CN108823089B - Nucleic acid detection chip based on multiple nucleic acid reactions - Google Patents
Nucleic acid detection chip based on multiple nucleic acid reactions Download PDFInfo
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- CN108823089B CN108823089B CN201810585842.6A CN201810585842A CN108823089B CN 108823089 B CN108823089 B CN 108823089B CN 201810585842 A CN201810585842 A CN 201810585842A CN 108823089 B CN108823089 B CN 108823089B
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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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- 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]
Abstract
The invention relates to a nucleic acid detection chip based on multiple nucleic acid reactions, which comprises an upper chip layer, a lower chip layer and a middle chip layer, wherein the upper chip layer, the middle chip layer and the lower chip layer are sequentially arranged, an FTA card is arranged in the middle chip layer, and the middle chip layer comprises: a sample chamber unit disposed at an intermediate position, storing the nucleic acid samples sequentially added, and dispersing the nucleic acid samples to the corresponding chambers under the rotation; a first siphon valve communicating with the sample chamber unit and introducing a prescribed nucleic acid sample into the reaction chamber unit by a siphon phenomenon; and a reagent storage and introduction unit which communicates with the reaction chamber unit and in which a reaction reagent corresponding to a predetermined nucleic acid is stored, wherein the reaction chamber unit is introduced with the nucleic acid sample, and then the reaction chamber unit is introduced with the reaction reagent to react the reaction reagent with the nucleic acid sample. The device has high automation degree, and the operator can realize the detection of nucleic acid only by single sample adding, thereby having high control precision.
Description
Technical Field
The invention relates to the field of nucleic acid detection, in particular to a nucleic acid detection chip based on multiple nucleic acid reactions.
Background
In the prior art, with the rapid development of gene sequencing technology, gene sequencing has been developed from traditional gene chip sequencing to current high-throughput gene sequencing. Currently, gene sequencing systems and devices have made great progress in various aspects, but their development is still in the initial stage, and various aspects still need continuous improvement and innovation.
A gene sequencing device in the prior art is an instrument for measuring gene sequences, and comprises a data processing device and a nucleic acid detection device. The data processing device is connected with the nucleic acid detection device and used for providing a user interface, controlling the work of the nucleic acid detection device according to a control instruction and displaying the work state of the nucleic acid detection device; the nucleic acid detection device is used for carrying out gene sequencing. Wherein the nucleic acid detecting apparatus comprises: the sequencing reaction chamber is connected with the liquid transmission component, the temperature control component and the displacement component respectively and is used for carrying out sequencing reaction; the liquid transmission component is used for transmitting liquid to the sequencing reaction chamber according to a control instruction; the temperature control assembly is used for adjusting the temperature of the sequencing reaction chamber; the displacement assembly is used for adjusting the position of the sequencing reaction chamber; the image acquisition component is used for acquiring a sequencing image of the sequencing reaction chamber. In this technical solution, the user can only know the operating state of each component of the nucleic acid detecting apparatus by operating the data processing apparatus, which is inconvenient to use.
However, the nucleic acid detecting apparatus in the related art can mix and detect only a single kind of nucleic acid, and particularly cannot automatically control different reagents for different reaction temperatures and times so that different nucleic acids automatically react according to actual reaction timing and temperatures.
Disclosure of Invention
The present invention is directed to a nucleic acid detecting chip based on multiple nucleic acid reactions, which overcomes the above-mentioned drawbacks.
In order to achieve the above object, the present invention provides a nucleic acid detection chip based on multiple nucleic acid reactions, comprising an upper chip layer, a lower chip layer and a middle chip layer, wherein the upper chip layer, the middle chip layer and the lower chip layer are sequentially disposed, an FTA card is disposed in the middle chip layer, and the middle chip layer comprises: a sample chamber unit disposed at an intermediate position, storing the nucleic acid samples sequentially added, and dispersing the nucleic acid samples to the corresponding chambers under the rotation; a first siphon valve communicating with the sample chamber unit and introducing a prescribed nucleic acid sample into the reaction chamber unit by a siphon phenomenon; a reagent storage/introduction unit which is connected to the reaction chamber unit and in which a reaction reagent corresponding to a predetermined nucleic acid is stored, and which introduces the reaction reagent into the reaction chamber unit after the nucleic acid sample is introduced into the reaction chamber unit, thereby allowing the reaction reagent to react with the nucleic acid sample; the reaction chamber unit is arranged at the edge position, mixes the specified nucleic acid sample with the reagent and carries out nucleic acid reaction, the reaction chamber of the reaction chamber unit is also communicated with a second siphon valve, after the reaction chamber unit tests one nucleic acid sample, the reaction chamber unit is cleaned, and another nucleic acid sample is tested;
the sample chamber unit comprises a sample chamber arranged in the middle, a sample shunting channel communicated with the sample chamber and arranged on the periphery of the sample chamber, and a plurality of sample halving chambers arranged on the outer side of the sample shunting channel and communicated with the sample shunting channel, wherein a nucleic acid sample is subjected to centrifugal force from the sample chamber at a first preset rotating speed, is discharged into the sample shunting channel, flows in the sample shunting channel, continues to move outwards under the action of the centrifugal force, and respectively enters each sample halving chamber, the sample shunting channel is a multi-semicircular ring, a first liquid guide pipe is arranged at the tail end of the multi-semicircular ring, and the tail end of the first liquid guide pipe is communicated with a first waste liquid chamber;
the reagent storage and introduction unit comprises a paraffin chamber close to the central part, paraffin is loaded in the paraffin chamber, and the paraffin chamber plays a role of blocking at low temperature; one end of the paraffin chamber is also communicated with a third exhaust channel, so that liquid in the channel smoothly flows.
Furthermore, the sample chamber is of an eccentric structure, the nucleic acid samples are collected to the side with the larger diameter in the rotating process of the sample chamber, and the nucleic acid samples are collected to the direction of the notch and flow to the sample shunting channel from the notch; the outer side wall of the sample chamber is an arc-shaped wall, the outer side wall of the sample chamber gradually extends towards the circle center side from the opening along the clockwise direction to form an eccentric structure of the sample chamber, and the end part of the arc-shaped wall is the position closest to the circle center of the sample chamber.
Further, the nucleic acid sample flows upwards to the sample shunting channel through the gap, a step surface is arranged between the sample shunting channel and the sample chamber, the height of the sample shunting channel is higher than that of the sample chamber, and meanwhile, the height of the sample equal-division chamber is higher than that of the sample shunting channel.
Further, the tail end of each sample halving chamber is connected and communicated with a first siphon valve for introducing the nucleic acid sample in the sample halving chamber into the reaction chamber, when the sample chamber rotates at a first preset speed, the nucleic acid sample enters the sample halving chamber, and the redundant sample enters a first waste liquid chamber; the sample chamber stops rotating and the sample in the sample aliquoting chamber flows into and fills the first siphon valve.
Further, first siphon valve include with sample partition room intercommunication and the first siphon section of outside extension, with first siphon section intercommunication and along the second siphon section that circumference was arranged, with the second siphon section of second siphon section and the third siphon section of inside extension, set up side by side with the third siphon section and the fourth siphon section of outside extension, be the arc section between third siphon section and fourth siphon section, it realizes the transition between third siphon section and fourth siphon section, still include the fifth siphon section of outside extension of slope with fourth siphon section intercommunication, with the sixth siphon section of radial outside extension of fifth siphon section intercommunication, the end of sixth siphon section with the first reservoir intercommunication of reaction chamber.
Further, the device still include with the second siphon valve of reaction chamber intercommunication, the second siphon valve includes with the reaction chamber intercommunication and along the seventh siphon section of radial to outer extension, with the eighth siphon section intercommunication of seventh siphon section along circumference, with the ninth siphon section of eighth siphon section intercommunication and along radial inside side extension to and set up side by side with the ninth siphon section, along the tenth siphon section of radial outside extension, still be provided with the arc section of transition between ninth siphon section and tenth siphon section, the end and the second waste liquid room intercommunication of tenth siphon section.
Further, the reagent storage introduction unit includes a paraffin chamber near a central portion, in which paraffin is loaded, as a driving source at a low temperature; and one end of the paraffin chamber is also communicated with a third exhaust channel for exhausting the gas in the paraffin chamber.
Further, a second mineral oil chamber, an LAMP reagent chamber and a primer chamber are sequentially arranged on the second branch from inside to outside along the radial direction of the chip, the second mineral oil chamber, the LAMP reagent chamber and the primer chamber are communicated through pipelines, mineral oil, the LAMP reagent and primers are respectively stored in the second mineral oil chamber, one end of the primer chamber is communicated with the reaction chamber through a third pipeline, the mineral oil is melted after paraffin is heated, the mineral oil is driven by centrifugal force to flow, and then the mineral oil pushes the LAMP reagent and the primers to enter the reaction chamber.
Furthermore, the first siphon valve is arranged on the bottom surface of the middle layer of the chip, and the second siphon valve is arranged on the top surface of the middle layer of the chip.
Compared with the prior art, the reaction chip has the advantages that the reaction chip comprises a sample chamber unit arranged in the middle position, stores the added nucleic acid sample, and equally divides the nucleic acid sample under the rotation action; at least two first siphon valves communicating with the sample chamber unit and introducing a nucleic acid sample into the reaction chamber unit by a siphon phenomenon, wherein the first siphon valves are disposed on a lower side of the layer in the chip; and a reaction chamber unit which is arranged at the edge position of the middle layer of the chip and is communicated with the reagent storage and introduction unit. The chip can realize the function of nucleic acid detection, can detect various blood viruses, has high automation degree, and can realize the detection of nucleic acid only by single sample adding of an operator; a single chip can detect multiple virus nucleic acids simultaneously, and researchers can increase or decrease detection units according to actual use requirements; the integrated sample pretreatment system on the chip has low requirements on the skills of operators and the environment for use, and does not need a special nucleic acid detection laboratory; a paraffin valve and a capillary valve are designed on the chip, and the opening and closing of the valves can be realized by controlling the temperature and the rotating speed, so that the sequential flow of different samples is controlled; the temperature control system arranged on the detection equipment can realize temperature control aiming at LAMP reaction and PCR reaction, the temperature required by LAMP reaction is kept at 65 ℃, the PCR reaction needs to realize periodic change of the temperature, and the heating chip and the cooling chip are informed to realize the alternate change of the temperature under the combined action.
Particularly, the sample chamber has an eccentric structure by arranging the involute shape on the side wall of the sample chamber, so that the movement of the nucleic acid sample under the centrifugal action is enhanced, and particularly, the nucleic acid sample in the flow rapidly rushes into the sample shunting channel in a superfine narrow channel by forming gaps at two ends of the involute-shaped side wall of the sample chamber so as to rapidly shunt the nucleic acid sample. Furthermore, the height that the sample shunted the passageway is higher than the height of sample room, simultaneously, the height that the sample partition room is higher than the height that the sample shunted the passageway, through setting up the structure that rises in proper order, avoid the nucleic acid sample to produce irregular flow when not rotating, moreover, through the structure that rises gradually, the rotational speed through controlling the sample room that can be convenient realizes appointed nucleic acid sample flow, if discharge the nucleic acid sample into sample partition passageway from the sample room through first predetermined rotational speed, discharge sample partition room into the sample partition room through the second predetermined rotational speed, convenient control.
Further, the first siphon valve of the present invention extends from the sample aliquoting chamber through to the radially inner side of the chip and the radially outer side of the chip until communicating with the reaction chamber; the second siphon valve extends from the reaction chamber to the radial inner side and the radial outer side of the chip until the second siphon valve is communicated with the second waste liquid chamber. According to the invention, a nucleic acid sample enters a sample halving chamber at a first preset speed, the sample enters a first siphon valve by stopping, the sample in the sample halving chamber enters a reaction chamber through the first siphon valve by stopping, waste liquid cannot pass through a second siphon valve at a second preset speed, the waste liquid is filled in the second siphon valve by stopping, capillary force is used for discharging the waste liquid into a second waste liquid chamber through the second siphon valve by stopping, therefore, the nucleic acid sample sequentially reaches preset positions by intermittent rotation and stopping, and the designated action is sequentially completed, so that the control is accurate and continuous.
Further, the paraffin wax is melted at low temperature and high temperature, the plugging reaction reagent and the primers are respectively stored, after melting, the reaction reagent and the primers can be moved by centrifugal force to enter the reaction chamber for reaction, and meanwhile, the mineral oil at the two ends of the reaction chamber is used for plugging, so that the normal reaction of the reaction chamber is ensured. Because only need can accomplish the state conversion through the heating, the automatic reaction that reacts, simultaneously, set up the second siphon valve on reaction chip intermediate level, the mineral oil of one of them branch road passes through the second siphon valve shutoff reaction chamber from one end, and another branch road pushes reaction reagent and primer into the reaction chamber reaction under centrifugal force's promotion to through the mineral oil shutoff, realize automatic reaction, self-sealing's effect.
Further, in the invention, a first siphon valve for introducing a nucleic acid sample is arranged on the bottom surface of the middle layer of the chip, a reagent storage and introduction unit for storing and introducing a reaction reagent and a second siphon valve are arranged on the top surface of the middle layer of the chip, and liquid introduction structures are arranged on the front surface and the back surface of the middle layer of the chip, so that the space of the middle layer of the chip is saved, meanwhile, the interference among liquids is avoided, and the reaction control is accurate.
Drawings
FIG. 1 is a schematic diagram showing the structure of the upper layer of a nucleic acid detecting chip according to the present invention, which is based on a plurality of nucleic acid reactions;
FIG. 2 is a schematic diagram showing the structure of the lower layer of the nucleic acid detecting chip according to the present invention;
FIG. 3 is a schematic diagram of the overall structure of the chip of the present invention;
FIG. 4 is a schematic diagram showing a first structure of a layer in a nucleic acid detecting chip according to the present invention;
FIG. 5 is a schematic diagram showing the structure of the middle layer of the chip with the first siphon valve of the nucleic acid detecting chip based on multiple nucleic acid reactions according to the present invention;
FIG. 6 is a diagram showing a second structure of a layer in the chip of the nucleic acid detecting chip according to the present invention;
FIG. 7 is a schematic diagram of the construction of a sample cell unit of the present invention;
FIG. 8 is a schematic view of a first siphon valve according to the present invention;
FIG. 9 is a schematic diagram of a reagent storage and introduction unit in a layer of a chip according to the present invention;
FIG. 10 is a schematic view of the structure of a reaction chamber according to the present invention;
FIG. 11 is a first schematic diagram of a chip reaction according to the present invention;
FIG. 12 is a second schematic diagram of a chip reaction according to the present invention;
FIG. 13 is a third schematic diagram of a chip reaction according to the present invention;
FIG. 14 is a fourth schematic diagram of a chip reaction according to the present invention;
FIG. 15 is a schematic view showing the structure of a nucleic acid detecting apparatus according to the present invention;
FIG. 16 is a schematic view of a half-sectional structure of a nucleic acid detecting apparatus of the present invention;
FIG. 17 is a schematic structural view of a temperature control module of the nucleic acid detecting apparatus according to the present 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.
Please refer to fig. 1, 2, and 3, which are schematic structural diagrams of an upper chip layer and a lower chip layer of the nucleic acid detecting reagent mixing and adding device of the present invention, respectively; and the overall structure schematic diagram of the chip. The whole structure of the chip of this embodiment includes chip upper strata 1, chip lower floor 3 and chip middle floor 2, and chip upper strata 1, chip middle floor 2 and chip lower floor 3 set up in order to set up FTA card 4 and the relevant structure of loading reagent, nucleic acid in chip middle floor 2. An upper layer positioning hole 13 is arranged on the upper layer 1 of the chip, a lower layer positioning hole 33 is arranged on the lower layer 3 of the chip, and three layers of chips are fixed into a whole through vacuum thermal bonding. The upper layer 1 of the chip is provided with an upper layer exhaust hole 12 for exhausting gas in the chip. The middle part of the upper layer chip 1 is also provided with a sample adding hole 11 for adding a nucleic acid sample into the layer 2 of the chip, and the chip mixes the nucleic acid with a reagent to be added through rotation.
FIGS. 4, 5 and 6 are schematic diagrams showing a first structure of a middle layer of a chip, a middle layer of a chip with a first siphon valve, and a second structure of a middle layer of a chip of a nucleic acid detecting reagent mixing and adding device according to the present invention. The chip layer 2 of the present embodiment includes: a sample chamber unit 21 disposed at an intermediate position, which stores the added nucleic acid sample and equally divides the nucleic acid sample under rotation; at least two first siphon valves 26 communicating with the sample chamber unit and introducing the nucleic acid sample into the reaction chamber unit by a siphon phenomenon, wherein the first siphon valves 26 are provided on the lower side of the layer 2 in the chip; a reaction chamber unit provided at an edge position of the layer 2 in the chip, which mixes the nucleic acid sample with the reagent and performs a nucleic acid reaction; a reagent storage and introduction unit 25 which communicates with the reaction chamber unit and in which a reaction reagent is stored, and which introduces the nucleic acid sample into the reaction chamber unit and then introduces the reaction reagent into the reaction chamber unit so that the reaction reagent reacts with the nucleic acid sample.
FIG. 7 is a schematic diagram of a sample cell according to the present invention; the sample cell unit comprises a sample cell 211 arranged at a middle position, a sample diversion channel 212 communicated with the sample cell and arranged at the periphery of the sample cell, and a plurality of sample aliquot chambers 213 arranged at the outer side of the sample diversion channel and communicated with the sample diversion channel; the nucleic acid sample in this embodiment is centrifuged at a certain rotation speed from the sample chamber 211, discharged into the sample distribution channel 212, flowed through the sample distribution channel 212, and moved outward by the centrifugal force to enter the sample aliquot chambers 213. Of course, the sample aliquoting chamber 213 of this embodiment is provided with at least three, for example, four or six, so that the sample aliquoting effect of this embodiment can be achieved, and the sequential and separate addition of multiple nucleic acid samples in this embodiment can also be achieved.
With continued reference to fig. 7, in order to further enhance the movement of the sample chamber 211 under the off-line force, the sample chamber 211 of this embodiment is of an eccentric structure, that is, the edge of the horizontal cross section of the sample chamber 211 is of an involute type or a curve with an increasing radius, so as to enhance the centrifugal effect. The nucleic acid sample is collected to the larger diameter side during the rotation of the sample chamber 211, and in this embodiment, the nucleic acid sample is collected in the direction of the slit 215 and flows from there to the sample distribution channel 212. The outer sidewall 210 of the sample chamber 211 is an arc-shaped wall, and the outer sidewall of the sample chamber gradually extends from the notch 215 to the center side in the clockwise direction to form an eccentric structure of the sample chamber, and the end 214 of the arc-shaped wall 210 is the position closest to the center of the sample chamber. Referring to FIG. 6, the nucleic acid sample flows upward to the sample distribution channel 212 through the gap 215, in this embodiment, a step surface 216 is disposed between the sample distribution channel 212 and the sample chamber 211, the height of the sample distribution channel 212 is higher than that of the sample chamber 211, and the height of the sample aliquot chamber 213 is higher than that of the sample distribution channel 212. In this embodiment, the sample aliquoting chamber 213 is a rectangular groove with an arc-shaped end, and of course, an elliptical groove or a rectangular groove is also provided, and an arc-shaped structure is provided at the end, so that on one hand, the nucleic acid sample can be buffered under the centrifugal action, and on the other hand, no liquid is left by the arc-shaped structure, so as to meet the actual requirement.
In this embodiment, the sample diversion channel 212 is a multi-half ring, and a first liquid guide tube 217 is disposed at a distal end of the multi-half ring, as shown in fig. 4, a distal end of the first liquid guide tube 217 is communicated with the first waste liquid chamber 202, and the first waste liquid chamber 202 is used for loading waste liquid. The first exhaust channel 243 is provided at the edge of the first waste liquid chamber 202, and since the sample chamber is a chip middle layer and sealed up and down, the first exhaust channel is provided to exhaust high-pressure gas when the nucleic acid sample flows, thereby ensuring smooth flow of the nucleic acid sample.
Specifically, the sample chamber side wall of the sample chamber is provided with an involute shape, so that the sample chamber has an eccentric structure, the movement of a nucleic acid sample under the centrifugal action is enhanced, and particularly, gaps are formed at two ends of the involute-shaped sample chamber side wall, so that the flowing nucleic acid sample quickly flows into a sample shunting channel in a superfine narrow channel, and the nucleic acid sample is quickly shunted. Furthermore, the height that the sample shunted the passageway is higher than the height of sample room, simultaneously, the height that the sample aliquoting room is higher than the height that the sample shunted the passageway, through setting up once ascending structure, avoid nucleic acid sample to produce irregular flow when not rotating, moreover, through the structure that risees one by one, can be convenient realize appointed nucleic acid sample flow through the rotational speed of control sample room, if discharge the nucleic acid sample into sample shunting passageway from the sample room through first predetermined rotational speed, discharge sample aliquoting room into the sample with sample shunting passageway through second predetermined rotational speed, convenient control.
FIG. 8 is a schematic view of a first siphon valve according to the present invention; the end of each sample aliquot chamber 213 is connected to and communicated with a first siphon valve for introducing the nucleic acid sample in the sample aliquot chamber 213 into the reaction chamber 257, after the chip is rotated at a first predetermined speed, the nucleic acid sample enters the sample aliquot chamber 213, and the excess sample enters the first waste chamber 202. The chip stops rotating and the sample in the sample aliquoting chamber flows into and fills the first siphon valve 26, whereas when the chip rotates at a first preset speed, the sample cannot fill the first siphon valve due to the centrifugal force. The chip of the invention prevents the sample from flowing disorderly by rotating and stopping in sequence. In order to make the amount of the nucleic acid sample stored in the first siphon valve 26 equal to the amount of the stored liquid in the sample halving chamber 213, i.e. the reaction requirement of the reaction chamber 257, the first siphon valve 26 of the present embodiment is provided with a bent structure, which can further enhance the siphon effect. The first siphon valve 26 includes a first siphon segment 261 communicated with the sample aliquoting chamber 213 and extending to the outside of the chip, a second siphon segment 262 communicated with the first siphon segment 261 and arranged along the circumferential direction of the chip, a third siphon segment 263 communicated with the second siphon segment 262 and extending to the inside of the chip, a fourth siphon segment 264 juxtaposed with the third siphon segment 263 and extending to the outside of the chip, an arc-shaped segment between the third siphon segment and the fourth siphon segment, which makes a transition between the third siphon segment and the fourth siphon segment, a fifth siphon segment 265 communicated with the fourth siphon segment 264 and extending obliquely to the outside of the chip, a sixth siphon segment 266 communicated with the fifth siphon segment 265 and extending radially to the outside of the chip, and a tip of the sixth siphon segment 266 is communicated with the first reservoir 2571 of the reaction chamber 257. The first reservoir 2571 serves as a terminal of the first siphon valve 26, and the nucleic acid sample fills the entire first siphon valve 26 by the siphon principle after the rotation of the chip is stopped at the first predetermined rotation speed. The chip rotates at a second predetermined speed, and due to the centrifugal force, the nucleic acid sample in the first siphon valve 26 enters the reaction chamber 257 via the first reservoir 2571, and the FTA card 4 extracts and adsorbs the nucleic acid sample in the sample. The chip stops rotating, the sample in the reaction chamber 257 flows into the second siphon valve 27, the chip rotates at the third preset speed, and the liquid in the reaction chamber 257 flows into the second waste chamber 201.
With continued reference to fig. 8, the second siphon valve 27 of the present embodiment includes a seventh siphon segment 274 communicating with the reaction chamber 257 and extending radially toward the outer layer of the chip, an eighth siphon segment 273 communicating with the seventh siphon segment 274 and extending circumferentially of the chip, a ninth siphon segment 272 communicating with the eighth siphon segment 273 and extending radially inward of the chip, and a tenth siphon segment 271 arranged side by side and extending radially outward of the chip, wherein a transition arc segment is further arranged between the ninth siphon segment and the tenth siphon segment, and a terminal end of the tenth siphon segment 271 communicates with the second waste chamber 201. Referring to fig. 4, since the second waste liquid chamber 201 of the present embodiment collects the waste liquid from which the acid sample is extracted, the amount of the collected waste liquid is large, the second waste liquid chamber 201 is an annular groove, and a second exhaust channel 242 is further provided at one end of the annular groove 201 so that the liquid can enter the second waste liquid chamber. As shown in fig. 9, a temporary reservoir 2421 is further provided at a communication position of the second waste liquid chamber 201 and the second air exhaust passage 242 to smoothly exhaust air. A second communication channel 254 communicating with the reagent storage introduction unit is further provided in the tenth siphon section 271.
Specifically, the first siphon valve of the present embodiment extends from the sample aliquoting chamber to the radially inner side and the radially outer side of the chip until communicating with the reaction chamber; the second siphon valve extends from the reaction chamber to the radial inner side and the radial outer side of the chip until the second siphon valve is communicated with the second waste liquid chamber. According to the invention, a nucleic acid sample enters a sample equal-division chamber through a first preset speed, the chip is made of a hydrophilic material, after the chip stops rotating, the sample enters a first siphon valve through capillary force, the sample of the first siphon valve enters a reaction chamber through a second preset speed, waste liquid enters a second siphon valve through the second preset speed, the waste liquid is filled in the second siphon valve through the capillary force through stopping, and then the sample of the second siphon valve is discharged into a second waste liquid chamber through a third preset speed. The two siphon valves are grooves formed in the upper surface and the lower surface of the middle layer 2 of the chip, and mutual interference is avoided.
FIG. 9 is a schematic structural diagram of a reagent storage and introduction unit in a layer of a chip according to the present invention; the reagent storage and introduction unit 25 of the embodiment can store LAMP or PCR reaction reagents and primers, the LAMP or PCR reaction reagents and the primers are sealed by mineral oil and paraffin, the LAMP or PCR reaction reagents and the primers are stored in the grooves when the melting temperature of the paraffin is not reached, the LAMP or PCR reaction reagents and the primers are driven by centrifugal force to enter the reaction chamber after the paraffin is heated and melted, and the LAMP or PCR reaction reagents and the primers are blocked by the mineral oil to smoothly react in the reaction chamber. Specifically, the reagent storage introduction unit 25 includes a paraffin chamber 251 near the center of the chip, in which paraffin is loaded and which functions as a stopper at low temperatures; one end of the paraffin chamber 251 is also communicated with a third air passage 241, so that the reagents in the paraffin chamber 251, the first mineral oil chamber 252, the second mineral oil chamber 253, the LAMP reagent chamber 255 and the primer chamber 256 can smoothly flow into the designated chambers. The paraffin chamber 251 is connected with two branches which are arranged side by side, wherein a first mineral oil chamber 252 is arranged on a first branch, mineral oil is loaded in the first mineral oil chamber 252 and is communicated with a second siphon valve 27 through a second communication pipeline 254, after paraffin is heated, the mineral oil flows to the second siphon valve 27 along the second communication pipeline 254 and is filled in the second siphon valve 27 to seal one end of the reaction chamber 257, in the embodiment, the first mineral oil chamber 252 is arranged along the direction which is far away from the center of the chip relative to the paraffin chamber 251, and the second communication pipeline 254 is arranged along the direction which is far away from the center of the chip relative to the first mineral oil chamber 252, so when the chip rotates, the mineral oil can flow along the radial centrifugal force direction of the chip. The second mineral oil chamber 253, the LAMP reagent chamber 255 and the primer chamber 256 are sequentially arranged on the second branch from inside to outside along the radial direction of the chip and are communicated through pipelines, mineral oil, LAMP reagents and primers are respectively stored in the second branch, one end of the primer chamber 256 is communicated with the reaction chamber 257 through a third pipeline 258, after paraffin is heated, the mineral oil, the LAMP reagents and the primers are driven to enter the reaction chamber by centrifugal force, and the mineral oil is blocked in the pipeline outside the reaction chamber to ensure normal reaction of the reaction chamber.
Specifically, the paraffin wax is melted at low temperature and high temperature, the plugging reaction reagent and the primers are respectively stored, the reaction reagent and the primers enter the reaction chamber for reaction by centrifugal force after melting, and meanwhile, the mineral oil at two ends of the reaction chamber is used for plugging, so that normal reaction of the reaction chamber is ensured. Because only need can accomplish the state conversion through the heating, the automatic reaction that reacts, simultaneously, set up the second siphon valve on reaction chip middle level, the mineral oil of one of them branch road passes through the second siphon valve shutoff reaction chamber from one end, and another branch road is under the effect of centrifugal force, and reaction reagent and primer flow in reaction chamber reaction to through the mineral oil shutoff, realize automatic reaction, self-sealing's effect.
FIG. 10 is a schematic view of a reaction chamber according to the present invention; the reaction chamber 257 is communicated with a first siphon valve 26 for introducing a nucleic acid sample, a reagent storing and introducing unit for introducing a reaction reagent and a primer, and a second siphon valve 27 for discharging a waste liquid and blocking, respectively, to introduce the respective components in sequence, thereby carrying out a reaction.
FIGS. 11-14 are schematic diagrams of chip reactions according to the present invention; the chip reaction process in the embodiment of the invention is as follows:
step a, adding a nucleic acid sample to a sample chamber 211 in a layer 2 of a chip in a sample adding hole 11 of a layer 1 of the chip;
b, rotating the chip at a first preset rotating speed, sequentially discharging the nucleic acid sample from the sample chamber into the sample shunting channel and the sample equal-dividing chamber, and allowing the redundant nucleic acid sample to enter the first waste liquid chamber;
c, stopping the chip from rotating, and enabling the sample in the sample halving chamber to flow into the first siphon valve;
d, the chip rotates in a second preset mode, liquid in the first siphon valve enters the reaction chamber under the action of centrifugal force due to the action of centrifugal force, and the FTA card extracts and adsorbs nucleic acid in the sample;
step e, stopping the rotation of the chip, enabling the nucleic acid sample waste liquid in the reaction chamber to flow into the second siphon valve, enabling the chip to rotate at a third preset speed, and enabling the nucleic acid sample waste liquid in the reaction chamber to flow into the second waste liquid chamber;
f, automatically adding a first cleaning solution and a second cleaning solution to the middle layer of the chip according to the steps a-e, and sequentially circulating and cleaning the FTA card according to the sample shunting channel, the sample equal-dividing chamber and the second siphon valve;
step g, the chip rotates and heats according to a fourth preset speed, paraffin is melted, LAMP or PCR reaction reagents and primers in the second branch flow into the reaction chamber under the action of centrifugal force, the reaction chamber is sealed through mineral oil, the mineral oil in the first branch enters the second siphon valve, and the waste liquid chamber is sealed;
and h, heating the chip to a temperature suitable for LAMP or PCR reaction, detecting by using fluorescence after the reaction is finished, and reading a reaction result.
In the invention, the first siphon valve for introducing the nucleic acid sample is arranged on the bottom surface of the middle layer of the chip, the reagent storage and introduction unit for storing and introducing the reaction reagent and the second siphon valve are arranged on the top surface of the middle layer of the chip, and the liquid introduction structures are arranged on the front surface and the back surface of the middle layer of the chip, so that the space of the middle layer of the chip is saved, meanwhile, the interference among liquids is avoided, and the reaction control is accurate.
FIG. 15 is a schematic view showing the structure of the nucleic acid detecting apparatus according to the present invention; the device of the embodiment comprises: a chip chamber for loading chips, the chip chamber comprising a chip chamber upper cover 51, a chip chamber middle ring 52 and a chip chamber lower ring 54, which are sequentially connected from top to bottom; the device also comprises a motor 57 and two sample adding pumps 56 which are arranged on one side of the chip chamber, wherein the motor 57 drives the sample adding pumps 56 to act so as to add nucleic acid samples into the reaction chip; the device also comprises a radiator 50 arranged on one side of the chip chamber, and the radiator is used for cooling the reaction chamber so as to control the temperature of the reaction chamber within a preset range; the reaction test device further comprises a controller 6, wherein the controller is provided with a display screen 61 and a control button 62, and the controller completes a reaction test through controlling the rotating speed, the temperature and the motor of the reaction chip. The chamber of this embodiment is disposed on a base 58, and the lower end of the base is provided with support legs 59 for supporting the base. Meanwhile, a camera 531 for recording the reaction chip, and an ultraviolet light source 53 are provided on the chip chamber upper cover 51.
FIG. 16 is a schematic view showing a half-sectional structure of a nucleic acid detecting apparatus according to the present invention; the apparatus of this embodiment further includes a cleaning solution 71 for cleaning the reaction chip, and the cleaning solution is applied or added to the reaction chip through an application tube 561. The output end of the motor 57 of this embodiment is provided with a first belt wheel 571, which is connected with a second belt wheel 573 through a belt 572, the second belt wheel is connected with a rotating shaft 576, the rotating shaft 576 is connected with the chip tray 521 and drives the chip tray 521 to rotate according to a preset speed, and the reaction chip of this embodiment is fixed on the chip tray. A positioning sensor 63 is arranged at the lower end of the rotating shaft 576, and the position of the rotating shaft is detected to ensure the accuracy and stability of the movement of the rotating shaft; a bearing 574 is provided on the rotating shaft to support the rotating shaft, and a conductive slip ring 575 is provided on the rotating shaft to allow a conductive wire to pass therethrough. In order to monitor the temperature of the reaction chip in real time, a temperature sensor 64 is provided on the chip tray to detect the temperature of the reaction chip in real time.
FIG. 16 is a schematic structural diagram of a temperature control module of the nucleic acid detecting apparatus according to the present invention; in this embodiment, the reaction chip is disposed in the middle ring 52 of the chip chamber, and a support plate is disposed between the middle ring 52 of the chip chamber and the lower ring 54 of the chip chamber, and a cooling module and a heating module are disposed on the support plate to control the temperature of the reaction chip on the upper side thereof. First heat exchange fins 82 and second heat exchange fins 84 are provided on the support plate, and heating fins 83 are provided on both heat exchange fins; a first refrigeration module 85 is arranged on the support plate; the first refrigeration module 85 is also communicated with a heat conduction circulating pump 86 through a connecting pipe 80, and the heat conduction circulating pump 86 is communicated with a second refrigeration module 88; a circulation fan 81 is further included to generate air convection for cooling or heating.
Specifically, the two heating modules and the refrigerating module are both connected with the controller, the temperature sensor transmits a real-time detection result to the controller, and the temperature of the reaction chip is controlled by controlling the heating modules and the refrigerating module so as to realize paraffin melting and other operations; meanwhile, the controller controls the starting and stopping of the motor, controls the adding of the sample and the rotation of the reaction chip, and further controls the reaction process.
In order to control the temperature more accurately, a set of three temperature sensors is disposed on the chip tray, the three temperature sensors respectively detect the temperature of the reaction chip and transmit the temperature to the controller, the controller 6 is provided with a selection module, and the selection module determines a first comparison value P of the first temperature sensor and a first comparison value P of the second temperature sensor according to the following average value operation formula21：
In the formula, P21A first comparison value r representing the positions of the first and second temperature sensors1Representing real-time sampled values of the first temperature sensor, r2Representing real-time sampled values of a second temperature sensor; r is3Representing real-time sampled values of a third temperature sensor; t denotes the mean square error operation and I denotes the integration operation.
Where I represents an arbitrary integral operation based on a quadratic function, the above formula is ratio information for obtaining the integral, and the following two formulas are the same, for example, based on a function y ═ ax2In the case that x takes the value (a, b), a<b is an arbitrary number.
The basic algorithm of the above average operation is: the method comprises the steps of carrying out integral operation and mean square error operation on all values in a certain time period by obtaining position values of all sampling points in the certain time period, and then obtaining a ratio to obtain a comparative average value.
The selected module judges the second comparison value P of the first temperature sensor and the third temperature sensor according to the following formula31：
In the formula, P31A second comparison value r representing the positions of the first and second temperature sensors1Representing real-time sampled values of the first temperature sensor, r2Representing real-time sampled values of a second temperature sensor; r is3Representing real-time sampled values of a third temperature sensor; t denotes the mean square error operation and I denotes the integration operation.
The selected module judges a third comparison value P of the second temperature sensor and the third temperature sensor according to the following formula23：
In the formula, P23A third comparison value r representing the second temperature sensor and the second temperature sensor1Representing real-time sampled values of the first temperature sensor, r2Representing real-time sampled values of a second temperature sensor; r is3Representing real-time sampled values of a third temperature sensor; t denotes the mean square error operation and I denotes the integration operation.
P obtained through the method21、P31、P23And obtaining difference values of the three comparison values for comparison, judging whether the difference values exceed a threshold value P stored in the selected module, if one difference value exceeds the threshold value P, the temperature is deviated, the reaction chip needs to be heated and regulated again to prevent the chip from being deviated due to local heating, particularly, the temperature control for paraffin melting needs to be accurate so that the paraffin can be melted at a proper temperature, the paraffin is melted to push mineral oil to a proper position, and the mineral oil pushes a reaction reagent and a primer to a reaction chamber to finish the detection reaction.
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 (9)
1. The utility model provides a nucleic acid detection chip based on multiple nucleic acid reaction which characterized in that, includes chip upper strata, chip lower floor and chip middle level, and chip upper strata, chip middle level and chip lower floor set up in order, set up the FTA card in the chip middle level, the chip middle level includes:
a sample chamber unit disposed at an intermediate position, storing the nucleic acid samples sequentially added, and dispersing the nucleic acid samples to the corresponding chambers under the rotation;
a first siphon valve communicating with the sample chamber unit and introducing a prescribed nucleic acid sample into the reaction chamber unit by a siphon phenomenon;
a reagent storage/introduction unit which is connected to the reaction chamber unit and in which a reaction reagent corresponding to a predetermined nucleic acid is stored, and which introduces the reaction reagent into the reaction chamber unit after the nucleic acid sample is introduced into the reaction chamber unit, thereby allowing the reaction reagent to react with the nucleic acid sample;
the reaction chamber unit is arranged at the edge position, mixes the specified nucleic acid sample with the reagent and carries out nucleic acid reaction, the reaction chamber of the reaction chamber unit is also communicated with a second siphon valve, after the reaction chamber unit tests one nucleic acid sample, the reaction chamber unit is cleaned, and another nucleic acid sample is tested;
the sample chamber unit comprises a sample chamber arranged in the middle, a sample shunting channel communicated with the sample chamber and arranged on the periphery of the sample chamber, and at least three sample halving chambers arranged on the outer side of the sample shunting channel and communicated with the sample shunting channel, wherein a nucleic acid sample is subjected to centrifugal force from the sample chamber at a first preset rotating speed, is discharged into the sample shunting channel, flows in the sample shunting channel, continues to move outwards under the action of the centrifugal force, and respectively enters each sample halving chamber, the sample shunting channel is a multi-semicircular ring, a first liquid guide pipe is arranged at the tail end of the multi-semicircular ring, and the tail end of the first liquid guide pipe is communicated with a first waste liquid chamber;
the reagent storage and introduction unit comprises a paraffin chamber close to the central part, paraffin is loaded in the paraffin chamber, and the paraffin chamber plays a role of blocking at low temperature; one end of the paraffin chamber is also communicated with a third exhaust channel, so that liquid in the channel smoothly flows.
2. The nucleic acid detecting chip according to claim 1, wherein the sample chamber has an eccentric structure, and the nucleic acid samples are collected toward a larger diameter side during rotation of the sample chamber, and the nucleic acid samples are collected toward the slit and flow therefrom toward the sample distribution channel;
the outer side wall of the sample chamber is an arc-shaped wall, the outer side wall of the sample chamber gradually extends towards the circle center side from the opening along the clockwise direction to form an eccentric structure of the sample chamber, and the end part of the arc-shaped wall is the position closest to the circle center of the sample chamber.
3. The nucleic acid detecting chip according to claim 2, wherein the nucleic acid sample flows upward through the opening to the sample diversion channel, a step surface is provided between the sample diversion channel and the sample chamber, the height of the sample diversion channel is higher than the height of the sample chamber, and the height of the sample aliquot chamber is higher than the height of the sample diversion channel.
4. The nucleic acid detecting chip based on multiple nucleic acid reactions of claim 1, wherein a first siphon valve is connected and communicated with the end of each sample aliquot chamber for introducing the nucleic acid sample in the sample aliquot chamber into the reaction chamber, and when the sample chamber rotates at a first preset speed, the nucleic acid sample enters the sample aliquot chamber, and the redundant sample enters the first waste chamber; the sample chamber stops rotating and the sample in the sample aliquoting chamber flows into and fills the first siphon valve.
5. The nucleic acid detecting chip based on multiple nucleic acid reactions according to claim 4, wherein the first siphon valve includes a first siphon segment communicating with the sample aliquot chamber and extending outward, a second siphon segment communicating with the first siphon segment and arranged in a circumferential direction, a third siphon segment communicating with the second siphon segment and extending inward, a fourth siphon segment juxtaposed with the third siphon segment and extending outward, an arc-shaped segment between the third siphon segment and the fourth siphon segment, which makes a transition between the third siphon segment and the fourth siphon segment, a fifth siphon segment communicating with the fourth siphon segment and extending obliquely outward, a sixth siphon segment communicating with the fifth siphon segment and extending radially outward, and a tip of the sixth siphon segment communicates with the first reservoir of the reaction chamber.
6. The nucleic acid detecting chip based on multiple nucleic acid reactions according to claim 1, wherein the middle layer of the chip further comprises a second siphon valve communicated with the reaction chamber, the second siphon valve comprises a seventh siphon segment communicated with the reaction chamber and extending radially outward, an eighth siphon segment communicated with the seventh siphon segment and extending circumferentially inward, a ninth siphon segment communicated with the eighth siphon segment and extending radially inward, and a tenth siphon segment arranged side by side with the ninth siphon segment and extending radially outward, a transition arc-shaped segment is further arranged between the ninth siphon segment and the tenth siphon segment, and the end of the tenth siphon segment is communicated with the second waste liquid chamber.
7. The nucleic acid detecting chip according to claim 1, wherein the paraffin chamber is connected to two branches arranged side by side, wherein a first mineral oil chamber is provided in a first branch, and mineral oil is loaded therein and communicated with the second siphon valve through a second communication pipeline, and after the paraffin is heated, the mineral oil flows along the second communication pipeline to the second siphon valve and fills the second siphon valve to block one end of the reaction chamber.
8. The nucleic acid detection chip based on multiple nucleic acid reactions according to claim 7, wherein the second branch is provided with a second mineral oil chamber, an LAMP reagent chamber and a primer chamber in sequence from inside to outside along the radial direction of the chip, the second mineral oil chamber, the LAMP reagent chamber and the primer chamber are communicated through a pipeline, mineral oil, an LAMP reagent and primers are stored in the second mineral oil chamber, one end of the primer chamber is communicated with the reaction chamber through a third pipeline, the mineral oil is melted when paraffin is heated, the mineral oil is pushed to flow by centrifugal force, and then the LAMP reagent and the primers are pushed to enter the reaction chamber by the mineral oil.
9. The nucleic acid detecting chip according to claim 3, wherein the first siphon valve is disposed on a bottom surface of the middle layer of the chip, and the second siphon valve is disposed on a top surface of the middle layer of the chip.
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| CN108642569B (en) * | 2018-06-07 | 2021-10-08 | 国家纳米科学中心 | Nucleic acid detection chip |
| CN109266518A (en) * | 2018-11-29 | 2019-01-25 | 奥然生物科技（上海）有限公司 | It is a kind of to be provided with micro-fluidic or nanometer flow control structure biological reaction apparatus |
| CN111558402B (en) * | 2020-03-10 | 2021-05-11 | 青岛英赛特生物科技有限公司 | Air pressure driven centrifugal micro-fluidic detection chip |
| CN113176790B (en) * | 2021-05-11 | 2022-02-15 | 北京中科生仪科技有限公司 | Liquid path control device based on PCR |
| CN114480587A (en) * | 2022-01-29 | 2022-05-13 | 中创科瑞(北京)生物科技有限公司 | Integrated detection chip and detection method based on CRISPR technology |
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