CN111812167A - A chemical indirect toxicity detection platform and its application - Google Patents

Abstract

The present invention is a chemical indirect toxicity detection platform, comprising a microfluidic system, a bioreactor, a cell metabolism detection chip and a waste liquid collector connected in sequence through microtubes, and the bioreactor includes a cell culture chamber and a cell impedance A detection unit, one end of the cell impedance detection unit is in electrical contact with the liquid in the cell culture chamber, and the other end is electrically connected with an electrochemical impedance detector, and the cell metabolism detection chip includes a glucose detection unit and a lactic acid detection unit. The invention combines electrochemical biosensor analysis technology and microfluidic chip, and integrates multiple microarray analysis technologies on the basis of microfluidic technology. It realizes a highly integrated, automated and mature chemical indirect toxicity detection platform.

Description

A chemical indirect toxicity detection platform and its application

technical field

The invention relates to the field of electrochemical sensing and the field of microfluidics, in particular to a chemical indirect toxicity detection platform and its application.

Background technique

Chemicals often enter the environment through various ways during their use, thereby causing a certain degree of damage to the environment and even endangering human life and health. In recent years, people have gradually realized the importance of chemical toxicity evaluation. High-sensitivity, rapid and non-destructive detection technology has become a problem that researchers pay close attention to. Replacing traditional whole animal experimental models by culturing cells, tissues and organs or lower organisms in vitro has become an important and meaningful research direction for chemical toxicity testing. The toxicity evaluation of commonly used traditional chemicals is mainly based on the integrity of the cell membrane. Sulfur rhodamine (SRB) method, neutral red (NR) staining method, etc.

At present, the development of new protocols for in vitro cytotoxicity evaluation and the effective evaluation of chemical toxicity or specific toxicity endpoints have also become research hotspots in in vitro cytotoxicity testing experiments. As a new type of research method, biosensors have many advantages such as rapid response, high specificity and sensitivity, and simple operation compared with traditional biological analysis methods. Electrochemical biosensors, as the earliest and most widely used branch of biosensors, combine the advantages of high sensitivity and high selectivity of electroanalytical methods, and have obvious advantages in the field of life analysis. In order to meet the requirements of miniaturization, high throughput and simultaneous monitoring of multiple cell lines in the development of cellular multi-parameter biosensors, microfluidic technology has the advantages of low reagent consumption, short analysis time, easy integration and parallel processing due to microfluidic technology. The advantages have been widely used in the field of biosensing and become one of the important technologies for drug metabolism and cytotoxicity analysis.

However, the existing technology still lacks a highly integrated, automated and mature chemical indirect toxicity detection platform.

SUMMARY OF THE INVENTION

The purpose of the invention is to combine electrochemical biosensor analysis technology and microfluidic chip, and integrate multiple microarray analysis technologies on the basis of microfluidic technology to realize high-throughput quantitative analysis; micromachining technology and nanotechnology can reduce biological The size of the sensor can be miniaturized, and portable detectors and diagnosis and treatment instruments can be realized; through the channel structure of micron size, the construction of in vitro cell culture, drug delivery, monitoring and other units are integrated into the chip platform, thereby providing an indirect toxicity detection platform for chemicals. Preparation.

A chemical indirect toxicity detection platform, comprising a microfluidic system, a bioreactor, a cell metabolism detection chip and a waste liquid collector connected in sequence through microtubes, the bioreactor comprising a cell culture chamber and a cell impedance detection unit, One end of the cell impedance detection unit is in electrical contact with the liquid in the cell culture chamber, and the other end is electrically connected to the electrochemical impedance detector. The cell metabolism detection chip includes a glucose detection unit and a lactic acid detection unit. The glucose detection unit and the lactic acid detection unit The unit is electrically connected to the electrochemical detector, and the microfluidic system drives the liquid to pass through the cell culture chamber, the electrochemical impedance detection unit, the glucose detection unit, and the lactic acid detection unit in sequence, and enter the waste liquid collector.

The core of the present invention is two sensor chips, each chip has different types of sensor elements, respectively measuring different cell parameters. The cell impedance sensor measures the change of cell impedance and reflects the cell adhesion rate; two electrochemical biosensors simultaneously monitor the glucose uptake rate and the lactate production rate online for the investigation of cell metabolism. After the cells are cultured in the cell culture chamber for a period of time, the cell metabolism indicators are detected by inputting toxic substances, thereby judging the survival of the cells and detecting the toxicity of chemicals.

The present invention is an on-line detection platform. Contrary to the traditional established cell viability endpoint determination, the on-line toxicity monitoring platform designed in the present invention can monitor the entire process of chemicals acting on cells, rather than a simple endpoint, so it can provide cell exposure More details on the chemical to facilitate the study of its toxicity mechanism. In addition, the chemical toxicity monitoring platform designed in this study has many advantages and features. Cells can be cultured on the sensor surface for a long time, and the chip can be used with only simple pretreatment, and can be used for the study of different adherent cell lines.

Preferably, the cell metabolism detection chip further includes a PBS injector, one end of the PBS injector is connected to the microfluidic system, and the other end is connected to the glucose detection unit and the lactic acid detection unit in turn, injecting PBS to clean the glucose detection unit, Lactic acid detection unit.

Preferably, the cell metabolism detection chip further includes an air injector, one end of the air injector is connected to the microfluidic system, and the other end is connected to the glucose detection unit and the lactic acid detection unit in turn. Lactic acid detection unit.

Preferably, the cell metabolism detection chip further includes a calibration solution syringe, one end of the PBS syringe is connected to the microfluidic system, and the other end is connected to the glucose detection unit and the lactic acid detection unit in turn, and the calibration solution is injected for calibration detection.

In this application, combined with the microfluidic technology, the process of cell exchange, sample addition, and self-cleaning can be automatically controlled.

Preferably, the bioreactor sequentially includes an upper clamp layer, a cell culture cavity layer, an interdigital electrode layer, an insulating base layer and a lower clamp layer from top to bottom, and the upper clamp layer is provided with a reactor inlet and a reactor outlet , the middle of the cell culture cavity layer is hollowed out to form a cylindrical culture cavity, the reactor inlet and the reactor outlet are located in the space just above the cylindrical culture cavity, and the bottom of the cylindrical culture cavity is provided with an interdigital electrode layer, so The interdigital electrode layer is connected to the insulating base layer, and the insulating base layer seals the cylindrical culture chamber.

Preferably, the insulating base layer is provided with a wedge-shaped structure matching with the interdigital electrode layer. The insulating base layer is provided with a wedge-shaped structure matched with the interdigitated electrode layer, and the wedge-shaped structure fixes the interdigitated electrode layer. Specifically, when the insulating substrate is printed, a space matching the shape of the interdigital electrode layer will be formed, so that the interdigital electrode layer can be fixed.

Preferably, the lower clamp layer is provided with an observation hole, and the cross-sectional area of the observation hole is consistent with the cross-sectional area of the cylindrical culture cavity.

Preferably, the cell metabolism detection chip includes a top clamp layer, a PDMS chip layer and a bottom clamp layer in order from top to bottom, and the top clamp layer is provided with a first liquid inlet, a second liquid inlet, and a third liquid inlet port, a fourth liquid inlet and a waste liquid outlet, the PDMS chip layer is provided with a glucose detection unit and a lactic acid detection unit, the glucose detection unit and the lactic acid detection unit are connected through a microporous channel, and the bottom clamp layer is provided with a wedge-shaped structure for fixing The glucose detection unit detection electrode and the lactic acid detection sensing electrode detection electrode.

Preferably, the first liquid inlet, the second liquid inlet, the third liquid inlet and the fourth liquid inlet are respectively used for the liquid inlet of PBS, air, calibration solution and sample solution, and the waste liquid outlet is connected to Waste pool connection.

The invention also protects the application of the toxicity detection platform, characterized in that the application includes the cytotoxicity assessment of chemicals.

The beneficial effects of the present invention are:

(1) The present invention combines electrochemical biosensor analysis technology and microfluidic chip, and integrates multiple microarray analysis technologies on the basis of microfluidic technology. It can realize a highly integrated, automated and mature chemical indirect toxicity detection platform;

(2) The present invention is an on-line detection platform. Contrary to the traditional established cell viability endpoint determination, the on-line toxicity monitoring platform designed in the present invention can monitor the entire process of chemicals acting on cells, rather than a simple endpoint. Provide more details on the exposure of cells to chemicals to facilitate the study of their toxicity mechanisms;

(3) The cells of the present invention can be cultured on the surface of the sensor for a long time, the chip can be used with only simple pretreatment, and can be used for the research of different adherent cell lines.

Description of drawings

Figure 1 is a schematic diagram of the overall structure;

Figure 2 is a schematic diagram of the structure of a bioreactor;

Figure 3 Schematic diagram of the structure of the cell metabolism detection chip;

Fig. 4 application embodiment 1 test result diagram;

Fig. 5 application embodiment 2 test result diagram;

Reference number:

Microfluidic system 1, bioreactor 2, cell metabolism detection chip 3, waste liquid collector 4, incubator 5, electrochemical detector 6, electrochemical impedance detector 7, upper fixture layer 201, cell culture chamber layer 202 , interdigital electrode layer 203, insulating base layer 204, lower clamp layer 205, reactor inlet 206, reactor outlet 207, observation hole 208, top clamp layer 301, PDMS chip layer 302, bottom clamp layer 303, first liquid inlet Port 304, second liquid inlet 305, third liquid inlet 306, fourth liquid inlet 307, waste liquid outlet 308, glucose detection unit 309, lactic acid detection unit 310, microporous channel 311, glucose detection unit detection electrode 312 . The lactic acid detection unit detects the electrode 313 .

Detailed ways

The specific embodiments of the present invention will be further described below in conjunction with the accompanying drawings:

A chemical indirect toxicity detection platform, as shown in Figure 1, Figure 2 and Figure 3, includes a microfluidic system 1, a bioreactor 2, a cell metabolism detection chip 3 and a waste liquid collector 4 connected in sequence through microtubes . Microfluidic system 1 is controlled by Lange TS-1B/4*W0109-1B four-channel push-pull mode syringe pump, Lange TS-1B/4*W0109-1B controller and execution unit are separate structures, and the execution unit is parallel syringe pump. Using independent actuators, which can be installed and combined at will, it is used to monitor the inflow of the system cell culture chip, as well as the electrode cleaning and calibration of the cell metabolism chip. The shown bioreactor 2 is placed in an incubator 5 using a Thermo CO2 incubator to culture and measure cells in real time. The interdigital electrode layer 203 in the bioreactor 2 is connected to the electrochemical impedance detector 7 through a DuPont wire. The electrochemical impedance detector 7 adopts a precision impedance analyzer and is controlled by a computer. After the response signal is collected, data processing is performed, Finally, the impedance spectrum data is obtained. The cell metabolism detection chip includes a glucose detection unit 309 and a lactic acid detection unit 310. In this embodiment, the glucose detection unit 309 is located in front of the lactic acid detection unit 310, but the sequence of glucose and lactic acid detection does not affect the detection result. The glucose detection unit 309 and the lactic acid detection unit 310 are electrically connected to the electrochemical detector 6, and the electrochemical detector 6 is a Chenhua multi-channel electrochemical workstation, which is controlled on a computer interface. The glucose detection unit 309 and the lactic acid detection unit 310 in the cell metabolism detection chip 3 can perform online real-time monitoring of glucose uptake in aerobic metabolism and lactic acid production in anaerobic metabolism in the cellular metabolism process. Through the analysis of these cellular parameters, indirect prediction of chemical toxicity is achieved. The waste liquid collector 4 is realized by a beaker in the figure.

The core of the present invention is two sensor chips, each chip has different types of sensor elements, respectively measuring different cell parameters. The cell impedance sensor measures the change of cell impedance and reflects the cell adhesion rate; two electrochemical biosensors simultaneously monitor the glucose uptake rate and the lactate production rate online for the investigation of cell metabolism. After the cells are cultured in the cell culture chamber for a period of time, the cell metabolism indicators are detected by inputting toxic substances, thereby judging the survival of the cells and detecting the toxicity of chemicals.

As shown in FIG. 2, the bioreactor includes an upper clamp layer 201, a cell culture cavity layer 202, an interdigital electrode layer 203, an insulating base layer 204 and a lower clamp layer 205 in order from top to bottom. The upper clamp layer 201 is provided with The reactor inlet 206 and the reactor outlet 207 are hollowed out in the middle of the cell culture chamber layer 202 to form a cylindrical culture chamber. The reactor inlet 206 and the reactor outlet 207 are located in the space just above the cylindrical culture chamber. The bottom of the culture chamber is provided with an interdigital electrode layer 203, the interdigital electrode layer 203 is connected with the insulating base layer 204, and the insulating base layer 204 seals the cylindrical culture chamber. The insulating base layer 204 is provided with a wedge-shaped structure matching with the interdigital electrode layer 204 . The lower clamp layer 205 is provided with an observation hole 208, and the cross-sectional area of the observation hole is consistent with the cross-sectional area of the cylindrical culture chamber.

The cell metabolism detection chip is shown in Fig. 3, and includes a top fixture layer 301, a PDMS chip layer 302 and a bottom fixture layer 303 in order from top to bottom. The top fixture layer is provided with a first liquid inlet 304 and a second liquid inlet hole. 305, the third liquid inlet 306, the fourth liquid inlet 307 and the waste liquid outlet 308, the PDMS chip layer is provided with a glucose detection unit 309 and a lactic acid detection unit 310, and the glucose detection unit and the lactic acid detection unit are connected through the microporous channel 311 , the bottom clamp layer is provided with a wedge-shaped structure to fix the glucose detection unit detection electrode 312 and the lactic acid detection sensor electrode detection electrode 313 .

As a preferred embodiment, the first liquid inlet 12 , the second liquid inlet hole 13 , the third liquid inlet 14 and the fourth liquid inlet 15 are respectively used for the liquid inlet of PBS, air, calibration solution and sample solution. , the waste liquid outlet 19 is connected to the waste liquid pool.

Application Example

Application Example 1 Acetaminophen toxicity detection

In order to accelerate cells into stationary phase, the optimal cell seeding density was determined to be 5×10 ⁵ cells. Data recording started 5 min after seeding the cells. After the cells entered the stationary phase, the toxicity exposure test was performed, and 1% DMSO (v/v) was added to the perfused medium to stop the growth of the cells and maintain normal metabolism. The signal generated under these conditions served as the reference signal (0% baseline value) for the experimental platform. After 3 hours, a certain concentration of chemical-acetaminophen was perfused into the platform, and the exposure experiment was performed, and the cells were exposed to the test compound for 19 hours. Acetaminophen was washed out by perfusing the medium for 2 hours after the end of the toxicity exposure experiment to restore the initial state of the cells.

The test results are shown in Figure 4. Figure 4 (a) The signal changes of the cell adhesion rate after treatment of HepG2 cells with different concentrations of acetaminophen in the chemical indirect toxicity monitoring platform; (b) The chemical indirect toxicity monitoring platform in the monitoring platform for different concentrations of paracetamol Signal changes of glucose uptake rate after acetaminophen treatment in HepG2 cells; (c) Signal changes of lactate production rate after treatment of HepG2 cells with different concentrations of acetaminophen monitored in the chemical indirect toxicity monitoring platform.

Studies have shown that (1) acetaminophen: after the cells were treated with high concentration (10mM) of acetaminophen for 3 hours, the cell impedance and cell adhesion rate were reduced to about 80% of the control group, indicating that the cell morphology shrank, and then It remained at about 80-90% for the remaining time, and the exposure was stopped after 22 h, and the recovery phase was entered, and the impedance reversibly returned to the baseline level similar to that before acetaminophen administration (Fig. 4a). The glucose uptake rate reached 110% of the control group after 3 h of drug administration, and the glucose uptake rate returned to the initial baseline level after the exposure was stopped (Fig. 4b). At the same time, extracellular lactate production increased to 130% after 10 mM acetaminophen administration, and the lactate production rate returned to initial baseline levels after cessation of exposure (Fig. 4c). The reasons for the above results may be: acetaminophen can cause mitochondrial damage in cells, which seriously affects cellular respiration. In addition, the metabolism of acetaminophen produces toxic benzoquinone, which consumes intracellular glutathione. Peptide (Glutathione, GSH) [100,101]. At 10 mM acetaminophen, cellular glucose uptake was increased by 110%, lactate production was increased by 130%, and aerobic respiration was calculated to decrease by approximately 5%. This indicated that after acetaminophen induced mitochondrial damage, the oxidative phosphorylation pathway to generate ATP was inhibited, while the glycolytic pathway increased the amount of ATP to compensate for the lack of energy. (2) Cyclophosphamide: The indirect toxicity monitoring platform showed that cyclophosphamide can cause HepG2 cell damage, and the degree of cell damage caused by different concentrations (10-400 μM) is also different.

Application Example 2 Cyclophosphamide Toxicity Detection

In order to accelerate cells into stationary phase, the optimal cell seeding density was determined to be 5×10 ⁵ cells. Data recording started 5 min after seeding the cells. After the cells entered the stationary phase, the toxicity exposure test was performed, and 1% DMSO (v/v) was added to the perfused medium to stop the growth of the cells and maintain normal metabolism. The signal generated under these conditions served as the reference signal (0% baseline value) for the experimental platform. After 3 hours, a certain concentration of chemical-cyclophosphamide was perfused into the platform, and the exposure experiment was carried out. The cells were exposed to the test compound for 19 hours. Cyclophosphamide was washed out by perfusing the medium for 2 hours after the toxicity exposure experiment to restore the initial state of the cells.

The test results are shown in Figure 5. Figure 5 (a) the signal changes of the cell adhesion rate after treatment of HepG2 cells with different concentrations of cyclophosphamide detected in the chemical indirect toxicity monitoring platform; (b) the chemical indirect toxicity monitoring platform. Monitored signal changes of glucose uptake rate after treatment of HepG2 cells with different concentrations of cyclophosphamide; (c) The monitored signal changes of lactate production rate after treatment of HepG2 cells with different concentrations of cyclophosphamide in the chemical indirect toxicity monitoring platform.

Under the action of 400 μM cyclophosphamide, the cell impedance decreased to 80% of the baseline value, and after the end of administration, the cell impedance only recovered to about 90% (Fig. 5a). Glucose uptake increased by 40% after simultaneous administration (Fig. 5b), while lactate levels decreased by around 15% (Fig. 5c). The above results indicate that high concentrations of cyclophosphamide can cause HepG2 cell death. Studies have shown that a large amount of acrolein is produced during the metabolism of cyclophosphamide, causing reactive oxygen species to produce lipid peroxidation, resulting in a metabolic stress response in tumor cells. The increase in glucose uptake and the decrease in lactate content may be related to the inhibition of the activity of enzymes related to glycolytic pathway in the cells after cyclophosphamide acts on HepG2 cells, or due to the reduced pressure in the interstitium, which increases the availability of O ₂ and makes HepG2 cells The shift from initially glycolytic-based cellular metabolism to the oxidative phosphorylation pathway results in an increased rate of glucose uptake.

Based on the disclosure and teaching of the above specification, those skilled in the art to which the present invention pertains can also make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the invention should also fall within the protection scope of the claims of the present invention. In addition, although some specific terms are used in this specification, these terms are only for convenience of description and do not constitute any limitation to the present invention.

Claims (10)

1. a chemical indirect toxicity detection platform, is characterized in that, comprises the microfluidic control system, the bioreactor, the cell metabolism detection chip and the waste liquid collector connected successively by the micropipe, and the described bioreactor comprises the cell culture cavity and a cell impedance detection unit, one end of the cell impedance detection unit is in electrical contact with the liquid in the cell culture chamber, and the other end is electrically connected with an electrochemical impedance detector, the cell metabolism detection chip includes a glucose detection unit and a lactic acid detection unit, the The glucose detection unit and the lactic acid detection unit are electrically connected to the electrochemical detector, and the microfluidic system drives the liquid to pass through the cell culture chamber, the electrochemical impedance detection unit, the glucose detection unit, and the lactic acid detection unit in sequence, and enter the waste liquid collector . 2. toxicity detection platform according to claim 1, is characterized in that, described cell metabolism detection chip also comprises PBS syringe, one end of described PBS syringe is connected with microfluidic system, and the other end is successively connected with glucose detection unit, The lactic acid detection unit is connected, and PBS is injected to clean the glucose detection unit and the lactic acid detection unit. 3. The toxicity detection platform according to claim 2, wherein the cell metabolism detection chip further comprises an air injector, one end of the air injector is connected with the microfluidic system, and the other end is connected with the glucose detection unit, The lactic acid detection unit is connected, and the glucose detection unit and the lactic acid detection unit are emptied by injecting air. 4. The toxicity detection platform according to claim 2 or 3, wherein the cell metabolism detection chip further comprises a calibration solution syringe, one end of the PBS syringe is connected with the microfluidic system, and the other end is sequentially connected with the glucose The detection unit and the lactic acid detection unit are connected, and the calibration solution is injected for calibration detection. 5. The toxicity detection platform according to claim 4, wherein the bioreactor sequentially comprises an upper clamp layer, a cell culture cavity layer, an interdigital electrode layer, an insulating base layer and a lower clamp layer from top to bottom, The upper clamp layer is provided with a reactor inlet and a reactor outlet, the middle of the cell culture cavity layer is hollowed out to form a cylindrical culture cavity, the reactor inlet and the reactor outlet are located in the space just above the cylindrical culture cavity, the The bottom of the cylindrical culture chamber is provided with an interdigital electrode layer, the interdigitated electrode layer is connected with the insulating base layer, and the insulating base layer seals the cylindrical culture chamber. 6 . The toxicity detection platform according to claim 5 , wherein the insulating base layer is provided with a wedge-shaped structure matched with the interdigital electrode layer, and the wedge-shaped structure fixes the interdigital electrode layer. 7 . 7 . The toxicity detection platform according to claim 5 , wherein the lower clamp layer is provided with an observation hole, and the cross-sectional area of the observation hole is consistent with the cross-sectional area of the cylindrical culture cavity. 8 . 8. The toxicity detection platform according to claim 4, wherein the cell metabolism detection chip comprises a top clamp layer, a PDMS chip layer and a bottom clamp layer in sequence from top to bottom, and the top clamp layer is provided with the first clamp layer. A liquid inlet, a second liquid inlet hole, a third liquid inlet, a fourth liquid inlet and a waste liquid outlet, the PDMS chip layer is provided with a glucose detection unit and a lactic acid detection unit, and the glucose detection unit and the lactic acid detection unit pass through the micropores The channel is connected, and the bottom clamp layer is provided with a wedge-shaped structure to fix the detection electrode of the glucose detection unit and the detection electrode of the lactic acid detection sensing electrode. 9. The toxicity detection platform according to claim 8, wherein the first liquid inlet, the second liquid inlet, the third liquid inlet, and the fourth liquid inlet are respectively used for PBS, air, calibration The inlet of liquid and sample solution, the waste liquid outlet is connected with the waste liquid pool. 10. The application of the toxicity detection platform according to any one of claims 1-9, wherein the application includes cytotoxicity detection of chemicals.

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