CN102728422B - Microfluidic chip apparatus and application thereof - Google Patents
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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 detected for quantum dot cytotoxicity, and its application detected in quantum dot cytotoxicity.
Background technology
Quantum dot has great using value as a kind of emerging nano material in bio-imaging, but its cytotoxicity had but limits widely using of it.For the toxic mechanism of quantum dot, numerous scientist gives different explanations, such as, discharge heavy metal ion, to produce reactive oxygen species and different surface naturies caused etc.
The Cytotoxic research of current quantum dot mostly to adopt in culture dish cultured cell or carries out zoopery.Document Li Y.; Zhou Y.; Wang H.; Perrett S.; Zhao Y.; TangZ.; Nie G.Angew Chem Int Edit, 2011,50,5860-5864 cause cytotoxicity in various degree by the HepG2 cell proof CdTe quantum of the glutathione bag quilt of different chirality of cultivating in culture dish.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 suffer from the cytotoxicity of the PbSe quantum dot that the animal model of breast cancer have detected fatty acid ester bag quilt at one.But cell is in a complicated cell-matrix and the interactional microenvironment of cell-soluble factor in vivo, the cell in this and 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 can the platform of simulated in vivo environment more accurately for the Cytotoxic research of quantum dot.
Micro-total analysis system is also known as micro-fluidic chip (microfluidic chip) or chip lab (lab on a chip, LOC), refer to integrated on the chip of a piece several square centimeters (even less) or basic integrated chemical with sample preparation, the reaction involved by biological grade in field, be separated, the basic operation unit such as detection and cell chulture, sorting, cracking, network is formed by microchannel, whole system is run through, in order to replace a kind of technology platform of the various functions of conventional chemical or biology laboratory with controlled fluid.In early 1990s, the concept of the micro-total analysis system (μ TAS) proposed first by Manz and Widmer, has adapted to life science and has carried out demand that is more efficient, highly sensitive, quick separating analysis to biological sample.Micro-fluidic chip is used widely in cellular metabolism, drug screening and stem cell tissue engineering in the past few decades.
Another potential application of micro-fluidic chip is the dimensional culture and the simulation human organ that carry out cell.Dimensional culture farthest can retain the specific function of organization of cell in live body under in vitro conditions, differentiation state, and can reappear spatial concentration gradient and body mechanics's microenvironment of tissue-tissue interaction interface, chemical substance in 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 utilize the microchannel of two close apposition on micro-fluidic chip, simulate the contact surface of alveolar and capillary and 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 utilize multichannel micro-fluidic chip to simulate the metabolism of medicine in human body and storage process.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, by comparing growth and the migration situation of cell diverse location in dimensional culture matrix, simulate the cell of different parts in tissue.As can be seen here, dimensional culture micro-fluidic chip is the experiment in vitro platform of a good drug screening and toxicity detection, be expected to replace zoopery, but, at present not yet by this platform application in the Cytotoxic research of quantum dot.
Summary of the invention
For above-mentioned present situation, the invention provides a kind of dimensional culture micro flow control chip device detected for quantum dot cytotoxicity.This apparatus structure is simple, microminiaturized, cost is low, be easy to preparation and operation.This device can realize the dimensional culture of cell and quantum dot act on cell after toxicity detection, and certain quantum dot cytotoxic mechanism can be disclosed.
The invention provides a kind of micro flow control chip device, wherein this device comprises the main channel and cell chulture chamber that are interconnected, and described cell culture chamber comprises the abluminal compartment and chamber far away that are arranged on both sides, main channel.Wherein, main channel can be used for transfusion cell culture medium and quantum dot solution by simulated blood vessel, and cell chulture chamber then can simulate the circumvascular adjacent tissue of quantum dot effect.
Described micro flow control chip device can be formed by polydimethylsiloxanechip chip and substrate of glass bonding.
Described abluminal compartment distance main channel 0.6-1.2mm, described chamber distance main channel 1.5-2.2mm far away.
Difference in height is had, the height height 30-60 μm of the aspect ratio cell chulture chamber of main channel between the main channel of described micro flow control chip device and cell chulture chamber.Main channel can not be leaked into when surface tension effects can be utilized like this to make the mixture of cell and dimensional culture matrix inject cell chulture chamber.
Medicine is transported to the tissue that will act on from administration blood vessel needs two steps: the first step, and medicine flows in the blood vessel of contiguous target location by the blood vessel that whole body distributes; Second step, medicine passes blood vessel target approach tissue by diffusion.Therefore, medicine diffusion is in the tissue the key factor affecting drug effect.
Therefore, present invention also offers a kind of method utilizing diffusion in above-mentioned micro flow control chip device simulated tissue, the bovine serum albumin(BSA) (FITC-BSA) of fluorescein sodium and marked by fluorescein isothiocyanate is injected in the main channel of described device, same position fluorescence intensity respectively in abluminal compartment and chamber far away, the diffusion transport process between simulated blood vessel and tissue.
With fluorescein sodium and FITC-BSA for model drug, wherein fluorescein sodium represents small-molecule substance, and FITC-BSA represents large biological molecule.Because these two kinds of materials all have fluorescence, after being injected into main channel, the same position fluorescence intensity of timing respectively in abluminal compartment and chamber far away.Relatively can prove the impact of distance on diffusion process by fluorescence intensity, the fluorescence intensity in synchronization abluminal compartment is always higher than chamber far away, and the diffusion velocity of the small-molecule substance of fluorescein sodium representative will faster than the large biological molecule of FITC-BSA representative.This method simulates the diffusion process of material in the tissue of distance blood vessel different distance.
The invention provides a kind of method utilizing above-mentioned micro flow control chip device to realize the dimensional culture of cell, the matrix that described 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 material of a kind of thermal reversion and bio-compatibility, and the copolymerization of (3-6) that the β-D-gala furanose linked by (1-3) and (1-4) link-dehydration-α-L-gala furanose forms, and can be used for biology sensor.The fusing point of agarose and gelation temperature can by changing molecular weight and modify functional group.Low melting-point agarose solidifies at 25 DEG C, is applicable to carrying out cell capture lower than the gelation temperatures of 37 DEG C, and what use in the present invention is low melting-point agarose.There is a certain size aperture in Ago-Gel, be applicable to passing through of the nutriment needed for cell chulture etc.In cell chulture chamber, cell is wrapped up by agarose, simulates cell well in vivo by three-dimensional environment that extracellular matrix wraps up.Therefore dimensional culture micro-fluidic chip provided by the invention can microenvironment in analogue body well, is suitable as the experiment in vitro platform of quantum dot Study of cytotoxicity.
Present invention also offers one utilizes described micro flow control chip device to detect the Cytotoxic method of quantum dot, detects the change of Apoptosis, intracellular reactive oxygen species (ROS) and glutathion inside cell (GSH) with fluorescence probe.Described fluorescence probe is the specificity fluorescent probe that can identify Apoptosis, intracellular reactive oxygen species or glutathion inside cell respectively specifically.
Apoptosis, the increase of intracellular reactive oxygen species and the minimizing of glutathione detect the conventional several indexs of quantum dot cytotoxicity, and the increase of intracellular reactive oxygen species and the minimizing of glutathione all show that quantum dot toxicity strengthens.After the quantum dot solution effect of the cell variable concentrations in chip under dimensional culture, quantum dot solution in main channel is replaced with the Cytotoxic specificity fluorescent probe of corresponding detection, due to the diffusion function of dimensional culture matrix, after hatching a period of time, imaging under fluorescence microscope, to take pictures, its toxicity can be analyzed.
Present invention also offers a kind of method utilizing micro flow control chip device of the present invention to prove quantum dot cytotoxic mechanism, using 3-MA as a kind of cell autophagy inhibitor, before quantum dot solution function cells, first inject main channel, after acting on cell, carry out quantum dot cytotoxicity experiment again, with transmission electron microscope contrast through the cell of 3-MA effect and the situation not forming phagocytic vesicle in the cell of 3-MA effect, explain that autophagocytosis is on the Cytotoxic impact of quantum dot in conjunction with apoptosis rate.More the speak more cytotoxicity of bright quantum dot of the phagocytic vesicle formed in cell is larger, otherwise then little.
The autophagocytosis of cell is a kind of approach of cell degradation bulk cytoplasm, long-lived proteins and whole organelle, is a kind of important protection mechanism in apoptosis.There are some researches show that the autophagocytosis of cell is very important equally in quantum dot cytotoxic mechanism.
Micro flow control chip device of the present invention can also application in drug screening or environmental poisonous substance detect.
As fully visible, beneficial effect of the present invention is:
Dimensional culture micro flow control chip device for the detection of quantum dot cytotoxicity of the present invention illustrates some significant advantages, comprise: make simple, be easy to operation, material therefor bio-compatibility is good, cell culture condition is closer to microenvironment in body, diffusion between energy simulated blood vessel and surrounding tissue, and the proof of quantum dot toxic mechanism can be used for.
Accompanying drawing explanation
Fig. 1 is the structural representation according to dimensional culture micro flow control chip device of the present invention.
Fig. 2 utilizes micro flow control chip device according to the present invention to take fluorescein sodium as the fluorescence intensity photo that model drug is simulated diffusion in tissue.
The fluorescence intensity photo that Fig. 3 utilizes micro flow control chip device according to the present invention to simulate diffusion in tissue for model drug with the bovine serum albumin(BSA) of marked by fluorescein isothiocyanate (FITC-BSA).
Fig. 4 is the fluorescence intensity change data analysis utilizing micro flow control chip device according to the present invention to simulate diffusion in tissue for model drug with the bovine serum albumin(BSA) (FITC-BSA) of fluorescein sodium and marked by fluorescein isothiocyanate, with most high fluorescent for 100%; What show in figure is the result of three groups of parallel laboratory tests.
Fig. 5 utilizes micro flow control chip device according to the present invention to characterize three days inner cell active fluoros that HepG2 cell carries out dimensional culture.
Fig. 6 is the three days inner cell survival rate analysis figure utilizing micro flow control chip device according to the present invention HepG2 cell to be carried out to dimensional culture; What show in figure is the result of three groups of parallel laboratory tests.
Fig. 7 utilizes the CdTe quantum (CdTe-COOH) of micro flow control chip device according to the present invention to carboxyl bag quilt to act on the Apoptosis fluorescence photo after HepG2 cell; What send bright blue fluorescence is apoptotic cell.
Fig. 8 utilizes the CdTe quantum (CdTe-COOH) of micro flow control chip device according to the present invention to carboxyl bag quilt to act on the apoptosis rate data analysis after HepG2 cell; What show in figure is the result of three groups of parallel tests.
Fig. 9 is that intracellular reactive oxygen species (ROS) after utilizing the CdTe quantum (CdTe-COOH) of micro flow control chip device according to the present invention to carboxyl bag quilt to act on HepG2 cell and glutathione (GSH) change fluorescence photo; The red fluorescence of ROS shows that more by force the cytotoxicity that quantum dot produces is stronger, and the more weak cytotoxicity showing that quantum dot produces of the green fluorescence of GSH is stronger.
Figure 10 utilizes the CdTe quantum (CdTe-COOH) of micro flow control chip device according to the present invention to carboxyl bag quilt to act on the intracellular reactive oxygen species (ROS) after HepG2 cell and the analysis of glutathione (GSH) delta data; What show in figure is the result of three groups of parallel tests.
Figure 11 utilizes micro flow control chip device according to the present invention to be the checking of one of quantum dot cytotoxic mechanism to autophagocytosis: transmission electron microscope characterizes formation and the mitochondrial deformation (marking with white arrow) that cell endocytic bites vesicle (marking with black arrow).
Figure 12 utilizes micro flow control chip device according to the present invention to be the checking of one of quantum dot cytotoxic mechanism to autophagocytosis: 3-MA effect whether Apoptosis contrast fluorescence photo.
Figure 13 utilizes micro flow control chip device according to the present invention to be the checking of one of quantum dot cytotoxic mechanism to autophagocytosis: 3-MA effect whether Apoptotic data analysis in abluminal compartment and chamber far away, what show in figure is the result of three groups of parallel tests, in figure, * indicates marked difference, and * * indicates highly significant difference.
Detailed description of the invention
Below by embodiment, the present invention is described further by reference to the accompanying drawings.
If without specified otherwise, it is experimentally normal experiment method; The reagent used and material etc. all obtain by commercial sources.
Micro-fluidic chip in the present invention adopts " re-expose " technology soft lithography process to be made.Its preparation method is as follows:
The making of mould: the acid solution process of silicon chip use water tiger, clean, dry after, the negative optical cement of one deck SU-8 2050 is got rid of with the rotating speed of 2000rpm on surface with sol evenning machine, at 65 DEG C of baking 10min, after 95 DEG C of baking 4min, after uv-exposure, development, nitrogen dry up, namely obtain the pattern of ground floor cell chulture chamber.Then get rid of the negative optical cement of second time with 1100rpm rotating speed, after above-mentioned operation equally, can obtain the pattern of second layer main channel, namely mould is successful.
The making of chip: silanization makes die surface hydrophobic, dimethyl silicone polymer (PDMS) prepolymer and initator mix with mass ratio 10:1 to be poured in mould, places 2h after bubble removing at 75 DEG C.Carefully taken off by PDMS sheet after polymerization, excision forming, with the punching of tack syringe needle, then through oxygen plasma treatment, bonding irreversible with slide, namely chip is successful.
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 chulture chamber.Wherein, main channel can be used for transfusion cell culture medium and quantum dot solution by simulated blood vessel, and cell chulture chamber then can simulate the circumvascular adjacent tissue of quantum dot effect.
This device comprises two parts: main channel and cell chulture chamber, main channel is communicated with cell culture chamber.The main channel of described micro flow control chip device is different with the height of cell chulture chamber, is respectively 71 μm and 38 μm.Main channel can not be leaked into when surface tension effects can be utilized like this to make the mixture of cell and dimensional culture matrix inject cell chulture chamber.The cell culture chamber of described micro flow control chip device is divided into two kinds, and the abluminal compartment and the distance main channel that are respectively distance main channel 0.8mm are the chamber far away of 2mm.
Embodiment 2: the diffusion in simulated tissue
Diffusion in tissue with the bovine serum albumin(BSA) (FITC-BSA) of fluorescein sodium and marked by fluorescein isothiocyanate for model drug.In the cell chulture chamber of both sides, implantation quality volume ratio is the agarose solution of 0.5%, after its condensation, injects the Fluress of 100 μMs and the FITC-BSA solution of 2.75mg/mL respectively from main channel.Every 5min, the fluorescence intensity of fluorescein sodium is taken pictures at the same position fluorescence microscope (Leica DMI 4000B) of chamber roof, every 10min, the fluorescence intensity of FITC-BSA is taken pictures.The Therapy lasted 300min of the Therapy lasted 120min of fluorescein sodium, FITC-BSA, result as shown in Figure 2, Figure 3 and Figure 4.The growth along with the time can be found out from Fig. 2-4, the obvious grow of fluorescence intensity, show that these two kinds of materials have diffused in agarose really, and the fluorescence intensity in same time abluminal compartment be higher than chamber far away.Three times parallel laboratory test result is similar, shows that the difference of distance causes the difference of diffusion.
Embodiment 3:HepG2 cell dimensional culture in the chips
Two dish 60cm
2the cell covered with in culture dish is cleared up, and cell suspension is centrifugal, and after removing 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.The cell chulture chamber of micro-fluidic chip of the present invention is injected in equal-volume cell suspension and the mixing of dimensional culture matrix, and chip places 10min to accelerate solidifying of agarose at 4 DEG C, by cell encapsulation wherein.Finally, culture medium is injected main channel and is coated with one deck at chip surface, in case the volatilization of culture medium in stop-pass road.Chip is placed in cell culture incubator, and culture medium is changed every day, detects cell survival rate every day with life or death kit (Calcein-AM/EthD-1).As shown in Figure 5 and Figure 6, cell all keeps normal in first three day activity of cultivating and form to result, still reaches more than 85% at the 3rd day cell survival rate.
Embodiment 4: to CdTe quantum (CdTe-COOH) cytotoxicity analysis of carboxyl bag quilt
The increase of Apoptosis, intracellular reactive oxygen species (ROS) and the minimizing of glutathione (GSH) are Cytotoxic three indexs of quantum dot, can with three species specificity fluorescence probe: Hoechst 33342, dihydro second ingot (DHE) and 2,3-naphthalene dicarbaldehyde (NDA) detect respectively.With the CdTe quantum of carboxyl bag quilt (CdTe-COOH) for this experiment.Storing solution (5mg/mL) stepwise dilution to 0 of CdTe-COOH quantum dot, 10,20,30,40,50 μ g/mL.With embodiment 3 by after HepG2 three-dimensional cell cultivation 24h, the quantum dot solution of variable concentrations injects main channel respectively, and after static lower effect 24h, the above-mentioned fluorescence probe solution of 100 μMs injects main channel, in 37 DEG C of incubators, hatch 1h.Fluorescence microscope (LeicaDMI 4000B) is taken pictures, Apoptosis software I mage-Pro Plus 6.0 analyzing and processing, and as shown in Figure 7 and Figure 8, what wherein in Fig. 7, sapphirine represented is apoptotic cell to result.The increase of ROS and GSH minimizing software QCapture Pro(version: 5.1.1.14 in cell) process, result as shown in Figure 9 and Figure 10, the red fluorescence of the ROS wherein in Fig. 9 in showed cell increases along with the increase of quantum dot concentration, and the green fluorescence of GSH in cell reduces along with the increase of quantum dot concentration.Can find out from Fig. 7-10, the cytotoxicity of CdTe-COOH quantum dot has obvious concentration dependent, increases with concentration, and cytotoxicity strengthens.Under the quantum dot solution effect of same concentration, apoptosis rate in abluminal compartment is apparently higher than chamber far away, the fluorescence intensity of ROS is also higher in abluminal compartment, in abluminal compartment, intracellular GSH reduces more, and the result of three parallel laboratory tests is similar, these results show that different diffusion length causes quantum dot cytotoxicity in various degree really.
The sign of one of toxic mechanism of embodiment 5:CdTe-COOH quantum dot---autophagocytosis
With embodiment 3 by after HepG2 three-dimensional cell cultivation 24h, the PBS of 3mM 3-MA injects main channel, act on cell 5h, and then with the CdTe-COOH quantum dot solution function cells 24h of variable concentrations, detect apoptosis rate by the method for same embodiment 4.Transmission electron microscope is used for the formation (as shown in Figure 10) of phagocytic vesicle in observation of cell, cell after the quantum dot solution effect of 20 μ g/mL is used for this experiment, cell is used in advance respectively and without 3-MA process, the cell without any process is used as control group.As can be seen from the Electronic Speculum figure in Figure 11, the quantity of the phagocytic vesicle in the cell of 3-MA process will be starkly lower than the cell (marking by black arrow) without 3-MA process, and the mitochondria number of damage also (marking by white arrow) on the low side.The formation of phagocytic vesicle is not then had, without injury of mitochondria yet in cell as a control group.In Figure 12, sapphirine is apoptotic cell, and as can be seen from Figure 12 and Figure 13, when low concentration quantum dot solution (<30 μ g/mL) function cells, 3-MA obviously can reduce apoptosis rate.
Claims (3)
1. the method utilizing micro flow control chip device to prove quantum dot cytotoxic mechanism, it is characterized in that, described micro flow control chip device comprises the main channel and cell chulture chamber that are interconnected, described cell culture chamber comprises the abluminal compartment and chamber far away that are arranged on both sides, main channel, wherein, using 3-MA as a kind of cell autophagy inhibitor, before quantum dot solution function cells, first inject main channel, after acting on cell, carry out quantum dot cytotoxicity experiment again, with transmission electron microscope contrast through the cell of 3-MA effect and the situation forming phagocytic vesicle in the cell of 3-MA effect, and the cytotoxicity of quantum dot is judged in conjunction with apoptosis rate.
2. method according to claim 1, is characterized in that, described abluminal compartment distance main channel 0.6-1.2mm, described chamber distance main channel 1.5-2.2mm far away.
3. method according to claim 1 and 2, is characterized in that, the height height 30-60 μm of the aspect ratio cell chulture chamber of described main channel.
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| CN112973814B (en) * | 2021-03-03 | 2022-03-18 | 北京理工大学 | Interlayer automatic alignment bonding device and method for multilayer microfluidic chip |
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