CN101376908B - Method for studying medicament metabolism based on molecule and cell level - Google Patents
Method for studying medicament metabolism based on molecule and cell level Download PDFInfo
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- CN101376908B CN101376908B CN200710012625XA CN200710012625A CN101376908B CN 101376908 B CN101376908 B CN 101376908B CN 200710012625X A CN200710012625X A CN 200710012625XA CN 200710012625 A CN200710012625 A CN 200710012625A CN 101376908 B CN101376908 B CN 101376908B
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Abstract
The invention relates to a method for researching drug metabolism based on molecular and cellular level, which is characterized by using a special microfluidic chip to make research on drug metabolism based on molecular and cellular level at the same time. In the method, the research on drug metabolism based on molecular and cellular level can be completed on the same microfluidic chip, to obtain the molecular structure information and the cell toxicity effect of drug metabolin; therefore, the method is applicable in low-effect consumption, high-throughput and high-content drug metabolism screening and drug interaction research.
Description
Technical field
The present invention relates to micro-fluidic chip and drug metabolism study platform technology, provide a kind of especially and used special micro-fluidic chip to be used for method based on molecule and cell levels research drug metabolism.
Background technology
Drug metabolism is meant that medicine is in vivo through the bio-transformation of drug metabolism enzyme.Medicine is after metabolism, and variation has taken place physico-chemical property, thereby has caused the change of its pharmacology and toxicological activity.Therefore, research drug metabolism and clear and definite its pathways metabolism all have important directive significance to formulating rational clinical application scheme, dosage form design and new drug development work, also are hot research fields such as clinical pharmacology, toxicology, high-flux medicaments sifting.Current, domestic and international generation and the definite pathways metabolism that the research of drug metabolism is mainly concentrated on meta-bolites.Under the promotion of Protocols in Molecular Biology, the research in drug metabolism enzyme field has positive meaning because of its research to the clinical medicine interphase interaction, is paid attention to widely.External metabolism research can be got rid of the interference of body intrinsic factor, and the direct viewing enzyme is to the selection metabolic of substrate, for bulk testing provides reliable theoretical foundation; The internal metabolism transformation efficiency is low, reaches the medicine that lacks the Sensitive Detection means greatly for toxicity, and external metabolism research becomes good research means.Liver is the vitals of drug metabolism; It is the main place that body carries out bio-transformation; Be rich in the mixed function oxidase system of a huge dependent cells cytochrome p 450 of participating in drug metabolism, the I of most drug reaches the II phase reaction mutually and depends on the liver enzyme system and take place.The drug metabolism study strategy generally be the medicine off-line or online be drug metabolite through the metabolic enzyme bio-transformation, the on the one hand separable detection of drug metabolite is obtained its molecular structure information, and then is studied its pathways metabolism; The toxic side effect of medicine after metabolism or the interaction of medicine are investigated in drug metabolite and cell (like liver cell) hatching on the other hand.Method commonly used at present such as HPLC-MS and microwell plate all can't realize obtaining simultaneously the molecular structure information and its CDCC information of drug metabolite.If on same platform, can satisfy these two requirements simultaneously, this development to high-throughput drug metabolism screening certainly will have great importance.
The micro-fluidic chip laboratory is claimed chip lab (Lab-on-A-Chip) or micro-fluidic chip (Microfluidic) again; Refer to specimen preparation, reaction related in chemistry and the field such as biology, separate, basic operation units such as detection, cell cultures, sorting, cracking are integrated or be integrated into basically on the chip of more than square centimeters (even littler); Form network by the microchannel; Run through total system with controlled fluid, in order to a kind of technology of the various functions that replace conventional chemical or biology laboratory.Breadboard essential characteristic of micro-fluidic chip and sharpest edges are that multiple monotechnics flexible combination, scale on the controlled small platform of integral body are integrated.
Lab-on-chip technology is applied in the drug metabolism study; Make full use of the integrated characteristics of its flexible combination and scale; Unit operations such as drug metabolism enzyme reactor, cell cultures, cell hatching, drug metabolite separation detection are integrated on the micro-fluidic chip; Certainly will be able to realize the poor efficiency consumption, the drug metabolism screening research of high-throughput, high intension.
Summary of the invention
The object of the present invention is to provide and a kind ofly used special micro-fluidic chip to be used for method based on molecule and cell levels research drug metabolism.
The invention provides a kind of method of using special micro-fluidic chip from molecule and cell levels drug metabolism to be studied simultaneously;
Described micro-fluidic chip is formed by upper strata, lower floor and sandwich of layers three part sealing-ins, and microfluidic channel is contained in the upper and lower of chip, and sandwich of layers is the substrate that is etched with micro through hole array, is fixed with drug metabolism enzyme in the micro through hole;
Procedure is:
Cell is charged into chip lower floor to be cultivated; Medicine gets into from the upper strata; Produce metabolite through the effect of sandwich of layers immobilization drug metabolism enzyme, metabolite has two paths: one is the direct entering separation detection passage from the upper strata, obtains the molecular structure information of metabolite; Another is for getting into the hatching of lower floor and cell, and estimate its cellulotoxic effect: per three micropores are one group, are respectively drug metabolism enzyme blank, medicine blank and drug metabolite and cytosis, and the drug metabolite CDCC is obtained in three groups of data contrasts exactly.
The invention provides a kind of micro-fluidic chip that is specifically designed to based on molecule and cell levels research drug metabolism; Described micro-fluidic chip is formed by upper strata, lower floor and sandwich of layers three part sealing-ins; Microfluidic channel is contained in the upper and lower of chip; Sandwich of layers is the substrate that is etched with micro through hole array, is fixed with drug metabolism enzyme in the micro through hole.
The chip material that the invention provides the upper and lower of described micro-fluidic chip is elastic silicone rubber PDMS; The sandwich of layers material is elastic silicone rubber, glass, quartz, polymetylmethacrylate or other chip material.
The micropore number that the invention provides micro through hole array is 3～90.
The invention provides the making method of micro-fluidic chip:
On the upper and lower layer template of making in advance, pour into a mould PDMS respectively, polymerization was peeled off the PDMS layer after solidifying after 20～60 minutes from template in baking oven, cut into upper and lower layer by desired size, got the aperture that supplies fluid to come in and go out on the upper strata;
Prepare the micro through hole array of sandwich of layers through the method for optical etching or machine drilling, utilize silicon sol-gel or hydrogel method that drug metabolism enzyme is fixed in this micropore;
The PDMS of lower floor is attached on the sandwich of layers substrate, closely sticks together with upper strata PDMS again, make two-layer passage alignment, form complete three-layer stereo sandwich chip.
The invention provides the drug metabolism enzyme that is fixed in the micro through hole is hepatomicrosome or recombination drug metabolism enzyme.
Gel provided by the invention is transparent, can directly examine under a microscope down the state of confluent monolayer cells; Through-hole surfaces is through the PVAc coating, and gel combines with through-hole surfaces closely, is difficult for embrittlement; PDMS surface silicon alkanisation after immobilization is accomplished, is easy to break away from, and can not influence the structure of gel.
The present invention can make based on the drug metabolism study of molecule and cell levels and on same micro-fluidic chip, accomplish.Can obtain the molecular structure information and its cellulotoxic effect of drug metabolite simultaneously, be applicable to the poor efficiency consumption, the drug metabolism screening of high-throughput, high intension and drug interaction research.
Micro-fluidic chip flexible design among the present invention can be selected multiple different chip material for use according to different application requirements.Flexible interface with multiple test set is provided.
Description of drawings
Fig. 1 is the structural representation of drug metabolism micro-fluidic chip,
Fig. 2 prepares the process synoptic diagram for the drug metabolism micro-fluidic chip,
Fig. 3 is the cell imaging figure of sol-gel place on the chip,
Fig. 4 is the online metabolism separation detection of a Paracetamol USP23,BP98 electrophoresis spectrogram,
Fig. 5 is cell fluorescence microscope imaging figure after drug metabolite-cytosis;
Embodiment
Embodiment 1: the making of micro-fluidic chip
Micro-fluidic chip (like Fig. 1) is to have the sandwich of layers substrate of drug metabolism enzyme in micro through hole array and the upper and lower layer PDMS sealing-in that is etched with microfluidic channel to form by immobilization.
Concrete steps are (like Fig. 2) as follows:
(1) on the upper and lower layer template of making in advance, pour into a mould PDMS respectively, polymerization was peeled off the PDMS layer after solidifying after 20～60 minutes from template in baking oven, cut into upper and lower layer by desired size, got the aperture that supplies fluid to come in and go out on the upper strata.
(2) be the ultrasonic punching of quartz plate of the sandwich of layers of 0.5-2.0mm at thickness, the diameter in hole is 1-3mm, and number can be decided according to the flux of research.Clean and drying after, each micropore splashes into an amount of PVAc (5%m/v toluene) respectively, dry up, with one in advance the PDMS of surface silicon alkanisation be attached under the quartz plate, form semi-enclosed microwell array.
(3) drug metabolism enzyme immobilization: utilize silicon sol-gel or hydrogel method with the tetramethoxy-silicane of ultrasonic hydrolysis and hepatomicrosome by volume 1:2 mix, add successively in the micropore with microsyringe then, airtight, place 4 ℃ of refrigerators to solidify 24 hours.
(4) chip sealing: tear the PDMS layer off, the PDMS chip that upper and lower layer is etched with microfluidic channel with stick together after the quartz plate of sandwich of layers is aimed at, two-layer passage is alignd, form complete three-layer stereo sandwich chip.Firm for guaranteeing sealing-in, need to keep chip clean.
Embodiment 2: the metabolism research of Paracetamol USP23,BP98 medicine
Add damping fluid in the liquid pool of embodiment 1 described micro-fluidic chip, power up flushing 30 minutes, in lower floor's chip channel, add cultured liver cancer HEPG2 cell then, add nutritive medium again, cell attachment spend the night (like Fig. 3); In the chip liquid pool, add 5mM Paracetamol USP23,BP98 medicine, 20mMMgCl and 10mM UDPGA coenzyme then; Voltage sample introduction 60 seconds; Make it the effect with drug metabolism enzyme Artogicurol transferring enzyme UGT, after reaction for some time, drug metabolite glycosylation Paracetamol USP23,BP98 and unreacted medicine are powered up the split tunnel of introducing the upper strata chip; And with the UV-detector detection, separation detection result is as shown in Figure 4.Simultaneously in order to estimate the cellulotoxic effect of drug metabolism; Drug metabolite is introduced into lower floor's cell passage: per three micropores are one group in the experiment; Two micropore sol-gel immobilization drug metabolism enzyme UGT wherein, sol-gel immobilization bovine serum albumin in another micropore and do not have the UGT enzyme; In the micropore of no UGT enzyme and immobilization have the micropore of UGT enzyme, power up then and charge into the medicine Paracetamol USP23,BP98, and another immobilization has to power up in the micropore of UGT enzyme and charges into the not damping fluid of drug Paracetamol USP23,BP98; Form the result of three groups of cell survival rates like this; Be that drug metabolism enzyme is blank, medicine is blank and drug metabolite and HEPG2 cytosis; Add optical dye iodate pyridine PI and bifurcation indigo orange AO at last; Under fluorescent microscope through dye marker anyway cell count confirm the survival rate (as shown in Figure 5) of its cell, three groups of data contrast just can obtain the drug metabolite CDCC exactly.
The metabolism research of embodiment 3:4-nitrophenol medicine
Add damping fluid in the liquid pool of embodiment 1 described micro-fluidic chip, power up flushing 30 minutes, in lower floor's chip channel, add cultured liver cancer HEPG2 cell then, add nutritive medium again, cell attachment spends the night; In the chip liquid pool, add 5mM4-nitrophenol, 20mM MgCl and 10mM UDPGA coenzyme then; Voltage sample introduction 60 seconds; Make it effect with drug metabolism enzyme UGT; After reaction for some time, drug metabolite Artogicurol 4-nitrophenol and unreacted medicine are powered up the split tunnel of introducing the upper strata chip, and detect with UV-detector.Simultaneously in order to estimate the cellulotoxic effect of drug metabolism; Drug metabolite is introduced into lower floor's cell passage: per three micropores are one group in the experiment; Two micropore sol-gel immobilization drug metabolism enzyme UGT wherein, sol-gel immobilization bovine serum albumin in another micropore and do not have the UGT enzyme; In the micropore of no UGT enzyme and immobilization have the micropore of UGT enzyme, power up then and charge into medicine 4-nitrophenol, and another immobilization has to power up in the micropore of UGT enzyme and charges into the not damping fluid of drug 4-nitrophenol; Form the result of three groups of cell survival rates like this; Be that drug metabolism enzyme is blank, medicine is blank and drug metabolite and HEPG2 cytosis; Add optical dye iodate pyridine PI and bifurcation indigo orange AO at last; Under fluorescent microscope through dye marker anyway cell count confirm the survival rate (as shown in Figure 5) of its cell, three groups of data contrast just can obtain the drug metabolite CDCC exactly.
Claims (4)
1. the method based on molecule and cell levels research drug metabolism is characterized in that: use special micro-fluidic chip to carry out drug metabolism study from molecule and cell levels simultaneously;
Described micro-fluidic chip is formed by upper strata, lower floor and sandwich of layers three part sealing-ins, and microfluidic channel is contained in the upper and lower of chip, and sandwich of layers is the substrate that is etched with micro through hole array, is fixed with drug metabolism enzyme in the micro through hole;
The chip material of the upper and lower of said micro-fluidic chip is elastic silicone rubber PDMS; The sandwich of layers material is elastic silicone rubber, glass, quartz or polymethylmethacrylate PMM;
The making method of said micro-fluidic chip is: on the upper and lower layer template of making in advance, pour into a mould PDMS respectively; Polymerization is after 20～60 minutes in baking oven; PDMS layer after solidifying is peeled off from template, cut into upper and lower layer, get the aperture that supplies fluid to come in and go out on the upper strata by desired size;
Prepare the micro through hole array of sandwich of layers through the method for optical etching or machine drilling, utilize silicon sol-gel or hydrogel method that drug metabolism enzyme is fixed in this micropore;
The PDMS of lower floor is attached on the sandwich of layers substrate, closely sticks together with upper strata PDMS again, make two-layer passage alignment, form complete three-layer stereo sandwich chip;
Procedure is:
Cell is charged into chip lower floor to be cultivated; Medicine gets into from the upper strata; Produce metabolite through the effect of sandwich of layers immobilization drug metabolism enzyme, metabolite has two paths: one is the direct entering separation detection passage from the upper strata, obtains the molecular structure information of metabolite; Another is for getting into the hatching of lower floor and cell, and estimate its cellulotoxic effect: per three micropores are one group, are respectively drug metabolism enzyme blank, medicine blank and drug metabolite and cytosis, and the drug metabolite CDCC is obtained in three groups of data contrasts exactly.
2. one kind is specifically designed to the said micro-fluidic chip based on molecule and cell levels drug metabolism study method of claim 1; It is characterized in that: described micro-fluidic chip is formed by upper strata, lower floor and sandwich of layers three part sealing-ins; Microfluidic channel is contained in the upper and lower of chip; Sandwich of layers is the substrate that is etched with micro through hole array, is fixed with drug metabolism enzyme in the micro through hole;
The chip material of the upper and lower of said micro-fluidic chip is elastic silicone rubber PDMS; The sandwich of layers material is elastic silicone rubber, glass, quartz or polymetylmethacrylate;
The making method of said micro-fluidic chip is: on the upper and lower layer template of making in advance, pour into a mould PDMS respectively; Polymerization is after 20～60 minutes in baking oven; PDMS layer after solidifying is peeled off from template, cut into upper and lower layer, get the aperture that supplies fluid to come in and go out on the upper strata by desired size;
Prepare the micro through hole array of sandwich of layers through the method for optical etching or machine drilling, utilize silicon sol-gel or hydrogel method that drug metabolism enzyme is fixed in this micropore;
The PDMS of lower floor is attached on the sandwich of layers substrate, closely sticks together with upper strata PDMS again, make two-layer passage alignment, form complete three-layer stereo sandwich chip.
3. according to the described micro-fluidic chip of claim 2, it is characterized in that: the described drug metabolism enzyme that is fixed in the micro through hole is hepatomicrosome or recombination drug metabolism enzyme.
4. according to the described micro-fluidic chip of claim 2, it is characterized in that: the micropore number of said micro through hole array is 3～90.
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| CN200710012625XA CN101376908B (en) | 2007-08-29 | 2007-08-29 | Method for studying medicament metabolism based on molecule and cell level |
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| CN101376908B true CN101376908B (en) | 2012-01-25 |
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| JP6170431B2 (en) * | 2010-09-29 | 2017-07-26 | マサチューセッツ インスティテュート オブ テクノロジー | Devices for high-throughput studies of cell-cell interactions |
| US20140274763A1 (en) * | 2013-03-15 | 2014-09-18 | Pathway Genomics Corporation | Method and system to predict response to pain treatments |
| CN104931551B (en) * | 2015-05-21 | 2017-12-26 | 西安交通大学 | Screen paper substrate micro-fluidic chip and the application of soil activation bacterium and composition |
| CN110117540B (en) * | 2019-05-29 | 2022-09-13 | 山东钛铖医疗器械有限公司 | Cell culture platform and manufacturing method thereof |
| CN112626025A (en) * | 2021-01-20 | 2021-04-09 | 温州医科大学附属第一医院 | Three-dimensional tumor cell drug resistance model and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1996014A (en) * | 2006-12-12 | 2007-07-11 | 中国人民解放军第二军医大学 | Array micro-fluidic chip device for use in drug metabolism screening |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1996014A (en) * | 2006-12-12 | 2007-07-11 | 中国人民解放军第二军医大学 | Array micro-fluidic chip device for use in drug metabolism screening |
Non-Patent Citations (4)
| Title |
|---|
| Petra S. Dittrich et.al..Lab-on-a-chip: microfluidics in drug discovery.《Nature Reviews Drug Discovery》.2006,第5卷210-218. * |
| 方肇伦等.微流控芯片发展与展望.《现代科学仪器》.2001,(第4期),3-6. * |
| 田玉平等.一体化微流控芯片的酶固定化技术.《高等学校化学学报》.2004,第25卷(第5期),847-849. * |
| 田玉平等.用于蛋白质酶解的一体化PDMS微流控芯片.《化学世界》.2005,(第10期),590-592. * |
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