CN114231408A - Nucleic acid detection chip and nucleic acid detection method - Google Patents
Nucleic acid detection chip and nucleic acid detection method Download PDFInfo
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- CN114231408A CN114231408A CN202111679524.4A CN202111679524A CN114231408A CN 114231408 A CN114231408 A CN 114231408A CN 202111679524 A CN202111679524 A CN 202111679524A CN 114231408 A CN114231408 A CN 114231408A
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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 and a nucleic acid detection method. The nucleic acid detection chip comprises a chip body, wherein the chip body is provided with a plurality of reagent cavities, detection cavities, control cavities and a first flow channel; the plurality of reagent chambers are sequentially communicated with each other, one reagent cavity is communicated with the detection cavity through a first flow channel, and the control cavity is communicated with the first flow channel; the first blocking piece capable of being switched between a flow state and a solid state under the triggering condition is preset in the control cavity, and the first blocking piece in the control cavity can flow to the first flow channel under the centrifugal action after being converted into the flow state and is converted into the solid state in the first flow channel so as to block the first flow channel. So, flow to first runner after under the effect of centrifugal force when first shutoff piece changes the flow state, change into solid-state and shutoff first runner into, play the palirrhea purpose of nucleic acid detection liquid in the restriction detection intracavity, avoided needing the integration to set up the check valve to be favorable to reducing and make the degree of difficulty and manufacturing cost, and the less portable of volume.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a nucleic acid detection chip and a nucleic acid detection method.
Background
The PCR (Polymerase Chain Reaction) technique is a molecular biology technique for amplifying and amplifying specific DNA (deoxyribonucleic acid) sequences in vitro. The PCR technology has the characteristics of strong specificity, high sensitivity, low purity requirement, simplicity, convenience and rapidness, so that the PCR technology is widely applied to nucleic acid detection and analysis.
The detection of the sample mainly comprises nucleic acid extraction, detection pretreatment and PCR detection. In order to realize the point-of-care testing (POCT) of nucleic acid and improve the testing accuracy, various reagents need to be preset in the reagent kit, and the mixing and transferring of various reagents and samples in the reagent kit are realized by centrifugal motion, so as to realize the steps of nucleic acid extraction, testing pretreatment and the like. However, in the prior art, the flow direction of the reagents in the reagent kit is difficult to control, which increases the operation difficulty of the operator, and a plurality of one-way valves need to be integrated on the reagent kit to control the flow direction of each reagent, thereby increasing the manufacturing difficulty and the manufacturing cost, and having a large volume and being inconvenient to carry. For example, in the patent application No. 202010737913.7, a check valve for allowing a liquid to flow from a sample addition part a to a sample addition part B is provided between a filtration membrane and the sample addition part a, and the flow direction of a reagent is controlled by the check valve.
Disclosure of Invention
Therefore, it is necessary to provide a nucleic acid detecting chip and a nucleic acid detecting method that improve the above-mentioned drawbacks, in order to solve the problems that the flow direction of the reagents in the reagent cartridge in the prior art is difficult to control, the operation difficulty of the operator is increased, and a plurality of check valves need to be integrated on the reagent cartridge to control the flow direction of each reagent, thereby increasing the manufacturing difficulty and the manufacturing cost, and having a large volume and being inconvenient to carry.
A nucleic acid detection chip comprises a chip body, wherein the chip body is provided with a plurality of reagent cavities, detection cavities, control cavities and first flow channels; the plurality of reagent chambers are sequentially communicated with each other, one reagent cavity is communicated with the detection cavity through the first flow channel, and the control cavity is communicated with the first flow channel;
the first blocking piece capable of being switched between a flow state and a solid state under a trigger condition is preset in the control cavity, and the first blocking piece in the control cavity can flow to the first flow channel under the centrifugal action after being converted into the flow state and is converted into the solid state in the first flow channel so as to block the first flow channel.
In one embodiment, when the temperature of the first plugging piece is higher than a first preset temperature or temperature range, the first plugging piece is switched from a solid state to a fluid state; and when the temperature of the first plugging piece is lower than the first preset temperature or temperature range, the flow state is switched into a solid state.
In one embodiment, the first closure member comprises a paraffin member.
In one embodiment, the plurality of reagent chambers and the detection chamber are sequentially arranged at intervals along a preset direction.
In one embodiment, the chip body further has a plurality of communication channels, each two adjacent reagent chambers are communicated through the communication channels, each communication channel is preset with a second blocking piece for closing the communication channel, and the second blocking piece can be converted from a solid state to a fluid state under a trigger condition.
In one embodiment, when the temperature of the second plugging member is higher than a second preset temperature or temperature range, the second plugging member is switched from a solid state to a fluid state.
In one embodiment, the second closure member comprises a paraffin member.
In one embodiment, the chip body comprises a first surface and a second surface opposite to the first surface;
the first surface is provided with a plurality of first adding holes which are communicated with the plurality of reagent cavities in a one-to-one correspondence manner; the nucleic acid detecting chip further comprises a first cover sheet covering the first surface.
In one embodiment, the first surface is provided with a second adding hole communicated with the control cavity and a third adding hole communicated with the plurality of communicating channels in a one-to-one correspondence manner.
In one embodiment, each of the reagent chamber, the control chamber, the first flow channel and each of the communication channels is formed by the second surface being recessed inward;
the nucleic acid detection chip also comprises a second cover plate covered on the second surface, and the second cover plate is transparent.
In one embodiment, the detection cavity penetrates through the first surface and the second surface of the chip body;
the first cover plate is at least transparent in the area corresponding to the detection cavity.
A nucleic acid detecting method using the nucleic acid detecting chip as described in any of the above embodiments, comprising the steps of:
each reagent cavity is respectively preloaded with a nucleic acid extraction reagent and a nucleic acid reaction reagent; adding a sample into the reagent chamber preloaded with the nucleic acid extraction reagent;
driving the chip body to do centrifugal motion by using a centrifugal machine so that a sample sequentially flows into the reagent cavities pre-filled with the nucleic acid reaction reagent to be uniformly mixed and enters the detection cavity through the first flow channel;
the first plugging piece which is in a solid state in the control cavity is converted into a fluid state under the triggering condition;
driving the chip body to do centrifugal motion by using a centrifugal machine so as to enable the first plugging piece in a fluid state to flow into the first flow channel;
the first blocking piece in the first flow channel is converted into a solid state under a triggering condition so as to block the first flow channel;
and carrying out nucleic acid detection on the liquid in the detection cavity by using a nucleic acid detection module.
When the nucleic acid detection chip and the nucleic acid detection method are actually used, each reagent is preset in each reagent cavity, the first solid blocking piece is preset in the control cavity, and a sample to be detected is placed in the corresponding reagent cavity. Then, the chip body is placed on a centrifuge to do centrifugal motion, so that the sample is sequentially and uniformly mixed with the reagents in the reagent cavities under the action of centrifugal force and reacts, and thus nucleic acid extraction and detection pretreatment are completed, and the nucleic acid detection solution is obtained. Then, the nucleic acid detection solution continues to flow into the detection cavity through the first flow channel under the action of centrifugal force. Then, the first plugging piece in the control cavity is converted from a solid state into a fluid state under the triggering condition, and flows to the first flow channel under the action of centrifugal force. And then converting the first blocking piece in a fluid state in the first flow channel into a solid state under a triggering condition so as to block the first flow channel, thereby preventing the nucleic acid detection liquid in the detection cavity from flowing back to the reagent cavity through the first flow channel. Finally, the nucleic acid detecting chip is transferred to a detecting device to perform nucleic acid detection (e.g., PCR fluorescence detection) on the nucleic acid detecting solution in the detection chamber.
So, through presetting first shutoff piece in the control intracavity at the chip body, flow to first runner under the effect of centrifugal force when utilizing this first shutoff piece to convert the flow state, the first shutoff piece that reachs first runner converts into solid-state under the trigger condition, realize the first runner of shutoff, play the palirrhea purpose of nucleic acid detection liquid in the restriction detection intracavity, avoided needing integrated setting check valve as among the prior art, thereby be favorable to reducing the manufacturing degree of difficulty and manufacturing cost, and the less portable of volume. In addition, nucleic acid extraction, pretreatment for detection and nucleic acid detection are carried out in a closed space of the nucleic acid detection chip, so that artificial and environmental pollution is avoided, detection precision is ensured, and the requirement on professionals and the dependence on a standard laboratory are reduced.
Drawings
FIG. 1 is an exploded view of a nucleic acid detecting chip according to an embodiment of the present invention;
FIG. 2 is a top view of the chip body of the nucleic acid detecting chip shown in FIG. 1;
FIG. 3 is a bottom view of the chip body of the nucleic acid detecting chip shown in FIG. 1;
FIG. 4 is a flowchart illustrating steps of a method for detecting nucleic acid according to an embodiment of the present invention;
FIG. 5 is a flowchart showing the detailed procedure of step S20 of the nucleic acid detecting method shown in FIG. 4.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.
In the description of the present invention, it is to be understood that the terms "central," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," "circumferential," and the like are used in the orientations and positional relationships indicated in the drawings for convenience in describing the invention and to simplify the description, and are not intended to indicate or imply that the referenced device or element must have a particular orientation, be constructed and operated in a particular orientation, and are not to be considered limiting of the invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
In the present invention, unless otherwise expressly stated or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally formed; can be mechanically or electrically connected; they may be directly connected or indirectly connected through intervening media, or they may be connected internally or in any other suitable relationship, unless expressly stated otherwise. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
In the present invention, unless otherwise expressly stated or limited, the first feature "on" or "under" the second feature may be directly contacting the first and second features or indirectly contacting the first and second features through an intermediate. Also, a first feature "on," "over," and "above" a second feature may be directly or diagonally above the second feature, or may simply indicate that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature may be directly under or obliquely under the first feature, or may simply mean that the first feature is at a lesser elevation than the second feature.
It will be understood that when an element is referred to as being "secured to" or "disposed on" another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may also be present. The terms "vertical," "horizontal," "upper," "lower," "left," "right," and the like as used herein are for illustrative purposes only and do not denote a unique embodiment.
Referring to fig. 1 to 3, an embodiment of the invention provides a nucleic acid detecting chip, including a chip body 10, the chip body 10 having a plurality of reagent chambers 11, a detecting chamber 12, a control chamber 14 and a first flow channel 13. The plurality of reagent chambers 11 are sequentially communicated with each other, and one of the reagent chambers 11 is communicated with the detection chamber 12 through the first flow channel 13, and the control chamber 14 is communicated with the first flow channel 13.
A first blocking piece capable of switching between a fluid state and a solid state under a trigger condition is preset in the control cavity 14. The first blocking member in the control chamber 14 can flow to the first flow channel 13 under centrifugal action after being converted into a fluid state, and is converted into a solid state in the first flow channel 13 to block the first flow channel 13.
When the nucleic acid detection chip is actually used, each reagent is preset in each reagent cavity 11, the first blocking piece in a solid state is preset in the control cavity 14, and a sample to be detected is placed in the corresponding reagent cavity 11. Then, the chip body 10 is placed on a centrifuge to perform centrifugal motion, so that the sample is sequentially mixed with the reagents in the reagent chambers 11 under the action of centrifugal force and reacts, thereby completing nucleic acid extraction and detection pretreatment, and obtaining the nucleic acid detection solution. Then, the nucleic acid detecting solution continues to flow into the detection chamber 12 through the first flow path 13 by the centrifugal force. Then, the first blocking member in the control chamber 14 is converted from a solid state to a fluid state under the triggering condition, and flows to the first flow channel 13 under the centrifugal force. Then, the first blocking member in a fluid state in the first flow channel 13 is converted into a solid state under the trigger condition to block the first flow channel 13, thereby preventing the nucleic acid detection solution in the detection chamber 12 from flowing back to the reagent chamber 11 through the first flow channel 13. Finally, the nucleic acid detecting chip is transferred to a detecting device to perform nucleic acid detection (e.g., PCR fluorescence detection) on the nucleic acid detecting solution in the detection chamber 12.
So, through presetting first shutoff piece in the control chamber 14 at chip body 10, flow to first runner 13 under the effect of centrifugal force when utilizing this first shutoff piece to convert the flow state, the first shutoff piece that reachs first runner 13 converts into solid-state under the trigger condition, realize shutoff first runner 13, play the palirrhea purpose of nucleic acid detection liquid in the restriction detection chamber 12, avoided needing integrated setting check valve as among the prior art, thereby be favorable to reducing the manufacturing difficulty and manufacturing cost, and the less portable of volume. In addition, nucleic acid extraction, pretreatment for detection and nucleic acid detection are carried out in a closed space of the nucleic acid detection chip, so that artificial and environmental pollution is avoided, detection precision is ensured, and the requirement on professionals and the dependence on a standard laboratory are reduced.
It should be noted that the first block piece and the various reagents may be preset in the control chamber 14 and the reagent chamber 11, respectively, at the time of manufacturing the nucleic acid detecting chip. Of course, the first closure and the various reagents may also be added by the user at the time of use, and are not limited thereto. The reagent may be in a capsule particle or a freeze-dried state, or may be a liquid, and is not limited herein.
Specifically, in the embodiment, when the temperature of the first blocking piece is higher than the first preset temperature or temperature range, the first blocking piece is switched from the solid state to the fluid state. When the temperature of the first plugging piece is lower than a first preset temperature or temperature range, the flow state is switched into a solid state. So, after nucleic acid detects liquid and gets into detection chamber 12, heat the first shutoff piece in the control chamber 14 for the temperature of first shutoff piece is greater than first predetermined temperature or temperature range, and then makes first shutoff piece convert the flow state into by solid-state. After the first blocking piece in a flow state enters the first flow channel 13 under the action of centrifugal force, the first blocking piece in the first flow channel 13 is gradually cooled (cooling measures can be adopted and natural cooling can also be adopted, and limitation is not made herein) until the temperature of the first blocking piece is smaller than a first preset temperature or temperature range, so that the first blocking piece is converted into a solid state, and the purpose of blocking the first flow channel 13 is achieved. It is to be understood that the first predetermined temperature or temperature range is set according to the phase transition temperature of the first blocking member, and is not limited thereto.
It should be noted that the triggering condition is not limited to temperature, and in other embodiments, the first blocking member may also be made of, for example, a photosensitive material, and can be switched between a solid state and a fluid state under the illumination condition, which is not limited herein.
It should be noted that the first blocking member needs to be made of an inert material to ensure that the first blocking member does not react with the sample, each reagent and the like, so as to avoid adverse effects on nucleic acid extraction, pretreatment for detection and nucleic acid detection. Alternatively, the first blocking member may be a paraffin member.
In the embodiment, the reagent chambers 11 and the detection chambers 12 are sequentially arranged at intervals along a preset direction. Thus, when the chip body 10 is loaded on a centrifuge for centrifugal motion, the rotation center is close to one end of the chip body 10 in the preset direction, so that the sample can sequentially reach each reagent chamber 11 along the preset direction by controlling the rotation speed, and finally reach the detection chamber 12. Alternatively, the preset direction may be a longitudinal direction of the chip body 10.
In the embodiment shown in the drawings, the number of the reagent chambers 11 is three, and the reagent chambers are respectively named as a first reagent chamber 11a, a second reagent chamber 11b and a third reagent chamber 11c for convenience of description. The first reagent chamber 11a is for accommodating a nucleic acid extracting reagent and a sample, and the second reagent chamber 11b and the third reagent chamber 11c are for accommodating a nucleic acid reaction reagent. The first reagent cavity 11a, the second reagent cavity 11b, the third reagent cavity 11c and the detection cavity 12 are sequentially arranged at intervals along the preset direction. The first reagent chamber 11a communicates with the second reagent chamber 11b, the second reagent chamber 11b communicates with the third reagent chamber 11c, the third reagent chamber 11c communicates with the detection chamber 12 via the first channel 13, and the control chamber 14 communicates with the first channel 13 via the third reagent chamber 11 c. It is understood that the nucleic acid extracting reagent may be a lysis reagent or the like, and the nucleic acid reaction reagent may be a MIX reagent or a TAQ (Thermus Aquaticus) enzyme. The MIX reagent may include, but is not limited to, a PCR buffer and/or a bio-enzyme.
In this way, in actual use, the sample and the nucleic acid extracting reagent are mixed uniformly in the first reagent chamber 11a by the vibration of the centrifuge, and the reaction is performed sufficiently. Then the mixed liquid in the first reagent cavity 11a enters the second reagent cavity 11b under the action of centrifugal motion, and is uniformly mixed with the nucleic acid reaction liquid in the second reagent cavity 11b and fully reacts. Then the mixed liquid in the second reagent cavity 11b enters the third reagent cavity 11c under the action of centrifugal motion, and is uniformly mixed with the nucleic acid reaction liquid in the third reagent cavity 11 c. Then the mixed liquid in the third reagent chamber 11c enters the detection chamber 12 through the first flow channel 13 under the action of centrifugal motion. Then, the first blocking piece in the control chamber 14 is heated, so that the temperature of the first blocking piece is greater than a first preset temperature or temperature range, and further converted into a flow state. Then, under the action of centrifugal motion, the first blocking member in the control chamber 14 in a fluid state enters the first flow channel 13 through the third reagent chamber 11 c. The temperature of the first blocking member entering the first flow channel 13 is reduced to be less than a first preset temperature or temperature range, so that the first blocking member in the first flow channel 13 is converted into a solid state, the first flow channel 13 is blocked, and the nucleic acid detection solution in the detection cavity 12 is prevented from flowing back to the third reagent cavity 11c through the first flow channel 13 in the subsequent transportation or detection process. Finally, the chip body 10 is transferred to a nucleic acid detection module for nucleic acid detection.
In the embodiment of the present invention, the chip body 10 further has a plurality of communication channels 15, and each two adjacent reagent chambers 11 are communicated through the communication channel 15, that is, the two adjacent reagent chambers 11 are communicated through the communication channel 15. Each communication channel 15 is predisposed with a second obturating member for closing the communication channel 15, which second obturating member is capable of being transformed from a solid state to a fluid state under triggering conditions. Thus, when the mixed liquid is uniformly mixed in the current reagent chamber 11 and reacts, the communication channel 15 between the current reagent chamber 11 and the next reagent chamber 11 adjacent to the current reagent chamber is blocked by the second blocking piece in a solid state, so that the mixed liquid in the current reagent chamber 11 cannot flow into the next reagent chamber 11. When the mixed liquid in the current reagent cavity 11 needs to be transferred to the next reagent cavity 11 adjacent to the current reagent cavity, the second blocking piece in the communication channel 15 between the two reagent cavities 11 is converted from a solid state into a flow state under the triggering condition, so that the mixed liquid can enter the next reagent cavity 11 from the current reagent cavity 11 through the flow channel under the action of centrifugal motion, and the transfer of the mixed liquid is realized.
Specifically, in the embodiment, when the temperature of the second plugging member is higher than a second preset temperature or temperature range, the second plugging member is switched from the solid state to the fluid state. And when the temperature of the second plugging piece is lower than a second preset temperature or temperature range, the flow state is switched into a solid state. So, when need not mix the liquid and shift between two adjacent reagent chambeies 11, the second shutoff piece in the intercommunication passageway 15 between these two reagent chambeies 11 is solid-state to play the effect of this intercommunication passageway 15 of shutoff, and then avoid mixing the liquid and shift between two adjacent reagent chambeies 11. When the mixed liquid is required to be transferred to the next reagent cavity 11 from the current reagent cavity 11, the second plugging piece in the communication channel 15 between the two reagent cavities 11 is heated, so that the temperature of the second plugging piece is greater than the second preset temperature or temperature range, the second plugging piece is converted into a flow state, and the mixed liquid in the current reagent cavity 11 can enter the next reagent cavity 11 through the communication channel 15 under the action of centrifugal motion. It is to be understood that the second predetermined temperature or temperature range is set according to the phase transition temperature of the second blocking member, and is not limited thereto.
It should be noted that the triggering condition is not limited to temperature, and in other embodiments, the second blocking member may also be made of, for example, a photosensitive material, and can be switched between a solid state and a fluid state under the illumination condition, which is not limited herein.
It should be noted that the second plugging member needs to be made of an inert material to ensure that the second plugging member does not react with the sample, each reagent and the like, so as to avoid adverse effects on nucleic acid extraction, pretreatment for detection and nucleic acid detection. Alternatively, the second blocking member may be a paraffin member.
In the embodiment shown in the drawings, the first reagent chamber 11a is communicated with the second reagent chamber 11b through a communication channel 15, the second reagent chamber 11b is communicated with the third reagent chamber 11c through a communication channel 15, and a solid second blocking piece is preset in each of the two communication channels 15 to block the communication channel 15. That is, the second blocking piece is used for blocking at the beginning, so that reagent cannot be transferred between the first reagent chamber 11a and the second reagent chamber 11b, and reagent cannot be transferred between the second reagent chamber 11b and the third reagent chamber 11c, and the problem that reagent in each reagent chamber 11 is mixed by mistake before use to cause poor use effect or incapability of use is avoided.
In this manner, when the reagent is used, the centrifuge vibrates, and the sample and the nucleic acid extraction reagent are mixed uniformly in the first reagent chamber 11a and sufficiently reacted. The second block in the communication passage 15 between the first reagent chamber 11a and the second reagent chamber 11b is then heated to be converted into a fluid state. The mixed solution in the first reagent chamber 11a smoothly enters the second reagent chamber 11b under the action of centrifugal motion, and is uniformly mixed with the nucleic acid reaction reagent in the second reagent chamber 11b for sufficient reaction. Then, the second blocking member in the communication passage 15 between the second reagent chamber 11b and the third reagent chamber 11c is heated to be converted into a fluid state. The mixed solution in the second reagent chamber 11b smoothly enters the third reagent chamber 11c under the action of centrifugal motion, and is uniformly mixed with the nucleic acid reaction reagent in the third reagent chamber 11 c. Then the mixed liquid in the third reagent chamber 11c enters the detection chamber 12 through the first flow channel 13 under the action of centrifugal motion. Then, the first blocking piece in the control cavity 14 is heated, so that the temperature of the first blocking piece is greater than a first preset temperature or temperature range, and further converted into a flow state. Then, under the action of centrifugal motion, the first blocking member in the control chamber 14 in a fluid state enters the first flow channel 13 through the third reagent chamber 11 c. The temperature of the first blocking member entering the first flow channel 13 is reduced to be less than a first preset temperature or temperature range, so that the first blocking member in the first flow channel 13 is converted into a solid state, the first flow channel 13 is blocked, and the nucleic acid detection solution in the detection cavity 12 is prevented from flowing back to the third reagent cavity 11c through the first flow channel 13 in the subsequent transfer or nucleic acid detection process. Finally, the chip body 10 is transferred to a nucleic acid detection module for nucleic acid detection.
In the embodiment of the present invention, the chip body 10 includes a first surface a1 and a second surface a2 opposite to the first surface a 1. The first surface a1 is provided with a plurality of first addition holes 111 that are in one-to-one correspondence with the plurality of reagent chambers 11. The nucleic acid detecting chip further comprises a first cover sheet 20 placed over the first surface A1 so that each of the first addition wells 111 is closed by the first cover sheet 20. In this manner, each reagent can be added into each reagent chamber 11 through each first addition hole 111. The first cover sheet 20 is then placed over the first surface a1 to close each of the first adding holes 111. Alternatively, the first cover sheet 20 may be a film material such as a sealing film. The first cover sheet 20 can be attached to the first surface a1 by hot melt adhesive packaging, etc., so as to close the first adding holes 111.
Further, the nucleic acid detecting chip further comprises a plunger which passes through the first cover plate 20 and is plugged in one first addition hole 111 correspondingly communicated with the reagent chamber 11 pre-filled with the nucleic acid extracting reagent. Thus, when it is necessary to use, the plunger is withdrawn, and the sample is added to the reagent chamber 11 pre-filled with the nucleic acid reaction reagent through the first addition hole 111, and then the plunger is sealed in the first addition hole 111. In the embodiment shown in the drawings, the plunger penetrates the first cover plate 20 and blocks the first adding hole 111 (i.e. the rightmost first adding hole 111 in fig. 2) communicating with the first reagent chamber 11 a.
In specific embodiments, the first surface a1 may further be provided with a second filling hole 142 communicating with the control chamber 14, and a third filling hole 151 communicating with the plurality of communication channels 15 in a one-to-one correspondence manner. In this manner, the first plugging member can be added into the control chamber 14 by the second adding hole 142, and the second plugging member can be added into each of the communication passages 15 by each of the third adding holes 151 to plug each of the communication passages 15. After the first and second block-out elements are added, the first flap 20 is placed over the first surface a 1.
Specifically, in the embodiment, each of the reagent chamber 11, the control chamber 14, the first flow channel 13 and each of the communication channels 15 is formed by the second surface a2 being recessed inward. That is, each reagent chamber 11, the control chamber 14, the first flow channel 13 and each communication channel 15 have an opening on the second surface a 2. The nucleic acid detecting chip further comprises a second cover sheet 30 covering the second surface A2, and the second cover sheet 30 is transparent. Thus, each reagent chamber 11, the control chamber 14, the first flow channel 13 and each communication channel 15 are closed by the second cover sheet 30 and are open at the second surface A2. Furthermore, the flow direction of the liquid in each reagent chamber 11, the control chamber 14, the first flow channel 13 and each communication channel 15 can be observed through the second cover sheet 30, so as to adjust the rotation speed and/or the rotation direction of the centrifugal movement in time. Alternatively, the second cover sheet 30 may be a film material such as a sealing film. Second lidding sheet 30 can be attached to second surface A2 by hot melt adhesive encapsulation, or the like.
Further, the chip body 10 further has a second channel 141, one end of the second channel 141 is communicated with the control chamber, and the other end of the second channel 141 is communicated with the first channel 13 or the reagent chamber 11 communicated with the first channel 13. Alternatively, the second flow path 141 is formed by the second surface a2 being inwardly depressed so that the flow of the first blocking member can be observed through the second covering sheet 30.
Further, the detection chamber 12 penetrates the first surface a1 and the second surface a2 of the chip body 10. The first cover sheet 20 is transparent at least in the region corresponding to the detection chamber 12. In this manner, the nucleic acid detecting module can perform PCR fluorescence detection on the nucleic acid detecting solution in the detection chamber 12 through the region of the first cover sheet 20 corresponding to the detection chamber 12 and the second cover sheet 30.
Further, the first surface a1 is divided into a first area and a second area. The reagent chambers 11, the control chamber 14, the communication passages 15, and the second channel 141 are located in a first region, the detection chamber 12 is located in a second region, and the first channel 13 extends from the first region to the second region. The first cover sheet 20 includes a first sub-cover sheet 21 and a second sub-cover sheet 22 which are separately provided. The first sub-coversheet 21 is intended to cover the first area and the second sub-coversheet 22 is intended to cover the second area. It will be appreciated that the first sub-cover sheet 21 has a through-going hole for the plunger to pass through.
Based on the nucleic acid detection chip, the invention also provides a nucleic acid detection method, which comprises the following steps:
s10, pre-filling the nucleic acid extraction reagent and the nucleic acid reaction reagent in each reagent chamber 11; the sample is added to the reagent chamber 11 preloaded with the nucleic acid extraction reagent. Specifically, the plunger in the first addition hole 111 communicating with the first reagent chamber 11a is pulled out, and then the sample is added from the first addition hole 111 into the first reagent chamber 11a containing the nucleic acid extracting reagent. The plunger is then re-plugged into the first additive hole 111.
S20, driving the chip body 10 to do centrifugal motion by using a centrifugal machine, so that the samples sequentially flow into the reagent chambers 11 pre-filled with the nucleic acid reaction reagent to be uniformly mixed, thereby reacting, and then enter the detection chamber 12 through the first flow channel 13, and thus, the nucleic acid extraction and the detection pretreatment are realized. Specifically, the rotational speed and/or rotational direction of the centrifugal motion is controlled so that the sample is mixed with each reagent in sequence to complete the nucleic acid extraction and detection pretreatment.
S30, the first blocking member in solid state in the control chamber 14 is transformed into fluid state under the triggering condition. Specifically, the control chamber 14 is heated, so that the temperature of the first blocking piece in the control chamber 14 is higher than a first preset temperature or temperature range, and the first blocking piece is converted from a solid state to a fluid state.
S40, driving the chip body 10 to perform a centrifugal motion by using a centrifugal machine, so that the first blocking member in a fluid state flows into the first flow channel 13.
S50, the first blocking piece in the first flow passage 13 is converted into a solid state under the trigger condition to block the first flow passage 13, so that the nucleic acid detection liquid in the detection cavity 12 is prevented from flowing back to the reagent cavity 11. Alternatively, the first blocking member in the first flow passage 13 may be cooled to a temperature lower than the first preset temperature or temperature range by a cooling measure or natural cooling, so that the first blocking member in the first flow passage 13 is transformed into a solid state, thereby blocking the first flow passage 13.
S60, the nucleic acid detection module is used for detecting the nucleic acid (such as PCR fluorescence detection) of the liquid in the detection cavity 12.
In an embodiment, step S20 specifically includes:
s21, mixing the sample and the reagent in the current reagent cavity 11 uniformly by using centrifugal motion, and fully reacting;
s22, under the trigger condition, the second block piece in the communication channel 15 between the current reagent chamber 11 and the next reagent chamber 11 adjacent to the current reagent chamber is converted from solid state to fluid state.
S23, driving the chip body 10 to do centrifugal motion by using a centrifugal machine so as to transfer the mixed liquid in the current reagent cavity 11 to the next reagent cavity 11;
s24, repeating the steps S21 to S23 until the mixed liquid reaches the reagent cavity 11 communicated with the first flow channel 13, and uniformly mixing with the reagent in the reagent cavity 11 to fully react to obtain the nucleic acid detection liquid;
s25, driving the chip body 10 to do centrifugal motion by the centrifugal machine, so that the nucleic acid detection liquid enters the detection cavity 12 through the first flow channel 13.
Specifically, in the embodiment, step S10 further includes:
adding various nucleic acid extraction reagents and nucleic acid reaction reagents to the respective reagent chambers 11 through the respective first addition holes 111, adding first plugs into the control chamber 14 through the second addition holes 142, and adding second plugs into the respective communication passages 15 through the respective third addition holes 151, respectively;
covering the first sub-cover sheet 21 on the first area of the first surface A1 to close each of the first addition wells 111, the second addition wells 142, and each of the third addition wells 151, and sealing the plunger in one of the first addition wells 111 communicating with the reagent chamber 11 in which the nucleic acid extracting reagent is previously placed;
when in use, the plunger is pulled out, and a sample is added into the reagent chamber 11 in which the nucleic acid extracting reagent is preliminarily placed through the first addition hole 111;
the plunger is again inserted into the first addition hole 111 communicating with the reagent chamber 11 in which the nucleic acid extracting reagent is preliminarily placed.
It should be noted that, the rotation speed and the rotation direction of the centrifugal motion can be adjusted according to actual needs, so as to achieve uniform mixing or transfer of the liquid in the reagent chamber 11 or transfer of the first blocking member in the control chamber 14, and the specific rotation speed value can be set according to actual conditions, which is not limited herein.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (12)
1. A nucleic acid detection chip is characterized by comprising a chip body (10), wherein the chip body (10) is provided with a plurality of reagent cavities (11), a detection cavity (12), a control cavity (14) and a first flow channel (13); the plurality of reagent cavities (11) are communicated with each other in sequence, one reagent cavity (11) is communicated with the detection cavity (12) through the first flow channel (13), and the control cavity (14) is communicated with the first flow channel (13);
the first blocking piece capable of being switched between a fluid state and a solid state under a trigger condition is preset in the control cavity (14), and the first blocking piece in the control cavity (14) can flow to the first flow channel (13) under the centrifugal action after being converted into the fluid state and is converted into the solid state in the first flow channel (13) so as to block the first flow channel (13).
2. The nucleic acid detecting chip according to claim 1, wherein the first block piece is switched from a solid state to a fluid state when the temperature is higher than a first predetermined temperature or temperature range; and when the temperature of the first plugging piece is lower than the first preset temperature or temperature range, the flow state is switched into a solid state.
3. The nucleic acid detecting chip according to claim 2, wherein the first stopper includes a paraffin member.
4. The nucleic acid detecting chip according to claim 1, wherein the plurality of reagent chambers (11) and the detecting chamber (12) are arranged at intervals in sequence along a predetermined direction.
5. The nucleic acid detecting chip according to claim 1, wherein the chip body (10) further has a plurality of communicating channels (15), each adjacent two of the reagent chambers (11) communicate with each other through the communicating channels (15), each of the communicating channels (15) is preset with a second block piece for closing the communicating channel (15), and the second block piece is capable of being converted from a solid state to a fluid state under a trigger condition.
6. The nucleic acid detecting chip according to claim 5, wherein the second block piece is switched from a solid state to a fluid state when the temperature is higher than a second predetermined temperature or temperature range.
7. The nucleic acid detecting chip according to claim 6, wherein the second sealing member includes a paraffin member.
8. The nucleic acid detecting chip according to claim 5, wherein the chip body (10) includes a first surface (A1) and a second surface (A2) facing away from the first surface (A1);
the first surface (A1) is provided with a plurality of first adding holes (111) which are communicated with the plurality of reagent cavities (11) in a one-to-one correspondence manner; the nucleic acid detecting chip further comprises a first cover sheet (20) which covers the first surface (A1).
9. The nucleic acid detecting chip according to claim 8, wherein the first surface (A1) is provided with a second addition hole (142) communicating with the control chamber (14) and a third addition hole (151) communicating with the plurality of communicating channels (15) in a one-to-one correspondence.
10. The nucleic acid detecting chip according to claim 8, wherein each of the reagent chamber (11), the control chamber (14), the first flow channel (13), and each of the communicating channels (15) is formed by inwardly recessing the second surface (A2);
the nucleic acid detecting chip further comprises a second cover sheet (30) which covers the second surface (A2), and the second cover sheet (30) is transparent.
11. The nucleic acid detecting chip according to claim 10, wherein the detection chamber (12) penetrates the first surface (A1) and the second surface (A2) of the chip body (10);
the first cover sheet (20) is transparent at least in the region corresponding to the detection chamber (12).
12. A nucleic acid detecting method using the nucleic acid detecting chip according to any one of claims 1 to 11, comprising the steps of:
each reagent cavity is respectively preloaded with a nucleic acid extraction reagent and a nucleic acid reaction reagent; adding a sample to the reagent chamber (11) pre-loaded with the nucleic acid extracting reagent;
a centrifugal machine is used for driving the chip body (10) to do centrifugal motion, so that a sample sequentially flows into the reagent cavities (11) pre-filled with the nucleic acid reaction reagent to be uniformly mixed, and enters the detection cavity (12) through the first flow channel (13);
the first plugging piece in the control cavity (14) in a solid state is converted into a fluid state under a triggering condition;
driving the chip body (10) to do centrifugal motion by using a centrifugal machine so as to enable the first plugging piece in a fluid state to flow into the first flow channel (13);
the first blocking piece in the first flow passage (13) is converted into a solid state under a triggering condition so as to block the first flow passage (13);
and detecting nucleic acid in the liquid in the detection cavity (12) by using a nucleic acid detection module.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111679524.4A CN114231408B (en) | 2021-12-31 | Nucleic acid detection chip and nucleic acid detection method | |
| PCT/CN2022/118158 WO2023124229A1 (en) | 2021-12-31 | 2022-09-09 | Molecular detecting device, nucleic acid detecting chip, method of processing and detecting molecular |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111679524.4A CN114231408B (en) | 2021-12-31 | Nucleic acid detection chip and nucleic acid detection method |
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| CN114231408A true CN114231408A (en) | 2022-03-25 |
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20080022035A (en) * | 2006-09-05 | 2008-03-10 | 삼성전자주식회사 | Centrifugal force-based microfluidic device for nucleic acid detection and microfluidic system comprising the device |
| WO2019061816A1 (en) * | 2017-09-26 | 2019-04-04 | 南京岚煜生物科技有限公司 | Multi-flux microfluidic nucleic acid detection chip for actively controlling flow path, and usage method thereof |
| CN110029052A (en) * | 2019-04-18 | 2019-07-19 | 深圳市刚竹医疗科技有限公司 | Micro-fluidic chip and analysis system |
| CN110452802A (en) * | 2019-08-07 | 2019-11-15 | 深圳市刚竹医疗科技有限公司 | It is complete to extract molecular diagnosis micro-fluidic chip and microfluidic system |
| CN111218395A (en) * | 2020-04-18 | 2020-06-02 | 博奥生物集团有限公司 | Full-flow biological detection device |
| WO2020192742A1 (en) * | 2019-03-27 | 2020-10-01 | 深圳市尚维高科有限公司 | Self-driven microfluidic chip and method for using same |
| CN111808721A (en) * | 2020-07-28 | 2020-10-23 | 宁波爱基因科技有限公司 | Nucleic acid extraction chip and method for rapidly extracting blood DNA by using same |
| CN111944682A (en) * | 2020-08-14 | 2020-11-17 | 上海前瞻创新研究院有限公司 | Nucleic acid detection chip, preparation method and nucleic acid detection method |
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20080022035A (en) * | 2006-09-05 | 2008-03-10 | 삼성전자주식회사 | Centrifugal force-based microfluidic device for nucleic acid detection and microfluidic system comprising the device |
| EP2026074A2 (en) * | 2007-07-31 | 2009-02-18 | Samsung Electronics Co., Ltd. | Centrifugal force-based microfluidic device for nucleic acid detection and microfluidic system including the same |
| WO2019061816A1 (en) * | 2017-09-26 | 2019-04-04 | 南京岚煜生物科技有限公司 | Multi-flux microfluidic nucleic acid detection chip for actively controlling flow path, and usage method thereof |
| WO2020192742A1 (en) * | 2019-03-27 | 2020-10-01 | 深圳市尚维高科有限公司 | Self-driven microfluidic chip and method for using same |
| CN110029052A (en) * | 2019-04-18 | 2019-07-19 | 深圳市刚竹医疗科技有限公司 | Micro-fluidic chip and analysis system |
| CN110452802A (en) * | 2019-08-07 | 2019-11-15 | 深圳市刚竹医疗科技有限公司 | It is complete to extract molecular diagnosis micro-fluidic chip and microfluidic system |
| CN111218395A (en) * | 2020-04-18 | 2020-06-02 | 博奥生物集团有限公司 | Full-flow biological detection device |
| CN111808721A (en) * | 2020-07-28 | 2020-10-23 | 宁波爱基因科技有限公司 | Nucleic acid extraction chip and method for rapidly extracting blood DNA by using same |
| CN111944682A (en) * | 2020-08-14 | 2020-11-17 | 上海前瞻创新研究院有限公司 | Nucleic acid detection chip, preparation method and nucleic acid detection method |
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