged at the liquid outlet
The method comprises the following steps of manufacturing a die of a lower-layer chip according to a standard soft lithography process, manufacturing the lower-layer die through five steps of substrate cleaning, gluing, prebaking, exposing and developing, mixing a PDMS prepolymer and a curing agent according to a ratio of 10, then casting and molding on the die, carrying out vacuum degassing after casting to eliminate generated bubbles, heating in an oven for 1 h, carrying out demolding operation after PDMS is cured, cutting off redundant parts, punching at reserved liquid inlet and outlet positions by using a puncher, wherein the die is in a convex shape at a channel position, and after PDMS is cast and molded, the strip-shaped opening position of the lower-layer chip after demolding is in a position contacting with the upper surface of the convex shape of the die. And (3) placing the punched lower chip in isopropanol, cleaning the lower chip by an ultrasonic cleaning instrument, and drying the lower chip for later use.
4. A bottom substrate:
cutting a polymethyl methacrylate (PMMA) plate with the thickness of 2 mm by a laser engraving machine according to the size to manufacture a bottom substrate layer, wherein the bottom substrate 4 is used for fixing and supporting the whole chip, and the material and the thickness can be selected according to the requirement.
5. Bonding the upper, middle and lower chip structures and the bottom substrate to form a complete chip, wherein the specific bonding steps are as follows:
the bottom surface of the lower chip is upward, the PMMA substrate surface to be modified is upward, and the two are put into a plasma surface treatment instrument together for modification (gas: O) 2 Power: 100 W, pressure: 600 mTorr, time: 40 And s), taking out the lower chip and the substrate, aligning and contacting (namely aligning and clinging the shapes of the lower chip and the substrate), and putting the lower chip and the substrate into a 70 ℃ oven to heat for about 2 hours to complete the bonding between the lower chip and the substrate. And after heating, taking out the composite chip from the oven, cooling at room temperature, putting the composite chip cooled to room temperature and the to-be-bonded surface of the processed intermediate layer porous film into a plasma surface treatment instrument together for modification (conditions are the same as above), carefully taking out the composite chip and the porous film, aligning and contacting, putting the composite chip into the 70 ℃ oven, and heating for about 2 hours to complete bonding between the composite chip and the porous film. And after heating, taking out the composite chip from the oven, cooling at room temperature, putting the composite chip and the upper chip which are cooled to the room temperature and have the bonding surfaces to be bonded upwards into a plasma surface treatment instrument for modification (the conditions are the same), carefully taking out the composite chip and the upper chip, aligning and contacting, putting the composite chip into the 70 ℃ oven, and heating for about 2 hours to complete bonding between the composite chip and the upper chip.
The resulting completed microchip device structure is shown in FIG. 7.
6. Characterization of gas concentration gradient formation experiment for upper chip
According to the condition that bromothymol blue solution meets CO 2 The change of the concentration gradient of the upper layer gas is converted into the change of the solution color by the color change phenomenon of blue changing into yellow. Selecting CO 2 The gas is used as an indicator gas, the alkaline solution of bromothymol blue is used as an indicator to indicate the formation of gas concentration gradient, and the alkaline bromothymol blue solution meets CO 2 The color of the gas channel changed to yellow and green (in the specific experiment, blue bromothymol blue alkaline solution was filled in the gas channel, and then CO was introduced into one side of the gas channel 2 A gas. Synthetic air is introduced into the other side of the gas channel, and CO in the gas channel is generated along with the mixing of the two gases in the gas channel 2 The gas concentration forms a gradient and the bromothymol blue solution is utilized to meet CO 2 Characterization of upper chip gas concentration gradient formation by blue-to-yellow discoloration), with CO 2 The color of the liquid in the channel shows a continuous change, and CO 2 The yellowing phenomenon is more remarkable as the concentration is higher. Introducing CO 2 Photographing at the time points of 4 min and 6 min of gas to monitor the color change of the liquid in the channel, and taking the result as shown in FIG. 8A, wherein no CO exists in the channel at 0 min 2 In the presence of all four channels blue, with CO 2 The color of the liquid in the channel gradually turns yellow, a concentration gradient is stably formed in 4 min, and CO flows from the yellow channel to the blue channel 2 The concentration is from maximum to minimum. The results of the colorimetric quantitative analysis are shown in fig. 8B, and the fitted linear equations for the 4 min and 6 min time are: y =34.014x-36.634, R 2 =0.9947，y=33.779x-25.314、R 2 =0.9433, the gas concentration gradients of the four channels are in a better linear relationship, which indicates that the upper chip gas layer can meet the design target of forming the gas concentration gradients.
7. Experimental characterization for forming liquid concentration gradient of lower chip
The method for characterizing the liquid concentration gradient is to introduce sterile water and bromothymol blue solution, and convert the concentration gradient change into blue shade change, as shown in FIG. 9A, the concentration distribution diagram after the liquid is introduced for 15 min can show that the four channels have blue with different shadesColor, four concentration gradients were formed, and a linear equation was fitted by quantitative analysis of the chromaticity: y =0.3539x-0.3025, R 2 =0.9315, the linear dependence is good (fig. 9B), indicating that the underlying chip liquid layer can meet the design goal of liquid concentration gradient formation.
Claims (9)
1. A microchip device capable of two-dimensional exposure of aerosol and liquid, comprising: the chip layer comprises an upper chip provided with an airflow channel, a middle chip and a lower chip provided with a liquid channel; the channel in the lower chip close to the liquid inlet end is zigzag, and the liquid channel at the rear part is an alternate elliptic structure and is used as a cell culture area; the middle chip layer takes a porous film with the aperture of 0.2-12 mu m as a cell inoculation culture growth support; strip-shaped openings are formed in the bottom of the airflow channel of the upper chip and the top of the airflow channel of the oval structure of the lower chip and used for being embedded/attached to the middle chip.
2. The aerosol and liquid two-dimensional exposure enabled microchip device of claim 1, wherein: the porous film is made of polycarbonate, polyester, polyethylene terephthalate, polydimethylsiloxane and polytetrafluoroethylene.
3. The aerosol and liquid exposure enabling microchip device of claim 1, wherein: the number of the airflow channels of the upper chip is four.
4. The aerosol and liquid exposure enabling microchip device of claim 1, wherein: the liquid channels of the lower chip are four.
5. The aerosol and liquid two-dimensional exposure enabled microchip device of claim 1, wherein: the direction of the air flow channel in the upper chip is vertical to that of the liquid channel in the lower chip.
6. The aerosol and liquid two-dimensional exposure realizable microchip device according to claim 1 or 4, characterized in that: the height of the airflow channel is 0.8 mm, and the width of the airflow channel is 2 mm.
7. The aerosol and liquid exposure enabling microchip device of claim 1, wherein: the height of the liquid channel is 0.15 mm, the width of the liquid channel is 0.6 mm, the oval major diameter of the liquid channel cell culture area is 3 mm, and the minor diameter of the liquid channel cell culture area is 2 mm.
8. The aerosol and liquid exposure enabling microchip device of claim 1, wherein: two gas inlets and one gas outlet are arranged in the upper chip.
9. The aerosol and liquid exposure enabling microchip device of claim 1, wherein: two liquid inlets and a liquid outlet are arranged in the lower chip.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202220927432.7U CN217922153U (en) | 2022-04-21 | 2022-04-21 | Microchip device capable of realizing two-dimensional exposure of aerosol and liquid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202220927432.7U CN217922153U (en) | 2022-04-21 | 2022-04-21 | Microchip device capable of realizing two-dimensional exposure of aerosol and liquid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN217922153U true CN217922153U (en) | 2022-11-29 |
Family
ID=84176217
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202220927432.7U Active CN217922153U (en) | 2022-04-21 | 2022-04-21 | Microchip device capable of realizing two-dimensional exposure of aerosol and liquid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN217922153U (en) |
-
2022
- 2022-04-21 CN CN202220927432.7U patent/CN217922153U/en active Active
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9739699B2 (en) | Device for the study of living cells | |
| Young et al. | Fundamentals of microfluidic cell culture in controlled microenvironments | |
| US9778153B2 (en) | Microfluid device and method of producing diffusively built gradients | |
| CN101629143B (en) | Microfluidic cell array chip for high-throughput medicament screening, method and use | |
| TWI438273B (en) | High-throughput perfusative microfluidic cell culture wafers for miniaturized three-dimensional cell culture | |
| US20160333298A1 (en) | Microfluidic Cell Culture Systems | |
| US9975118B2 (en) | Device for the study of living cells | |
| US20110236970A1 (en) | Chamber of a bioreactor platform | |
| CN107497503A (en) | For studying the micro-fluidic chip of the unicellular invasion and attack of tumour and Epithelial and stromal conversion | |
| CN109682962A (en) | Immunofluorescence test system and detection method based on micro-fluidic chip | |
| CN104561286B (en) | A kind of novel polymeric enzyme chain reaction micro-fluid chip control system and preparation method thereof | |
| CN108117968A (en) | A kind of single celled method of high-throughput automatic capture based on drop micro-fluidic chip | |
| Park et al. | Microchemostat array with small-volume fraction replenishment for steady-state microbial culture | |
| CN112920951B (en) | Cell screening chip and manufacturing and cell screening and collecting method thereof | |
| CN217922153U (en) | Microchip device capable of realizing two-dimensional exposure of aerosol and liquid | |
| CN113083386A (en) | Simple and rapid liquid sample discretization chip and using method thereof | |
| US20230193182A1 (en) | Multiplanar microfluidic devices with multidirectional direct fluid communication among adjacent microfluidic channels | |
| EP2811013A1 (en) | Hanging network plate | |
| JP7078918B2 (en) | Biochip and bioassay device and bioassay method | |
| CN113564034A (en) | Detachable micro-fluidic chip and application method thereof | |
| Demming | Disposable lab-on-chip systems for biotechnological screening | |
| FI129674B (en) | Apparatus and method for cell cultivation | |
| CN215440452U (en) | Detachable micro-fluidic chip | |
| CN112955537A (en) | Cell culture substrate and method for producing same | |
| CN219079546U (en) | Cell and tissue culture organ chip |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| GR01 | Patent grant | ||
| GR01 | Patent grant |