CN111812167A - Chemical indirect toxicity detection platform and application thereof - Google Patents
Chemical indirect toxicity detection platform and application thereof Download PDFInfo
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Abstract
The invention relates to a chemical indirect toxicity detection platform, which comprises a microfluidic system, a bioreactor, a cell metabolism detection chip and a waste liquid collector which are sequentially connected through a micro-tube, wherein the bioreactor comprises a cell culture cavity and a cell impedance detection unit, one end of the cell impedance detection unit is electrically contacted with liquid in the cell culture cavity, the other end of the cell impedance detection unit is electrically connected with an electrochemical impedance detector, and the cell metabolism detection chip comprises a glucose detection unit and a lactic acid detection unit. The invention combines the electrochemical biosensor analysis technology and the microfluidic chip, integrates a plurality of microarray analysis technologies on the basis of the microfluidic technology, and realizes a highly integrated, automated and mature chemical indirect toxicity detection platform through a channel structure with micron-sized dimensions and a cell culture cavity made of high-biocompatibility materials.
Description
Technical Field
The invention relates to the field of electrochemical sensing and micro-fluidic, in particular to a chemical indirect toxicity detection platform and application thereof.
Background
Chemicals often enter the environment through various routes during their use, causing some damage to the environment and even endangering human life and health. In recent years, the importance of chemical toxicity evaluation has been increasingly recognized. The high-sensitivity, rapid and nondestructive detection technology becomes a problem which is closely concerned by researchers. The replacement of traditional whole animal experimental models by in vitro cultured cells, tissues and organs or lower organisms has become an important and meaningful research direction for the toxicity test of chemicals. The toxicity evaluation of common traditional chemicals is mainly based on the integrity of cell membranes, and common methods comprise a tetramethyl azodicarbonyl chloride (MTT) colorimetric method, a staining counting method, a Lactate Dehydrogenase (LDH) leakage method, a flow cytometry method, a Sulforhodamine (SRB) method, a Neutral Red (NR) staining method and the like.
At present, the development of a new scheme for evaluating in vitro cytotoxicity and the effective evaluation of the toxicity or specific toxicity end point of chemicals also become research hotspots of in vitro cytotoxicity test experiments. As a novel research method, compared with the traditional biological analysis means, the biosensor has the advantages of rapid response, high specificity and sensitivity, simple and convenient operation and the like. The electrochemical biosensor is used as a branch of biosensors which are the earliest and most widely applied, combines the advantages of high sensitivity and high selectivity of an electroanalytical method, and has obvious advantages in the field of life analysis. In order to meet the requirements of development of cell multi-parameter biosensors on miniaturization, high flux, simultaneous monitoring of multiple cell lines and the like, the microfluidic technology has the advantages of low reagent consumption, short analysis time, easy integration, parallel processing and the like, is widely applied to the field of biosensing and becomes one of important technologies for drug metabolism and cytotoxicity analysis.
However, the prior art still lacks a highly integrated, automated and mature chemical indirect toxicity detection platform.
Disclosure of Invention
The invention aims to combine the electrochemical biosensor analysis technology with a microfluidic chip, and fuse a plurality of microarray analysis technologies on the basis of the microfluidic technology to realize high-throughput quantitative analysis; the size of the biosensor can be reduced by micro-processing technology and nano-technology, so that miniaturization is achieved, and a portable detector and a diagnosis instrument are realized; the preparation method of the chemical indirect toxicity detection platform is further provided by building in-vitro cell culture, drug administration, monitoring and other units to be integrated into a chip platform through a channel structure with micron-sized dimensions.
The utility model provides an indirect toxicity testing platform of chemicals, includes microfluidic system, bioreactor, cell metabolism detection chip and the waste liquid collector that connects gradually through the microtubule, bioreactor includes cell culture chamber and cell impedance detecting element, cell impedance detecting element one end and the liquid electrical contact in cell culture chamber, the other end is connected with electrochemistry impedance detector electricity, cell metabolism detection chip includes glucose detecting element and lactic acid detecting element, glucose detecting element and lactic acid detecting element are connected with electrochemistry detector electricity, microfluidic system drive liquid loops through cell culture chamber, electrochemistry impedance detecting element, glucose detecting element, lactic acid detecting element, gets into the waste liquid collector.
The core of the invention is two sensor chips, each chip has different types of sensing elements, and different cell parameters are respectively measured. The cell impedance sensor measures the change of cell impedance and reflects the adhesion rate of cells; the two electrochemical biosensors simultaneously monitor the glucose uptake rate and the lactic acid production rate on line for the investigation of cell metabolism. After the cells are cultured in the cell culture cavity for a period of time, the cell metabolism indexes are detected by inputting toxic substances, so that the survival condition of the cells is judged, and the toxicity of chemicals is detected.
The invention is an on-line detection platform, and is opposite to the traditional established cell viability endpoint determination, the on-line toxicity monitoring platform designed by the invention can monitor the whole process of the chemical acting on the cell, but not a simple endpoint, so that more details of the chemical exposure of the cell can be provided, and the toxicity mechanism research is convenient. In addition, the chemical toxicity monitoring platform designed by the research has many advantages and characteristics, cells can be cultured on the surface of the sensor for a long time, and the chip can be used only by simple pretreatment and can be used for researching different adherent cell lines.
Preferably, the cell metabolism detection chip further comprises a PBS injector, one end of the PBS injector is connected with the microfluidic system, the other end of the PBS injector is sequentially connected with the glucose detection unit and the lactic acid detection unit, and the PBS injector is used for injecting PBS to clean the glucose detection unit and the lactic acid detection unit.
Preferably, the cell metabolism detection chip further comprises an air injector, one end of the air injector is connected with the microfluidic system, the other end of the air injector is sequentially connected with the glucose detection unit and the lactic acid detection unit, and the glucose detection unit and the lactic acid detection unit are emptied by injecting air.
Preferably, the cell metabolism detection chip further comprises a calibration solution injector, one end of the PBS injector is connected with the microfluidic system, the other end of the PBS injector is sequentially connected with the glucose detection unit and the lactic acid detection unit, and the calibration solution is injected for calibration detection.
This application combines micro-fluidic technology, and cell trades liquid, application of sample, self-cleaning process can realize automated control.
As preferred, bioreactor includes anchor clamps layer, cell culture chamber layer, interdigital electrode layer, insulating stratum basale and anchor clamps layer down from last in proper order, it is provided with reactor entry and reactor export to go up the anchor clamps layer, the fretwork forms cylindrical cultivation chamber in the middle of the cell culture chamber layer, reactor entry and reactor export are located cylindrical cultivation chamber directly over the space, cylindrical bottom of cultivateing the chamber is provided with the interdigital electrode layer, the interdigital electrode layer with insulating stratum basale is connected, the sealed cylindrical cultivation chamber of insulating stratum basale.
Preferably, the insulating substrate layer is provided with a wedge-shaped structure matched with the interdigital electrode layer. And a wedge-shaped structure matched with the interdigital electrode layer is arranged on the insulating substrate layer, and the wedge-shaped structure fixes the interdigital electrode layer. Specifically, the insulating substrate forms a space matching the shape of the interdigital electrode layer during printing, thereby fixing the interdigital electrode layer.
Preferably, the lower clamp layer is provided with an observation hole, and the cross-sectional area of the observation hole is consistent with that of the cylindrical culture chamber.
Preferably, the cell metabolism detection chip sequentially comprises a top clamp layer, a PDMS chip layer and a bottom clamp layer from top to bottom, the top clamp layer is provided with a first 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, the glucose detection unit and the lactic acid detection unit are connected through a micropore channel, and the bottom clamp layer is provided with a wedge-shaped structure fixed glucose detection unit detection electrode and a lactic acid detection sensing electrode detection electrode.
Preferably, the first liquid inlet, the second liquid inlet hole, the third liquid inlet and the fourth liquid inlet are respectively used for feeding the PBS, the air, the calibration solution and the sample solution, and the waste liquid outlet is connected with the waste liquid pool.
The invention also protects the use of a toxicity detection platform, characterized in that said use comprises the assessment of the cytotoxicity of a chemical.
The invention has the following beneficial effects:
(1) the invention combines the electrochemical biosensor analysis technology and the microfluidic chip, integrates a plurality of microarray analysis technologies on the basis of the microfluidic technology, and realizes a highly integrated, automated and mature chemical indirect toxicity detection platform through a channel structure with micron-sized dimensions and a cell culture cavity made of a high-biocompatibility material;
(2) the invention is an on-line detection platform, contrary to the traditional established cell viability end point determination, the on-line toxicity monitoring platform designed by the invention can monitor the whole process of the chemical acting on the cell, but not a simple end point, thereby providing more details of the chemical exposure of the cell and facilitating the toxicity mechanism research;
(3) the cells can be cultured on the surface of the sensor for a long time, and the chip can be used only by simple pretreatment and can be used for the research of different adherent cell lines.
Drawings
FIG. 1 is a schematic view of the overall structure;
FIG. 2 is a schematic view of the structure of a bioreactor;
FIG. 3 is a schematic diagram of a cell metabolism detection chip;
FIG. 4 is a graph showing the test results of application example 1;
FIG. 5 is a graph showing the test results of application example 2;
reference numerals:
the device comprises a microfluidic system 1, a bioreactor 2, a cell metabolism detection chip 3, a waste liquid collector 4, an incubator 5, an electrochemical detector 6, an electrochemical impedance detector 7, an upper clamp layer 201, a cell culture cavity layer 202, an interdigital electrode layer 203, an insulating substrate layer 204, a lower clamp layer 205, a reactor inlet 206, a reactor outlet 207, an observation hole 208, a top clamp layer 301, a PDMS chip layer 302, a bottom clamp layer 303, a first liquid inlet 304, a second liquid inlet 305, a third liquid inlet 306, a fourth liquid inlet 307, a waste liquid outlet 308, a glucose detection unit 309, a lactic acid detection unit 310, a micropore channel 311, a glucose detection unit detection electrode 312 and a lactic acid detection unit detection electrode 313.
Detailed Description
The following further describes embodiments of the present invention with reference to the accompanying drawings:
an indirect toxicity testing platform for chemicals is shown in fig. 1, fig. 2 and fig. 3, and comprises a microfluidic system 1, a bioreactor 2, a cell metabolism testing chip 3 and a waste liquid collector 4 which are connected in sequence through a microtube. The microfluidic system 1 is controlled by a Lange TS-1B/4W 0109-1B four-channel push-pull mode injection pump, a Lange TS-1B/4W 0109-1B controller and an execution unit are of a split structure, and the execution unit is a parallel injection pump. Independent actuators are used, and the independent actuators can be freely installed and combined, and are used for monitoring the liquid feeding of the cell culture chip of the system and cleaning and calibrating the electrodes of the cell metabolism chip. Bioreactor 2 is shown placed in incubator 5, which uses a Thermo CO2 incubator to culture and measure cells in real time. The interdigital electrode layer 203 in the bioreactor 2 is connected with the electrochemical impedance detector 7 through a DuPont lead, the electrochemical impedance detector 7 adopts a precise impedance analyzer and is controlled by a computer, and after response signals are collected, data processing is carried out to finally obtain impedance spectrum data. The cell metabolism detection chip comprises a glucose detection unit 309 and a lactate detection unit 310, wherein the glucose detection unit 309 is positioned in front of the lactate detection unit 310 in the embodiment, but the detection result is not influenced by the detection sequence of glucose and lactate. The glucose detection unit 309 and the lactic acid detection unit 310 are electrically connected with the electrochemical detector 6, and the electrochemical detector 6 is a Chenghua multi-channel electrochemical workstation and is controlled on a computer interface. The glucose detecting unit 309 and the lactate detecting unit 310 in the cell metabolism detecting chip 3 can perform online real-time monitoring on glucose uptake in aerobic metabolism and lactate production in anaerobic metabolism during cell metabolism. Through the analysis of the cell parameters, the indirect prediction of the toxicity of the chemical is realized. The waste liquid collector 4 is realized by a beaker in the figure.
The core of the invention is two sensor chips, each chip has different types of sensing elements, and different cell parameters are respectively measured. The cell impedance sensor measures the change of cell impedance and reflects the adhesion rate of cells; the two electrochemical biosensors simultaneously monitor the glucose uptake rate and the lactic acid production rate on line for the investigation of cell metabolism. After the cells are cultured in the cell culture cavity for a period of time, the cell metabolism indexes are detected by inputting toxic substances, so that the survival condition of the cells is judged, and the toxicity of chemicals is detected.
Bioreactor is as shown in fig. 2, from last to including in proper order down last anchor clamps layer 201, cell culture chamber layer 202, interdigital electrode layer 203, insulating substrate layer 204 and lower anchor clamps layer 205, it is provided with reactor entry 206 and reactor export 207 to go up anchor clamps layer 201, fretwork forms cylindrical cultivation chamber in the middle of the cell culture chamber layer 202, reactor entry 206 and reactor export 207 are located cylindrical cultivation chamber directly over the space, cylindrical bottom of cultivateing the chamber is provided with interdigital electrode layer 203, interdigital electrode layer 203 with insulating substrate layer 204 connects, insulating substrate layer 204 seals up cylindrical cultivation chamber. The insulating substrate layer 204 is provided with a wedge-shaped structure matched with the interdigital electrode layer 204. The lower jig layer 205 is provided with a viewing hole 208 having a cross-sectional area that coincides with the cross-sectional area of the cylindrical culture chamber.
The cell metabolism detection chip sequentially comprises a top clamp layer 301, a PDMS chip layer 302 and a bottom clamp layer 303 from top to bottom, the top clamp layer is provided with a first liquid inlet 304, a second liquid inlet 305, a third liquid inlet 306, a fourth liquid inlet 307 and a waste liquid outlet 308, the PDMS chip layer is provided with a glucose detection unit 309 and a lactic acid detection unit 310, the glucose detection unit and the lactic acid detection unit are connected through a micropore channel 311, and the bottom clamp layer is provided with a wedge-shaped structure fixed glucose detection unit detection electrode 312 and a lactic acid detection sensing electrode detection electrode 313.
In 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 feeding PBS, air, calibration solution and sample solution, and the waste liquid outlet 19 is connected to a waste liquid pool.
Application examples
Application example 1 acetaminophen toxicity test
To accelerate the cells into stationary phase, the optimal cell seeding density was determined to be 5X 105And (4) respectively. Data recording was started 5min after cell inoculation. After the cells have reached a stationary phase, toxicity exposure tests are performed, and 1% DMSO (v/v) is added to the perfused medium to stop the cells from growing and maintain normal metabolism. The signal generated under these conditions was used as the reference signal (0% baseline) for the experimental platform. After 3h, a chemical, acetaminophen, of a certain concentration was poured into the platform, and the cells were exposed to the test compound for 19 h. 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 fig. 4. FIG. 4(a) signal changes in cell adhesion rates after acetaminophen treatment at different concentrations monitored in a chemical indirect toxicity monitoring platform for HepG2 cells; (b) the monitored signal change of the glucose uptake rate after acetaminophen treatment of different concentrations in a chemical indirect toxicity monitoring platform to HepG2 cells; (c) signal change in lactate production rate after acetaminophen treatment at various concentrations monitored in the chemical indirect toxicity monitoring platform for HepG2 cells.
Studies have shown that (1) acetaminophen: after 3h of high concentration (10mM) of acetaminophen in the cells, the cell impedance and cell adhesion rate decreased to approximately 80% of the control, indicating a cell morphology contraction, which then remained around 80-90% for the remaining time, and after 22h exposure ceased, leading to a recovery phase where the impedance returned reversibly to a level similar to the baseline level before acetaminophen administration (FIG. 4 a). Glucose uptake reached 110% of the control group after 3h dosing, and glucose uptake returned to the initial baseline level after exposure ceased (fig. 4 b). At the same time, extracellular lactate production increased to 130% after 10mM acetaminophen administration, and lactate production rate returned to the initial baseline level after exposure ceased (fig. 4 c). The reasons for the above results may be: acetaminophen causes mitochondrial damage to cells, which severely affects cellular respiration, and acetaminophen produces toxic benzoquinone during metabolism, which consumes intracellular Glutathione (GSH) [100,101 ]. At 10mM acetaminophen, cellular glucose uptake increased by 110%, while lactate production increased by 130%, which was calculated to decrease aerobic respiration by about 5%. It is shown that after acetaminophen causes mitochondrial damage, ATP production pathway from oxidative phosphorylation pathway is inhibited, and ATP production from glycolytic pathway is increased to compensate for the lack of energy. (2) Cyclophosphamide: an indirect toxicity monitoring platform shows that cyclophosphamide causes HepG2 cell damage, and the cell damage degree is different along with different concentrations (10-400 mu M).
Application example 2 detection of Cyclophosphamide toxicity
To accelerate the cells into stationary phase, the optimal cell seeding density was determined to be 5X 105And (4) respectively. Data recording was started 5min after cell inoculation. After the cells have reached a stationary phase, toxicity exposure tests are performed, and 1% DMSO (v/v) is added to the perfused medium to stop the cells from growing and maintain normal metabolism. The signal generated under these conditions was used as the reference signal (0% baseline) for the experimental platform. After 3h, a chemical, cyclophosphamide, with a certain concentration was perfused into the platform for exposure experiments, and the cells were exposed to the test compound for 19 h. Cyclophosphamide was washed out by perfusing the medium for 2 hours after the end of the toxicity exposure experiment to restore the original state of the cells.
The test results are shown in fig. 5, which is (a) the signal change of cell adhesion rate after different concentrations of cyclophosphamide treated HepG2 cells monitored in the chemical indirect toxicity monitoring platform; (b) the monitored signal change of the glucose uptake rate of the HepG2 cells treated by cyclophosphamide with different concentrations in a chemical indirect toxicity monitoring platform; (c) signal change in lactate production rate after different concentrations of cyclophosphamide treated HepG2 cells monitored in a chemical indirect toxicity monitoring platform.
The cellular impedance decreased to 80% of the baseline value with 400 μ M cyclophosphamide and only returned to around 90% after the end of the dose (fig. 5 a). While glucose uptake increased by 40% after administration (fig. 5b) and lactic acid levels decreased by around 15% (fig. 5 c). The above results indicate that high concentrations of cyclophosphamide lead to HepG2 cell death. Research shows that cyclophosphamide produces a large amount of acrolein in the metabolic process, and active oxygen is caused to produce lipid peroxidation, so that tumor cells generate metabolic stress reaction. Wherein the increased glucose uptake and decreased lactate levels may be associated with inhibition of glycolytic pathway-related enzyme activity in the cell after cyclophosphamide action on HepG2 cells, or increased O due to decreased pressure in the intercellular space2The availability of (a) makes HepG2 cells shift from cellular metabolism, in which the initial glycolytic pathway predominates, to the oxidative phosphorylation pathway, resulting in increased glucose uptake rates.
Variations and modifications to the above-described embodiments may occur to those skilled in the art, which fall within the scope and spirit of the above description. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and variations of the present invention should fall within the scope of the claims of the present invention. Furthermore, although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
Claims (10)
1. The indirect toxicity detection platform for the chemicals is characterized by comprising a micro-fluidic system, a bioreactor, a cell metabolism detection chip and a waste liquid collector which are sequentially connected through a micro-tube, wherein the bioreactor comprises a cell culture cavity and a cell impedance detection unit, one end of the cell impedance detection unit is electrically contacted with liquid in the cell culture cavity, the other end of the cell impedance detection unit is electrically connected with an electrochemical impedance detector, the cell metabolism detection chip comprises a glucose detection unit and a lactic acid detection unit, the glucose detection unit and the lactic acid detection unit are electrically connected with the electrochemical detector, and liquid is driven by the micro-fluidic system to sequentially pass through the cell culture cavity, the electrochemical impedance detection unit, the glucose detection unit and the lactic acid detection unit and enter the waste liquid collector.
2. The toxicity detection platform of claim 1, wherein the cell metabolism detection chip further comprises a PBS injector, one end of the PBS injector is connected to the microfluidic system, and the other end of the PBS injector is sequentially connected to the glucose detection unit and the lactate detection unit, and the PBS injector is used for cleaning the glucose detection unit and the lactate detection unit.
3. The toxicity detection platform of 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, the other end of the air injector is sequentially connected with the glucose detection unit and the lactate detection unit, and the glucose detection unit and the lactate detection unit are emptied by injecting air.
4. The toxicity detection platform of claim 2 or 3, wherein the cell metabolism detection chip further comprises a calibration solution injector, one end of the PBS injector is connected with the microfluidic system, the other end of the PBS injector is sequentially connected with the glucose detection unit and the lactic acid detection unit, and calibration solution is injected for calibration detection.
5. The toxicity detection platform of claim 4, wherein the bioreactor comprises an upper clamp layer, a cell culture cavity layer, an interdigital electrode layer, an insulating substrate layer and a lower clamp layer in sequence 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 to form a cylindrical culture cavity, the reactor inlet and the reactor outlet are located in a space right above the cylindrical culture cavity, the bottom of the cylindrical culture cavity is provided with the interdigital electrode layer, the interdigital electrode layer is connected with the insulating substrate layer, and the insulating substrate layer seals the cylindrical culture cavity.
6. The toxicity detection platform of claim 5, wherein the insulating substrate layer is provided with wedge-shaped structures which are matched with the interdigital electrode layer, and the wedge-shaped structures fix the interdigital electrode layer.
7. The toxicity detection platform of claim 5, wherein the lower clamp layer is provided with a viewing aperture having a cross-sectional area that is consistent with the cross-sectional area of the cylindrical culture chamber.
8. The toxicity detection platform of 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, the top clamp layer is provided with a first liquid inlet, a second liquid inlet, 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, 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 a detection electrode of the glucose detection unit and a detection electrode of the lactic acid detection sensor electrode.
9. The toxicity detection platform of claim 8, wherein the first liquid inlet, the second liquid inlet hole, the third liquid inlet and the fourth liquid inlet are used for feeding PBS, air, calibration solution and sample solution, respectively, and the waste liquid outlet is connected to a waste liquid pool.
10. Use of the toxicity test platform of any of claims 1-9, wherein the use comprises a cytotoxicity test of a chemical.
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