CN201628717U - Microfluidic chip used for detecting pathogenic microorganisms - Google Patents
Microfluidic chip used for detecting pathogenic microorganisms Download PDFInfo
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- CN201628717U CN201628717U CN2010201107319U CN201020110731U CN201628717U CN 201628717 U CN201628717 U CN 201628717U CN 2010201107319 U CN2010201107319 U CN 2010201107319U CN 201020110731 U CN201020110731 U CN 201020110731U CN 201628717 U CN201628717 U CN 201628717U
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Abstract
The utility model relates to a microfluidic chip used for detecting pathogenic microorganisms. The chip comprises a cover plate and a substrate. Fluid transmission channels, a recognition/enriching cavity, a bioluminescence detection unit and liquid storage tanks are arranged on the cover plate. The two ends of the recognition/enriching cavity are respectively communicated with the first and the second fluid transmission channels, the first and the second liquid storage tanks are respectively connected with the first and the second fluid transmission channels, and the first and the second liquid storage tanks are respectively arranged at the two ends of the chip. The liquid storage tanks are connected with an external transmission device, the bioluminescence detection unit is arranged at the bottom end of the second fluid transmission channel or the second liquid storage tank, and the second fluid transmission channel and the immune enriching cavity are step-shaped. The use of the microfluidic chip can realize the integral operation of immunoaffinity recognition/enriching, inclusion cracking, bioluminescence reaction and detection, etc, largely simplify the analysis detection process of pathogenic microorganisms, and obviously reduce sample usage and reagent consumption. The utility model has the advantages of small detection error, short time, low cost and portability.
Description
Technical field
The utility model relates to a kind of microflow controlled biochip, particularly a kind of integrated micro-flow control chip at the sorting/enrichment of pathogenic microorganism sample, inclusion cracking and bioluminescence detection by quantitative.
Background technology
Often pathogenicity is strong for strong pathogenic pathogenic microorganism, can be less than 10 pathogens as the minimum infective dose of helicobacter pylori Escherichia coli O 157: H7 (E.coli O157:H7).Therefore, development is the disease's spread of human society infection prevention to quick, sensitive, the special analyzing detecting method of pathogenic microorganism in environment, food and the clinical samples, guarantees pressing for of human health.At present, conventional pathogenic microorganism detection method mainly contains cultivation, immunological detection and molecular biology method, and all there are many deficiencies in the limitation because of himself research means: traditional bacterial cultivation and biochemical identification method specificity are low, time and effort consuming, strict to culture environment and operating personnel, are not suitable for the fast detecting of pathogen.Immunological detection method, as enzyme linked immunosorbent assay (ELISA), bioluminescence immunoassay method, immune colloidal gold technique etc., need provide a fairly large number of purification of bacterial, maybe need sample is carried out enrichment method, be difficult to obtain at short notice testing result equally.Though PCR and correlation technique thereof have the specificity and the sensitivity of height, but because method tetchiness, in testing process, because sample contamination, RNA are illegally transcribed, target RNA low expression level, design of primers is unreasonable and experiment condition is not optimized, false positive or false negative result usually appear in the limitation of drawing materials etc.
Defective with respect to existing detection technique, characteristics such as the micro-fluidic chip technology that phase early 1990s occurs is fast with integrated, microminiaturized, the variation of analysis means, the analysis speed of its height, accuracy height, present special advantages technically, be fit to the multinomial requirement of pathogenic microorganism fast detecting: at first, micro-fluidic chip system has multiple operating unit flexible combination, integral body is controlled and scale is integrated characteristics, can be with the sample preparation of whole pathogenic microorganism and check and analysis process conformity on chip piece; Second, process by MEMS, the intensive analysis channel array of etching on chip easily, increased analysis throughput greatly, and it is independent mutually between each passage of chip, avoided the cross interference of sample, the compartment analysis process is carried out in an airtight relatively passage fully, has reduced the infected danger of operator.The 3rd, in view of the structure (micro updating or the upgrading of receiving) of chip microchannel, the high-specific surface area under the microscale, fluid mass-transfer, heat transfer have reduced the consumption of sample and reagent soon effectively, shorten whole analysis time greatly, reach the purpose of express-analysis.Therefore, utilizing the micro-fluidic chip technology to carry out microbe research more and more is subject to people's attention.
At present, through the development of more than ten years, the various pathogenic microorganism pre-service of carrying out based on microflow control technique (Inami H, et al.Biosens Bioelectron.2009,24 (11): 3299-3305; KulinskiMD, et al.Biomed Microdevices.2009,11 (3): 671-678.), enrichment/sorting (Bao N, et al.J Chromatogr A, 2008,1181 (1-2): 153-158; Qiu J, et al.Talanta.2009,79 (3): 787-795.) and separation detection (Lee SJ, et al.Sensors and ActuatorsB, 2008,132 (2): 443448; Zordan MD, et al.Cytometry A.2009,75 (2): new method 155-162.) continues to bring out.But existing technology and method mainly are at a certain unit or step in the pathogenic microorganism analyzing and testing, launch as enrichment/sorting, pre-service or separation detection, usually detect to need to divide and finishes several times, have and detect shortcomings such as error is big, the time long, consume when detecting medicine and amount of reagent are big.
Therefore, realizing that on same micro-fluidic chip sorting/enrichment, inclusion cracking, bioluminescence reaction and the detection one of pathogenic microorganism goes on foot the chip of finishing, is to demand problem soon at present urgently.
Summary of the invention
The utility model purpose is to provide a kind of micro-fluidic chip that pathogenic microorganism detects that is used for, adopt the described micro-fluidic chip of this case, can realize integrated operations such as immune affine identification/enrichment, inclusion cracking, bioluminescence reaction and detection, simplify the analyte detection process of pathogenic microorganism greatly, significantly reduce amount of samples and reagent consumption, and have detect that error is little, the time is short, cost is low, easy to carry, the medicine and the few advantage of amount of reagent that consume.
For achieving the above object, the utility model is by the following technical solutions:
Be used to detect the micro-fluidic chip of pathogenic microorganism, described micro-fluidic chip comprises cover plate and substrate, cover plate and substrate sealing-in are fitted, cover plate is provided with two fluid delivery channel, hexagon identification/enrichment chamber, bioluminescent detection unit and two liquid storage tanks, the two ends in identification/enrichment chamber that are filled with the microballon of antibody modification are respectively with first, second fluid delivery channel is communicated with, first liquid storage tank is connected with the first fluid transmission channel, second liquid storage tank is connected with second fluid delivery channel, first, two liquid storage tanks lay respectively at the two ends of chip, liquid storage tank links to each other with the external transmission device, the bioluminescent detection unit is positioned at terminal or second liquid storage tank of second fluid delivery channel, and second fluid delivery channel and immunity enrichment chamber are step-like.
Described first fluid transmission channel is an access road, and second fluid delivery channel is an exit passageway; The wide 200 μ m of first fluid transmission channel, dark 70 μ m, long 1cm; The wide 200 μ m of second fluid delivery channel, dark 30 μ m, long 1.8cm.
The described second fluid delivery channel degree of depth is less than the immunity enrichment chamber degree of depth.
Described identification/long the 7mm of enrichment chamber cavity, wide 3mm, its cavity depth is greater than 1 times of bead diameter and less than 2 times of bead diameter.
Angle α between two hypotenuses that described identification/enrichment chamber links to each other with passage is 60 °.
Microballon in described identification/enrichment chamber, enrichment chamber is monolayer alignment.
The diameter of described first liquid storage tank and second liquid storage tank is respectively 1.5mm and 3mm.
Described microballon material is glass or polystyrene material, and described patch material is the polymer poly dimethyl siloxane.
The surperficial immobilized probe molecule antibody of described microballon is for having the organic antibody of all kinds of pathogenic microorganisms of target.
The utlity model has following advantage:
1. with the sample introduction of pathogenic microorganism sample, sorting/enrichment, the functions such as highly sensitive detection that add reagent, mixing, reaction and inclusion are integrated on the microchip, can simplify analysis process greatly, reduce the consumption of sample and reagent, the operate miss that minimizing is brought because of the repeated detection operation, the sample loss of avoiding sample transfer to produce improves the analysis speed, accuracy in detection and the sensitivity that detect.
2. be the identification molecule with antibody, can strengthen detection specificity, utilize the covalent modification can be expediently that corresponding antibodies is immobilized in bead surface.Microballon is cheap, can control the quantity of microballon according to the needs of sample, guarantees detection sensitivity and sensing range.Simultaneously, utilize microflow control technique, can be expediently with microballon importing/derivation microchip, pollution-free but the chip repeated multiple times is used, can guarantee the accuracy of mensuration.
3. micron order or nanophase microballon have bigger specific surface area, can increase antigen/antibody in conjunction with probability, help accelerating immune response, shorten analysis time, improve the speed that detects of pathogenic microorganism.
4. functions such as sample pretreatment (comprising identification/enrichment, inclusion cracking), bioluminescence reaction and detection are integrated on the single micro-fluidic chip, can make the micro flow control chip device structure more compact, be beneficial to the pathogenic microorganism detection system of development microminiaturization, portability.
Description of drawings
Fig. 1 is the structure diagram of the utility model chip;
Fig. 2 is the side view of the utility model chip;
Fig. 3 is the structure diagram of the utility model chip, and wherein α is the angle between identification/enrichment chamber hypotenuse and the extended line of passage in identification/enrichment chamber.
Among the figure, 1 is the first fluid transmission channel, and 2 are identification/enrichment chamber, and 3 is second fluid delivery channel, and 4 is microballon, and 5 is first liquid storage tank, and 6 is second liquid storage tank.
Embodiment
The utilization method of molding (Meng Fei etc. SCI .2002,23 (7): 1264-1268; Liu Changchun etc., Micrometer-Nanometer Processing Technology, 2004,1:58-61) produce the PDMS cover plate of microstructure as shown in Figure 1, after the ultraviolet light photochemical treatment, this cover plate and glass substrate sealing-in can be obtained complete micro-fluidic chip.Referring to Fig. 1, Fig. 2 and Fig. 3, this chip is made up of fluid delivery channel 1,3, identification/enrichment chamber 2, bioluminescent detection unit 4 and first, second liquid storage tank 5,6.Cover plate is provided with first, second fluid delivery channel 1,3, hexagon identification/enrichment chamber 2, bioluminescent detection unit 4 and first, second liquid storage tank 5,6, and the two ends in identification/enrichment 2 chambeies that are filled with the microballon 4 of antibody modification are communicated with first, second fluid delivery channel 1,3 respectively.Microballon in the enrichment chamber, identification/enrichment chamber is monolayer alignment.The first fluid transmission channel is an access road, and second fluid delivery channel is an exit passageway; The wide 200 μ m of first fluid transmission channel, dark 70 μ m, long 1cm; The wide 200 μ m of second fluid delivery channel, dark 30 μ m, long 1.8cm.The diameter of first liquid storage tank and second liquid storage tank is respectively 1.5mm and 3mm.First liquid storage tank 5 is connected with first fluid transmission channel 1, second liquid storage tank 6 is connected with second fluid delivery channel 3, and first and second liquid storage tank lays respectively at the two ends of chip.Described identification/long the 7mm of enrichment chamber cavity, wide 3mm, dark 70 μ m.Angle α between two hypotenuses that identification/enrichment links to each other with passage is 60 °, and its cavity depth is greater than 1 times of bead diameter and less than 2 times of bead diameter.Adopt the hexagon microcavity of this structure, make it have bigger effective cavity area, can have higher microballon filling rate; Hexagonal rock-steady structure can make the microballon of filling be arranged as stable hexagonal lattice accumulation mode, is not vulnerable to liquid and flows the influence of shearing and keep the stability of its arrangement; And the hexagon cavity body structure has bigger radical length than geometric configuratioies such as the circle of equal volume, square, rhombuses, liquid flows to/when going out cavity, the linear velocity of fluid radially is linear along cavity and changes, and has reduced the possibility that produces dead volume when flow velocity is big in the cavity.Be filled with the microballon through antibody modification in the chamber, the microballon particle diameter is a micron order, is slightly less than cavity depth, makes it be monolayer alignment in microcavity.Microballon adopts glass or polystyrene material.The surperficial immobilized probe molecule antibody of microballon is for having the organic antibody of all kinds of pathogenic microorganisms of target, and described patch material is the polymer poly dimethyl siloxane.Liquid storage tank links to each other with the external transmission device, and the bioluminescent detection unit is positioned at terminal or second liquid storage tank 6 of second fluid delivery channel 3, and the luminous detection site of present embodiment is arranged in outlet liquid storage tank 6.The second fluid delivery channel degree of depth is less than the enrichment chamber degree of depth, and it is step-like making second fluid delivery channel 3 and immunity enrichment chamber 2, prevents that microballon from flowing into exit passageway.
The method of the surperficial immobilized probe molecule antibody of microballon is as follows:
Accurately take by weighing 10mg diameter 50 μ m glass microballoons in centrifuge tube, with Piranha solution (H
2SO
4: H
2O
2=3: 1) after the soaked overnight, with aseptic ultrapure water washing 5 times, place 70 ℃ of dry 30min then again.In centrifuge tube, add 2%APTES acetone soln reaction 1min, clean 5 times with acetone and aseptic ultrapure water successively.At 10 μ L 1mgmL
-1Add 50 μ L MES damping fluids, 8 μ L 4mgmL in the antibody working fluid
-1EDC (
-2mmolml
-1) and 12 μ L 4mgmL
-1NHS (
-2mmolmL
-1), behind the room temperature reaction 15min, add 120 μ L 0.1molL
-1The PBS damping fluid stops NHS-antibody activation ester and forms reaction, and promptly obtaining antibody concentration is 50 μ gmL
-1Reactant liquor.Reactant liquor is added in the above-mentioned centrifuge tube that bead is housed room temperature reaction 2h.After finishing, reaction uses 0.1molL
-1PBS cleans 3 times, obtains the immune microballon modified through anti-E.coli O157:H7 polyclonal antibody, place 4 ℃ standby.
Chip successively with after 75% alcohol, the cleaning of aseptic ultrapure water, with 1% bSA (BSA) sealing 30min, is used the 0.01molL of pH=7.20 again
-1The PBS buffer solution for cleaning.0.01molL has been filled with in microballon adding behind antibody modification
-1In the chip microchannel inlet liquid storage tank of PBS, utilize syringe pump with 10 μ lmin
-1The flow velocity malleation drives and is packed in the immune microcavity.Microballon in this chip immunity enrichment chamber is easy to import and derive, and becomes individual layer closely to arrange in the enrichment chamber.Present embodiment adopts reference culture E.coli O157:H7 and through anti-E.coli O157:H7 polyclonal antibody (Kirkegaard﹠amp; Perry LaboratoriesInc, USA) microballon of Xiu Shiing carries out the analyzing and testing of micro-fluidic chip.
Said method is selected different reagent and reaction conditions according to different antibody, can obtain the microballon of different antibody modifications.
Adopt the method for chip detection pathogenic microorganism described in the utility model as follows:
The sample that will contain pathogenic microorganism enters in the immunity identification/enrichment chamber 2 by first fluid transmission channel 1 under pump pressure drives by first liquid storage tank 5; Be filled with the microballon through antibody modification in the chamber, by specific immune recognition reaction, the pathogenic microorganism in the sample is identified, catches, and is enriched in bead surface; After reaction finishes, drive bioluminescent reagents and enter immunity identification/enrichment chamber 2 through first liquid storage tank 5, first fluid transmission channel 1, the pathogenic microorganism of enrichment is by after the decomposition agent cracking wherein, discharge inclusion and generate light-emitting composite, the light-emitting composite that generates is eluted to second fluid delivery channel 3 and second liquid storage tank 6, detects bioluminescence intensity as luminous detection point with second liquid storage tank 6.According to the size of luminous intensity can judgement sample in whether the existing and what of content of target pathogenic microorganism, thereby the testing result of obtaining.
As: 37 ℃ of LB nutrient culture media are increased the 0.01molL of the E.coli O157:H7 bacterium liquid of bacterium 8h with pH=7.20
-110 times of PBS damping fluid dilutions.Getting this bacterial suspension 1 μ L is added in the chip inlet liquid storage tank, with 5 μ Lmin
-1The flow velocity malleation is urged in the chip, uses the 0.01molL of pH=7.20 again
-1The PBS damping fluid is with 10 μ Lmin
-1Flow velocity flushing 5min is to reduce or to eliminate non-specific adsorption that chip wall, microballon gap produce or residual.With bioluminescent reagents (BacTiter-Glo
TMMicrobial CellViability Assay, Promega is USA) with 1 μ Lmin
-1The flow velocity malleation transfers in the chip, is that luminous detection point detects luminous intensity in chip outlet liquid storage tank.
Claims (9)
1. micro-fluidic chip that is used to detect pathogenic microorganism, described micro-fluidic chip comprises cover plate and substrate, cover plate and substrate sealing-in are fitted, it is characterized in that: cover plate is provided with two fluid delivery channel, hexagon identification/enrichment chamber, bioluminescent detection unit and two liquid storage tanks, the two ends in identification/enrichment chamber that are filled with the microballon of antibody modification are respectively with first, second fluid delivery channel is communicated with, first liquid storage tank is connected with the first fluid transmission channel, second liquid storage tank is connected with second fluid delivery channel, first, two liquid storage tanks lay respectively at the two ends of chip, liquid storage tank links to each other with the external transmission device, the bioluminescent detection unit is positioned at terminal or second liquid storage tank of second fluid delivery channel, and second fluid delivery channel and immunity enrichment chamber are step-like.
2. the micro-fluidic chip that is used to detect pathogenic microorganism according to claim 1 is characterized in that: described first fluid transmission channel is an access road, and second fluid delivery channel is an exit passageway; The wide 200 μ m of first fluid transmission channel, dark 70 μ m, long 1cm; The wide 200 μ m of second fluid delivery channel, dark 30 μ m, long 1.8cm.
3. the micro-fluidic chip that is used to detect pathogenic microorganism according to claim 1 is characterized in that: the described second fluid delivery channel degree of depth is less than the immunity enrichment chamber degree of depth.
4. the micro-fluidic chip that is used to detect pathogenic microorganism according to claim 1 is characterized in that: the described identification/long 7mm of enrichment chamber cavity, wide 3mm, its cavity depth is greater than 1 times of bead diameter and less than 2 times of bead diameter.
5. the micro-fluidic chip that is used to detect pathogenic microorganism according to claim 1 is characterized in that: the angle α between two hypotenuses that described identification/enrichment chamber links to each other with passage is 60 °.
6. the micro-fluidic chip that is used to detect pathogenic microorganism according to claim 1 is characterized in that: the microballon in described identification/enrichment chamber, enrichment chamber is monolayer alignment.
7. the micro-fluidic chip that is used to detect pathogenic microorganism according to claim 1 is characterized in that: the diameter of described first liquid storage tank and second liquid storage tank is respectively 1.5mm and 3mm.
8. the micro-fluidic chip that is used to detect pathogenic microorganism according to claim 1 is characterized in that: described microballon material is glass or polystyrene material, and described patch material is the polymer poly dimethyl siloxane.
9. the micro-fluidic chip that is used to detect pathogenic microorganism according to claim 1 is characterized in that: the surperficial immobilized probe molecule antibody of described microballon is for having the organic antibody of all kinds of pathogenic microorganisms of target.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106198499A (en) * | 2016-07-03 | 2016-12-07 | 厦门大学 | A kind of micro-fluidic chip for chemiluminescence detection and detection method thereof |
| CN109870567A (en) * | 2019-02-20 | 2019-06-11 | 广州睿辰生物科技有限公司 | Micro-fluidic chip and its method for separating based on immunization sorting ApoE albumen |
| CN110331096A (en) * | 2019-07-19 | 2019-10-15 | 东北大学 | Simulate the micro-fluidic chip of tumor microenvironment and the construction method of tumor microenvironment |
| CN112076806A (en) * | 2019-06-14 | 2020-12-15 | 中国科学院青岛生物能源与过程研究所 | Centrifugal enrichment microfluidic chip for low-concentration liquid sample |
| CN112316994A (en) * | 2020-11-06 | 2021-02-05 | 江南大学 | Integrated detection chip and method for saccharomycetes |
| CN113413933A (en) * | 2021-07-02 | 2021-09-21 | 山东大学第二医院 | Micro-fluidic chip based on glass microspheres and application thereof |
| CN114252602A (en) * | 2021-12-22 | 2022-03-29 | 清华大学深圳国际研究生院 | Micro-fluidic chip, detection system based on micro-fluidic chip and detection method of bacteria |
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2010
- 2010-02-09 CN CN2010201107319U patent/CN201628717U/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106198499A (en) * | 2016-07-03 | 2016-12-07 | 厦门大学 | A kind of micro-fluidic chip for chemiluminescence detection and detection method thereof |
| CN109870567A (en) * | 2019-02-20 | 2019-06-11 | 广州睿辰生物科技有限公司 | Micro-fluidic chip and its method for separating based on immunization sorting ApoE albumen |
| CN112076806A (en) * | 2019-06-14 | 2020-12-15 | 中国科学院青岛生物能源与过程研究所 | Centrifugal enrichment microfluidic chip for low-concentration liquid sample |
| CN110331096A (en) * | 2019-07-19 | 2019-10-15 | 东北大学 | Simulate the micro-fluidic chip of tumor microenvironment and the construction method of tumor microenvironment |
| CN112316994A (en) * | 2020-11-06 | 2021-02-05 | 江南大学 | Integrated detection chip and method for saccharomycetes |
| CN115364911A (en) * | 2021-05-18 | 2022-11-22 | 重庆大学 | Aerosol microorganism sampling enrichment chip and preparation method thereof |
| CN115364911B (en) * | 2021-05-18 | 2023-12-05 | 重庆大学 | Aerosol microorganism sampling enrichment chip and preparation method thereof |
| CN113413933A (en) * | 2021-07-02 | 2021-09-21 | 山东大学第二医院 | Micro-fluidic chip based on glass microspheres and application thereof |
| CN114252602A (en) * | 2021-12-22 | 2022-03-29 | 清华大学深圳国际研究生院 | Micro-fluidic chip, detection system based on micro-fluidic chip and detection method of bacteria |
| CN114252602B (en) * | 2021-12-22 | 2023-09-12 | 清华大学深圳国际研究生院 | Microfluidic chip, detection system based on microfluidic chip and detection method of bacteria |
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Granted publication date: 20101110 Termination date: 20140209 |