CN102728422A - Microfluidic chip apparatus and application thereof - Google Patents
Microfluidic chip apparatus and application thereof Download PDFInfo
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- CN102728422A CN102728422A CN201210191266XA CN201210191266A CN102728422A CN 102728422 A CN102728422 A CN 102728422A CN 201210191266X A CN201210191266X A CN 201210191266XA CN 201210191266 A CN201210191266 A CN 201210191266A CN 102728422 A CN102728422 A CN 102728422A
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Abstract
The invention provides a three-dimensional cultivation microfluidic chip apparatus used for quantum dot cytotoxicity detection. The microfluidic chip apparatus is manufactured by bonding a dimethyl silicone polymer chip and a glass substrate. The chip has two parts, which are a main channel and cell cultivation chambers. The main channel is used for simulating a blood vessel. The cell cultivation chambers communicate with the main channel with different distances, and are used for simulating adjacent tissues with different distances from the blood vessel. The heights of the main channel and the cell cultivation chambers are different, such that a mixture of cells and a three-dimensional cultivation substrate is prevented from leaking to the main channel when injected into the cell cultivation chambers. The apparatus can further be used for proofing that one of the quantum dot cytotoxicity cell mechanisms is the cell autophagia effect. The microfluidic chip apparatus provided by the invention is advantaged in simple manufacturing and easy operation. The apparatus can be used for simulating the diffusion process of the blood vessel and the adjacent tissues. With the apparatus, three-dimensional cultivation and quantum dot cytotoxicity detection of cells can be realized. Therefore, the apparatus is suitable for medicine screening and environmental toxicology analysis.
Description
Technical field
The present invention relates to a kind of micro-fluidic chip, specifically relate to a kind of dimensional culture micro flow control chip device that the quantum dot cytotoxicity detects that is used for, with and the application that detects in the quantum dot cytotoxicity.
Background technology
Quantum dot has great using value as a kind of emerging nano material aspect the bio-imaging, but the cytotoxicity that it had has but limited being widely used of it.For the toxic mechanism of quantum dot, numerous scientists have provided different explanation, for example discharge heavy metal ion, produce reactive oxygen species and different surface character and cause etc.
The Cytotoxic research of quantum dot at present mostly is employed in the culture dish cultured cell or carries out zoopery.Document Li Y.; Zhou Y.; Wang H.; Perrett S.; Zhao Y.; Tang Z.; Nie G.Angew Chem Int Edit, 2011,50, the HepG2 cell proof of 5860-5864 through in culture dish, cultivating causes cytotoxicity in various degree with the CdTe quantum dot that the glutathione of different chiralitys encapsulates.Document Shuhendler A.J.; Prasad P.; Chan H.C.; Gordijo C.R.; Soroushian B.; Kolios M.; Yu K.; O Brien P.J.; Rauth A.M.; Wu X.Y.ACS Nano, 2011,5,1958-1966 has detected the cytotoxicity of the PbSe quantum dot that fatty acid ester encapsulates on an animal model of suffering from breast cancer.Yet cell is in the cell-matrix and the interactional microenvironment of cell-soluble factor of a complicacy in vivo, and the cell in this and the culture dish has a great difference.And zoopery length consuming time, cost are high.Therefore, be badly in need of a kind of high flux, low cost, save time and more accurately the platform of simulated in vivo environment be used for the Cytotoxic research of quantum dot.
Micro-total analysis system is claimed micro-fluidic chip (microfluidic chip) or chip lab (lab on a chip again; LOC); Be meant on the chip of more than square centimeters (even littler) sample preparation, reaction related in the integrated or basic integrated chemical and fields such as biology, separate, basic operation unit such as detection and cell cultivation, sorting, cracking; Form network by the microchannel; Run through whole system with controlled fluid, in order to a kind of technology platform of the various functions that replace conventional chemical or biology laboratory.In early 1990s, the notion of the micro-total analysis system (μ TAS) that is proposed first by Manz and Widmer has adapted to life science biological sample has been carried out demand more efficient, highly sensitive, quick compartment analysis.Micro-fluidic chip is used widely in cellular metabolism, drug screening and stem cell organizational project in decades in the past.
The potential application of another of micro-fluidic chip is to carry out the dimensional culture of cell and simulation human organ.Dimensional culture can farthest keep the specific function of organization of cell in live body under vitro conditions, differentiation state, and can reappear spatial concentration gradient and body mechanics's microenvironment of tissue-tissue interaction interface, chemical substance in the organs of living beings.Document Huh D.; Matthews B.D.; Mammoto A.; Montoya-Zavala M.; Hsin H.Y.; Ingber D.E.Science, 2010,328,1662-1668 utilizes two tight microchannels arranged side by side on micro-fluidic chip, to simulate alveolar and contact-making surface capillaceous and has reproduced the respiratory function of alveolar.Document Zhang C.; Zhao Z.; Abdul Rahim N.A.; Van Noort D.; Yu H.Lab Chip, 2009,9,3185-3192 utilizes multichannel micro-fluidic chip to simulate metabolism and the storage process of medicine in human body.Document Derda R.; Tang S.K.; Laromaine A.; Mosadegh B.; Hong E.; Mwangi M.; Mammoto A.; Ingber D.E.; Whitesides G.M.PLoS One, 2011,6, e18940 has simulated the cell of different parts in the tissue through comparing the growth and the migration situation of cell diverse location in dimensional culture matrix.This shows that the dimensional culture micro-fluidic chip is the experiment in vitro platform that a good drug screening and toxicity detect, and is expected to replace zoopery, still, at present as yet not with this platform application in the Cytotoxic research of quantum dot.
Summary of the invention
To above-mentioned present situation, the invention provides a kind of dimensional culture micro flow control chip device that the quantum dot cytotoxicity detects that is used for.This apparatus structure is simple, microminiaturized, cost is low, be easy to preparation and operation.This device can realize that the toxicity that dimensional culture and the quantum dot of cell act on behind the cell detects, and can disclose certain quantum dot cytotoxic mechanism.
The invention provides a kind of micro flow control chip device, wherein this device comprises main channel and the cell culture chamber chamber that is interconnected, and said cell culture chamber comprises the abluminal compartment and chamber far away that is arranged on the both sides, main channel.Wherein, the main channel can be used for transfusion cell culture medium and quantum dot solution by simulated blood vessel, and the circumvascular adjacent tissue of quantum dot effect then can be simulated in the cell culture chamber chamber.
Said micro flow control chip device can be formed by polydimethylsiloxanechip chip and substrate of glass bonding.
Said abluminal compartment is apart from main channel 0.6-1.2mm, and said chamber far away is apart from main channel 1.5-2.2mm.
Between the main channel of said micro flow control chip device and the cell culture chamber chamber difference in height is arranged, the high 30-60 μ of the height m of the aspect ratio cell culture chamber chamber of main channel.Can utilize surface tension effects to make the mixture of cell and dimensional culture matrix can not leak into the main channel when injecting the cell culture chamber chamber like this.
Medicine from the administration blood vessel be transported to needed for two steps the tissue that will act on: the first step, medicine flows in the blood vessel of contiguous target location through the blood vessel that whole body distributes; In second step, medicine passes blood vessel target approach tissue through diffusion.Therefore, the diffusion of medicine in tissue is a key factor that influences drug effect.
Therefore; The present invention also provides a kind of method of utilizing diffusion in the above-mentioned micro flow control chip device simulated tissue; In the main channel of said device, inject the bovine serum albumin(BSA) (FITC-BSA) of fluorescein sodium and marked by fluorescein isothiocyanate; Same position fluorescence intensity in abluminal compartment and chamber far away respectively, the diffusion transport process between simulated blood vessel and tissue.
With fluorescein sodium and FITC-BSA is model drug, and wherein fluorescein sodium is represented small-molecule substance, and FITC-BSA represents large biological molecule.Because these two kinds of materials all have fluorescence, be injected into the main channel after, the same position fluorescence intensity in abluminal compartment and chamber far away respectively regularly.Comparison through fluorescence intensity can prove the influence of distance to diffusion process, and the fluorescence intensity in the synchronization abluminal compartment always is higher than chamber far away, and the diffusion velocity of the small-molecule substance of fluorescein sodium representative will be faster than the large biological molecule of FITC-BSA representative.This method has been simulated material apart from the diffusion process in the tissue of blood vessel different distance.
The invention provides a kind of method of utilizing above-mentioned micro flow control chip device to realize the dimensional culture of cell; The matrix that said dimensional culture adopts is agarose; Mix with PBS (PBS) and hyclone (FBS) during use, the mass volume ratio of agarose and final mixture is 0.5%-3%.
Agarose is the reversible material with bio-compatibility of a kind of heat, is formed by (1-3) β-D-gala furanose that links and (3-6)-dehydration-α-L-gala furanose copolymerization that (1-4) links, and can be used for biology sensor.The fusing point of agarose and gelation temperature can be regulated and control with modification functional group through changing molecular weight.Low melting-point agarose solidifies at 25 ℃, and the gelation temperature that is lower than 37 ℃ is fit to carry out cell capture, and that uses among the present invention is low melting-point agarose.The aperture that has a certain size in the Ago-Gel is fit to cell and cultivates passing through of required nutriment etc.In the cell culture chamber chamber, cell is wrapped up by agarose, has simulated cell well in vivo by the three-dimensional environment of extracellular matrix parcel.Therefore dimensional culture micro-fluidic chip provided by the invention microenvironment in the analogue body well is suitable as the experiment in vitro platform of quantum dot Study of cytotoxicity.
The present invention also provides a kind of and has utilized said micro flow control chip device to detect the Cytotoxic method of quantum dot, detects the variation of Apoptosis, intracellular reactive oxygen species (ROS) and glutathion inside cell (GSH) with fluorescence probe.Described fluorescence probe is to distinguish the specificity fluorescent probe of recognizing cells apoptosis, intracellular reactive oxygen species or glutathion inside cell specifically.
The increase of Apoptosis, intracellular reactive oxygen species and the minimizing of glutathione are to detect quantum dot cytotoxicity several indexs commonly used, and the increase of intracellular reactive oxygen species and the minimizing of glutathione show that all quantum dot toxicity strengthens.After the quantum dot solution effect of cell in the chip under the dimensional culture with variable concentrations; Quantum dot solution in the main channel is replaced with the Cytotoxic specificity fluorescent probe of relevant detection; Because the diffusion function of dimensional culture matrix; After hatching a period of time, under fluorescence microscope, form images, take pictures, can analyze its toxicity.
The present invention also provides a kind of method of utilizing micro flow control chip device proof quantum dot cytotoxic mechanism of the present invention; With the 3-methyl adenine as a kind of cell autophagy inhibitor; Before the quantum dot solution function cells; Inject earlier the main channel, act on cell after, carry out the quantum dot cytotoxicity experiment again; Through the cell of 3-methyl adenine effect and the situation that in the cell of 3-methyl adenine effect, does not form phagocytic vesicle, explain that in conjunction with apoptosis rate autophagocytosis is to the Cytotoxic influence of quantum dot with the transmission electron microscope contrast.Speak more the more cytotoxicity of bright quantum dot of the phagocytic vesicle that forms in the cell is big more, otherwise then little.
The autophagocytosis of cell is a kind of approach of cell degradation bulk cytoplasm, long-life albumen and whole organelle, is a kind of important protection mechanism in the apoptosis.Autophagocytosis that there are some researches show cell is very important equally in the quantum dot cytotoxic mechanism.
Micro flow control chip device of the present invention can also be used in drug screening or environmental poisonous substance detection.
To sum up visible, beneficial effect of the present invention is:
The dimensional culture micro flow control chip device that is used for the detection of quantum dot cytotoxicity of the present invention has been showed some significant advantages; Comprise: make simple; Easy operating, the material therefor bio-compatibility is good, and cell culture condition more approaches microenvironment in the body; Can simulated blood vessel and surrounding tissue between diffusion, and can be used for the proof of quantum dot toxic mechanism.
Description of drawings
Fig. 1 is the structural representation according to dimensional culture micro flow control chip device of the present invention.
Fig. 2 is that utilization micro flow control chip device according to the present invention is the fluorescence intensity photo that model drug is simulated diffusion in the tissue with the fluorescein sodium.
It is the fluorescence intensity photo that model drug is simulated diffusion in the tissue with the bovine serum albumin(BSA) (FITC-BSA) of marked by fluorescein isothiocyanate that Fig. 3 utilizes micro flow control chip device according to the present invention.
Fig. 4 is that to utilize micro flow control chip device according to the present invention be the fluorescence intensity delta data analysis that model drug is simulated diffusion in organizing with the bovine serum albumin(BSA) (FITC-BSA) of fluorescein sodium and marked by fluorescein isothiocyanate, is 100% with high fluorescent; That show among the figure is the result of three groups of parallel laboratory tests.
Fig. 5 utilizes micro flow control chip device according to the present invention that three days inner cell active fluoros that the HepG2 cell carries out dimensional culture are characterized.
Fig. 6 utilizes micro flow control chip device according to the present invention the HepG2 cell to be carried out three days inner cell survival rate analysis figure of dimensional culture; That show among the figure is the result of three groups of parallel laboratory tests.
Fig. 7 is the Apoptosis fluorescence photo after the CdTe quantum dot (CdTe-COOH) that utilizes micro flow control chip device according to the present invention that carboxyl is encapsulated acts on the HepG2 cell; Send the apoptotic cell that is of sapphirine fluorescence.
Fig. 8 is the apoptosis rate data analysis after the CdTe quantum dot (CdTe-COOH) that utilizes micro flow control chip device according to the present invention that carboxyl is encapsulated acts on the HepG2 cell; That show among the figure is the result of three groups of parallel tests.
Fig. 9 is the intracellular reactive oxygen species (ROS) and glutathione (GSH) variation fluorescence photo after the CdTe quantum dot (CdTe-COOH) that utilizes micro flow control chip device according to the present invention that carboxyl is encapsulated acts on the HepG2 cell; The red fluorescence of ROS shows that more by force the cytotoxicity that quantum dot produces is strong more, and the green fluorescence of GSH shows that the cytotoxicity that quantum dot produces is strong more a little less than more.
Figure 10 is intracellular reactive oxygen species (ROS) and the analysis of glutathione (GSH) delta data after the CdTe quantum dot (CdTe-COOH) that utilizes micro flow control chip device according to the present invention that carboxyl is encapsulated acts on the HepG2 cell; That show among the figure is the result of three groups of parallel tests.
Figure 11 is that utilization micro flow control chip device according to the present invention is the checking of one of quantum dot cytotoxic mechanism to autophagocytosis: the formation and the mitochondrial deformation (marking with white arrow) of phagocytic vesicle (marking with black arrow) in the transmission electron microscope characterize cells.
Figure 12 is that utilization micro flow control chip device according to the present invention is the checking of one of quantum dot cytotoxic mechanism to autophagocytosis: 3-methyl adenine effect whether Apoptosis contrast fluorescence photo.
Figure 13 is that utilization micro flow control chip device according to the present invention is the checking of one of quantum dot cytotoxic mechanism to autophagocytosis: 3-methyl adenine effect whether Apoptosis data analysis in abluminal compartment and the chamber far away; That show among the figure is the result of three groups of parallel tests; * representes that marked difference is arranged among the figure, and * * representes to have the highly significant difference.
The specific embodiment
Combine accompanying drawing that the present invention is done further explanation through embodiment below.
If no specified otherwise, used experimental technique is the normal experiment method; Employed reagent and material etc. all can obtain through commercial sources.
Micro-fluidic chip among the present invention adopts " re-expose " technology to be made with the soft lithographic method.Its preparation method is following:
The making of mould: after silicon chip water tiger acid solution is handled, cleans, dried; Get rid of the negative optical cement of one deck SU-82050 with sol evenning machine on the surface with the rotating speed of 2000rpm; At 65 ℃ of baking 10min; Behind 95 ℃ of baking 4min, after uv-exposure, development, nitrogen dry up, promptly obtain the pattern of ground floor cell culture chamber chamber.Get rid of for the second time negative optical cement with the 1100rpm rotating speed then, after above-mentioned same operation, can obtain the pattern of second layer main channel, mould is promptly made success.
The making of chip: silanization makes die surface hydrophobic, and dimethyl silicone polymer (PDMS) prepolymer mixes with mass ratio 10%1 with initator to be poured in the mould, except that placing 2h at 75 ℃ behind the bubble.PDMS sheet after the polymerization is carefully taken off, excision forming, with the punching of tack syringe needle, then through oxygen plasma treatment, with the irreversible bonding of slide, chip is promptly made success.
Embodiment 1: the structure of chip
As shown in Figure 1, micro flow control chip device of the present invention is made up of main channel and cell culture chamber chamber.Wherein, the main channel can be used for transfusion cell culture medium and quantum dot solution by simulated blood vessel, and the circumvascular adjacent tissue of quantum dot effect then can be simulated in the cell culture chamber chamber.
This device comprises two parts: main channel and cell culture chamber chamber, main channel and cell culture chamber are communicated with.The main channel of said micro flow control chip device is different with the height of cell culture chamber chamber, is respectively 71 μ m and 38 μ m.Can utilize surface tension effects to make the mixture of cell and dimensional culture matrix can not leak into the main channel when injecting the cell culture chamber chamber like this.The cell culture chamber of said micro flow control chip device is divided into two kinds, is respectively apart from the abluminal compartment of main channel 0.8mm and is the chamber far away of 2mm apart from the main channel.
Embodiment 2: the diffusion in the simulated tissue
Diffusion in the tissue is a model drug with the bovine serum albumin(BSA) (FITC-BSA) of fluorescein sodium and marked by fluorescein isothiocyanate.The implantation quality volume ratio is 0.5% agarose solution in the cell culture chamber chamber of both sides, treat that it condenses after, from the main channel, inject the Fluress of 100 μ M and the FITC-BSA solution of 2.75mg/mL respectively.Same position in chamber roof is taken pictures to the fluorescence intensity of fluorescein sodium at a distance from 5min with fluorescence microscope (Leica DMI 4000B) is every, and every separated 10min takes pictures to the fluorescence intensity of FITC-BSA.The Therapy lasted 120min of fluorescein sodium, the Therapy lasted 300min of FITC-BSA, result such as Fig. 2 and shown in Figure 3.From Fig. 2-4, can find out the growth along with the time, the obvious grow of fluorescence intensity show that these two kinds of materials have diffused in the agarose really, and the fluorescence intensity in the same time abluminal compartment will be higher than chamber far away.Three times the parallel laboratory test result is similar, and the difference that shows distance has caused the difference of diffusion.
The dimensional culture of embodiment 3:HepG2 cell in chip
Two dish 60cm
2The cell that covers with in the culture dish is cleared up, and cell suspension is centrifugal, and behind the removal supernatant liquor, it is 10 that remaining cell is dispersed to cell density again
6/ mL.Dimensional culture matrix is mixed by 100 μ L 3% (w/v) low melting-point agarose solution, 100 μ L hyclones and 100 μ L PBSs.Equal-volume cell suspension and dimensional culture matrix are mixed the cell culture chamber chamber of injecting micro-fluidic chip of the present invention, and chip is placed 10min to quicken solidifying of agarose, with the cell parcel wherein at 4 ℃.At last, culture medium is injected the main channel and is coated with one deck at chip surface, to prevent the volatilization of culture medium in the passage.Chip places cell culture incubator, and culture medium every day is changed, and detects cell survival rate every day with life or death kit (Calcein-AM/EthD-1).Active and form all keeps normally in first three day of cultivating for result such as Fig. 5 and shown in Figure 6, cell, still reaches more than 85% at the 3rd day cell survival rate.
Embodiment 4: CdTe quantum dot (CdTe-COOH) cytotoxicity analysis that carboxyl is encapsulated
The minimizing of the increase of Apoptosis, intracellular reactive oxygen species (ROS) and glutathione (GSH) is Cytotoxic three indexs of quantum dot; Can use three species specificity fluorescence probe: Hoechst 33342; Dihydro second ingot (DHE) and 2,3-naphthalene dicarbaldehyde (NDA) detects respectively.The CdTe quantum dot (CdTe-COOH) that encapsulates with carboxyl is used for this experiment.The storing solution of CdTe-COOH quantum dot (5mg/mL) stepwise dilution to 0,10,20,30,40,50 μ g/mL.After with embodiment 3 the HepG2 cell three-dimensional being cultivated 24h, the quantum dot solution of variable concentrations injects the main channel respectively, and behind the static 24h of effect down, the above-mentioned fluorescence probe solution of 100 μ M injects the main channel, in 37 ℃ of incubators, hatches 1h.Fluorescence microscope (Leica DMI 4000B) is taken pictures, and Apoptosis is with software I mage-Pro Plus 6.0 analyzing and processing, result such as Fig. 7 and shown in Figure 8, and what wherein sapphirine was represented among Fig. 7 is apoptotic cell.The increase of ROS and GSH reduce with software QCapture Pro (version: 5.1.1.14) handle in the cell; Result such as Fig. 9 and shown in Figure 10; Wherein the red fluorescence of the ROS in the showed cell increases along with the increase of quantum dot concentration among Fig. 9, and the green fluorescence of the GSH in the cell reduces along with the increase of quantum dot concentration.Can find out that from Fig. 7-10 cytotoxicity of CdTe-COOH quantum dot has the significant concentration dependence, increase that cytotoxicity strengthens with concentration.Under the quantum dot solution effect of same concentration; Apoptosis rate in the abluminal compartment is apparently higher than chamber far away; The fluorescence intensity of ROS also is higher in the abluminal compartment; Intracellular GSH reduces manyly in the abluminal compartment, and the result of three parallel laboratory tests is similar, and these results show that different diffusion length has caused quantum dot cytotoxicity in various degree really.
One of toxic mechanism of embodiment 5:CdTe-COOH quantum dot---the sign of autophagocytosis
After with embodiment 3 the HepG2 cell three-dimensional being cultivated 24h, the PBS of 3mM 3-methyl adenine injects the main channel, acts on cell 5h.Behind the CdTe-COOH of variable concentrations quantum dot solution function cells 24h, use method to detect apoptosis rate with embodiment 4.Transmission electron microscope is used for the formation (shown in figure 10) of phagocytic vesicle in the observation of cell; Cell after the quantum dot solution effect of 20 μ g/mL is used for this experiment; Cell in advance respectively with and handle without the 3-methyl adenine, without the cell of any processing as control group.Can find out among the Electronic Speculum figure from Figure 11; The quantity of the phagocytic vesicle in the cell that the 3-methyl adenine is handled will be starkly lower than the cell of handling without the 3-methyl adenine (marking with black arrow), and the mitochondria number (marking with white arrow) also on the low side of damage.As the formation that does not then have phagocytic vesicle in the cell of control group, there is not the mitochondria damage yet.Sapphirine is apoptotic cell among Figure 12, from Figure 12 and Figure 13, can find out, when low concentration quantum dot solution (< 30 μ g/>mL) function cells, the 3-methyl adenine can obviously reduce apoptosis rate.
Claims (9)
1. micro flow control chip device, wherein this device comprises main channel and the cell culture chamber chamber that is interconnected, said cell culture chamber comprises the abluminal compartment and chamber far away that is arranged on the both sides, main channel.
2. device according to claim 1 is characterized in that, said abluminal compartment is apart from main channel 0.6-1.2mm, and said chamber far away is apart from main channel 1.5-2.2mm.
3. device according to claim 1 and 2 is characterized in that, the high 30-60 μ of the height m of the aspect ratio cell culture chamber chamber of said main channel.
4. a utilization is according to the method for diffusion in claim 1 or the 2 said device simulated tissues; It is characterized in that; In the main channel of said device, inject the bovine serum albumin(BSA) of fluorescein sodium and marked by fluorescein isothiocyanate; Same position fluorescence intensity in abluminal compartment and chamber far away respectively, the diffusion transport process between simulated blood vessel and tissue.
5. method of utilizing device according to claim 1 and 2 to realize the dimensional culture of cell; It is characterized in that; The matrix that said dimensional culture adopts is agarose; Mix with PBS and hyclone during use, the mass volume ratio of said agarose and final mixture is 0.5%-3%.
6. one kind is utilized device according to claim 1 and 2 to realize the method that the quantum dot cytotoxicity detects; It is characterized in that the cytotoxicity of quantum dot is judged in the variation that detects Apoptosis, intracellular reactive oxygen species and glutathion inside cell with fluorescence probe.
7. method according to claim 5 is characterized in that, described fluorescence probe is to distinguish the specificity fluorescent probe of recognizing cells apoptosis, intracellular reactive oxygen species or glutathion inside cell specifically.
8. method of utilizing device according to claim 1 and 2 proof quantum dot cytotoxic mechanism; It is characterized in that; As a kind of cell autophagy inhibitor, before the quantum dot solution function cells, inject the main channel earlier with the 3-methyl adenine; After acting on cell; Carry out the quantum dot cytotoxicity experiment again,, and combine apoptosis rate to judge the cytotoxicity of quantum dot with the cell and the situation that does not have in the cell of 3-methyl adenine effect, to form phagocytic vesicle of transmission electron microscope contrast through the effect of 3-methyl adenine.
9. claim 1 or the 2 described devices application in drug screening or environmental poisonous substance detect.
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