CN204945045U - Catch and the active dynamic monitor of single microalgae cell that chlorophyll fluorescence characterizes based on liquid-gas interface is unicellular - Google Patents
Catch and the active dynamic monitor of single microalgae cell that chlorophyll fluorescence characterizes based on liquid-gas interface is unicellular Download PDFInfo
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- CN204945045U CN204945045U CN201520701417.0U CN201520701417U CN204945045U CN 204945045 U CN204945045 U CN 204945045U CN 201520701417 U CN201520701417 U CN 201520701417U CN 204945045 U CN204945045 U CN 204945045U
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Abstract
The utility model discloses and a kind ofly to catch and the active dynamic monitor of single microalgae cell that chlorophyll fluorescence characterizes based on liquid-gas interface is unicellular, this device comprises single microalgae cell capturing unit and the active dynamic monitoring unit of single microalgae cell; Described single microalgae cell capturing unit comprises bubble generator and micro-fluidic chip assembly; Described bubble generator comprises digital injection pump, syringe and sealing duct; Described microfluidic components comprises micro-fluidic chip carrying platform, micro-fluidic chip; The active dynamic monitoring unit of described single microalgae cell comprises the transmission of LASER Light Source, fluoroscopic examination assembly, ruddiness optical filter, constant voltage dc source, data handling component and data and display module; The utility model adopts micro-fluidic chip as the detection platform detecting activity of microalgae, relevant photoelectric detection component and data handling component small volume, relative to large-scale valuable checkout equipment, the features such as the utility model has that structure is simple, processing ease and portability.
Description
Technical field
The utility model relates to catch and chlorophyll fluorescence characterizes single algae cell activity dynamic monitor based on liquid-gas interface is unicellular.
Background technology
Micro-algae is the ancient rudimentary plant of a class, is distributed in the waters such as ocean, freshwater lake widely.In recent years, the invasion with the water-borne adventive of ballast for cruising worldwide causes serious environmental pollution and huge economic loss, and micro-algae, as one of primary biological in adventive, also receives increasing concern.And the nutritional labeling containing multiple high value in microalgae cell and industrial chemicals, algae has more and more been utilized in the middle of experiment.At present, the method for detecting activity of microalgae mainly contains: optical microscopy, dye fluorescence microscopic method, Flow cytometry, molecule and biochemical method etc.
Optical microscopy from water, separates Phytoplankton Cells with cell immobile liquid is fixing, and directly observe the form of Phytoplankton Cells under the microscope, the form different according to cell judges the activity of microalgae cell.This method requires that experimenter must possess abundant hydrobiont and gain knowledge, and the energy common algae of Identification also judges activity, and human factor impact and error are comparatively large, and working strength is large, efficiency is low.
Dye fluorescence microscopic method is by carrying out the dyeing of fluorescent dye to micro-algae sample, fluorescent dye and interior some active substance (as DNA etc.) of micro-frond combine, and judge the activity of micro-algae according to contaminated fluorescence intensity.This method overcomes artificial shortcoming of observing activity of microalgae, improves accuracy to a certain extent; But often kind of dyestuff is according to the difference of respective dyeing mechanism, can only set up some algae, universality is poor, and dyeing course is loaded down with trivial details, need the personnel with professional knowledge to dye, dyeing course is subject to the interference of extraneous various factors, and some dyestuff itself exists toxicity.
Flow cytometry with fluorochrome label microalgae cell and the suspension be made into, is utilized measured by flow cytometry by the fluorescence signal of microalgae cell in the aaerosol solution of laser-irradiated domain and scattered light signal, is judged the activity of micro-algae by these signals.This method work efficiency is high, has advantage fast and accurately, but commercial flow cytometer is expensive, bulky, sample pre-treatment is loaded down with trivial details, complicated operation, in addition, the judgement of algae activity still relies on fluorescent dye to carry out, and the shortcoming that fluorescent dye exists is resolved not yet.
Molecule and biochemical method, mainly comprise: nucleic acid hybridization technique, polymerase chain reaction (PCR) technology, biochip technology, DNA fingerprint technology, enzyme activity assay etc.The method has the high advantage of detection sensitivity.Its shortcoming is: helpless for micro-algae cubing, and equipment needed thereby is expensive, heavy, and bring back laboratory after sample is collected in basic employing and carry out culture identification, time and effort consuming, efficiency is not high, and the kind carrying out detecting is also relatively limited.
An above-mentioned detection method detected object cell colony often, this difference often existed between fuzzy individual cells, statistical average result can not reflect the truth of single algae cell activity change very exactly.In order to resolve characteristics of cell biology better, the technology developed for studying individual cells is imperative.
The prerequisite realizing individual cells detection is catching of cell and fixes.Existing cell capture technology mainly handles the active manipulation of means realization to cell by means of electric field force, dielectric power, static pressure, magnetic field force etc., and by cell capture in the specific position of micro-fluidic chip passage.
It is by repeatedly switching one group of low-voltage that electric field force catches cell, make charged cell constantly change flow velocity and direction under electric field force effect, adherent bottom final settlement to microchannel, can control cell capture like this near cross junction in certain limit.
It is utilize dielectrophoretic force to be captured in by individual cells on microelectrode dot matrix that dielectric power catches cell, when individual cells be trapped in occupy the maximum position of dielectrophoretic force in the middle of electrode time, all the other cells are washed away by the solution flowed because dielectrophoretic force is less.
It is that cell travels forward and taken to by the liquid stream of wing passage and catches single celled position under fluid pressure action that static pressure catches cell, individual cells is trapped in wing passage mouth, main channel liquid is stoped to flow to the flowing of this direction, then all the other cells can be brought to next cell capture mouth by main channel liquid stream, thus form that array is unicellular catches.
It is utilize magnetic field force in chip, adsorb magnetic bead with specific antibodies that magnetic field force catches cell, completes catching cell by the interaction of magnetic bead and cell.
These cell capture methods often need the equipment of external complexity, and sample pre-treatment is loaded down with trivial details, and operation steps is complicated, can not accomplish to catch single algae cell quickly and easily, be unfavorable for the carrying out of follow-up single microalgae cell Activity determination work.
Utility model content
For solving the problems referred to above that prior art exists, the utility model will provide a kind of active dynamic monitor of single microalgae cell utilizing interfacial tension to characterize to the technology and chlorophyll fluorescence of catching single microalgae cell.
To achieve these goals, technology incidence of criminal offenses of the present utility model is as follows: catch and the active dynamic monitoring new method of single microalgae cell that chlorophyll fluorescence characterizes and device based on liquid-gas interface is unicellular, it comprises single microalgae cell capturing unit and the active dynamic monitoring unit of single microalgae cell.Single microalgae cell capturing unit is used for providing platform for microalgae cell detects, and catches single microalgae cell from the multiple microalgae cells flowing through capture region.The chlorophyll fluorescence signal of single microalgae cell of the active dynamic monitoring unit of single microalgae cell for detecting single microalgae cell capturing unit and capturing, and the fluorescence signal detected is converted to electric signal, electric signal is amplified, filtering, noise reduction, and gather by data handling component the signal detected, process, thus the chlorophyll fluorescence signal of single microalgae cell is clearly illustrated out over the display.
Described single microalgae cell capturing unit comprises bubble generator and micro-fluidic chip assembly.Described bubble generator comprises digital injection pump, syringe and sealing duct.Described digital injection pump is made up of stepper motor and driver, screw mandrel and support etc., and have the screw mandrel, the nut that move back and forth, nut is connected with the piston of described syringe, and described sealing duct is connected with the injection port of described syringe.Described microfluidic components comprises micro-fluidic chip carrying platform, micro-fluidic chip.Described micro-fluidic chip comprises injection port, outlet, gas injection port, passage and cell capture region.Described gas injection port is connected with the other end of described sealing duct.Described sample intake passage two ends are respectively equipped with injection port and outlet, and described sample intake passage is provided with the perpendicular and inlet channel communicated, and described inlet channel is provided with gas injection port, and described gas injection port is connected with the other end of sealing duct; The junction of described sample intake passage and inlet channel forms cell capture region.
The active dynamic monitoring unit of described single microalgae cell comprises the transmission of LASER Light Source, fluoroscopic examination assembly, ruddiness optical filter, constant voltage dc source, data handling component and data and display module.Described fluoroscopic examination assembly to be in the light shell and support composition primarily of photoelectric reflection negative electrode, electron optics input system, electron multiplication system, electron collector, totally enclosed type.Described LASER Light Source and support are fixed together, the anode and cathode of described fluoroscopic examination assembly is connected with described constant voltage dc source, the output terminal of described fluoroscopic examination assembly is connected with described data handling component, described data handling component transmits with described data and display module is connected, and described ruddiness optical filter is arranged on described totally enclosed type and is in the light the upper surface of shell.
Further, in technique scheme, described micro-fluidic chip carrying platform is microslide.
Further, in technique scheme, described sample intake passage width is 200um.
Further, in technique scheme, described inlet channel width is 200um.
Catch and the method for operating of the active dynamic monitoring new method of single microalgae cell that chlorophyll fluorescence characterizes and device based on liquid-gas interface is unicellular, comprise the following steps:
1.. under the made micro-fluidic chip performed is placed on inverted microscope, itself and bubble generator are connected.
2.. in micro-fluidic chip, inject the damping fluid prepared, open syringe pump, depended on pressure gets a bubble near micro-fluidic chip capture region.
3.. micro-algae (algae that salt algae, flat algae and other physiologically actives are similar) cell suspending liquid is injected the passage of micro-fluidic chip, control the speed of syringe pump, when microalgae cell flows through capture region, bubble and microalgae cell are fully collided, due to capillary existence, single microalgae cell can be adsorbed on bubble.
4.. after successfully catching cell, micro-fluidic chip is placed on micro-fluidic chip carrying platform, is fixed on the active dynamic monitoring unit of single microalgae cell.
5.. open the every switch on the active dynamic monitoring unit of single microalgae cell, the single microalgae cell of catching is detected.
6.. data handling component carries out signal extraction, acquisition and processing to testing result, and by result display on a display screen.
Further, in technique scheme, described buffer solution is phosphate buffer solution 1 × PBS.
Utility model beneficial effect
1, because the utility model adopts micro-fluidic chip as the detection platform detecting activity of microalgae, relevant photoelectric detection component and data handling component small volume, relative to large-scale valuable checkout equipment, the features such as the utility model has that structure is simple, processing ease and portability;
2, relative to need realize catching cell by means of manipulation means such as electric field force, dielectric power, static pressure, magnetic field force and spaces in the past, the utility model utilizes the fast Acquisition of liquid-gas interface realization to individual cells, not only overcome the equipment complexity that classic method has, the shortcomings such as complex operation, efficiency are not high, and simplify the channel architecture of micro-fluidic chip, there is stronger practicality and broader application;
3, the utility model adopts the method for induced with laser chlorophyll fluorescence to detect the activity of static microalgae cell, overcomes the shortcoming that the method that in the past dyes to micro-algae sample is before detection brought, has the advantages that workload is few, accuracy is high;
4, the utility model achieves the active dynamic monitoring of single microalgae cell, and this is a much progress in cell detection field.The existing method that algae cell activity is analyzed, usually be all obtain statistical average result from the cell colony specimen in use, the difference existed between the fuzzy individual cells of such meeting, statistical average result can not reflect the truth of single algae cell activity change very exactly.Single cell analysis can resolve characteristics of cell biology better, for many new biological informations, all has great importance for the rule of the stuctures and properties between research cell, vital movement and essence, preclinical medicine and medicament research and development.
Accompanying drawing explanation
Fig. 1 is the structural representation of single microalgae cell capturing unit;
Fig. 2 is microfluidic chip structure schematic diagram;
Fig. 3 is the structural representation of the active dynamic monitoring unit of single microalgae cell;
Fig. 4 is the fluorescence signal variation diagram of single flat algae cell after liquor natrii hypochloritis's process;
Fig. 5 is the fluorescence signal variation diagram of single Dunaliella salina cell after liquor natrii hypochloritis's process;
In figure: 1, microslide, 2, micro-fluidic chip, 3, sealing duct, 4, syringe, 5, syringe pump, 6, injection port, 7, gas injection port, 8, outlet, 9, microalgae cell capture region, 10, micro-fluidic chip assembly, 11, LASER Light Source, 12, ruddiness optical filter, 13, photoelectric reflection negative electrode, 14, electron optics input system, 15 electron multiplication systems, 16, electron collector (anode), 17, fluoroscopic examination assembly, 18, constant voltage dc source, 19, data handling component, 20, data transmission and display module.
Embodiment
Below in conjunction with accompanying drawing and example, the utility model is further described.Wherein micro-algae laboratory sample is provided by Research Institute of Ocean Fishery Science, Liaoning Province.
Embodiment 1
Catch and the active dynamic monitor of single microalgae cell that chlorophyll fluorescence characterizes based on liquid-gas interface is unicellular, comprise single microalgae cell capturing unit and the active dynamic monitoring unit of single microalgae cell.
As shown in Figure 1 and Figure 2, single microalgae cell capturing unit comprises bubble generator and micro-fluidic chip assembly.
Bubble generator comprises digital injection pump, syringe and sealing duct; Digital injection pump is by stepper motor and driver thereof, screw mandrel and be configured to, and have the screw mandrel, the nut that move back and forth, nut is connected with the piston of syringe, and sealing duct one end is connected with the injection port of syringe.
Microfluidic components comprises micro-fluidic chip carrying platform, micro-fluidic chip; Micro-fluidic chip comprises sample intake passage, and sample intake passage two ends are respectively equipped with injection port 6 and outlet 8, and sample intake passage is provided with the perpendicular and inlet channel communicated, and inlet channel is provided with gas injection port, and gas injection port is connected with the other end of sealing duct; The junction of sample intake passage and inlet channel forms cell capture region.
Micro-fluidic chip 2 microslide 1 is bonded together securely by plasma cleaner, the gas injection port 7 of micro-fluidic chip 2 is connected with one end of sealing duct 3, the other end of sealing duct 3 is connected with syringe 4, syringe 4 is accurately controlled by syringe pump 5, thus realizes the object accurately controlling bubble in microchannel.When catching cell, microslide 1 and micro-fluidic chip 2 are put under the microscope, when microalgae cell suspending liquid flows through capture region 9 by injection port 6, by controlling syringe pump 5, the bubble of generation and microalgae cell are bumped against, because liquid-gas interface tension force microalgae cell can be adsorbed on bubble, thus the single microalgae cell of catching is fixed on certain position of capture region 9.
The active dynamic monitoring unit of single microalgae cell comprises the transmission of LASER Light Source, fluoroscopic examination assembly, ruddiness optical filter, constant voltage dc source, data handling component and data and display module.
Fluoroscopic examination assembly comprises photoelectric reflection negative electrode, electron optics input system, electron multiplication system, electron collector, totally enclosed type are in the light shell and support.
LASER Light Source and support are fixed together, the anode and cathode of fluoroscopic examination assembly is connected with constant voltage dc source, the output terminal of fluoroscopic examination assembly is connected with data handling component, data handling component transmits with data and display module is connected, and ruddiness optical filter is arranged on totally enclosed type and is in the light the upper surface of shell.
When completing after single microalgae cell catches, micro-fluidic chip assembly 10 is fixed on the active dynamic monitoring unit of single microalgae cell by micro-fluidic chip carrying platform.LASER Light Source 11 adopts wavelength to be the blue light of 488nm, and the sealing being fixed on fluoroscopic examination assembly 17 by support is in the light case surface.The fluorescence that the microalgae cell at capture region 9 place sends due to laser excitation is got on fluoroscopic examination assembly 17 after ruddiness optical filter 12 filters, fluoroscopic examination assembly 17 can convert fluorescence signal to electric signal to data handling component 19 by photoelectric reflection negative electrode 13, electron optics input system 14, electron multiplication system 15 and electron collector (anode) 16 etc. and carry out analyzing and processing, finally the signal after process is delivered to data transmission and display module 20 and presents on a display screen.
Catch and the active dynamic monitoring method of single microalgae cell that chlorophyll fluorescence characterizes based on liquid-gas interface is unicellular, comprise the following steps:
A, micro-fluidic chip 2 and microslide 1 to be bonded together by plasma cleaning, syringe 4 and syringe pump 5 are coupled together by the gas injection port 7 of sealing duct 3 with micro-fluidic chip 2, after connecting by micro-fluidic chip 2 with microslide 1 as under fluorescent microscope.Inject phosphate buffer that 10 μ L prepare (1 × PBS, PH=9.0) with digital pipettor toward chip injection port 6, regulate syringe pump (ATOM – 1235N), make it get a bubble at capture region 9 place.Inject the flat algae cell suspending liquid (laboratory sample is provided by Research Institute of Ocean Fishery Science, Liaoning Province) of 10 μ L toward chip injection port 6 with digital pipettor, when flat algae stream of cells is through capture region 9, the bubble of generation and flat algae cell is bumped against by controlling syringe pump 5 thus catches single flat algae cell.
B, micro-fluidic chip 2 and microslide 1 to be taken off from microscope, be fixed on the active dynamic monitoring unit of single microalgae cell by micro-fluidic chip carrying platform.Open LASER Light Source, capture region 9 place that adjustment angle makes it be irradiated to micro-fluidic chip assembly 10.Inject the reagent treatment of 10 μ L toward chip injection port 6 with digital pipettor.Around closing after light source, open the switch of fluoroscopic examination assembly 17, the single flat algae cell started catching carries out Activity determination.
C, detect after a period of time, close the switch of fluoroscopic examination assembly 17, institute surveys single microalgae cell fluorescence signal and transmits through data handling component 19 and data and present on a display screen, as Fig. 4 after display module 20.
Embodiment 2
Be to replace flat algae cell suspending liquid (laboratory sample is provided by Research Institute of Ocean Fishery Science, Liaoning Province) with Dunaliella salina cell suspending liquid with the difference of embodiment 1, Fig. 5 is shown in the fluorescence signal change of single Dunaliella salina cell after liquor natrii hypochloritis's process.
The various embodiments described above are only for illustration of the utility model; wherein the structure of each parts, connected mode and manufacture craft etc. all can change to some extent; every equivalents of carrying out on the basis of technical solutions of the utility model and improvement, all should not get rid of outside protection domain of the present utility model.
Claims (6)
1. catch and the active dynamic monitor of single microalgae cell that chlorophyll fluorescence characterizes based on liquid-gas interface is unicellular, it is characterized in that:
Comprise single microalgae cell capturing unit and the active dynamic monitoring unit of single microalgae cell;
Described single microalgae cell capturing unit comprises bubble generator and micro-fluidic chip assembly;
Described bubble generator comprises digital injection pump, syringe and sealing duct; Described microfluidic components comprises micro-fluidic chip carrying platform, micro-fluidic chip; Described micro-fluidic chip comprises sample intake passage, described sample intake passage two ends are respectively equipped with injection port and outlet, described sample intake passage is provided with the perpendicular and inlet channel communicated, and described inlet channel is provided with gas injection port, and described gas injection port is connected with the other end of sealing duct; The junction of described sample intake passage and inlet channel forms cell capture region;
The active dynamic monitoring unit of described single microalgae cell comprises the transmission of LASER Light Source, fluoroscopic examination assembly, ruddiness optical filter, constant voltage dc source, data handling component and data and display module;
Described fluoroscopic examination assembly comprises photoelectric reflection negative electrode, electron optics input system, electron multiplication system, electron collector, totally enclosed type are in the light shell and support.
2. device according to claim 1, it is characterized in that: described digital injection pump is by stepper motor and driver thereof, screw mandrel and be configured to, there is the screw mandrel, the nut that move back and forth, nut is connected with the piston of described syringe, and described sealing duct one end is connected with the injection port of described syringe.
3. device according to claim 1, it is characterized in that: described LASER Light Source and support are fixed together, the anode and cathode of described fluoroscopic examination assembly is connected with described constant voltage dc source, the output terminal of described fluoroscopic examination assembly is connected with described data handling component, described data handling component transmits with described data and display module is connected, and described ruddiness optical filter is arranged on described totally enclosed type and is in the light the upper surface of shell.
4. device according to claim 1, is characterized in that: described micro-fluidic chip carrying platform is microslide.
5. device according to claim 1, is characterized in that: described sample intake passage width is 200um.
6. device according to claim 1, is characterized in that: described inlet channel width is 200um.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105136763A (en) * | 2015-09-10 | 2015-12-09 | 大连海事大学 | Single microalgae cell activity dynamic monitoring novel method and device based on gas-liquid interface single cell capture and chlorophyll fluorescence characterization |
| CN107748138A (en) * | 2017-12-05 | 2018-03-02 | 山东交通学院 | Activity of microalgae detection means and method based on filter liquor principle |
| CN110308086A (en) * | 2019-07-09 | 2019-10-08 | 大连海事大学 | It is a kind of based on micro-fluidic particle surface partial region fluorescent marker device and method |
| CN113189180A (en) * | 2021-03-29 | 2021-07-30 | 大连海事大学 | Microalgae characterization and identification device and method based on alternating current-dielectrophoresis |
-
2015
- 2015-09-10 CN CN201520701417.0U patent/CN204945045U/en not_active Withdrawn - After Issue
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105136763A (en) * | 2015-09-10 | 2015-12-09 | 大连海事大学 | Single microalgae cell activity dynamic monitoring novel method and device based on gas-liquid interface single cell capture and chlorophyll fluorescence characterization |
| CN105136763B (en) * | 2015-09-10 | 2017-10-13 | 大连海事大学 | The active dynamic monitoring new method of single microalgae cell and device characterized based on the unicellular capture of gas-liquid interface and chlorophyll fluorescence |
| CN107748138A (en) * | 2017-12-05 | 2018-03-02 | 山东交通学院 | Activity of microalgae detection means and method based on filter liquor principle |
| CN107748138B (en) * | 2017-12-05 | 2023-11-28 | 山东交通学院 | Microalgae activity detection device and method based on filter liquid principle |
| CN110308086A (en) * | 2019-07-09 | 2019-10-08 | 大连海事大学 | It is a kind of based on micro-fluidic particle surface partial region fluorescent marker device and method |
| CN110308086B (en) * | 2019-07-09 | 2021-11-05 | 大连海事大学 | Particle surface partial-area fluorescence labeling device and method based on micro-fluidic |
| CN113189180A (en) * | 2021-03-29 | 2021-07-30 | 大连海事大学 | Microalgae characterization and identification device and method based on alternating current-dielectrophoresis |
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