CN108043478A - A kind of micro-fluidic chip, manual centrifugal device and nucleic acid detection method - Google Patents
A kind of micro-fluidic chip, manual centrifugal device and nucleic acid detection method Download PDFInfo
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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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- 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
- B01L3/502753—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 bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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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
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- 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
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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
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Abstract
The present invention relates to a kind of micro-fluidic chip, manual centrifugal device and nucleic acid detection method, including an at least sample-adding layer and the channel layer being arranged on the downside of the sample-adding layer；Wherein, set in the upper surface of the channel layer it is several along central distribution, to load test agent and test agent be transmitted to the test cell of a wherein chamber, each test cell passes through channel attached chamber between each other including at least two；The sample-adding layer is along the circumferentially disposed several wells corresponding with the channel layer in center, to inject reagent into a wherein chamber for the corresponding test cell.Invention manual centrifugal device, the portable unit of external power supply is not depended on, nucleic acid extraction, amplification and detection are integrated using microfluidic chip technology, quick, easily foranalysis of nucleic acids is realized, for the quick response and field diagnostic of the emergencies such as extensive infectious disease, food safety affair, Environment Pollution Event.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a micro-fluidic chip, a manual centrifugal device and a nucleic acid detection method.
Background
In the prior art, commonly used nucleic acid detection methods include rapid detection of nucleic acid based on amplification, gene chip technology based on hybridization, and the like; among them, the nucleic acid rapid detection technology based on amplification is widely applied to the fields of pathogen detection, genotyping and the like due to the advantages of sensitivity, high efficiency, rapidness, convenience and the like.
In the prior art, a commonly used nucleic acid detection device mainly comprises a PCR instrument, a constant-temperature nucleic acid amplification detector, a gel imaging system, a gene chip detection platform, a genomics sequencer and other large-scale instruments. The instruments have higher requirements on working environment and operators, and limit the application of the instruments in places with insufficient conditions such as the field, the basic level, the incident scene and the like; therefore, the conventional nucleic acid detection usually depends on a large-scale instrument, and needs to be improved in terms of integration, portability, environmental suitability, and the like.
Chinese patent publication No.: the publication 105823729A discloses an automatic nucleic acid detection device, which comprises a fixed seat, a main support plate vertically fixed on the fixed seat, an automatic sample reaction mechanism and a fixed frame thereof, a nucleic acid detection light path structure and a bearing frame thereof, and a controller; the sample reaction automatic mechanism fixing frame is fixed on the main supporting plate, and an outlet of the sample reaction automatic mechanism extends to the lower part of the fixing frame and is positioned outside the fixing frame body; the nucleic acid detection light path structure bearing frame is positioned below the outlet of the sample reaction automatic mechanism and is fixedly connected with the main supporting plate, and the inlet of the nucleic acid detection light path structure is connected with the outlet of the sample reaction automatic mechanism; the controller is arranged on the fixed seat and is respectively and electrically connected with the sample reaction automatic mechanism and the nucleic acid detection light path structure.
Among the above-mentioned technical scheme, on the one hand, detection device adopts automatic mechanism to test, supports through the support frame, and in the test procedure, it is higher to service environment requirement, and the measuring accuracy is difficult to guarantee.
On the other hand, since the detection structure employs a nucleic acid detection optical path structure and the inlet thereof is connected to the outlet of the sample reaction automation mechanism, it is difficult to mix and test the reaction solution as desired during actual testing because an integrated chip structure is not employed.
Finally, the detection device adopts the sample reaction automatic mechanism and the nucleic acid detection light path structure to be electrically connected, a power supply needs to be provided in real time, and the use environment is limited.
Disclosure of Invention
The invention aims to provide a micro-fluidic chip, a manual centrifugal device and a nucleic acid detection method, which are used for overcoming the technical defects.
In order to achieve the above object, the present invention provides a micro-control chip, which comprises at least one sample-adding layer and a channel layer disposed on the lower side of the sample-adding layer; wherein,
arranging a plurality of testing units which are distributed along the center and used for loading a reagent to be tested and transmitting the reagent to be tested to one chamber on the upper surface of the channel layer, wherein each testing unit comprises at least two chambers which are mutually connected through a channel;
the sample adding layer is provided with a plurality of sample adding holes corresponding to the channel layer along the central circumferential direction so as to inject a reagent into one chamber of the corresponding testing unit.
Further, the sample adding layer comprises an upper sample adding layer which is arranged at the upper side and is used for loading a reagent to a chamber close to the center in the test unit, and an upper sample adding hole is arranged on the upper sample adding layer;
the device also comprises a sample adding layer which is arranged on the lower side of the upper sample adding layer and is used for loading a reagent to the test unit far away from the central chamber, and a middle layer sample adding hole is arranged on the sample adding layer.
Furthermore, a plurality of first transfusion holes corresponding to one chamber of the testing unit of the channel layer and the upper sample adding hole of the upper sample adding layer are arranged in the sample adding layer along the central circumferential direction.
Furthermore, each test unit is provided with four chambers, a first chamber, a second chamber, a third chamber and a fourth chamber are sequentially arranged along the center of the channel layer towards the edge direction, the first chamber, the second chamber and the third chamber are respectively loaded with different reagents, and the reagents are introduced into the fourth chamber to be mixed under the action of rotating centrifugal force.
Further, the first chamber is loaded with medicines for pretreating the sample in the chip;
a primer for nucleic acid amplification is preloaded in the second chamber and is used for specific recognition of a target nucleic acid sequence;
the third chamber is preloaded with a mixture of reagents for nucleic acid amplification for specific amplification and detection of a target nucleic acid sequence.
Furthermore, a double-sided adhesive tape is arranged between the upper sample adding layer and the sample adding layer, the two layers are adhered, a second infusion hole for a reagent to pass through is formed in the double-sided adhesive tape, and the reagent injected from the upper sample adding layer is guided into the testing unit on the lower side.
Furthermore, the upper layer sample adding hole and the middle layer sample adding hole are both counter bores, the hole shoulders of the counter bores are provided with sample adding hole sealing materials, and the counter bores can prevent a gap between the sealing materials and the chip from generating a capillary action to enable a solution in the cavity to flow out.
Further, a channel is arranged between the adjacent chambers, and an expansion structure is arranged on the channel to prevent the added solution from being mixed in advance due to capillary action; the extension structure is a groove formed in the channel, the width of the groove is larger than that of the channel, and the depth of the groove is larger than that of the channel.
Furthermore, the first chamber close to the center of the circle in the chip can be pre-loaded with drugs such as zeolite and the like, and the drugs are used for combining substances such as protein, polysaccharide, lipid, inorganic salt, surfactant and the like in the sample lysate, so that the substances are prevented from inhibiting subsequent nucleic acid amplification, and the pretreatment of the sample in the chip is realized. The second chamber may be preloaded with nucleic acid amplification primers for specific recognition of a target nucleic acid sequence. The third chamber can be pre-loaded with a mixture of reagents for nucleic acid amplification, and the components of the reagents can comprise betaine, tween20, MgCl2, MnCl2, calcein, nucleic acid polymerase and the like, and the reagents can be used for specific amplification and detection of target nucleic acid sequences.
The invention also provides a manual centrifugal device with the microfluidic chip, which comprises a rotary disc for fixing the microfluidic chip, a centrifugal device body and a manual device arranged on the centrifugal device body; wherein,
the manual device includes: the micro-fluidic chip comprises a pull rod with teeth, an input gear and an output gear, wherein the input gear is meshed with the pull rod core, the output gear drives a rotating shaft to rotate, and the rotating shaft drives a micro-fluidic chip arranged on a rotary table to rotate.
The invention also provides a nucleic acid detection method with the microfluidic chip, which comprises the following steps:
step S1, in the liquid loading process, adding lysis solution consisting of alkali and sodium dodecyl sulfate and zeolite for preventing the lysis solution from inhibiting subsequent nucleic acid amplification into a sample to be detected, wherein a primer for nucleic acid amplification can be pre-loaded in the second chamber and used for specific identification of a target nucleic acid sequence, and a mixture of reagents for nucleic acid amplification can be pre-loaded in the third chamber and used for specific amplification and detection of the target nucleic acid sequence;
step S2, in the centrifugal process, the microfluidic chip is fixed in a manual centrifugal device, a pull rod of the manual pumping centrifugal device is rotated to enable a turntable to drive the chip to rotate, and in the rotation process, the liquids in the first chamber, the second chamber and the third recovery 123 are mixed and finally collected in a fourth chamber; then, mineral oil is used for filling the vacant space of each cavity, and adhesive tapes are used for sealing each sample adding hole;
step S3, in the reaction process, the portable warm paste is placed into the lower cavity of the manual centrifugal device and the upper space of the chip, the top cover of the device is covered, the temperature in the device is gradually increased and is maintained within the range of 63-68 ℃ for at least 2h, and the nucleic acid amplification reaction is realized in the process;
step S4, judging the process, opening the device after the reaction process, observing the color or fluorescence of the reaction liquid in the fourth chamber of the chip, and judging whether the amplification reaction occurs and whether the corresponding target sequence exists in the sample.
Compared with the prior art, the invention has the advantages that,
on one hand, the micro-fluidic chip provided with the sample adding layer and the channel layer is arranged, and sample adding is carried out through different sample adding layers according to different reagent loading requirements in the chip, so that the micro-fluidic chip is convenient to control. Furthermore, by arranging the plurality of sample adding holes distributed along the center and the testing units corresponding to the sample adding holes, different chambers correspond to different sample adding holes when reagents are loaded, and uniform sample adding is facilitated. In addition, the four chambers are arranged, different reagents are loaded respectively, centrifugal force is generated through rotation, the reagents are mixed according to the preset requirements, and the testing precision is high. The detection medicine can be preloaded in the cavity, so that the step of detecting the medicine adding sample is omitted, and the use is more convenient and flexible.
Furthermore, in order to prevent the additive reagent from flowing out of the sample adding holes, the upper layer sample adding hole and the middle layer sample adding hole are both counter-sunk holes, and liquid does not exist in the sinking part of the counter-sunk holes, so that the liquid in the solution cavity can be prevented from flowing out due to the capillary action generated by the gap between the sealing material and the chip. The invention arranges a channel between the adjacent chambers, and arranges an expansion structure on the channel to prevent the added solution from mixing in advance due to capillary action.
On the other hand, the manual centrifugal device disclosed by the invention is a portable device independent of an external power supply, integrates nucleic acid extraction, amplification and detection by utilizing a microfluidic chip technology, realizes rapid and convenient nucleic acid analysis, and is used for rapid response and field diagnosis of emergency events such as large-scale infectious diseases, food safety events, environmental pollution events and the like. The method has the advantages of no need of external power supply, good environmental adaptability, no complex operation and the like, and is particularly suitable for being applied to occasions with insufficient conditions such as the field, the basic level, the incident scene and the like.
Drawings
Fig. 1 is a schematic perspective view of a microfluidic chip according to the present invention;
FIG. 2 is a schematic diagram of an exploded structure of a microfluidic chip according to the present invention;
fig. 3 is a schematic front view of a channel layer of the microfluidic chip according to the present invention;
FIG. 4 is a schematic diagram of a front view of a single channel of the microfluidic chip of the present invention;
FIG. 5 is a schematic perspective view of a single channel of a microfluidic chip according to the present invention;
FIG. 6 is a schematic view of the internal perspective of the hand centrifuge of the present invention;
FIG. 7 is a schematic view of the internal perspective of the manual centrifuge with manual power means of the present invention;
FIG. 8 is a schematic diagram of the rotational speed test results of the manual centrifuge apparatus of the present invention;
FIG. 9 is a temperature profile of a microfluidic chip during nucleic acid amplification in the manual centrifugation apparatus according to the present invention;
FIG. 10 is a schematic diagram of the effect test of the manual centrifugal device and the microfluidic chip of the present invention on liquid manipulation;
FIG. 11 is a fluorescence photograph of the chip when the manual centrifugal device and the microfluidic chip of the present invention are used for nucleic acid extraction, amplification and detection of Staphylococcus aureus;
FIG. 12 is a schematic diagram showing the fluorescence intensity values after reaction in the process of extracting, amplifying and detecting nucleic acid of Pseudomonas aeruginosa by using the manual centrifugal device and the microfluidic chip of the invention.
Detailed Description
The above and further features and advantages of the present invention are described in more detail below with reference to the accompanying drawings.
Preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only for explaining the technical principle of the present invention, and are not intended to limit the scope of the present invention.
It should be noted that in the description of the present invention, the terms of direction or positional relationship indicated by the terms "upper", "lower", "left", "right", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings, which are only for convenience of description, and do not indicate or imply that the device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
Furthermore, it should be noted that, in the description of the present invention, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
Fig. 1-2 show a schematic diagram of a three-dimensional structure and an explosion structure of a microfluidic chip according to the present invention; comprises an upper sample adding layer 21, a double-sided adhesive tape 22, a sample adding layer 23 and a channel layer 11 which are arranged from top to bottom in sequence. Wherein, a plurality of testing units 12 distributed along the center are arranged on the upper surface 13 of the channel layer 11 to load the testing agent and transfer the testing agent to one chamber, and each testing unit 12 comprises at least two chambers connected with each other through a channel 125; the middle of the channel layer 11 is provided with a first through hole 15 for the rotation shaft to pass through and a first connection hole 14 connected with the middle sample adding layer 23.
With continued reference to fig. 2, the upper sample addition layer 21 includes: the third through hole 223 is arranged in the middle for the rotating shaft to pass through, and a plurality of upper sample adding holes 211 corresponding to the channel layer 11 are arranged along the central circumferential direction, so as to inject a reagent into one of the corresponding chambers of the test unit 12. In this embodiment, the transverse cross-section of the upper sample adding layer 21, the double-sided adhesive tape 22, the sample adding layer 23 and the channel layer 11 is circular, so that a plurality of sample adding holes or test units 12 are conveniently arranged along the central ring direction, enough space is provided for the arrangement of the sample adding holes, and the sample adding holes are arranged along the circumferential direction, so that the reagent is conveniently injected into the test units 12. It can be understood by those skilled in the art that the sample application holes or test units 12 correspond to each other, and the surface areas of the upper sample application layer 21, the double-sided tape 22, the sample application layer 23 and the channel layer 11 and the sample application holes or test units 12 can be adjusted according to the carrying capacity of the chip and the amount of the test reagents.
With continued reference to fig. 2, the middle sample-loading layer 23 includes: a plurality of middle layer sample adding holes 231 corresponding to the channel layer 11 are arranged along the central circumferential direction so as to inject a reagent into one chamber of the corresponding test unit 12; a plurality of first infusion holes 232 corresponding to one chamber of the test unit 12 of the channel layer 11 and the upper sample adding hole 211 of the upper sample adding layer 21 are arranged along the central circumferential direction; when reagent is injected through the upper sample addition hole 211, the reagent firstly passes through the upper sample addition layer 21, and then flows downwards through the first infusion hole 232 on the double-sided tape and the sample addition layer 23 to enter one chamber in the test unit 12. The second connection hole 234 is provided on the middle sample addition layer 23 corresponding to the first connection hole 14 connected to the middle sample addition layer 23. It can be understood by those skilled in the art that the upper sample adding layer and the channel layer can be directly disposed in the present embodiment, and there is no need to dispose the middle sample adding layer, and similarly, a plurality of middle sample adding layers 23 can be disposed, and it is only necessary to dispose the infusion hole for the upper sample adding layer to pass through and the sample adding hole for introducing liquid to the lower side on each middle sample adding layer.
Specifically, the surface area of the upper sample addition layer 21 is smaller than that of the middle sample addition layer 23, and the surface area of the sample addition layer 23 is the same as that of the channel layer 11; the upper layer sample adding hole 211 and the first infusion hole 232 which are circumferentially arranged are closer to the center of the chip than the middle layer sample adding hole 231, and different reagents can be loaded into different chambers when different reagents are injected into corresponding chambers of the test unit through the upper layer sample adding hole 211 and the middle layer sample adding hole 231 respectively, and the different chambers can be subjected to sequential mixing reaction along the arrangement sequence of the chambers.
Referring to fig. 2, a double-sided tape 22 is disposed between the upper sample addition layer 21 and the sample addition layer 23, the two layers are bonded to each other, a second infusion hole 221 through which a reagent passes is disposed thereon, and the reagent injected from the upper sample addition layer 21 is introduced into the lower test unit 12. The microfluidic chip of the embodiment can be manufactured by assembling patterned PDMS, PMMA and PC layer by layer, the top layer is reserved with a sample adding hole for introducing a subsequent sample, and the bottom channel layer can be made of glass, PMMA, PDMS, PC or silicon chip. In order to prevent the additive reagent from flowing out of the sample adding hole, the upper layer sample adding hole 211 and the middle layer sample adding hole 231 are both countersunk holes, and no liquid exists in the sunken part of the countersunk holes, so that the liquid in the solution cavity can be prevented from flowing out due to the capillary action generated by the gap between the sealing material and the chip.
Referring to fig. 3-5, there are shown a schematic front view structure diagram of a channel layer of the microfluidic chip, a schematic front view structure diagram of a single channel, and a schematic three-dimensional structure diagram of the single channel, respectively; each test unit 12 of this embodiment is provided with four chambers, which are a first chamber 121, a second chamber 122, a third chamber 123, and a fourth chamber 124 in sequence along the center to the edge direction of the channel layer, wherein the first, second, and third chambers are loaded with different reagents respectively, and the reagents are introduced into the fourth chamber 124 for mixing under the action of rotational centrifugal force. In order to extract, amplify and detect target nucleic acid, zeolite may be pre-loaded in the first chamber 121 for binding proteins, polysaccharides, lipids, inorganic salts, surfactants and other substances in the sample lysate, and preventing the substances from inhibiting subsequent nucleic acid amplification; a primer for nucleic acid amplification can be preloaded in the second chamber 122 for specific recognition of a target nucleic acid sequence; the third chamber 123 may be pre-loaded with a mixture of reagents for nucleic acid amplification, which may include betaine, tween20, MgCl2, MnCl2, calcein, nucleic acid polymerase, etc., for specific amplification and detection of a target nucleic acid sequence. And the reagent is centrifuged, and the mixed reagent in the three chambers is introduced into the fourth chamber for detection. The invention is not limited to the number of the pre-filled medicines and the number of the chambers, and technicians can change the types of the pre-filled medicines and the number of the chambers according to actual requirements, thereby achieving the purpose of detection.
With continued reference to fig. 4, a channel 125 is disposed between the adjacent chambers, and an expanding structure 126 is disposed on the channel 125 to prevent the added solution from being mixed in advance due to capillary action, in this embodiment, the expanding structure 126 is a groove formed on the channel 125, the width of the groove is greater than the width of the channel 125, and the depth of the groove is greater than the depth of the channel 125, so that the expanding structure can prevent the solution from being mixed in advance in the left-right direction and the lower direction.
Referring to fig. 6-7, there are shown schematic internal perspective views of a manual centrifuge apparatus of the present invention and a schematic internal perspective view of a manual centrifuge apparatus with a manual power device; manual centrifugal device is including fixed above-mentioned chip 1's carousel and centrifugal device body, the manual device of setting on the centrifugal device body, the centrifugal device body includes: the reaction device comprises a heat insulation layer 32 arranged on the periphery and a shell 33 arranged in the heat insulation layer 32, wherein an air inlet 31 is arranged in the middle of the shell 33, an air outlet (not shown) is arranged on the upper end surface of the shell, and the shell and the heat insulation layer control the temperature of the reaction environment. The chip 1 is arranged at the upper part of the shell, the upper layer warm paste 30 is arranged at the upper end of the chip 1, and the support frame 36 is arranged at the lower end of the chip 1 to support the chip; the lower layer of warm paste 34 is also arranged below the support frame 36, and can be the upper layer of warm paste 30 or a component with a heating function. The manual device is used for driving the chip to rotate, and comprises a support 38 arranged at the upper end of the heat insulation layer 32 and a pull rod 37 connected with the support 38 and driving the support to rotate, wherein teeth arranged on the pull rod are meshed with an input gear 351, an output gear (not shown in the figure) is arranged inside the support, a connecting shaft 352 of the output gear is connected with the support 38, the support 38 drives the output gear to rotate, and the output gear is meshed with the input gear 351 and drives the input gear to rotate; meanwhile, the input gear 351 is fixed on the turntable, the turntable rotates and drives the chip 1 to rotate along the connecting shaft 351, and the reagents in each chamber in the test unit 12 are all guided into the outermost chamber under the centrifugal rotation effect. As can be understood by those skilled in the art, the gear is driven to rotate by the rack; a hand wheel can be arranged at the upper end of the shell to drive the rotating shaft to rotate to provide power, or the power is provided through a coil spring, or the rotation of the driving gear directly drives the chip to rotate through a motor, so that centrifugal force is generated.
Referring to fig. 8, when the manual centrifugal device according to the embodiment of the present invention is used, the pull rod on the device is pulled to rotate the turntable, the rotational speed of the turntable in the device is measured in real time by using the tachometer, and the measurement result shows that the turntable continuously rotates within 8min, the maximum rotational speed can reach 2922rpm, which is shown as the change of the rotational speed of the turntable within 5 min.
Referring to fig. 9, the manual centrifugal device according to the embodiment of the present invention is used to fix the microfluidic chip 1 on the turntable, and the portable warming patch is placed in the device, and one warming patch is placed in the lower cavity, and three small warming patches are flatly laid on the chip. And placing the temperature measuring probe on the upper surface of the chip, and measuring the temperature of the chip in real time. The measurement result shows that the temperature of the chip can be raised to 63 ℃ within 1.5 hours, and the temperature can be continuously maintained within the range of 63-68 ℃ for more than 2 hours. The real-time temperature measurement results are shown in FIG. 6.
The process of realizing the nucleic acid detection method based on the microfluidic chip-level manual centrifugal device comprises the following steps:
step S1, in the liquid loading process, adding lysis solution consisting of alkali and sodium dodecyl sulfate and zeolite for preventing the lysis solution from inhibiting subsequent nucleic acid amplification into a sample to be detected, wherein a primer for nucleic acid amplification can be pre-loaded in the second chamber for specific identification of a target nucleic acid sequence, and a mixture of reagents for nucleic acid amplification can be pre-loaded in the third chamber 123 for specific amplification and detection of the target nucleic acid sequence;
step S2, in the centrifugal process, the microfluidic chip is fixed in a manual centrifugal device, a pull rod of the manual pumping centrifugal device is rotated to enable a turntable to drive the chip to rotate, and in the rotation process, the liquids in the first chamber 121, the second chamber 122 and the third recovery 123 are mixed and finally collected in a fourth chamber; thereafter, the empty space of each chamber was filled with mineral oil, and each well was closed with tape.
Step S3, in the reaction process, the portable warm paste is placed into the lower cavity of the manual centrifugal device and the upper space of the chip, the top cover of the device is covered, the temperature in the device is gradually increased and is maintained within the range of 63-68 ℃ for at least 2h, and the nucleic acid amplification reaction is realized in the process.
Step S4, a determination process, in which the device is turned on after the completion of the above reaction process, and the color or fluorescence of the reaction solution in the chip chamber 4 is observed, thereby determining whether the amplification reaction occurs and whether the target sequence exists in the corresponding sample.
The method for detecting a nucleotide according to this example is described below by way of examples.
Example one
The manual centrifugal device and the microfluidic chip of the embodiment of the invention were tested for the manipulation capability of liquid by using an aqueous pigment to simulate a nucleic acid detection reaction reagent.
Step S1, a liquid filling process, namely filling zeolite and a preinstalled simulation reagent into the first chamber, the second chamber and the third chamber, and introducing a simulation sample mixture into the first chamber;
step S2, in the centrifugation process, the microfluidic chip is fixed in the manual centrifugation device, and the pull rod of the centrifugation device is manually pulled to rotate the turntable, so that the chips are driven to rotate, and in the rotation process, the liquids in the first chamber 121, the second chamber 122, and the third recovery 123 are mixed and finally collected in the fourth chamber.
Referring to fig. 10, it is observed that the pre-loaded mock reagent and the introduced mock sample are present in their respective chambers, well separated from each other, without mixing of the liquids, prior to centrifugation. Indicating that the use of the chip allows good separation of the pre-loaded reagent and sample solution. After centrifugation, the liquids in the first, second and third chambers are collected in the chamber 4 and completely mixed, which indicates that the chip can realize complete mixing of the pre-loaded reagent and the sample solution after centrifugation, thereby ensuring the smooth operation of the nucleic acid amplification reaction on the chip. The results show that the manual centrifugal device and the microfluidic chip can realize better liquid control, and simultaneously, the sample and the reaction reagent in each test unit are almost simultaneously mixed by a centrifugal touch mixing mechanism, so that the nucleic acid amplification reaction in each test unit is ensured to be simultaneously carried out, and the influence of the reaction time among different samples on the nucleic acid amplification and detection results is eliminated.
Example two
To a sample containing Staphylococcus aureus, lysate (0.2N NaOH, 1% SDS) was added. A microfluidic chip pre-loaded with zeolite and reaction reagents is used, after which the sample mixture containing the lysis buffer is introduced into the first chamber. The above steps S1-S4 were repeated, using ultrapure water as a negative control. The microfluidic chip is fixed in a manual centrifugal device, and a pull rod of the centrifugal device is manually pulled, so that the rotary disc drives the chip to rotate. Thereafter, the empty space of each chamber was filled with mineral oil, and each well was closed with tape. The portable warm patch is placed into the lower cavity of the manual centrifugal device and the upper space of the chip, and the top cover of the device is covered to start the nucleic acid amplification reaction. After 4 hours, the apparatus was opened, and the fluorescence of the reaction solution in the chip chamber 4 was observed.
Referring to fig. 11, a fluorescence photograph of the chip when the manual centrifugation device and the microfluidic chip of the present invention are used for extracting, amplifying and detecting nucleic acid of staphylococcus aureus, wherein the sample containing staphylococcus aureus on the left side of the drawing shows green fluorescence, and the negative control group on the right side of the drawing does not show fluorescence.
EXAMPLE III
Lysis solution (0.2N NaOH, 1% SDS) was added to the samples containing P.aeruginosa. A microfluidic chip pre-loaded with zeolite and reaction reagents is used, after which the sample mixture containing the lysis buffer is introduced into the first chamber. The above steps S1-S4 were repeated, using ultrapure water as a negative control. Ultrapure water was used as a negative control. The microfluidic chip is fixed in a manual centrifugal device, and a pull rod of the centrifugal device is manually pulled, so that the rotary disc drives the chip to rotate. Thereafter, the empty space of each chamber was filled with mineral oil, and each well was closed with tape. The portable warm patch is placed into the lower cavity of the manual centrifugal device and the upper space of the chip, and the top cover of the device is covered to start the nucleic acid amplification reaction. After 4 hours, the device was turned on, the fluorescence of the reaction solution in the chip chamber 4 was photographed using a cell phone, and the fluorescence intensity was measured using image processing software.
Referring to FIG. 12, which is a schematic diagram of fluorescence intensity values after reaction when the manual centrifugal device and the microfluidic chip of the present invention are used for nucleic acid extraction, amplification and detection of Pseudomonas aeruginosa, the fluorescence intensity of a sample containing Pseudomonas aeruginosa is significantly higher than that of a negative control group, indicating the presence of a characteristic sequence in the sample.
So far, the technical solutions of the present invention have been described in connection with the preferred embodiments shown in the drawings, but it is easily understood by those skilled in the art that the scope of the present invention is obviously not limited to these specific embodiments. Equivalent changes or substitutions of related technical features can be made by those skilled in the art without departing from the principle of the invention, and the technical scheme after the changes or substitutions can fall into the protection scope of the invention.
Claims (10)
1. A microfluidic chip is characterized by comprising at least one sample adding layer and a channel layer arranged on the lower side of the sample adding layer; wherein,
arranging a plurality of testing units which are distributed along the center and used for loading a reagent to be tested and transmitting the reagent to be tested to one chamber on the upper surface of the channel layer, wherein each testing unit comprises at least two chambers which are mutually connected through a channel;
the sample adding layer is provided with a plurality of sample adding holes corresponding to the channel layer along the central circumferential direction so as to inject a reagent into one chamber of the corresponding testing unit.
2. The microfluidic chip according to claim 1, wherein the sample application layer comprises an upper sample application layer disposed on an upper side to apply a reagent to a chamber near the center in the test unit, on which an upper sample application hole is disposed;
the device also comprises a sample adding layer which is arranged on the lower side of the upper sample adding layer and is used for loading a reagent to the test unit far away from the central chamber, and a middle layer sample adding hole is arranged on the sample adding layer.
3. The microfluidic chip according to claim 2, wherein the sample loading layer is provided with a plurality of first infusion wells along a central circumferential direction, the first infusion wells corresponding to one of the chambers of the test unit of the channel layer and the sample loading well on the upper layer of the upper sample loading layer.
4. The microfluidic chip according to claim 3, wherein each of the test units has four chambers, and the four chambers are sequentially a first chamber, a second chamber, a third chamber and a fourth chamber along the center of the channel layer toward the edge, wherein the first chamber, the second chamber and the third chamber are loaded with different reagents respectively, and the reagents are introduced into the fourth chamber to be mixed under the action of a rotating centrifugal force.
5. The microfluidic chip according to claim 4, wherein the first chamber is loaded with a drug for pre-treatment of a sample in the chip;
a primer for nucleic acid amplification is preloaded in the second chamber and is used for specific recognition of a target nucleic acid sequence;
the third chamber is preloaded with a mixture of reagents for nucleic acid amplification for specific amplification and detection of a target nucleic acid sequence.
6. The microfluidic chip according to claim 2, wherein a double-sided adhesive tape is disposed between the upper sample loading layer and the sample loading layer, the two layers are adhered to each other, a second infusion hole is disposed thereon for passing a reagent, and the reagent injected from the upper sample loading layer is introduced into the lower test cell.
7. The microfluidic chip according to claim 2, wherein the upper layer well and the middle layer well are both counter-sunk holes, and a well sealing material is disposed on a shoulder of the counter-sunk holes, and the counter-sunk holes are used to prevent a gap between the sealing material and the chip from generating a capillary action to allow a solution in the chamber to flow out.
8. The microfluidic chip according to claim 2, wherein a channel is provided between the adjacent chambers, and an extension structure is provided on the channel to prevent premature mixing of the added solutions due to capillary action; the extension structure is a groove formed in the channel, the width of the groove is larger than that of the channel, and the depth of the groove is larger than that of the channel.
9. A manual centrifugation device having the microfluidic chip according to any one of claims 1 to 8, comprising a turntable for fixing the microfluidic chip, a centrifugation device body, and a manual device disposed on the centrifugation device body; wherein,
the manual device includes: the micro-fluidic chip comprises a pull rod with teeth, an input gear and an output gear, wherein the input gear is meshed with the pull rod core, the output gear drives a rotating shaft to rotate, and the rotating shaft drives a micro-fluidic chip arranged on a rotary table to rotate.
10. A method for detecting nucleic acid having the microfluidic chip according to any one of claims 1 to 8, comprising:
step S1, in the liquid loading process, adding lysis solution consisting of alkali and sodium dodecyl sulfate and zeolite for preventing the lysis solution from inhibiting subsequent nucleic acid amplification into a sample to be detected, wherein a primer for nucleic acid amplification can be pre-loaded in the second chamber and used for specific identification of a target nucleic acid sequence, and a mixture of reagents for nucleic acid amplification can be pre-loaded in the third chamber and used for specific amplification and detection of the target nucleic acid sequence;
step S2, in the centrifugal process, fixing the microfluidic chip in a manual centrifugal device, rotating a pull rod for pulling the centrifugal device manually to enable a turntable to drive the chip to rotate, wherein in the rotation process, the liquids in the first chamber, the second chamber and the third chamber are mixed and finally collected in a fourth chamber; then, mineral oil is used for filling the vacant space of each cavity, and adhesive tapes are used for sealing each sample adding hole;
step S3, in the reaction process, the portable warm paste is placed into the lower cavity of the manual centrifugal device and the upper space of the chip, the top cover of the device is covered, the temperature in the device is gradually increased and is maintained within the range of 63-68 ℃ for at least 2h, and the nucleic acid amplification reaction is realized in the process;
step S4, judging the process, opening the device after the reaction process, observing the color or fluorescence of the reaction liquid in the fourth chamber of the chip, and judging whether the amplification reaction occurs and whether the corresponding target sequence exists in the sample.
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