CN105647789A - Real-time cell metabolite detection device based on micro-fluidic chip - Google Patents
Real-time cell metabolite detection device based on micro-fluidic chip Download PDFInfo
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- CN105647789A CN105647789A CN201610066768.8A CN201610066768A CN105647789A CN 105647789 A CN105647789 A CN 105647789A CN 201610066768 A CN201610066768 A CN 201610066768A CN 105647789 A CN105647789 A CN 105647789A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
Abstract
The invention provides a real-time cell metabolite detection device based on a micro-fluidic chip. The device comprises the micro-fluidic chip, an automatic sampling assembly, a pretreatment and enrichment assembly and a detector. Channels provided with cell culture rooms are arranged in the micro-fluidic chip, the automatic sampling assembly capable of conducting sampling at outlets of the channels is arranged around the micro-fluidic chip, and the pretreatment and enrichment assembly is connected to the down stream of the automatic sampling assembly and can conduct desalting and target product enriching on a cell metabolite obtained through sampling. The detector is arranged at the portion corresponding to an outlet of the pretreatment and enrichment assembly to conduct online detection on the target product. According to the detection device, the micro-fluidic chip, automatic sampling, pretreatment and enrichment and detection are integrated into a whole, and real-time online detection and analysis can be conducted on a biochemical reaction on the micro-fluidic chip and the drug metabolism process.
Description
Technical field
The present invention relates to cell research technical field, particularly relate to a kind of products of cellular metabolism real-time detection apparatus based on micro-fluidic chip.
Background technology
It is known that microfluidic chip technology is relative to traditional cell research platform, paid close attention to widely owing to having low reagent consumption, easy of integration, high flux, easily operation and good repeatability. In recent years, zoopery starts to be opposed by the mankind gradually, and microfluidic chip technology is then able to one of method replacing zoopery most potentiality, has broad application prospects. Passage in micro-fluidic chip is generally the micro scale channel suitable with cell size, and this makes research worker be possible not only to cell is accurately manipulated, additionally it is possible to reduce the consumption of cell consumption and reagent. In prior art, utilize the characteristic of micro-fluidic chip, successfully simulate cancer growing state in lactiferous ducts in the chips.
Drug metabolism is it is recognized that the structure of the molecule really worked in medicine, and this uses for clinical medicine can play guidance and supervisory function bit. In order to better carry out biochemical reaction and the drug metabolism simulation of complexity on micro-fluidic chip, it is necessary to more perfect detection and analytical equipment and method. Traditional drug metabolism detection right and wrong are online, and owing to can not carry out continuous print metabolism monitoring, this makes research staff can not understand the cell overall process to drug absorption, metabolism all sidedly. Therefore, how to set up one can online, quickly and the brief analytical equipment of process and method analyze for drug metabolism and just seem extremely important. It addition, detection of the prior art and analytical equipment are enriched with at product, detection limit and anti-interference in still suffer from a lot of deficiencies, this makes to carry out complex biochemical reaction on micro-fluidic chip and drug metabolism is subject to bigger restriction.
Summary of the invention
The invention provides a kind of products of cellular metabolism real-time detection apparatus based on micro-fluidic chip, including: micro-fluidic chip, automatic sampling assembly, pre-treatment and enrichment assembly and detector.The passage of cell culture chamber it is provided with in micro-fluidic chip. The automatic sampling assembly being provided around to sample from the exit of passage at micro-fluidic chip. Connect pre-treatment and enrichment assembly in the downstream of automatic sampling assembly, the products of cellular metabolism that sampling obtains can be carried out desalination and target product enrichment by it. Exit corresponding to pre-treatment with enrichment assembly is provided with detector, so that target product is implemented on-line checking. In this case, the detecting device collection micro-fluidic chip of the present invention, automatic sampling, pre-treatment and enrichment and be detected on one, it is possible to the biochemical reaction on micro-fluidic chip and drug metabolism processes are carried out real-time, online detection and analysis. I.e., it is possible to products of cellular metabolism is carried out continuous print monitoring, to understand the overall process of biochemical reaction on micro-fluidic chip and drug metabolism all sidedly.
Further, micro-fluidic chip includes the passage of multiple parallel connection, and the exit region at the passage near multiple parallel connections is equipped with cell culture chamber. Preferably, passage can be configured with the passage of dendroid bifurcated. Thus, it is possible to simultaneously, online carrying out organize experiment more, it is achieved the cellular metabolism situation of multiple passages is carried out monitoring continuously and contrasts, to reach the purpose multiple cellular metabolisms or biochemical reaction being monitored and detecting.
Further, micro-fluidic chip also includes being provided with cleanout fluid and stores the flushing channel of room. Thus, before automatic sampling assembly samples from different passages, it is possible to first use flushing channel that automatic sampling assembly is carried out, to avoid interfering between different passage.
Further, it is configured with at the port of export of parallel port and flushing channel and is perpendicular to the sample tap that passage is outwardly. Preferably, the shape of sample tap can be configured to match with automatic sampling assembly, to facilitate sampling.
Further, automatic sampling assembly includes pipette, mobile platform and pump. One end of pipette is being arranged in mobile platform in the way of the sample tap of aligned with channel, and the other end of pipette connects with pump. Thus, under the control of computer software, pipette up and down and can move left and right with mobile platform, and the different sample tap to be directed at micro-fluidic chip is sampled. In this case, moved back and forth by mobile platform, pipette can continuously, online, repeatedly from different sample tap samplings. Additionally. Under the effect of pump, it is possible to the micro-example of extraction is pumped into pre-treatment and is enriched with in assembly, to carry out next step processing procedure.
Further, pre-treatment includes enriching column and channel converter with enrichment assembly. The entrance of enriching column can connect with automatic sampling assembly with channel converter through conduit; The outlet of enriching column can connect with waste liquid bottle with channel converter through conduit. Thus, first the sample that sampling obtains carries out desalination process and target product enrichment in enriching column, is entered in waste liquid bottle by unwanted product afterwards.
Further, pre-treatment also includes syringe pump and detached dowel with enrichment assembly. Syringe pump can pass through conduit and connect with the entrance of enriching column with channel converter; Detached dowel can pass through the outlet of conduit and channel converter and enriching column. Thus, to injection eluent in enriching column, target product can being carried out de-washing by syringe pump, afterwards, target product enters the separation carrying out classification in detached dowel, in order to follow-up detection.
Further, channel converter is six-way valve. Thus, the transformation process of passage can be realized by rotating six-way valve, and then sample is carried out different pretreatment process.
Further, detector is electrospray mass spectrometer or chromatograph.Preferably, the outlet of detached dowel can be connected with spraying tip. Thus, it is possible to sample is carried out different detections.
It is an advantage of the current invention that: cell culture medium liquid directly can be carried out on-line real-time measuremen by (1). (2) in target product enrichment, detection limit and anti-interference in can meet the needs of people better. (3) can realize the cellular metabolism situation of multiple passages is monitored and is contrasted, it is simple to carry out organizing experiment more simultaneously. (4) there is higher detection sensitivity and the broader suitability.
Accompanying drawing explanation
Based on embodiment reference accompanying drawing, the present invention will be described in more detail hereinafter. Wherein:
Fig. 1 is the structural representation of the products of cellular metabolism real-time detection apparatus based on micro-fluidic chip of the embodiment of the present invention;
Fig. 2 is the structural representation of the micro-fluidic chip of the embodiment of the present invention;
Fig. 3 a and 3b is the pre-treatment schematic diagram from the different use state of enrichment assembly of the embodiment of the present invention; And
Fig. 4 is the most advanced and sophisticated part-structure schematic diagram with detector of spraying of the embodiment of the present invention.
In the accompanying drawings, identical parts use identical accompanying drawing labelling. Accompanying drawing is not according to actual ratio.
Detailed description of the invention
Below in conjunction with accompanying drawing, the invention will be further described.
As shown in figures 1 and 3, a kind of products of cellular metabolism real-time detection apparatus 100 based on micro-fluidic chip, including: micro-fluidic chip 6, automatic sampling assembly, pre-treatment and enrichment assembly 40 and detector 20. The passage 7 of cell culture chamber 7 ' it is provided with in micro-fluidic chip 6. The automatic sampling assembly being provided around to sample from the exit of passage 7 at micro-fluidic chip 6. Connect pre-treatment and enrichment assembly 40 in the downstream of automatic sampling assembly, the products of cellular metabolism that sampling obtains can be carried out desalination and target product enrichment by it. Exit corresponding to pre-treatment with enrichment assembly 40 is provided with detector 20, so that target product is implemented on-line checking. In this case, the detecting device collection micro-fluidic chip 6 of the present invention, automatic sampling, pre-treatment and enrichment and be detected on one, it is possible to the biochemical reaction on micro-fluidic chip 6 and drug metabolism processes are carried out real-time, online detection and analysis. I.e., it is possible to products of cellular metabolism is carried out continuous print monitoring, to understand the overall process of biochemical reaction on micro-fluidic chip 6 and drug metabolism all sidedly.
In a specific embodiment of the present invention, as illustrated in fig. 1 and 2, micro-fluidic chip 6 is positioned on removable article carrying platform 4. Removable article carrying platform 4 is connected with stage carrier 1. Micro-fluidic chip 6 includes the passage 7 of multiple parallel connection, it is preferable that passage 7 can be configured to dendroid. It is provided with two chip inlets 2 in the porch of passage 7, is configured with in the exit of passage 7 and is perpendicular to the outwardly sample tap of passage 9. The cross sectional shape of sample tap 9 can arbitrarily be arranged, it is preferable that in order to match with pipette 10, and sample tap 9 is arranged to the bore tubulose slightly larger than the bore of pipette 10. The main part of passage 7 is configured to craspedodrome passage or serpentine channel, it addition, be provided with cell culture chamber 7 ' on the craspedodrome passage of passage 7. Preferably, cell culture chamber 7 ' is positioned close to the region of sample tap 9.
When, after the cell culture medium liquid injecting variable concentrations or drug containing and non-drug containing from chip inlet 2, cell culture medium liquid forms certain Concentraton gradient through the main part of passage 7.The cell culture medium liquid of variable concentrations enters in cell culture chamber 7 ', cell carries out different stimulations and makes cell make corresponding response and then the change of generation metabolic activity, make the Related Component of the culture medium of surrounding change. Different products of cellular metabolism is drawn onto in pre-treatment and enrichment assembly by sample tap 9 by pipette 10 in base fluid, to carry out follow-up process.
In order to avoid interfering between different passages 7, micro-fluidic chip 6 is additionally provided with independent string passage, i.e. store the flushing channel 8 of room with cleanout fluid, and be furnished with corresponding inlet 3. In this case, before pipette 10 samples between different passages 7, it is possible to first use the liquid in flushing channel 8 that pipette 10 is carried out, to avoid detection error to greatest extent.
In a specific embodiment of the present invention, as it is shown in figure 1, automatic sampling assembly includes pipette 10, pipette fixed platform 5 and pump 12. Mobile platform 5 is connected with stage carrier 1, and is arranged at the top of micro-fluidic chip 6. Pipette 10 is vertically arranged in mobile platform 4, with the sample tap 9 of energy aligned with channel 7. The other end of pipette 10 is connected with pump 12 by conduit 17. In this case, by the control of computer software, pipette 10 can with mobile platform about 5 with move left and right, and the different sample tap 9 to be directed at micro-fluidic chip 6 is sampled or cleans. By the reciprocating motion of stage carrier 1, pipette 10 can continuously, sample online, repeatedly from different sample taps 9. It addition, under the effect of pump 12, it is possible to the micro-example of extraction is pumped into pre-treatment and is enriched with in assembly 40, to carry out next step processing procedure.
In a specific embodiment of the present invention, as shown in figures 1 and 3, pre-treatment is main with enrichment assembly 40 by six-way valve 14 enriching column 16 syringe pump 15 and detached dowel 18 one-tenth. Six-way valve 14 to play the effect of Channel-shifted. For the ease of illustrating, the hole that we are connected six-way valve 14 with pump is defined as a hole, and adjacent successively along rotation counterclockwise and a hole is No. two holes to No. six holes. When six-way valve 14 rotates, as shown in Figure 3 a, enriching column 16 entrance is by conduit and No. two hole connections, and No. two holes connect with a hole and finally connect with automatic sampling assembly 30. Enriching column 16 exports by conduit and No. five hole connections, and No. five holes connect with No. six holes and are ultimately connected to waste liquid bottle 17. After valve 14 rotates counterclockwise, as shown in Figure 3 b, now, enriching column 16 entrance is by conduit and No. two hole connections, and No. two holes connect with No. three holes, and No. three holes connect syringe pump 15 enriching column 16 and export by conduit and No. five hole connections, No. five holes connect with No. four holes, and No. four holes connect detached dowel 18
When six-way valve 14 rotates, entered to No. 14 holes of six-way valve through conduit 13 by the cell culture medium liquid of pump 12 sample, be then passed through the passage between six-way valve 14 and enter No. two holes, enter enriching column 16 through No. two holes. Products of cellular metabolism in cell culture medium liquid carries out desalination and target product enrichment at enriching column 16. The composition not being enriched with and liquid export by conduit from enriching column 16, are ultimately discharged into waste liquid bottle 17 through No. five holes and No. six holes. When, after six-way valve 14 turns, entering eluent by the syringe pump 15 being connected with No. three holes, the target product of enriching column 16 is eluted, it is connected to detached dowel 18 then through No. four holes and is easily separated.
In a specific embodiment of the present invention, as shown in Figure 4, same class target product after the separation of separated post 18 is flowed into spraying most advanced and sophisticated 19, mist connects high pressure with most advanced and sophisticated 19 places, spraying is ejected with the form of charged microlayer model with the liquid at most advanced and sophisticated 19 places, enters into and electrospray mass spectrometer 20 is detected.
The micro-fluidic chip of the present invention adopts the photoetching method of standard to make, and detailed process is as follows: first, and silicon chip is put in concentrated sulphuric acid and heating in the mixed solution that weight ratio is 3:1 of hydrogen peroxide, micro-boils about 30 minutes. Then, by silicon chip wash clean and dry, silicon chip is carried out by sol evenning machine spin coating, control the height of required passage. Afterwards, being put into by the silicon chip that spin coating is good in baking oven and carry out front baking process, holding temperature is 65 DEG C, and the time is 30 minutes. Using exposure machine that this silicon chip carries out uv-exposure by photo etched mask, then put into by silicon chip and carry out after bake in baking oven, holding temperature is 65 DEG C, and the time is about 15 minutes, so that required passage is reinforced further. After above-mentioned processing procedure, silicon chip is developed and silanization treatment, required mould can be obtained. Mould is put in baking oven, evaporate unnecessary developing agent and silylating reagent. Finally, polydimethylsiloxane (PDMS) and initiator are mixed with the weight ratio of 10:1, imports on mould, after degassed, put into and baking oven is dried 4-6 hour, obtain the solid, shaped PDMS module with micro-fluidic chip passage. This PDMS module is departed from from silicon chip, punches with card punch, then carry out the micro-fluidic chip just obtaining using in the present invention after plasma is bonded with sheet glass.
In sum, the products of cellular metabolism real-time detection apparatus based on micro-fluidic chip of the present invention, movement by mobile platform 5, can so that pipette 10 controllable precise arrive micro-fluidic chip 6 different sample taps 9, and moving up and down of pipette 10 can be regulated and controled so that pipette 10 can carry out compact siro spinning technology with sample tap 9 and separate. Under the regulation and control of pump 12, it is possible to the culture medium drawing designated volume detects, again through enrichment below, separation and detecting step, mass spectrum or the chromatograph detection signal of some materials in culture medium may finally be effectively obtained.
Although by reference to preferred embodiment, invention has been described, but without departing from the scope of the invention, it is possible to it is carried out various improvement and parts therein can be replaced with equivalent. Especially, as long as being absent from structural hazard, the every technical characteristic being previously mentioned in each embodiment all can combine in any way. The invention is not limited in the specific embodiment disclosed in literary composition, but include all technical schemes falling in scope of the claims.
Claims (10)
1. the products of cellular metabolism real-time detection apparatus based on micro-fluidic chip, it is characterised in that including:
Micro-fluidic chip, is provided with the passage of cell culture chamber;
Automatic sampling assembly, is arranged at around described micro-fluidic chip, and can sample from the exit of described passage;
Pre-treatment and enrichment assembly, be connected to the downstream of described automatic sampling assembly, and the products of cellular metabolism that described sampling is obtained carries out desalination and target product enrichment; And
Detector, is correspondingly arranged in the exit of described pre-treatment and enrichment assembly, so that described target product is implemented on-line checking.
2. real-time detection apparatus according to claim 1, it is characterised in that described micro-fluidic chip includes the passage of multiple parallel connection;
Exit region at the passage near multiple described parallel connections is equipped with cell culture chamber.
3. real-time detection apparatus according to claim 2, it is characterised in that described micro-fluidic chip also includes being provided with cleanout fluid and stores the flushing channel of room.
4. the real-time detection apparatus according to Claims 2 or 3, it is characterised in that the port of export at described parallel port and flushing channel is configured with and is perpendicular to the sample tap that passage is outwardly.
5. real-time detection apparatus according to claim 4, it is characterised in that described automatic sampling assembly includes pipette, mobile platform and pump;
One end of described pipette is arranged in described mobile platform in the way of being directed at the sample tap of described passage, and the other end of described pipette connects with described pump.
6. real-time detection apparatus according to claim 1, it is characterised in that described pre-treatment includes enriching column and channel converter with enrichment assembly;
The entrance of described enriching column can connect with described automatic sampling assembly with channel converter through conduit;
The outlet of described enriching column can connect with waste liquid bottle with channel converter through conduit.
7. real-time detection apparatus according to claim 6, it is characterised in that described pre-treatment also includes syringe pump and detached dowel with enrichment assembly;
Described syringe pump can pass through conduit and connect with the entrance of channel converter with described enriching column;
Described detached dowel can pass through the outlet of conduit and channel converter and described enriching column.
8. the real-time detection apparatus according to claim 6 or 7, it is characterised in that described channel converter is six-way valve.
9. real-time detection apparatus according to claim 1, it is characterised in that described detector is electrospray mass spectrometer or chromatograph.
10. real-time detection apparatus according to claim 9, it is characterised in that the outlet of described detached dowel can be connected with spraying tip.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610066768.8A CN105647789B (en) | 2016-01-29 | 2016-01-29 | A kind of products of cellular metabolism real-time detection apparatus based on micro-fluidic chip |
| PCT/CN2017/072258 WO2017129099A1 (en) | 2016-01-29 | 2017-01-23 | Real-time cell metabolite detection device based on microfluidic chip |
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| CN201610066768.8A CN105647789B (en) | 2016-01-29 | 2016-01-29 | A kind of products of cellular metabolism real-time detection apparatus based on micro-fluidic chip |
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| CN105647789A true CN105647789A (en) | 2016-06-08 |
| CN105647789B CN105647789B (en) | 2019-01-04 |
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| WO (1) | WO2017129099A1 (en) |
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| WO2017129099A1 (en) * | 2016-01-29 | 2017-08-03 | 清华大学 | Real-time cell metabolite detection device based on microfluidic chip |
| CN109682902A (en) * | 2018-12-19 | 2019-04-26 | 中国科学院成都生物研究所 | Micro-fluidic chip extracts fluidic chip-mass spectrometry analytical equipment and the method for declining |
| WO2022226920A1 (en) * | 2021-04-29 | 2022-11-03 | 京东方科技集团股份有限公司 | Immunoassay device and immunoassay method |
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| US20220288588A1 (en) * | 2019-08-05 | 2022-09-15 | Auer Precision Company, Inc. | Microfluidic passive plasma separation device and method |
| CN111172025B (en) * | 2020-01-14 | 2022-11-15 | 中国科学院烟台海岸带研究所 | Compound micro-fluidic chip device of hydrogen sulfide activity detection based on cell |
| CN114507593B (en) * | 2022-01-29 | 2023-10-17 | 扬州大学 | Integrated portable drug sensitive detection chip |
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| CN109682902A (en) * | 2018-12-19 | 2019-04-26 | 中国科学院成都生物研究所 | Micro-fluidic chip extracts fluidic chip-mass spectrometry analytical equipment and the method for declining |
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| Publication number | Publication date |
|---|---|
| WO2017129099A1 (en) | 2017-08-03 |
| CN105647789B (en) | 2019-01-04 |
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