CN113337398A - Micro-fluidic chip - Google Patents
Micro-fluidic chip Download PDFInfo
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- CN113337398A CN113337398A CN202110605187.8A CN202110605187A CN113337398A CN 113337398 A CN113337398 A CN 113337398A CN 202110605187 A CN202110605187 A CN 202110605187A CN 113337398 A CN113337398 A CN 113337398A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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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
Abstract
The scheme discloses a microfluidic chip which comprises at least one group of microfluidic detection structures, wherein each microfluidic detection structure comprises a sample loading port, a sample cavity, a washing cavity and a detection cavity, and the sample loading port is used for supplying materials to the sample cavity; a lysis solution, magnetic beads and an organic phase layer are arranged in the sample cavity, the magnetic beads are arranged in the lysis solution, and the organic phase layer is arranged above the lysis solution; the washing cavity is internally provided with a washing liquid and an organic phase layer, and the organic phase layer is arranged above the washing liquid; a detection object and an organic phase layer are arranged in the detection cavity, and the organic phase layer is arranged above the detection object; the organic phase layer of the sample cavity is communicated with the organic phase layer of the washing cavity through a micro-channel, and the organic phase layer of the washing cavity is communicated with the organic phase layer of the detection cavity through the micro-channel. The scheme is pre-filled in the production process, and the detection of the sample can be implemented without filling materials into each cavity at first in the use process, so that the non-contact use is realized.
Description
Technical Field
The application relates to the technical field of nucleic acid detection, in particular to a microfluidic chip.
Background
The POC molecular diagnosis has wide application prospect as an accurate in-vitro diagnosis technology which does not depend on professional laboratories and personnel. The core of the method lies in the integration and automation of the operation processes of sample cracking, nucleic acid separation, enrichment, purification, amplification, nucleic acid detection and the like of nucleic acid, the conventional POC molecular diagnosis platform is roughly divided into two types, one type is that the manual operation is simulated by using external automatic equipment comprising a mechanical arm so as to serially connect all the steps of the whole operation, and the scheme has more external equipment, high cost and is not beneficial to the popularization of a large-area basic layer. The other diagnosis platform takes a micro-fluidic chip as a main analysis carrier, and all operation steps are completed in series in the micro-fluidic chip in a fluid flow mode between chambers. Compared with a scheme of simulating manual operation by external equipment, the microfluidic technical scheme can greatly reduce the consumption of reagents and samples by virtue of the characteristics of fluid under microscale and the large-scale integration of detection channels on the chip, quickens the reaction rate, is easy to realize high-throughput analysis, and is beneficial to the miniaturization and automation of an analysis system.
The existing microfluidic chip usually needs to connect products or fill a plurality of reagents on site, so that the steps are complex to use and non-contact operation cannot be realized. Meanwhile, the prior art has a complex structure, higher requirements on the processing technology of the microfluidic chip, higher difficulty in mass production and high cost.
Disclosure of Invention
The embodiment of the application provides a micro-fluidic chip capable of realizing non-contact use.
In order to solve the above technical problem, an embodiment of the present application provides a microfluidic chip, which adopts the following technical scheme:
a microfluidic chip comprises at least one microfluidic detection structure, wherein the microfluidic detection structure comprises a sample loading port, a sample cavity, a washing cavity and a detection cavity, and the sample loading port is used for supplying materials to the sample cavity; a lysis solution, magnetic beads and an organic phase layer are arranged in the sample cavity, the magnetic beads are arranged in the lysis solution, and the organic phase layer is arranged above the lysis solution; the washing cavity is internally provided with a washing liquid and an organic phase layer, and the organic phase layer is arranged above the washing liquid; a detection object and an organic phase layer are arranged in the detection cavity, and the organic phase layer is arranged above the detection object; the organic phase layer of the sample cavity is communicated with the organic phase layer of the washing cavity through a micro-channel, and the organic phase layer of the washing cavity is communicated with the organic phase layer of the detection cavity through the micro-channel.
Furthermore, the detection object comprises an eluent, a primer probe and dehydrated particles of an amplification reagent, the primer probe and the dehydrated particles of the amplification reagent are fixed at the bottom of the detection cavity through paraffin, and the paraffin isolates the dehydrated particles of the primer probe and the dehydrated particles of the amplification reagent from the eluent.
Further, a shallow pool is arranged in the micro-channel, and a plugging object is arranged in the shallow pool.
Further, the plugging object is provided with a plunger formed by paraffin.
Furthermore, the micro-flow detection structure also comprises a base, wherein the sample cavity, the washing cavity, the detection cavity, the micro-channel and the shallow pool are integrally formed on the base.
Furthermore, the microfluidic chip also comprises a top cover, the top cover seals the openings of the sample cavity, the washing cavity, the detection cavity, the microchannel and the shallow pool, and the sample loading opening is formed in the top cover.
Further, the sample cavity is also provided with a sealing cap, the sealing cap is fixed with the top cover, and the sealing cap seals the sample loading port.
Furthermore, a conical body is arranged at the bottom of the sample cavity, and the conical body is filled with the lysis solution.
Furthermore, the washing chamber includes cylinder and cone, the cone sets up the bottom of cylinder, washing liquid is full of the cone, and under the magnetic bead drove nucleic acid and wash in the washing chamber and remove the washing settlement state of external magnetic field, the magnetic bead drove nucleic acid and gathers in the bottom of cone.
Furthermore, the surface of the detection cavity, into which nucleic acid enters, is a first surface, the surface adjacent to the first surface is a second surface and a third surface, the surface opposite to the first surface is a fourth surface, and the lengths of the first surface and the fourth surface are shorter than those of the second surface and the third surface.
Further, the first surface and the fourth surface include a straight portion and an inclined portion, and the inclined portions of the first surface and the fourth surface are connected at the bottom of the detection chamber.
Further, the included angle between the inclined parts of the first surface and the fourth surface is 45 degrees.
Furthermore, the micro-flow detection structures are provided with a plurality of groups, and the micro-flow detection structures are arranged side by side, flush and uniformly, wherein the sample loading ports are uniformly arranged on the top cover side by side.
Compared with the prior art, the embodiment of the application mainly has the following beneficial effects: by arranging the sample cavity, the washing cavity and the detection cavity in the microfluidic detection structure, the lysis solution, the washing solution and the detection object which are filled in the sample cavity, the washing cavity and the detection cavity in advance can be aqueous phase liquid, and an organic phase layer is arranged above the aqueous phase liquid in each cavity, wherein the organic phase layer is formed by organic phase liquid with density lower than that of the aqueous phase liquid. The organic phase layers among the three chambers are communicated through the micro-channel, so that an environment that the three chambers are communicated, but aqueous phase liquid among the three chambers cannot be communicated with each other is formed; when the device is used, a sample to be detected is filled in the sample cavity through the feeding hole, then the magnetic beads in the sample cavity are controlled by the external magnetic field to drive nucleic acid to enter the organic phase layer, the surface tension formed by the organic phase in the micro-channel is overcome, and the device moves among the sample cavity, the washing cavity and the detection cavity to realize operations of sample cracking, nucleic acid separation, amplification, detection and the like of the nucleic acid. The water phase liquid and the light organic phase layer are preset in the closed sample cavity, the washing cavity and the detection cavity, and the organic phase layer is communicated through the micro-channel, so that the water phase liquid between the cavities can not be subjected to crosstalk in the storage and transportation processes, the water phase liquid can be filled in advance in the production process, the detection of the sample can be realized without filling materials into the cavities in the using process, and the non-contact use is realized.
Drawings
In order to illustrate the solution of the present application more clearly, the drawings that are needed in the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and that other drawings can be obtained by those skilled in the art without inventive effort.
Fig. 1 is a schematic structural diagram of a microfluidic chip according to the present application;
FIG. 2 is a schematic structural diagram of a microfluidic chip according to the present application in a storage state;
fig. 3 is a cross-sectional view of a microfluidic chip according to the present application.
FIG. 4 is a flow chart of a method for using the microfluidic chip in this embodiment;
FIG. 5 is a flowchart illustrating step S200 in FIG. 4 according to the present embodiment;
FIG. 6 is a flowchart illustrating step S400 of FIG. 4 according to the present embodiment;
FIG. 7 is a flowchart illustrating step S600 in FIG. 4 according to the present embodiment;
reference numerals:
1-sample cavity, 11-sealing cap, 2-washing cavity, 3-detection cavity, 4-microchannel, 41-shallow pool structure, 5-base, 6-top cover.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "including" and "having," and any variations thereof, in the description and claims of this application and the description of the above figures are intended to cover non-exclusive inclusions. The terms "first," "second," and the like in the description and claims of this application or in the above-described drawings are used for distinguishing between different objects and not for describing a particular order.
Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the application. The appearances of the phrase in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. It is explicitly and implicitly understood by one skilled in the art that the embodiments described herein can be combined with other embodiments.
In order to make the technical solutions better understood by those skilled in the art, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings.
A microfluidic chip comprises at least one microfluidic detection structure, wherein the microfluidic detection structure comprises a sample loading port, a sample cavity 1, a washing cavity 2 and a detection cavity 3, and the sample loading port is used for supplying materials to the sample cavity 1; a lysis solution, magnetic beads and an organic phase layer are arranged in the sample cavity 1, the magnetic beads are arranged in the lysis solution, and the organic phase layer is arranged above the lysis solution; the washing cavity 2 is internally provided with washing liquid and an organic phase layer, and the organic phase layer is arranged above the washing liquid; a detection object and an organic phase layer are arranged in the detection cavity 3, and the organic phase layer is arranged above the detection object; the lysis solution, the washing solution and the detection object can be water phase liquid, the organic phase layer of the sample cavity 1 is communicated with the organic phase layer of the washing cavity 2 through the micro-channel 4, and the organic phase layer of the washing cavity 2 is communicated with the organic phase layer of the detection cavity 3 through the micro-channel 4.
Specifically, a sample chamber 1, a washing chamber 2 and a detection chamber 3 are arranged in the microfluidic detection structure, wherein the sample chamber 1, the washing chamber 2 and the detection chamber 3 are pre-filled with aqueous phase liquid corresponding to the functions of the chambers, and an organic phase layer is arranged above the aqueous phase liquid in each chamber, wherein the organic phase layer is formed by organic phase liquid with weight lower than that of the aqueous phase liquid. The organic phase layers among the three chambers are communicated through the micro-channel 4, so that an environment that the three chambers are communicated with each other but the water phase liquid among the three chambers cannot be communicated with each other is formed.
Wherein the lysis solution is used for lysing a sample to be detected and releasing nucleic acid. The washing liquid is used for cleaning a sample to be detected, so that other substances except the nucleic acid on the magnetic beads are removed. The detection solution is used for amplifying and detecting the purified nucleic acid.
When the device is used, a sample to be detected is filled in the sample cavity 1 through the feeding port, the sample to be detected is then converged with lysis solution and magnetic beads in the sample cavity 1, the magnetic beads in the sample cavity 1 are controlled by an external magnetic field to move so as to drive nucleic acid to enter the organic phase layer, the magnetic beads drive the nucleic acid to move so as to overcome surface tension formed by organic phases in the micro-channel, and the nucleic acid is transferred among the sample cavity 1, the washing cavity 2 and the detection cavity 3 so as to realize operations of sample lysis, nucleic acid separation, amplification, detection and the like of the nucleic acid.
The water phase liquid and the light organic phase layer are preset in the closed sample cavity 1, the washing cavity 2 and the detection cavity 3, and the organic phase layer is communicated through the micro channel 4, so that the water phase liquid between the cavities is prevented from crosstalk in the storage and transportation processes, the water phase liquid can be filled in advance in the processing process, the detection of the sample can be realized without filling materials or connecting pipelines to the cavities in the using process, and the non-contact use is realized. In addition, because the materials related to detection do not need subsequent filling, a plurality of filling ports do not need to be provided, or a splicing structure for realizing the assembly of the microfluidic detection structure is adopted, the product structure of the microfluidic chip is simpler, the process requirement is greatly reduced, and the microfluidic chip is suitable for large-scale batch production.
The magnetic beads are usually filled or stored in the form of a magnetic bead mixed solution, and the magnetic bead mixed solution includes a liquid phase capable of suspending the magnetic beads, pre-prepared magnetic beads capable of adsorbing nucleic acids, and a reagent component for promoting the adsorption of the magnetic beads and the nucleic acids.
Further, the detection object comprises an eluent, a primer probe and dehydrated particles of an amplification reagent, the primer probe and the dehydrated particles of the amplification reagent are fixed at the bottom of the detection cavity 3 through paraffin with a medium melting point (60-70 ℃), and the paraffin isolates the dehydrated particles of the primer probe and the dehydrated particles of the amplification reagent from the eluent.
The eluent is used for separating the washed sample to be detected from the magnetic beads for transferring the sample to be detected, and the primer probe and the amplification reagent are used for amplifying and detecting the sample to be detected. In the middle of the use, drive the magnetic bead through external magnetic field and drive the sample that awaits measuring and get into detection chamber 3, later cut off the magnetism adsorption connection between sample that awaits measuring and the magnetic bead through the eluant for the magnetic bead leaves detection chamber 3 through external magnetic field drive magnetic bead later. And then heating the paraffin in the detection cavity 3 to melt the paraffin, dissolving the amplification reagent in the eluent, and contacting the primer probe and the amplification reagent with a sample to be detected in the eluent to realize the process flow of enrichment and amplification.
The dehydration particles of the primer probe and the amplification reagent are isolated from the eluent by paraffin, the magnetic beads and the nucleic acid are firstly separated from the eluent, and then the paraffin is melted to enable the nucleic acid to be contacted with the primer probe and the amplification reagent, so that the process flow of enriching and amplifying the sample to be detected in the environment without the magnetic beads is facilitated. The enrichment and amplification effects of the sample to be detected in the detection cavity 3 are enhanced. And the above scheme is also realized under the non-contact operation.
Further, a shallow pool is arranged in the micro-channel 4, and a plugging object is arranged in the shallow pool.
A shallow pool structure 41 is arranged in the microchannel 4 to accommodate a plugging substance, and the microchannel 4 is plugged by the plugging substance to prevent mutual series flow of aqueous phase liquid between the adjacent sample cavity 1 and the washing cavity 2 or between the washing cavity 2 and the detection cavity 3 in the transportation process. The scheme can improve the detection success rate of nucleic acid detection.
Further, the stopper is arranged as a plunger formed of low melting (45-55 ℃) paraffin.
Specifically, the plugging substance is preferably paraffin with a low melting point, plugging is realized in the transportation process, and the paraffin is heated in the use process to be melted into a liquid state compatible with an organic phase, so that the passing performance of magnetic beads in the microchannel 4 is improved, and the scheme can improve the running efficiency of the magnetic beads in the nucleic acid detection process. And the scheme can realize the plugging and dredging of the micro-channel 4 under the non-contact condition. Preferably, the shallow pool structure 41 and the detection chamber 3 are simultaneously provided with paraffin, the paraffin in the shallow pool is firstly heated to be dissolved, the melting of the paraffin in the detection chamber 3 is carried out after the preset paraffin in the shallow pool is melted, the paraffin in the shallow pool structure 41 is set as low-melting-point paraffin, and the paraffin in the detection chamber 3 is set as medium-melting-point paraffin to prevent the paraffin in the detection chamber 3 from being melted in advance due to misoperation.
Further, the micro-flow detection structure further comprises a base 5, and the sample cavity 1, the washing cavity 2, the detection cavity 3, the micro-channel 4 and the shallow pool are integrally formed on the base 5.
Particularly, the sample cavity 1, the washing cavity 2, the detection cavity 3, the micro-channel 4 and the shallow pool are integrally formed through pouring, extruding and other forms on the base 5, so that the processing is convenient, the number of parts required by product assembly is reduced, the process is simplified, and the efficient processing of the microfluidic chip is facilitated.
Further, the microfluidic chip further comprises a top cover 6, the top cover 6 seals the openings of the sample cavity 1, the washing cavity 2, the detection cavity 3, the microchannel 4 and the shallow pool, and the sample loading opening is formed in the top cover 6.
Specifically, the top cover 6 is arranged to seal the openings of the sample cavity 1, the washing cavity 2, the detection cavity 3, the micro-channel 4 and the shallow pool, wherein the fixed relation between the top cover 6 and the base 5 can be set arbitrarily according to the production and use environments, the top cover 6 and the base 5 are separated and processed respectively, and the processing difficulty of the microfluidic chip is facilitated to be simplified.
Further, the sample cavity 1 is further provided with a sealing cap 11, the sealing cap 11 is fixed with the top cover 6, and the sealing cap 11 seals the sample loading port.
Specifically, after pre-loading of the aqueous phase liquid and the organic phase liquid of the microfluidic chip and other detection materials is realized, the sample loading port is sealed by the sealing cap 11, and then the sealing cap 11 is removed to unseal before use. So prevent spilling hourglass, anti-pollution is favorable to promoting the degree of accuracy that the sample that awaits measuring detected.
Further, a conical body is arranged at the bottom of the sample cavity 1, and the conical body is filled with the lysis solution.
It is specific, lysate and magnetic bead have been prestored in sample chamber 1, great vacant space has been reserved when the transport condition, when the user state, to letting in the solution that has the sample that awaits measuring in sample chamber 1, and the space of reserving through sample chamber 1 stores, the volume that contains the solution of the sample that awaits measuring is great, when design sample chamber 1, set up sample chamber 1 into long and narrow form cavity usually and make the degree of depth of cavity less, prevent that the cavity degree of depth from excessively causing the unable sufficient magnetic attraction of magnetic bead production to the cavity bottom of external magnetic field, the control efficiency to the magnetic bead is influenced. At this time, by arranging the conical body at the bottom of the sample cavity 1, wherein the conical body comprises the inclined surface, the surface area of the sample cavity 1 can be obviously improved, and the sample to be detected can be conveniently dispersed and stayed on the inclined surface. In the use, the sample that awaits measuring spreads on the inclined plane of conical body, and the magnetic bead rolls on the inclined plane and produces in the magnetism adsorption between the sample that awaits measuring with the sample intensive contact mixture that awaits measuring, and the combination of magnetic bead and the sample that awaits measuring is more rapid and abundant from this, and this scheme has promoted the efficiency that the magnetic bead adsorbs the sample that awaits measuring.
Further, the washing chamber 2 comprises a cylinder and a cone, the cone is arranged at the bottom of the cylinder, the washing liquid is filled in the cone, and the magnetic beads drive the nucleic acids to gather at the bottom of the cone under the washing sedimentation state that the magnetic beads drive the nucleic acids to be washed in the washing chamber 2 and the external magnetic field is removed.
It is specific, under the washing state, through applying external magnetic field, the drive magnetic bead drives the sample that awaits measuring and removes to the top of washing chamber 2, perhaps make the magnetic bead remove to the bottom of washing chamber 2 under the drive of gravity through removing external magnetic field, so reciprocal, the realization is to the washing of the sample that awaits measuring and with the separation of impurity, in the middle of this process, the liquid stream is setting up to the regional smooth formation in 2 upper portions of washing chamber of cylinder, and set up to 2 bottoms in washing chamber of conical body can concentrate the magnetic bead, be favorable to under the drive of external magnetic field, drive all magnetic beads to the top motion of washing chamber 2, this scheme can promote the washing efficiency of the sample that awaits measuring.
Further, the surface of the detection cavity 3, into which the nucleic acid enters, is a first surface, the surface adjacent to the first surface is a second surface and a third surface, the surface opposite to the first surface is a fourth surface, and the lengths of the first surface and the fourth surface are shorter than those of the second surface and the third surface.
Specifically, it sets up to flat thin wall shape to detect chamber 3 preferred, in the use, need to detect the sample that awaits measuring in the chamber 3 and heat up the cooling, and the detection chamber 3 of flat thin wall shape can provide a great surface area to the cavity of certain volume, can improve the thermal current power that detects chamber 3 like this for promote the efficiency of rising temperature and cooling when carrying out the heat exchange with the external world. The scheme is beneficial to accurate detection of the sample to be detected.
Further, the first surface and the fourth surface include a straight portion and an inclined portion, and the inclined portions of the first surface and the fourth surface are connected at the bottom of the detection chamber 3.
Specifically, through the slope portion of two connections in detection chamber 3 lower part, can produce a convection current vortex field in the region of detection chamber 3 lower part, the reinforcing detects 3 internal mobility in chamber, so, each component in the detection chamber 3 body can intensive mixing and reaction, and this scheme has promoted the reaction efficiency who detects chamber 3.
Further, the included angle between the inclined parts of the first surface and the fourth surface is 45 degrees.
Specifically, in the process of detecting the sample to be detected, the sample to be detected needs to be irradiated by the excitation light and the emission light, and the spectrum generated by the sample to be detected is generated and received outside the detection cavity 3 to complete the detection step. Excitation light and emission form the light path in detecting chamber 3 when to the illumination of the sample that awaits measuring, because the region is less in detecting chamber 3, the optical path is limited, sets up to 45 degrees through the angle with the rake contained angle to at the straight portion input excitation light and emission light, through the refraction, receive the biggest optical path that the record spectrum can utilize to detect chamber 3 and can provide in the slope, the accuracy that the sample that awaits measuring detected is promoted to this scheme.
Furthermore, the micro-flow detection structures are provided with a plurality of groups, and the micro-flow detection structures are arranged side by side, flush and uniformly, wherein the sample loading ports are uniformly arranged on the top cover side by side.
It is specific, detect the structure through set up multiunit miniflow on base 5, be favorable to the batch that the check sample detected to go on, multiunit miniflow detects the structure and sets up side by side, and flush each other, the interval between each miniflow detects the structure is even, make the interval between the filler on the apron also even, so can detect the notes of the detection sample of structure to a plurality of miniflows with the help of the disposable completion of external equipment, be favorable to carrying out the operation to miniflow detection mechanism simultaneously through corollary equipment.
The embodiment of the application also discloses a use method of the micro-fluidic chip.
A method of using a microfluidic chip, the method comprising:
step S100: and (3) injecting a sample to be detected into the sample cavity, and mixing the sample to be detected with a mixed solution containing lysis solution and magnetic beads.
The sample to be detected is filled into the sample cavity, so that the sample to be detected is mixed with the lysis solution and the magnetic beads.
Step S200: the magnetic beads are driven to move in the lysis solution to adsorb the nucleic acid.
Specifically, the sample to be tested is usually human body fluid such as blood, urine, saliva and the like, the components are usually complex, the components comprise mother liquor, organic matters such as proteins, cell membranes, cytoplasm, sugar, lipid and the like, and inorganic matters such as salt and the like, substances in the sample to be tested are cracked under a heating state, and flocculent nucleic acid and other impurities are present in a free state in the mother liquor of the sample to be tested. The magnetic beads are driven by external force to move in the lysis solution, nucleic acid in the mother solution of the sample to be detected can be adsorbed, and the nucleic acid in the sample to be detected can be gathered and purified by the magnetic beads through pretreatment.
Step S300, the magnetic beads absorbing the nucleic acid are driven to pass through the organic phase layer above the lysis solution and enter the organic phase layer of the washing cavity through the micro-channel.
The magnetic beads are driven by external force to drive the nucleic acid to move upwards and enter the organic phase layer, and because the mother liquor of the sample to be detected of the organic phase layer and the aqueous phase is mutually exclusive, the magnetic beads drive the nucleic acid and a small amount of impurities to enter the organic phase layer, and the aqueous phase liquid can hardly be driven to enter the organic phase layer.
Step S400: the magnetic beads adsorbing nucleic acid are driven to fall into the washing liquid and move in the washing liquid layer and the organic phase layer to remove impurities.
In the process that the magnetic beads are driven to enter the washing liquid through the organic phase layer, the affinity between the organic impurities and the organic phase layer is obviously greater than that between the organic impurities and the washing liquid in a water phase, so that a large amount of organic phase impurities moving along with the magnetic beads stay in the organic phase layer, the magnetic beads continuously move under the driving of external force in the washing liquid, and other organic impurities and inorganic impurities carried by the magnetic beads are filtered out, so that the nucleic acid carried by the magnetic beads is clean.
Step S500: and driving the washed magnetic beads to pass through an organic phase layer above the washing solution and enter the detection cavity through the micro-channel.
The specific steps of driving the magnetic beads to be transferred from the washing solution to the eluent by an external force are basically the same as the process of driving the magnetic beads from the lysis solution to the washing solution. In this process part of the remaining organic impurities can be removed by a second pass through the organic phase liquid.
Step S600: the washed magnetic beads are driven into an eluent and moved in the eluent to elute, amplify, and detect nucleic acids.
When the nucleic acid is adsorbed on the magnetic beads, it is difficult to mix and pair with the primer probe and to observe the nucleic acid. The eluent can remove the connection between the nucleic acid and the magnetic beads, so that the nucleic acid is separated from the magnetic beads and is dissociated in the eluent for subsequent amplification and detection. Adding primer probes for amplification and nucleic acid labeling and an amplification solution to the eluate to amplify the nucleic acid. And starting a thermal cycle program, rapidly heating and cooling the detection cavity to reach the temperature required by PCR thermal cycle, carrying out PCR reaction, exciting and collecting a fluorescence signal above the inclined plane of the detection cavity when each thermal cycle reaches the temperature required by primer extension, observing the change curve of the fluorescence signal intensity along with the cycle number, and finally forming a real-time fluorescence amplification curve.
In the embodiment, the magnetic beads adsorb nucleic acid in the lysis solution, and then are controlled to move above the lysis solution under the driving of an external force to drive the nucleic acid to enter the organic phase layer; and then overcoming the surface tension of an organic phase in the microchannel, passing through the microchannel, and breaking through an interface between the organic phase and the sample mother liquor to be detected of a water phase under the assistance of the microchannel structure of the organic phase, so that the magnetic beads drive the nucleic acid to be separated from the sample mother liquor to be detected. Then the magnetic beads are driven to drive the nucleic acid to enter a washing solution for washing. And (3) washing the nucleic acid, eluting and amplifying the nucleic acid in the eluent under the drive of the magnetic beads, and collecting a fluorescence amplification curve. Wherein the organic phase layer is also disposed on the upper layer of the eluent and the washing solution. The magnetic beads move in the organic phase layer above the lysis solution, the washing solution and the eluent, the organic phase layer is lighter than the aqueous phase layer, the organic phase layer is arranged on the upper layers of the lysis solution, the washing solution and the eluent of the aqueous phase layer, mutual diffusion between adjacent aqueous phase liquid and the organic phase layer can be effectively prevented, the organic phase layer is prevented from entering the aqueous phase liquid, and the efficiency of cracking, adsorption with the magnetic beads, washing, elution, amplification and optical detection of nucleic acid is influenced. The scheme improves the efficiency and the accuracy of nucleic acid detection through the microfluidic chip.
Another advantage of this solution is that: the method for completing the cracking, enrichment, purification, elution and amplification detection of the target analyte in a closed system by non-contact operation in the whole process is provided, and meanwhile, different aqueous phase liquids are effectively isolated, so that the chip can be preloaded with reagents required by nucleic acid analysis except for a sample to be detected for a long time, the operation efficiency of the nucleic acid analysis is finally improved, and the complexity of required external operation is reduced.
Further, the step S200: the method for driving the magnetic beads to move in the lysis solution to adsorb the nucleic acid specifically comprises the following steps:
step S201: and oscillating the chip lysate to ensure that the magnetic beads are fully contacted with the sample to be detected.
Step S202: and loading an external magnetic field to drive the magnetic bead assembly to move into the microchannel.
Specifically, the lysis solution mixed with magnetic beads and free nucleic acids is oscillated to enable the magnetic beads and the nucleic acids in the lysis solution to be fully distributed, then the magnetic beads are attracted to move to adsorb the nucleic acids through an external loading magnetic field and are gathered in an organic phase layer, the gathered magnetic beads are driven to move through the traction of the magnetic field and enter a microchannel, the surface tension of an organic phase in the microchannel is overcome through the gathered magnetic beads, and the gathered magnetic beads enter the microchannel.
Further, the step of driving the magnetic beads to move in the lysis solution to adsorb the nucleic acid further comprises:
step S203: loading the external magnetic field, adsorbing the magnetic beads to rise into an organic phase layer,
step S204: and removing the external magnetic field to drive the magnetic beads to settle and adsorb nucleic acid.
An external magnetic field is applied above the lysate, causing the beads to move upward under the attraction of the magnetic force to be transported to the top of the lysate. And then removing the external magnetic field, enabling the magnetic beads to fall under the action of gravity, realizing traversal of the region in the lysate in the falling process, and adsorbing the nucleic acid in the region. In one embodiment, in order to enhance the accuracy of controlling the magnetic beads, the magnetic field does not cover the whole area where the lysate is located, but covers one segment of the lysate, the magnetic beads in the influence range of the size of one segment are controlled to ascend or descend by sequentially loading and removing the magnetic field on different segments of the lysate to traverse the lysate in the segment, and the operation of driving the magnetic beads to ascend and descend is performed on each segment by repeatedly loading and removing the magnetic field to adsorb all the nucleic acids in the lysate. The scheme can drive the magnetic beads to move in all areas of the lysis solution by utilizing gravity so as to adsorb all free nucleic acids in the lysis solution, and avoids mutual permeation between an organic phase and the lysis solution, and the scheme has high adsorption rate on the nucleic acids.
Further, the driving magnetic beads converge, including: step S2021: and the step of collecting the magnetic beads comprises the step of collecting the magnetic beads into the next region after the magnetic beads move up and down for one time until all regions in the lysis solution are traversed, and finally collecting the magnetic beads into the microchannel.
In another embodiment of the scheme in step S200, the magnetic bead is driven by an external magnetic field to move upward in the segment, and then the magnetic bead is driven to move to the next segment, then the external magnetic field is removed, the magnetic bead moves downward under the drive of gravity, traverse the region where the segment is located, and adsorb nucleic acid in the segment, the magnetic bead is cyclically loaded with a magnetic field to adsorb the magnetic bead, the magnetic bead is moved to an adjacent segment, the magnetic bead is released to traverse the region where the segment is located, and adsorb nucleic acid in the segment, until all regions in the lysate are traversed and the nucleic acid in the lysate is adsorbed, at this time, all the magnetic beads are gathered in one segment, and can be simultaneously driven by the external magnetic field, which is beneficial to driving all the magnetic beads to pass through a microchannel by the subsequent magnetic field, and can improve the efficiency of nucleic acid detection.
Furthermore, the micro-channel is filled with an organic phase solution, and the converged magnetic beads are separated from the mother liquor when passing through the micro-channel.
Specifically, the sample to be tested comprises nucleic acid and mother liquor for storing the nucleic acid, various impurities exist in the mother liquor, the nucleic acid is not pure in the mother liquor, the mother solution is aqueous phase, the surface tension provided by the organic phase solution filled in the micro-channel only allows the magnetic beads to drive the nucleic acid to pass through, under the drive of an external magnetic field, the magnetic beads in the lysis solution move upwards to enter the organic layer, the magnetic field is moved, under the drive of magnetic attraction, the magnetic beads move to the port of the micro-channel and are gathered, the magnetic beads gathered at the port of the micro-channel can overcome the surface tension generated by the organic liquid in the micro-channel, pass through the micro-channel and enter the organic layer above the washing liquid, this scheme can block the mother liquor and the lysate of the sample that awaits measuring through the separation of microchannel to and the organic matter impurity that partial magnetic bead carried, makes magnetic bead and nucleic acid more pure, and this scheme can promote nucleic acid detection's the degree of accuracy.
Further, the method for using the microfluidic chip comprises the following steps: the method also comprises the step of heating the micro-channel communicated with the sample cavity, the washing cavity and the detection cavity to melt paraffin in the micro-channel and fuse organic phase liquid in the micro-channel. Through paraffin shutoff microchannel, prevent the cluster each other between lysate and the washing liquid to dredge the microchannel before the drive magnetic bead transports nucleic acid between lysate and washing liquid, will soak the microchannel through the similar compatibility between the paraffin of dissolving and the organic phase simultaneously, guarantee the trafficability characteristic of microchannel, this scheme can promote the degree of accuracy of detection.
Further, the step S400: the magnetic beads adsorbing the nucleic acid are driven to fall into the washing liquid and move in the washing liquid layer and the organic phase layer to remove impurities, and the method for removing the impurities specifically comprises the following steps:
step S401: an external magnetic field is applied to drive the magnetic beads adsorbing the nucleic acid to move to the upper part of the washing solution.
Step S402: and removing the external magnetic field to drive the magnetic beads adsorbing the nucleic acid to settle, and filtering impurities on the nucleic acid.
Step S403: and circularly loading the external magnetic field and removing the external magnetic field until the impurities on the nucleic acid are filtered out.
Specifically, through loading external magnetic field in the washing liquid, the drive magnetic bead drives nucleic acid upward movement in order to remove to the top of washing liquid, later withdraws external magnetic field for the magnetic bead falls under the drive of gravity, realizes wasing with the washing liquid contact, and later circulation drive magnetic bead reciprocates in the washing liquid, in order to realize the filtering to the organic impurity and the inorganic impurity that nucleic acid carried. The scheme does not need to stir the washing liquid, prevents mutual diffusion between the organic phase layer and the washing liquid, and has higher filtration rate for filtering impurities out of nucleic acid.
Further, the step of eluting nucleic acid specifically comprises:
step S601: and loading an external magnetic field to drive the washed magnetic beads to move to the upper part of the eluent.
Step S602: and removing the external magnetic field to drive the washed magnetic beads to settle, and filtering out impurities on the nucleic acid.
Step S603: and circularly loading the external magnetic field and removing the external magnetic field until the nucleic acid and the magnetic beads are completely eluted.
Specifically, through loading external magnetic field in the eluant, the drive magnetic bead drives nucleic acid and upwards moves in order to remove to the top of eluant, later withdraws external magnetic field for the magnetic bead falls under the drive of gravity, contacts with the eluant, and later circulation drive magnetic bead reciprocates in the eluant, with the separation of realization magnetic bead and nucleic acid. The scheme does not need to stir the eluent, prevents mutual diffusion between the organic phase layer and the eluent, and has higher elution efficiency on nucleic acid.
Further, the step of amplifying the nucleic acid specifically comprises:
step S604: an external magnetic field is loaded, and the adsorbed magnetic beads rise into the organic phase and are removed through the microchannel.
Step S605: heating the detection cavity to melt the nucleic acid, melting the wax seal in the detection cavity, and uniformly mixing the amplification reagent in the wax seal with the eluted nucleic acid.
Step S606: the temperature required for amplification in the detection chamber is maintained until amplification of the eluted nucleic acid is completed.
Specifically, after elution is achieved, the magnetic beads are separated from pure nucleic acid, an external magnetic field is loaded, the magnetic beads are moved to an organic phase layer at the top of eluent, the pure nucleic acid is remained in the eluent, then the eluent is heated, the nucleic acid in the eluent is melted, and a wax seal in the eluent is melted, so that the nucleic acid amplification reagent is exposed to be in contact with nucleic acid elution liquid in the detection cavity to be dissolved and release components, the melted paraffin rises to the organic phase layer above the eluent under the action of buoyancy generated by density difference between the paraffin and the eluent, the components released by the dissolved paraffin are dissolved in a water phase, and the melted nucleic acid is combined to form a nucleic acid amplification reaction system. Wherein the components released by dissolving the paraffin comprise nucleic acid fragments serving as primer probes and amplification reagents.
Further, the step of reacting the nucleic acid specifically comprises:
step S607: circularly heating and cooling the eluted nucleic acid to perform PCR reaction, and collecting a fluorescence amplification curve of the eluted nucleic acid under the PCR reaction.
In the process of nucleic acid amplification, according to the temperature required by PCR thermal cycle, the temperature of the detection cavity is circularly and rapidly increased and decreased to realize PCR reaction, when each thermal cycle reaches the temperature required by primer extension, a fluorescence signal is excited and collected at the upper part of the inclined plane of the detection cavity, the change curve of the fluorescence signal intensity along with the cycle number is observed, and finally a real-time fluorescence amplification curve is formed. The scheme can ensure that the amplification of the nucleic acid is generated in a pure environment and is not interfered by magnetic beads, and the accuracy of nucleic acid detection is improved.
It is to be understood that the above-described embodiments are merely illustrative of some, but not restrictive, of the broad invention, and that the appended drawings illustrate preferred embodiments of the invention and do not limit the scope of the invention. This application is capable of embodiments in many different forms and is provided for the purpose of enabling a thorough understanding of the disclosure of the application. Although the present application has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that the present application may be practiced without modification or with equivalents of some of the features described in the foregoing embodiments. All equivalent structures made by using the contents of the specification and the drawings of the present application are directly or indirectly applied to other related technical fields and are within the protection scope of the present application.
Claims (13)
1. A microfluidic chip, characterized in that: the device comprises at least one micro-flow detection structure, wherein the micro-flow detection structure comprises a sample loading port, a sample cavity, a washing cavity and a detection cavity, and the sample loading port is used for supplying materials to the sample cavity; a lysis solution, magnetic beads and an organic phase layer are arranged in the sample cavity, the magnetic beads are arranged in the lysis solution, and the organic phase layer is arranged above the lysis solution; the washing cavity is internally provided with a washing liquid and an organic phase layer, and the organic phase layer is arranged above the washing liquid; a detection object and an organic phase layer are arranged in the detection cavity, and the organic phase layer is arranged above the detection object; the organic phase layer of the sample cavity is communicated with the organic phase layer of the washing cavity through a micro-channel, and the organic phase layer of the washing cavity is communicated with the organic phase layer of the detection cavity through the micro-channel.
2. A microfluidic chip according to claim 1, wherein: the detection object comprises an eluent, a primer probe and dehydration particles of an amplification reagent, the primer probe and the dehydration particles of the amplification reagent are fixed at the bottom of the detection cavity through paraffin, and the paraffin isolates the dehydration particles of the primer probe and the amplification reagent from the eluent.
3. A microfluidic chip according to claim 1, wherein: a shallow pool is arranged in the micro-channel, and a plugging object is arranged in the shallow pool.
4. A microfluidic chip according to claim 3, wherein: the plugging substance is arranged as a plunger formed by paraffin.
5. A microfluidic chip according to claim 4, wherein: the micro-flow detection structure further comprises a base, and the sample cavity, the washing cavity, the detection cavity, the micro-channel and the shallow pool are integrally formed on the base.
6. A microfluidic chip according to claim 5, wherein: the micro-fluidic chip also comprises a top cover, wherein the top cover seals the openings of the sample cavity, the washing cavity, the detection cavity, the micro-channel and the shallow pool, and the sample loading opening is formed in the top cover.
7. A microfluidic chip according to claim 6, wherein: the sample cavity is also provided with a sealing cap, the sealing cap is fixed with the top cover, and the sealing cap seals the sample loading port.
8. A microfluidic chip according to claim 7, wherein: the bottom of the sample cavity is provided with a conical body, and the conical body is filled with the lysis solution.
9. A microfluidic chip according to claim 7, wherein: the washing chamber includes cylinder and cone, the cone sets up the bottom of cylinder, washing liquid is full of the cone, and magnetic bead drives nucleic acid and washs and remove the washing settlement state of external magnetic field in the washing chamber, and the magnetic bead drives nucleic acid and gathers in the bottom of cone.
10. A microfluidic chip according to claim 7, wherein: the surface that detects the interior nucleic acid of chamber and get into is first face, and the face adjacent with first face is second face and third face, with the face that the first face is relative is the fourth face, the length of first face and fourth face is shorter than the length of second face and third face.
11. A microfluidic chip according to claim 10, wherein: the first surface and the fourth surface comprise a straight part and an inclined part, and the inclined parts of the first surface and the fourth surface are connected at the bottom of the detection cavity.
12. A microfluidic chip according to claim 11, wherein: the angle of the included angle of the inclined parts of the first surface and the fourth surface is 45 degrees.
13. A microfluidic chip according to any one of claims 6 to 12, wherein: the micro-flow detection structures are provided with a plurality of groups, and the micro-flow detection structures are mutually arranged side by side, flush and uniformly distributed, wherein a plurality of sample loading ports are uniformly distributed on the top cover side by side.
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