CN114225978B - Micro-fluidic chip and microorganism detection method - Google Patents
Micro-fluidic chip and microorganism detection method Download PDFInfo
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- CN114225978B CN114225978B CN202111450397.0A CN202111450397A CN114225978B CN 114225978 B CN114225978 B CN 114225978B CN 202111450397 A CN202111450397 A CN 202111450397A CN 114225978 B CN114225978 B CN 114225978B
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- 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
- 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
- B01L3/50273—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 characterised by the means or forces applied to move the fluids
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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
- B01L3/502738—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 characterised by integrated valves
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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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
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/02—Adapting objects or devices to another
- B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
- B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
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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
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
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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 micro-fluidic chip and a microorganism detection method, comprising a chip body, wherein a mixing module is arranged on the chip body, and the mixing module comprises a first gas pumping structure, a mixing pneumatic valve, a mixing one-way valve, a liquid mixing valve and a mixing channel which are sequentially connected; the first gas pumping structure is used for controlling the mixing pneumatic valve and the liquid mixing valve to be switched between an opening state and a closing state; the mixing pneumatic valve is used for controlling whether the mixing one-way valve is communicated with the liquid conveying channel or not; the liquid mixing valve is used for storing liquid or gas when in an open state, and the liquid mixing valve is used for extruding the liquid or gas into the mixing channel when in a closed state. The invention also comprises a pumping module which is used for pumping the liquid to the mixing module and has a structure and a principle similar to those of the mixing module, so that the microorganism can be conveniently and rapidly detected, the skill requirement of operators is reduced, a complex pumping system is not needed, the field detection at the basic level is convenient, and the detection cost is saved.
Description
Technical Field
The invention relates to the technical field of microbial detection, in particular to a micro-fluidic chip and a microbial detection method.
Background
Background in food samples is complex, the concentration of food-borne pathogenic bacteria is usually low, and the conventional detection method is difficult to directly detect the food samples. The double-antibody sandwich technology is a biological detection technology based on antigen-antibody immune combination, a capture probe combined with a specific antibody is used for capturing target bacteria, then a signal probe combined with another antibody is used for marking the target bacteria to form an immune capture probe-target bacteria-immune signal probe double-antibody sandwich structure, and a signal probe is used for converting a corresponding bacteria concentration signal into a detectable physical signal such as light, heat, magnetism, force, sound, electricity and the like to indirectly detect the concentration of the target bacteria. However, when the existing double-antibody sandwich technology is applied to microbial detection, most of the existing double-antibody sandwich technology depends on various instruments and professional operators, the automation degree is not high, and the technology is not suitable for detection scenes with limited conditions such as a basic layer and the like.
In recent years, biosensors based on microfluidic chips have been developed greatly due to their advantages of simple operation, low cost, small sample size, high sensitivity, fast response speed, on-site detection and the like. However, the existing microfluidic biosensor still requires a complex pumping system when in use, and each reagent inlet generally requires an independent pump for sample application, which is difficult to be applied to a substrate environment. Therefore, how to detect microorganisms more conveniently and rapidly is an important issue to be solved urgently in the industry at present.
Disclosure of Invention
The invention provides a micro-fluidic chip and a microorganism detection method, which are used for solving the defects that a micro-fluidic biosensor in the prior art needs to be matched with a complex pumping system when in use, is troublesome to use, is not beneficial to basic site detection and the like, realizing convenient and rapid detection on microorganisms, reducing the skill requirement of operators, needing no complex pumping system, being convenient to detect on the basic site and saving the detection cost.
The invention provides a micro-fluidic chip which comprises a chip body, wherein a mixing module is arranged on the chip body, and the mixing module comprises a first gas pumping structure, a mixing pneumatic valve, a mixing one-way valve, a liquid mixing valve and a mixing channel which are sequentially connected;
the first gas pumping structure is used for controlling the mixing pneumatic valve and the liquid mixing valve to be switched between an opening state and a closing state;
one end of the mixing pneumatic valve is connected with the liquid conveying channel, the other end of the mixing pneumatic valve is connected with the mixing one-way valve, and the mixing pneumatic valve is used for controlling whether the mixing one-way valve is communicated with the liquid conveying channel or not;
the mixing one-way valve is used for controlling the unidirectional flow of liquid or gas along the direction from the mixing pneumatic valve to the liquid mixing valve;
the liquid mixing valve is used for storing liquid or gas when in an open state, and the liquid mixing valve is used for extruding the liquid or gas into the mixing channel when in a closed state.
According to the micro-fluidic chip provided by the invention, the hybrid pneumatic valve comprises a valve pneumatic cavity and a valve cavity, a first elastic membrane is arranged between the valve pneumatic cavity and the valve cavity, the valve pneumatic cavity is communicated with the first gas pumping structure, and the valve cavity is communicated with the liquid conveying channel.
According to the micro-fluidic chip provided by the invention, the mixing one-way valve comprises a valve top and a vent hole, the valve top is positioned above the vent hole, a second elastic membrane is arranged between the valve top and the vent hole, a first pipeline is arranged between one end of the valve top and the mixing pneumatic valve, and a second pipeline is arranged between the other end of the valve top and the liquid mixing valve.
According to the micro-fluidic chip provided by the invention, the liquid mixing valve comprises a mixing pneumatic cavity and a mixing cavity, a third elastic membrane is arranged between the mixing pneumatic cavity and the mixing cavity, the mixing pneumatic cavity is communicated with the first gas pumping structure, and when the liquid mixing valve is in an open state, the mixing cavity is respectively communicated with the mixing one-way valve and the mixing channel.
According to the invention, the micro-fluidic chip is provided, and the mixing cavity is of a hemispherical structure.
According to the micro-fluidic chip provided by the invention, the first gas pumping structure comprises a gas storage cavity and a fourth elastic membrane, the upper end of the gas storage cavity is open, and the fourth elastic membrane covers the upper end of the gas storage cavity.
According to the micro-fluidic chip provided by the invention, the micro-fluidic chip further comprises a waste liquid cavity, the waste liquid cavity is positioned in the chip body, one end of the mixing channel is connected with the liquid mixing valve, the other end of the mixing channel is positioned above the waste liquid cavity, and the other end of the mixing channel is communicated with the waste liquid cavity.
According to the micro-fluidic chip provided by the invention, the micro-fluidic chip further comprises a plurality of standard color comparison cards, and the standard color comparison cards are positioned in the chip body.
According to the micro-fluidic chip provided by the invention, the micro-fluidic chip further comprises a feeding hole and a pumping module, wherein the pumping module comprises a second gas pumping structure, a pumping pneumatic valve, a liquid pumping structure and a pumping one-way valve which are sequentially connected;
the second gas pumping structure is used for controlling the pumping pneumatic valve and the liquid pumping structure to be switched between an opening state and a closing state;
the pumping pneumatic valve is used for controlling whether the liquid pumping structure is communicated with the feed port or not;
the liquid pumping structure is used for storing liquid or gas when in an opening state, and the liquid pumping structure is used for extruding the liquid or gas to the pumping one-way valve when in a closing state
The pumping one-way valve is communicated with the liquid conveying channel and is used for enabling liquid or gas to flow in one direction along the direction from the liquid pumping structure to the liquid conveying channel.
The invention also provides a microorganism detection method, which comprises the following steps:
injecting a sample to be detected, immunomagnetic beads and labeled enzyme into the feeding hole, and pressing the second gas pumping structure to convey the three solutions to the mixing module;
pressing a first gas pumping structure to fully mix the three solutions, and standing and incubating to form an immunomagnetic bead-target bacterium-labeled enzyme double-antibody sandwich compound;
placing a magnetic separator above a liquid mixing valve, adsorbing magnetic beads and double-antibody sandwich compounds in the liquid mixing valve to the top of the liquid mixing valve, and pumping liquid into a waste liquid pool;
dropwise adding cleaning fluid into the sample inlet, pressing the second gas pumping structure to pump the cleaning fluid into the liquid mixing valve, cleaning the magnetic substance on the top of the liquid mixing valve to remove the unbound labeled enzyme, and pumping the cleaning fluid into the waste liquid cavity;
dropping a chromogenic substrate into the sample inlet, pressing the first gas pumping structure to pump the chromogenic substrate to the liquid mixing valve, removing the magnetic separator, and enabling the chromogenic substrate to resuspend the double-antibody sandwich compound;
standing for a period of time, and catalyzing a chromogenic substrate by the marker enzyme on the double-antibody sandwich to enable the original colorless and transparent solution to generate color; and comparing the generated color with a standard color comparison card to determine the concentration of the bacteria in the detection sample.
According to the micro-fluidic chip and the microorganism detection method provided by the invention, when the mixing pneumatic valve is in an open state, the mixing one-way valve is communicated with the liquid conveying channel, and when the mixing pneumatic valve is in a closed state, the mixing one-way valve is not communicated with the liquid conveying channel. Through the mixed pneumatic valve of first gas pumping structural control and liquid mixing chamber all be in the open mode, then will wait to detect sample and various required liquid solutions that detect and carry mixed check valve through liquid transfer passage, then liquid passes through the check valve and carries in the liquid mixing valve.
Then the mixing pneumatic valve and the liquid mixing valve are controlled to be closed through the first gas pumping structure, the mixing one-way valve and the mixing pneumatic valve are both in a closed state at the moment, and when the liquid mixing valve is closed, the solution in the liquid mixing valve is extruded into the mixing channel. Then open the liquid mixing valve through first gas pumping structure, the liquid in the mixing channel can be sucked back in the liquid mixing chamber, through opening and closing of continuous control liquid mixing valve for liquid reciprocating motion between liquid mixing valve and mixing channel makes the sample and detect operation such as incubation that stews after the solution intensive mixing, and then can realize on micro-fluidic chip that advance kind, mix in the conventional microorganism immunity detection experiment, hatch, enrichment etc. operate.
And then realized convenient, quick detecting the microorganism, reduced operating personnel skill requirement, need not complicated pumping system, be convenient for at basic unit field test, practiced thrift the detection cost.
Drawings
In order to more clearly illustrate the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic structural diagram of a microfluidic chip provided in the present invention;
fig. 2 is one of cross-sectional views of a mixing module of a microfluidic chip provided in the present invention;
fig. 3 is a second cross-sectional view of a hybrid module of the microfluidic chip provided in the present invention;
FIG. 4 is a second schematic structural diagram of a microfluidic chip according to the present invention;
FIG. 5 is a schematic diagram of a part of the structure of a microfluidic chip provided by the present invention;
fig. 6 is a second schematic diagram of a partial structure of a microfluidic chip provided in the present invention;
FIG. 7 is a flow chart of a method for detecting microorganisms provided by the present invention;
reference numerals:
1: a chip body; 2: a mixing module; 3: a first gas pumping arrangement;
4: a hybrid pneumatic valve; 5: a mixing one-way valve; 6: a liquid mixing valve;
7: a mixing channel; 8: a liquid delivery channel; 9: a feed inlet;
10: a second gas pumping arrangement; 11: a pumping pneumatic valve; 12: a liquid pumping arrangement;
13: a pumping check valve; 14: a waste fluid chamber; 15: a standard color comparison card;
31: a gas storage cavity; 32: a fourth elastic film; 41: a valve pneumatic cavity;
42: a valve cavity; 43: a first elastic film; 51: a valve top;
52, a vent hole; 53: a second elastic film; 61, a mixed pneumatic cavity;
62, a mixing cavity; and 63, a third elastic film.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention.
The microfluidic chip and the microorganism detection method according to the present invention will be described with reference to fig. 1 to 7.
As shown in fig. 1, fig. 2 and fig. 3, the microfluidic chip includes a chip body 1, a mixing module 2 is disposed on the chip body 1, and the mixing module 2 includes a first gas pumping structure 3, and a mixing pneumatic valve 4, a mixing one-way valve 5, a liquid mixing valve 6 and a mixing channel 7 connected in sequence.
Specifically, the first gas pumping structure 3 is used for controlling the hybrid pneumatic valve 4 and the liquid mixing valve 6 to be switched between an open state and a closed state;
one end of the hybrid pneumatic valve 4 is connected with the liquid conveying channel 8, the other end of the hybrid pneumatic valve 4 is connected with the hybrid one-way valve 5, and the hybrid pneumatic valve 4 is used for controlling whether the hybrid one-way valve 5 is communicated with the liquid conveying channel 8 or not;
the mixing check valve 5 is used for controlling the unidirectional flow of liquid or gas in the direction from the mixing pneumatic valve 4 to the liquid mixing valve 6;
the liquid mixing valve 6 is used to store liquid or gas in the open state, and the liquid mixing valve 6 squeezes liquid or gas into the mixing channel 7 in the closed state.
In use, when the mixing air-operated valve 4 is in the open state, the mixing check valve 5 is in communication with the liquid delivery passage 8, and when the mixing air-operated valve 4 is in the closed state, the mixing check valve 5 is not in communication with the liquid delivery passage 8. The mixing pneumatic valve 4 and the liquid mixing chamber 62 are both controlled to be in an open state by the first gas pumping structure 3, then the sample to be detected and various liquid solutions required for detection are conveyed to the mixing one-way valve 5 through the liquid conveying passage 8, and then the liquid is conveyed to the liquid mixing valve 6 through the one-way valve.
Then the first gas pumping structure 3 controls the mixing pneumatic valve 4 and the liquid mixing valve 6 to close, at this time, the mixing one-way valve 5 and the mixing pneumatic valve 4 are both in a closed state, and when the liquid mixing valve 6 is closed, the solution in the liquid mixing valve 6 is extruded into the mixing channel 7. Then open liquid mixing valve 6 through first gas pumping structure 3, the liquid in mixing channel 7 can be sucked back in liquid mixing chamber 62, through opening and closing of continuous control liquid mixing valve 6 for liquid reciprocating motion between liquid mixing valve 6 and mixing channel 7 makes the sample and carry out operations such as incubation of standing after the detection solution intensive mixing, and then can realize operations such as appearance, mixture, incubation, enrichment in the conventional microorganism immunodetection experiment on micro-fluidic chip.
Therefore, the microorganism can be conveniently and rapidly detected, the skill requirement of operators is reduced, a complex pumping system is not needed, the detection on the basic site is facilitated, and the detection cost is saved.
For example, when the microfluidic chip of the invention is applied to the double-antibody sandwich technology for microorganism detection:
firstly, the mixing pneumatic valve 4 and the liquid mixing valve 6 are opened, and the three solutions of the sample to be detected, the immunomagnetic beads and the labeled enzyme are conveyed into the liquid mixing valve 6 through the liquid conveying channel 8. Then closing the mixing pneumatic valve 4 and the liquid mixing valve 6, extruding the liquid in the liquid mixing valve 6 into the mixing channel 7, then opening the liquid mixing valve 6 to enable the liquid in the mixing channel 7 to return to the liquid mixing valve 6, enabling the liquid to flow back and forth between the mixing channel 7 and the liquid mixing valve 6 by continuously opening and closing the liquid mixing valve 6, further enabling the three solutions to be fully mixed, standing and incubating, and enabling the immunomagnetic beads, the target bacteria in the sample and the labeled enzyme to be combined to form an immunomagnetic bead-target bacteria-labeled enzyme double-antibody sandwich compound;
placing a magnetic separator above the liquid mixing valve 6, adsorbing the magnetic beads and the double-antibody sandwich compound in the liquid mixing valve 6 to the top of the liquid mixing valve 6, and discharging the liquid; conveying the cleaning solution to the liquid mixing valve 6, cleaning the magnetic substance on the top of the liquid mixing valve 6 to remove the unbound labeled enzyme, and then discharging the cleaning solution;
conveying the chromogenic substrate to a liquid mixing valve 6, removing the magnetic separator, enabling the chromogenic substrate to resuspend the double antibody sandwich compound, standing for a period of time, and enabling the labeled enzyme on the double antibody sandwich to catalyze the chromogenic substrate to enable the original colorless and transparent solution to generate color; the resulting color is compared to a standard color chart 15 to determine the concentration of bacteria in the test sample. The microfluidic chip disclosed by the invention is simple and convenient to operate, does not need a complex pumping system, reduces the professional requirement degree on operators, and is more suitable for basic site detection.
Further, as shown in fig. 1, fig. 2 and fig. 3, the hybrid pneumatic valve 4 includes a valve pneumatic chamber 41 and a valve chamber 42, a first elastic membrane 43 is disposed between the valve pneumatic chamber 41 and the valve chamber 42, the valve pneumatic chamber 41 is communicated with the first gas pumping structure 3, and the valve chamber 42 is communicated with the liquid delivery passage 8. When the pneumatic valve is used, the first elastic membrane 43 is attached to cover the upper end face of the valve pneumatic valve, gas is conveyed into the valve pneumatic cavity 41 through the first gas pumping structure 3, the gas pressure in the valve pneumatic cavity 41 is enhanced, the first elastic membrane 43 protrudes upwards to be attached to the valve cavity 42, the mixing pneumatic valve 4 is in a closed state, and the mixing one-way valve 5 is not communicated with the liquid conveying channel 8. Then the first gas pumping structure 3 stops delivering gas into the valve pneumatic cavity 41, the pressure in the valve pneumatic cavity 41 gradually falls back to the initial value, the first elastic membrane 43 deforms downwards to recover the shape due to the elastic force, so that the mixing pneumatic valve 4 is in the open state, and the mixing one-way valve 5 is communicated with the liquid delivery channel 8.
Further, as shown in fig. 1, 2 and 3, the mixing check valve 5 includes a valve top 51 and a vent 52, the valve top 51 is located above the vent 52, a second elastic membrane 53 is disposed between the valve top 51 and the vent 52, a first pipe is disposed between one end of the valve top 51 and the mixing pneumatic valve 4, and a second pipe is disposed between the other end of the valve top 51 and the liquid mixing valve 6. When in use, the mixing air-operated valve 4 and the liquid mixing valve 6 are in an opening state, the second elastic membrane 53 covers the upper end of the vent hole 52, the mixing air-operated valve 4 is controlled to be opened by the first air pumping structure 3, at this time, the mixing air-operated valve 4 is communicated with the gap, then the solution is conveyed to the mixing air-operated valve 4 through the liquid conveying channel 8, the liquid moves to the second elastic membrane 53 through the first pipeline, at this time, the pressure received above the second elastic membrane 53 is greater than the pressure exerted by the atmospheric pressure, then the second elastic membrane 53 protrudes downwards, then the solution can smoothly flow to the second pipeline and then flows into the liquid mixing valve 6, then the second elastic membrane 53 deforms upwards due to the action of the elastic force, the second elastic membrane 53 is attached to the valve top 51, and at this time, the mixing one-way valve 5 is closed. Then the mixing pneumatic valve 4 and the liquid mixing valve 6 are closed successively or simultaneously through the first gas pumping structure 3, at the moment, the liquid in the liquid mixing valve 6 can only flow to the mixing channel 7, and then the liquid one-way flowing function of the mixing one-way valve 5 is realized.
Further, as shown in fig. 1, fig. 2 and fig. 3, the liquid mixing valve 6 includes a mixing pneumatic cavity 61 and a mixing cavity 62, a third elastic membrane 63 is disposed between the mixing pneumatic cavity 61 and the mixing cavity 62, the mixing pneumatic cavity 61 is communicated with the first gas pumping structure 3, wherein when the liquid mixing valve 6 is in an open state, the mixing cavity 62 is respectively communicated with the mixing one-way valve 5 and the mixing channel 7. When the liquid mixing device is used, the third elastic membrane 63 covers the opening at the upper end of the mixing pneumatic cavity 61, the first gas pumping structure 3 conveys gas into the mixing pneumatic cavity 61, the gas pressure in the mixing pneumatic cavity 61 is increased, the third elastic membrane 63 protrudes upwards to be attached to the mixing cavity 62, the liquid mixing cavity 62 is in a closed state, and the solution in the mixing cavity 62 is extruded into the mixing channel 7. Then the first gas pumping structure 3 stops delivering gas into the mixing pneumatic valve 4, so that the gas pressure in the mixing pneumatic cavity 61 is restored to the initial value, the third elastic membrane 63 is deformed downwards due to the action of the elastic force, the liquid mixing valve 6 is in an open state at the moment, and the solution in the mixing channel 7 returns to the mixing cavity 62. By controlling the liquid mixing valve 6 to be continuously switched between the open state and the closed state, so that the solution can flow back and forth between the mixing channel 7 and the mixing chamber 62, the sample to be detected and the solution required for detection can be fully mixed.
In which the mixing chamber 62 has a hemispherical configuration, as shown in figures 2 and 3. In use, the hemispherical mixing chamber 62 can induce the liquid to swirl as the liquid in the mixing channel 7 flows to the mixing chamber 62, thereby achieving effective mixing of the sample to be tested and the solution required for testing.
Further, as shown in fig. 4, the first gas pumping structure 3 includes a gas storage chamber 31 and a fourth elastic membrane 32, an upper end of the gas storage chamber 31 is open, and the fourth elastic membrane 32 covers the upper end opening of the gas storage chamber 31. When the pneumatic valve is used, the gas storage cavity 31 is respectively communicated with the valve pneumatic cavity 41 and the mixed pneumatic cavity 61, a downward external force is applied to the fourth elastic membrane 32, the fourth elastic membrane 32 extrudes gas in the gas storage cavity 31, so that the gas in the gas storage cavity 31 is conveyed into the valve pneumatic cavity 41 and the mixed pneumatic cavity 61, the gas pressure in the valve pneumatic cavity 41 and the mixed pneumatic cavity 61 is enhanced, the first elastic membrane 43 is attached to the valve cavity 42, the third elastic membrane 63 is attached to the mixed cavity 62, and the mixed pneumatic valve 4 and the liquid mixing valve 6 are closed. When the external force applied to the fourth elastic membrane 32 is removed, the fourth elastic membrane 32 deforms upwards to recover the shape, the gas storage cavity 31 does not convey gas into the valve pneumatic cavity 41 and the mixed pneumatic cavity 61 any more, the gas pressure in the valve pneumatic cavity 41 and the mixed pneumatic cavity 61 falls gradually, the first elastic membrane 43 and the third elastic membrane 63 recover along with the original state, and then the valve pneumatic cavity 41 and the mixed pneumatic cavity 61 are opened. It is achieved that the control of both the mixing pneumatic valve 4 and the liquid mixing valve 6 by the first gas pumping arrangement 3 can be switched between an open state and a closed state.
Wherein in an alternative embodiment of the present invention, the first elastic membrane 43, the second elastic membrane 53, the third elastic membrane 63 and the fourth elastic membrane 32 are different parts of the same elastic membrane. It should be understood that the first elastic film 43, the second elastic film 53, the third elastic film 63, and the fourth elastic film 32 may be elastic films independent of each other.
Further, as shown in fig. 1, 5 and 6, the microfluidic chip further includes a feed port 9 and a pumping module, and the pumping module includes a second gas pumping structure 10, and a pumping pneumatic valve 11, a liquid pumping structure 12 and a pumping check valve 13 connected in sequence;
the second gas pumping structure 10 is used for controlling the switching of the pumping pneumatic valve 11 and the liquid pumping structure 12 between an opening state and a closing state;
the pumping pneumatic valve 11 is used for controlling whether the liquid pumping structure 12 is communicated with the feed port 9 or not;
the liquid pumping structure 12 is used for storing liquid or gas in an open state, and the liquid pumping structure 12 is used for pressing the liquid or gas to the pumping check valve 13 in a closed state
The pumping check valve 13 is communicated with the liquid delivery passage 8, and the pumping check valve 13 is used for enabling the liquid or gas to flow in one direction along the direction from the liquid pumping structure 12 to the liquid delivery passage 8.
When the pumping pneumatic valve 11 is in an open state, the liquid pumping structure 12 is communicated with the sample inlet, and when the pumping pneumatic valve 11 is in a closed state, the liquid pumping structure 12 is not communicated with the sample inlet. The second gas pumping structure 10 controls the pumping pneumatic valve 11 and the liquid pumping structure 12 to be both in an opening state, so that a sample to be detected and various solutions required by detection are injected into the feed port 9, and the solutions flow to the liquid pumping structure 12 through the pumping pneumatic valve 11. The pumping pneumatic valve 11 and the liquid pumping structure 12 are then controlled to close by the second gas pumping structure 10, and the liquid pumping structure 12 squeezes the liquid in the liquid pumping structure 12 to the pumping check valve 13 and is delivered to the liquid delivery channel 8 through the pumping check valve 13. The pumping pneumatic valve 11 and the liquid pumping structure 12 are then controlled to open again by the second gas pumping structure 10, at which time the gas pressure in the liquid pumping structure 12 is low, the liquid at the feed port 9 will be sucked to the liquid pumping structure 12 through the pumping pneumatic valve 11, and then the liquid in the liquid pumping structure 12 will be squeezed into the liquid transporting channel 8. Through controlling the continuous opening and closing of the pneumatic valve 11 of pumping and liquid pumping structure 12 promptly, just can carry the liquid delivery channel 8 department with the liquid of feed inlet 9 department, and then will wait to detect sample and detect required solution and carry and mix, hatch, operation such as enrichment to mixing module 2. Therefore, the microorganism can be conveniently and rapidly detected, the skill requirement of operators is reduced, a complex pumping system is not needed, the detection on the basic site is facilitated, and the detection cost is saved.
Wherein in an alternative embodiment of the present invention the second gas pumping arrangement 10 is of the same specific construction as the first gas pumping arrangement 3. It should be understood that second gas pumping arrangement 10 may be any other suitable arrangement having a gas delivery function.
Wherein, in an alternative embodiment of the present invention, the pumping pneumatic valve 11 has the same specific structure as the hybrid pneumatic valve 4. It should be appreciated that the pumping pneumatic valve 11 may be of any other suitable configuration.
Wherein, in an alternative embodiment of the present invention, the liquid pumping structure 12 is the same as the specific structure of the liquid mixing valve 6. It should be understood that liquid pumping arrangement 12 may be any other suitable arrangement.
Wherein in an alternative embodiment of the invention, the pumping check valve 13 is of the same specific construction as the mixing check valve 5. It should be understood that pumping check valve 13 may be any other suitable structure having a function of controlling the unidirectional flow of liquid or gas.
Further, as shown in fig. 1, fig. 2 and fig. 3, the microfluidic chip further includes a waste liquid chamber 14, the waste liquid chamber 14 is located in the chip body 1, one end of the mixing channel 7 is connected to the liquid mixing valve 6, the other end of the mixing channel 7 is located above the waste liquid chamber 14, and the other end of the mixing channel 7 is communicated with the waste liquid chamber 14. In use, a liquid such as a sample to be tested and a solution required for retrieval is first fed into the liquid mixing valve 6 and flows back and forth between the liquid mixing valve 6 and the mixing channel 7. When the reaction is completed or the reaction is required to be changed midway, when the waste liquid is discharged, and the like, gas is conveyed to the liquid conveying channel 8, and simultaneously, the mixing pneumatic valve 4 and the liquid mixing valve 6 are in an opening state through the first gas pumping structure 3, the gas passes through the mixing one-way valve 5 after passing through the mixing pneumatic valve 4, then the liquid in the liquid mixing valve 6 is extruded into the mixing channel 7, then the gas is continuously conveyed to the liquid conveying channel 8, so that the liquid is continuously extruded to the other end of the mixing channel 7, then the liquid flows into the waste liquid cavity 14 from the other end of the mixing channel 7, and further the discharge of the waste liquid is realized.
Wherein a water absorbing member is provided in the waste liquid chamber 14. When using, the waste liquid that waste liquid chamber 14 was arranged in can effectual absorption of the piece that absorbs water, and then improves waste liquid storage capacity of waste liquid chamber 14.
In an alternative embodiment of the present invention, the absorbent member is, for example, an absorbent pad. It should be understood that the absorbent pad can be any other suitable component having an absorbent function.
Further, as shown in fig. 1 and fig. 4, the microfluidic chip further includes a plurality of standard color cards 15, and the standard color cards 15 are located in the chip body 1. When the kit is used, the standard colorimetric card 15 can be used as a reference for judging the result of a microorganism detection experiment, so that the microorganism concentration of a sample to be detected can be conveniently and quickly judged, and the kit is convenient for field detection at a basic level.
In another aspect, as shown in fig. 7, the present invention also provides a method for detecting microorganisms, comprising:
s1, injecting a sample to be detected, immunomagnetic beads and labeled enzyme into a feed port, and pressing a second gas pumping structure to convey the three solutions to a mixing module;
s2, pressing a first gas pumping structure to fully mix the three solutions, and standing and incubating to form an immunomagnetic bead-target bacterium-labeled enzyme double-antibody sandwich compound;
s3, placing the magnetic separator above the liquid mixing valve, adsorbing the magnetic beads and the double-antibody sandwich compound in the liquid mixing valve to the top of the liquid mixing valve, and pumping the liquid into a waste liquid pool;
s4, dropwise adding cleaning fluid into the sample inlet, pressing the second gas pumping structure to pump the cleaning fluid into the liquid mixing valve, cleaning the magnetic substance on the top of the liquid mixing valve to remove the unbound labeled enzyme, and pumping the cleaning fluid into a waste liquid cavity;
s5, dropwise adding a chromogenic substrate into the sample inlet, pressing the first gas pumping structure to pump the chromogenic substrate to the liquid mixing valve, removing the magnetic separator, and enabling the chromogenic substrate to resuspend the double antibody sandwich compound;
s6, standing for a period of time, and catalyzing a chromogenic substrate by a marker enzyme on the double-antibody sandwich to enable an original colorless and transparent solution to generate color; comparing the generated color with a standard color comparison card, and determining the concentration of the bacteria in the detection sample.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (7)
1. A micro-fluidic chip is characterized by comprising a chip body, wherein a mixing module is arranged on the chip body, and the mixing module comprises a first gas pumping structure, a mixing pneumatic valve, a mixing one-way valve, a liquid mixing valve and a mixing channel which are sequentially connected;
the first gas pumping structure is used for controlling the mixing pneumatic valve and the liquid mixing valve to be switched between an opening state and a closing state;
one end of the mixing pneumatic valve is connected with the liquid conveying channel, the other end of the mixing pneumatic valve is connected with the mixing one-way valve, and the mixing pneumatic valve is used for controlling whether the mixing one-way valve is communicated with the liquid conveying channel or not;
the mixing one-way valve is used for controlling the unidirectional flow of liquid or gas along the direction from the mixing pneumatic valve to the liquid mixing valve;
the liquid mixing valve is used for storing liquid or gas in an opening state, and the liquid mixing valve is used for extruding the liquid or gas into the mixing channel in a closing state;
the pneumatic valve of mixing includes valve pneumatic chamber and valve pocket, valve pneumatic chamber with be provided with first elastic membrane between the valve pocket, valve pneumatic chamber with first gas pumping structure intercommunication, the valve pocket with liquid transport passageway intercommunication, the liquid mixing valve is including mixing pneumatic chamber and hybrid chamber, the pneumatic chamber of mixing with be provided with the third elastic membrane between the hybrid chamber, the pneumatic chamber of mixing with first gas pumping structure intercommunication, wherein, work as when the liquid mixing valve is in the open mode, the hybrid chamber respectively with mix check valve and hybrid passage intercommunication.
2. The microfluidic chip according to claim 1, wherein the mixing check valve comprises a valve top and a vent hole, the valve top is located above the vent hole, a second elastic membrane is arranged between the valve top and the vent hole, a first pipeline is arranged between one end of the valve top and the mixing pneumatic valve, and a second pipeline is arranged between the other end of the valve top and the liquid mixing valve.
3. The microfluidic chip according to claim 1, wherein the mixing chamber has a hemispherical structure.
4. The microfluidic chip according to any of claims 1 to 3, wherein the first gas pumping structure comprises a gas reservoir and a fourth elastic membrane, the upper end of the gas reservoir is open, and the fourth elastic membrane covers the upper end of the gas reservoir.
5. The microfluidic chip according to any of claims 1 to 3, further comprising a waste chamber, wherein the waste chamber is located in the chip body, one end of the mixing channel is connected to the liquid mixing valve, the other end of the mixing channel is located above the waste chamber, and the other end of the mixing channel is communicated with the waste chamber.
6. The microfluidic chip according to any of claims 1 to 3, further comprising a plurality of standard color cards, wherein the standard color cards are located in the chip body.
7. The microfluidic chip according to any one of claims 1 to 3, further comprising a feed port and a pumping module, wherein the pumping module comprises a second gas pumping structure, and a pumping pneumatic valve, a liquid pumping structure and a pumping check valve connected in sequence;
the second gas pumping structure is used for controlling the pumping pneumatic valve and the liquid pumping structure to be switched between an opening state and a closing state;
the pumping pneumatic valve is used for controlling whether the liquid pumping structure is communicated with the feed port or not;
the liquid pumping structure is used for storing liquid or gas when in an opening state, and the liquid pumping structure is used for extruding the liquid or gas to the pumping one-way valve when in a closing state
The pumping one-way valve is communicated with the liquid conveying channel and is used for enabling liquid or gas to flow in one direction along the direction from the liquid pumping structure to the liquid conveying channel.
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| CN103861668A (en) * | 2012-12-17 | 2014-06-18 | 中国科学院沈阳自动化研究所 | Microfluidic chip based on pneumatic micro pumps and micro mixer |
| CN110208520A (en) * | 2019-06-27 | 2019-09-06 | 深圳华迈兴微医疗科技有限公司 | A kind of active liquid stream control micro-fluidic detection system |
| CN210720414U (en) * | 2019-06-27 | 2020-06-09 | 深圳华迈兴微医疗科技有限公司 | Magnetic particle luminous micro-fluidic chip |
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| TWI691593B (en) * | 2016-11-24 | 2020-04-21 | 國立清華大學 | Automatic microfluidic system for antibiotic susceptibility testing and method of operating thereof |
| JP6574538B2 (en) * | 2017-09-25 | 2019-09-11 | 株式会社テイエルブイ | Liquid pressure feeding device state determination device and liquid pressure feeding device |
| CN109647553B (en) * | 2018-12-29 | 2020-11-20 | 北京化工大学 | Multi-index disease joint detection microfluidic device |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103861668A (en) * | 2012-12-17 | 2014-06-18 | 中国科学院沈阳自动化研究所 | Microfluidic chip based on pneumatic micro pumps and micro mixer |
| CN110208520A (en) * | 2019-06-27 | 2019-09-06 | 深圳华迈兴微医疗科技有限公司 | A kind of active liquid stream control micro-fluidic detection system |
| CN210720414U (en) * | 2019-06-27 | 2020-06-09 | 深圳华迈兴微医疗科技有限公司 | Magnetic particle luminous micro-fluidic chip |
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