CN115814863A - Closed micro-fluidic chip for nucleic acid amplification detection - Google Patents

Abstract

The invention provides a closed microfluidic chip for nucleic acid amplification detection, which comprises a gland and at least one chip; the first surface of the gland is provided with at least one mounting groove, and the second surface is provided with at least one gland sample inlet and a gland gas outlet corresponding to each mounting groove; at least one amplification part is formed in the chip, and the chip is arranged in the mounting groove; an elastic adhesive tape is arranged at the bottom of the mounting groove and fixes the chip in the mounting groove; at least two micro-cavity holes are formed in the elastic adhesive tape, and the at least two micro-cavity holes seal the communication position of the gland injection port and the amplification part and the communication position of the gland air outlet and the amplification part; the second surface of the gland is provided with at least one sealing mechanism which is used for sealing the sample inlet of the gland and the gas outlet of the gland. In the process of nucleic acid amplification detection, the leakage of a liquid sample can be prevented, so that a totally-enclosed PCR environment is realized, the manufacturing process is simple, the cost is low, and the method is convenient for large-scale application and popularization.

Description

Closed micro-fluidic chip for nucleic acid amplification detection

Technical Field

The invention relates to the field of in-vitro diagnosis nucleic acid detection, in particular to a closed microfluidic chip for nucleic acid amplification detection.

Background

As one of the main detection modes in the field of in vitro diagnosis, nucleic acid detection is the most direct, reliable and sensitive method for realizing early, rapid and specific detection of pathogens, can rapidly detect pathogen nucleic acid in a detection sample, and provides scientific detection basis for accurate diagnosis of infectious cases. The nucleic acid amplification detection is to amplify a nucleic acid sequence to be detected through the action of enzyme, wherein the PCR (polymerase chain reaction) technology is most widely applied due to the advantages of good specificity, low cost and the like. PCR consists of three basic reaction steps of denaturation, annealing and extension, the reaction time is always a limiting factor of the application of the PCR technology, and an instrument for improving the PCR reaction time has appeared at present, for example, patent document CN 111269825A discloses a rapid nucleic acid instant detector with a rapid temperature rise and fall module, but the current PCR reaction vessel has low heat conduction efficiency, and for the PCR reaction needing cyclic temperature rise and fall, the heat conduction efficiency of the PCR reaction vessel is low, so that the temperature rise and fall speed is slow, and the whole process takes longer.

The micro-fluidic chip technology integrates the traditional biochemical analysis on a chip with the size of a few square centimeters or even smaller, and completes detection and analysis in a micro-nano scale channel and a micro-chamber in the chip. However, the microfluidic chip requires a suitable heat conducting material in nucleic acid detection, so that the manufacturing difficulty and cost are greatly increased, and a totally-enclosed PCR amplification environment is difficult to realize, thereby limiting the further clinical application of the microfluidic chip. Therefore, a closed microfluidic chip with high thermal conductivity is urgently needed, the thermal conductivity of the microfluidic chip is improved, the detection time is shortened, the totally closed PCR amplification environment can be realized, and the manufacturing cost of the chip is reduced.

Disclosure of Invention

In view of all or part of the defects of the prior art, the invention provides the closed microfluidic chip for nucleic acid amplification detection, which can prevent a liquid sample from leaking in the nucleic acid amplification detection process, thereby realizing a totally closed PCR environment, and has the advantages of simple manufacturing process, low cost and convenience for large-scale application and popularization.

In order to achieve the above object, the present invention provides the following technical solutions: a closed microfluidic chip for nucleic acid amplification detection comprises a gland and at least one chip; the first surface of the gland is provided with at least one mounting groove, and the second surface is provided with at least one gland sample inlet and a gland gas outlet corresponding to each mounting groove; at least one amplification part is formed inside the chip, and the chip is installed in the installation groove; an elastic adhesive tape is arranged at the bottom of the mounting groove and fixes the chip in the mounting groove; at least two micro-cavity holes are formed in the elastic adhesive tape, and the micro-cavity holes seal the communication position of the gland sample inlet and the amplification part and the communication position of the gland gas outlet and the amplification part; the second face of gland is provided with at least one sealing mechanism, sealing mechanism is used for sealing the gland inlet with the gland gas outlet.

The elastic adhesive tape has an elastic buffering function, so that on one hand, the chip is attached in the mounting groove through the adhesive tape with viscosity, and the chip can be fixed; on the other hand, when the microfluidic chip is used by being attached to an external device (for example, a PCR device), the connection between the inlet of the gland and the amplification part of the chip and the connection between the outlet of the gland and the amplification part of the chip can be sealed by using the elastic buffer action of the elastic adhesive tape. The liquid sample enters the chip amplification part from the gland sample inlet, gas in the chip is discharged to the gland gas outlet from the chip amplification part, and the sample can be prevented from leaking under the sealing action of the elastic adhesive tape. The sealing mechanism seals the gland sample inlet and the gland gas outlet, so that a totally closed PCR amplification environment can be realized. One or more sealing mechanisms can be provided, and one sealing mechanism can be used for sealing one or more gland sample inlets and gland gas outlets.

The first surface of the gland is provided with four mounting grooves, and the second surface is provided with one gland sample inlet and one gland gas outlet corresponding to each mounting groove; each mounting groove is internally provided with one chip, and an amplification part is formed in each chip. One amplification part can perform a sample test, and one chip can perform a sample test when one amplification part exists in one chip. A chip can be selectively installed in one installation groove, and when four installation grooves exist in one gland, four samples can be detected simultaneously.

The first face of gland is provided with at least one iron sheet, iron sheet and the magnet interact on the external equipment produce suction. The iron sheet can be formed on the gland through adopting an embedding process in the injection molding process of the gland, the iron sheet arranged and the magnet assembled on the external equipment interact to generate suction force, so that the elastic adhesive tape in the installation groove is extruded under an action force (the elastic adhesive tape and the chip must have certain thickness, and the elastic adhesive tape can be extruded when the iron sheet on the gland and the magnet of the external equipment generate suction force). The sealing effect of the elastic adhesive tape is further enhanced under the action of the suction force between the iron sheet and the magnet, so that the reaction liquid cannot leak into the air to cause aerosol pollution in the temperature rising and falling process.

The iron sheet that the first face of gland set up totally two, two the iron sheet symmetry sets up the both sides of gland the periphery of mounting groove. Two iron sheet settings can make a plurality of position of chip all receive the effort that suction produced between iron sheet and the magnet on every side in the both sides of gland to produce diversified pressure to the elastic adhesive tape, further strengthen sealed effect.

The sealing mechanism comprises a fixing structure fixed on the gland and a plurality of sealing plugs connected with the fixing structure, and the sealing plugs are used for sealing the gland sample inlet and the gland gas outlet. A plurality of the sealing plugs can be arranged in a row and taken as a whole, and the row of the sealing plugs can be connected with a fixed structure to simultaneously seal a plurality of gland sample inlets and gland gas outlets. The sealing plug is connected with the gland through the fixing structure, so that the sealing of the gland sample inlet and the gland gas outlet can be facilitated. The sealing plug is made of an elastic base material of silica gel or a rubber-coated layer, and the silica gel sealing plug can prevent the sealing material from reacting with the sample.

The elastic adhesive tape is a high-temperature-resistant foam adhesive tape, but is not limited to the material, and the elastic and high-temperature-resistant material such as polyacrylate material and silica gel material is the main chemical material within the protection scope of the patent. The high temperature resistance means that the temperature can be maintained for more than 30min under the temperature condition of 100 ℃. A plurality of temperature rise and drop stages exist in the nucleic acid amplification process, and the high-temperature-resistant foam adhesive tape is selected, so that the reaction liquid cannot leak into the air to cause aerosol pollution in the rapid temperature rise and drop process, and the totally-closed PCR environment is realized.

The chip comprises a first material layer and a second material layer, wherein the first material layer is positioned between the elastic adhesive tape and the second material layer; the first material layer is a sample inflow and outflow circulation layer, and the second material layer is an amplification layer. The second material layer is the main part of the amplification part, the sample is subjected to amplification reaction on the second material layer, the first material layer is used as a channel for sample introduction and discharge, and the composite double-layer chip main body structure enables the processing technology of the chip to be simpler and more convenient.

The chip further comprises a heat conducting sheet, the heat conducting sheet is located on the surface, on one side, of the second material layer, and the surface is far away from the first material layer, and the heat conducting sheet is connected with an external heat source. The conducting strip can be non-metal conducting strip or metal conducting strip, and non-metal conducting strip includes conducting strip or heat conduction graphite flake etc. that heat conduction silica gel piece, heat conduction phase change material constitute, and the metal conducting strip includes metal conducting strips such as copper or aluminium. The second material layer has an amplification part formed therein, and a plurality of temperature raising and lowering processes are performed in the nucleic acid amplification process. The heat-conducting sheet is directly contacted with the second material layer on which the amplification part is formed, heat is transferred to the heat-conducting sheet through an external heat source (for example, the heat-conducting sheet is placed on a heat transfer seat of the PCR equipment and the heat transfer seat is connected with the external heat source), and the heat-conducting sheet transfers the heat to the second material layer of the chip.

At least one sample inlet hole and at least one gas outlet hole are formed in the first material layer, and at least one liquid inlet micro-channel, an amplification cavity and a liquid outlet micro-channel are formed in the second material layer; the gland sample inlet, the sample inlet hole, the liquid inlet micro-channel and the amplification cavity are communicated in sequence, and the gland gas outlet, the gas outlet hole, the liquid outlet micro-channel and the amplification cavity are communicated in sequence. The micro-cavity hole formed in the elastic adhesive tape is respectively positioned between the gland sample inlet and the sample inlet and between the gland gas outlet and the gas outlet, and the micro-cavity hole is respectively sealed between the gland sample inlet and the sample inlet and between the gland gas outlet and the gas outlet. The formation of the amplification cavity, the liquid inlet micro-channel and the liquid outlet micro-channel can be completed through an etching process, a structure similar to a ditch is formed in the second material layer, the amplification cavity, the liquid inlet micro-channel and the liquid outlet micro-channel are formed by matching the first material layer, the process is simple, and the preparation cost of the chip is low.

The amplification cavity is a coiled pipe amplification cavity. The amplification cavity is in a snake-shaped trend, a longer reaction cavity can be formed in a smaller space, and the path of sample circulation is prolonged, so that the detection sensitivity and reliability of the microfluidic chip are improved. The amplification cavity is set into a plurality of reciprocating curved cavity structures such as snakes, and the volume of the chip can be reduced.

The first material layer is a glass sheet, and the second material layer is a silicon wafer. The multi-material composite chip can meet the requirements of target chips by utilizing the characteristics of different materials, and silicon-based chips can be obtained through a wafer etching process. The chip is a silicon-based chip, the adopted material has high thermal conductivity, the temperature of the amplification cavity in the chip is almost the same as that of a heat transfer seat of external equipment, the heat conduction is faster, and the rapid temperature rise and fall of the microfluidic chip can be realized.

The pressing cover is provided with at least one positioning hole, and the pressing cover is positioned on external equipment through the positioning hole. The position to be heated on the chip can be accurately positioned at the heat source of an external instrument through the positioning hole, so that the temperature rising and reducing efficiency is improved.

And hand-held structures are symmetrically arranged on two sides of the gland. The micro-fluidic chip is convenient for operators to use, and is safer and more convenient in the use process of nucleic acid amplification detection.

The technical scheme of the invention has the beneficial effects that: the closed microfluidic chip for nucleic acid amplification detection provided by the invention has the advantages that the elastic adhesive tape is arranged between the mounting groove and the chip, the chip is fixed by using the viscosity of the elastic adhesive tape, and the sample inlet of the gland, the gas outlet of the gland and the chip are sealed by using the elastic buffering effect of the elastic adhesive tape. The sealing mechanism is used for sealing the gland sample inlet and the gland gas outlet, and the elastic adhesive tape is further extruded under the suction action of the iron sheet and the magnet, so that the sealing effect of the elastic adhesive tape is improved, and the totally-enclosed PCR environment can be realized. Further utilize the conducting strip to heat the chip that high thermal conductivity material formed, can realize the quick temperature rise and fall of amplification portion, greatly shorten nucleic acid detection consuming time. The invention has simple manufacturing process and low cost, and is convenient for large-scale application and popularization.

Drawings

In order to more clearly illustrate the technical solutions in the specific embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without inventive efforts.

Fig. 1 is a front perspective view of a microfluidic chip of example 1 of the present invention.

Fig. 2 is a schematic perspective view of a gland according to embodiment 1 of the present invention.

Fig. 3 is a top view of a silicon-based chip according to embodiment 1 of the present invention.

Fig. 4 is a front view of a silicon-based chip according to embodiment 1 of the present invention.

Fig. 5 is a front perspective view of a silicon-based chip according to embodiment 1 of the present invention.

Fig. 6 is a schematic perspective structural view of a silicon-based chip and a foam tape in embodiment 1 of the present invention.

Fig. 7 is a side perspective view of a microfluidic chip according to example 1 of the present invention.

Fig. 8 is a top perspective view of a microfluidic chip (including a heat transfer mount) according to example 1 of the present invention.

Fig. 9 is a schematic perspective view of a microfluidic chip according to example 1 of the present invention.

Fig. 10 is a rear view (second side, back side) of the microfluidic chip of example 1 of the present invention.

Fig. 11 is a front view (first side, front side) of a microfluidic chip (including a heat transfer seat) according to example 1 of the present invention.

Reference numerals: 1-pressing a cover; 101-pressing a cover sample inlet; 102-gland outlet; 103-pressing cover liquid inlet micropores; 104-pressing and liquid outlet micropores; 2-a sealing mechanism; 201-a fixed structure; 202-a sealing plug; 301. 302-iron sheet; 4-positioning holes; 5-chip; 501-an amplification cavity; 502-liquid inlet microchannel; 503-liquid outlet micro-channel; 504-sample wells; 505-air outlet; 506-a first material layer; 507-a second material layer; 508-thermally conductive fins; 6-elastic adhesive tape; 601-liquid inlet hole; 602-liquid outlet holes; 7-heat transfer seat; 8-mounting grooves; 110-a handheld structure.

Detailed Description

The technical solutions in the specific embodiments of the present invention will be clearly and completely described below, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

This example provides a closed microfluidic chip for nucleic acid amplification detection, as shown in fig. 1 and 2, comprising a cover 1, an elastic tape 6 and a chip 5. The gland 1 is a plastic gland, in particular a polycarbonate material (PC plastic) and is manufactured by an injection molding process. The PC plastic belongs to a colorless and transparent amorphous thermoplastic material, has physical and mechanical properties such as high-quality heat resistance, good transparency and extremely high impact resistance, and has the characteristics of high-efficiency light transmittance, refractive index, easiness in processing and forming and the like. In other embodiments, the base material of the gland 1 may also be made of other high molecular materials, such as Polystyrene (PS), polypropylene (PP), cyclic Olefin Polymer (COP), polyvinyl chloride (PVC), and other heat-resistant materials. The gland 1 comprises a first surface (a front surface, the front surface of which is used as the front surface of the microfluidic chip) and a second surface (a back surface, the back surface of which is used as the back surface of the microfluidic chip, and can be back-up when in use), the base material of the gland 1 adopted in the embodiment is colorless and transparent, and a front perspective view of the microfluidic chip is shown in fig. 1, namely, partial structures of the first surface and the second surface of the gland 1 are simultaneously shown.

Four mounting grooves 8 for mounting the chip 5 are formed on the first face (front face) of the cover 1 by an injection molding process. The four mounting grooves 8 are distributed in the middle of the gland 1 and can be distributed in a straight line and in parallel or in a shape like Chinese character 'tian'. In this embodiment, the four mounting grooves 8 are distributed in a shape like a Chinese character 'tian'. A gland sample inlet 101 and a gland gas outlet 102 are formed on a second surface (back surface) of the gland 1 corresponding to the mounting groove 8. A gland liquid inlet micropore 103 communicated with the gland sample inlet 101 and a gland liquid outlet micropore 104 communicated with the gland gas outlet 102 are correspondingly formed in the mounting groove 8.

At least one chip 5 can be mounted in each mounting groove 8, and one chip 5 can be used for detecting at least one sample, so that a plurality of samples can be detected simultaneously by providing a plurality of mounting grooves 8. The mounting grooves 8 are provided with a corresponding number of chips 5 according to requirements, and the chips 5 can be selectively assembled. In other embodiments, more or less than four mounting grooves 8 may be disposed on the pressing cover 1, for example, only one to two mounting grooves 8 are disposed, and for example, eight mounting grooves 8 may be disposed to simultaneously detect more samples.

As shown in fig. 3, the chip 5 is a silicon-based chip and is composed of a first material layer 506, a second material layer 507 and a heat conducting fin 508, wherein the first material layer 506 is a glass sheet, and the second material layer 507 is a silicon wafer. And stacking and connecting the sheet structures in sequence according to the sequence of the glass sheet, the silicon wafer and the heat conducting sheet 508 to form the silicon-based chip 5. The second material layer 507 is made of silicon wafers, so that an amplification part can be formed in the silicon wafers conveniently through a wafer etching process, and the temperature rising and falling efficiency of the PCR reagent can be almost the same as the temperature rising and falling efficiency of a heat source on external equipment by utilizing the semiconductor heat conduction property of the silicon wafers. In other embodiments, the material of the first material layer 506 and the second material layer 507 can also be selected from other materials, such as high thermal conductivity materials like aluminum nitride, silicon carbide, etc. The heat-conducting plate 508 is preferably an elastic heat-conducting material, which can reduce the air cavity generated between the chip 5 and the heat-conducting seat of the external device.

As shown in fig. 5, a serpentine amplification chamber 501, a liquid inlet microchannel 502 and a liquid outlet microchannel 503 are formed in the second material layer 507 (silicon wafer) by a wafer etching process, and the liquid inlet microchannel 502, the amplification chamber 501 and the liquid outlet microchannel 503 are sequentially communicated. Referring to fig. 3 and 4, a sample inlet hole 504 is formed in the first material layer 506 (glass sheet), and the sample inlet hole 504 is communicated with the liquid inlet microchannel 502; an air outlet 505 is formed in the first material layer 506 (glass sheet), and the air outlet 505 is communicated with the liquid outlet micro-channel 503.

As shown in fig. 6 and 7, an elastic adhesive tape 6 is applied to the side of the first material layer 506 (glass sheet) of the chip 5 facing away from the second material layer 507 (silicon wafer). Two micro-cavity holes are formed inside the elastic adhesive tape 6, wherein one micro-cavity hole is a liquid inlet hole 601, and the other micro-cavity hole is a liquid outlet hole 602. The liquid inlet 601 of the elastic tape 6 is communicated with the sample inlet 504 of the chip 5, and the liquid outlet 602 of the elastic tape 6 is communicated with the gas outlet 505 of the chip 5. The other side of the elastic adhesive tape 6 is attached to the mounting groove 8 in a matched manner through a tool, the liquid inlet hole 601 is coated on the outer side of the gland liquid inlet micropore 103, and the liquid outlet hole 602 is coated on the outer side of the gland liquid outlet micropore 104, so that the gland liquid inlet micropore 103 is connected to the sample inlet hole 504 of the chip 5 in a sealed manner to realize communication, and the gland liquid outlet micropore 104 is connected to the gas outlet hole 505 of the chip 5 in a sealed manner to realize communication. Thereby realizing the sealing function of the elastic adhesive tape 6 on the gland sample inlet 101, the gland gas outlet 102 and the chip 5.

The elastic tape 6 is an elastic tape, and may be a tape having a sticky surface or a double-sided tape, and may be used by peeling off a release film on the surface. High temperature resistant foam adhesive tape is selected for use to elastic adhesive tape 6, and in PCR nucleic acid amplification detection, there are a plurality of stages of heating and cooling, adopt high temperature resistant foam adhesive tape can make reaction liquid can not leak in the air and lead to the aerosol pollution at quick heating and cooling in-process to realize totally closed PCR environment. In other embodiments, other high temperature resistant elastic tapes 6 may be selected, such as a high temperature resistant organic silica gel layer, an acrylic adhesive layer, etc., and an elastic and high temperature resistant material, such as a polyacrylate material, a silica gel material, etc., may be selected as the main chemical material. Elastic adhesive tape 6 has viscidity, and the one side laminating of bubble cotton sticky tape is on the glass piece, and shape, size and the mounting groove 8 phase-match of bubble cotton sticky tape, the another side of bubble cotton sticky tape match the laminating in mounting groove 8 of gland 1 through the frock to fix chip 5 in mounting groove 8.

Referring to fig. 1, 7 to 9, two seal mechanisms 2 are formed on the second surface (back surface) of the gland 1, and the two seal mechanisms 2 are disposed on both sides of the gland 1, and one of the seal mechanisms 2 is described as an example (fig. 7 isbase:Sub>A side perspective view of the microfluidic chip, and an observation direction is indicated bybase:Sub>A linebase:Sub>A-base:Sub>A in fig. 1). The sealing mechanism 2 comprises a fixing structure 201 fixed on the gland 1, and a group of sealing plugs 202 connected with the fixing structure 201. The sealing plugs 202 are silica gel sealing plugs, and the group of sealing plugs 202 has four silica gel plugs, and the four silica gel plugs are connected with the fixing structure 201 as a whole. The four silica gel plugs are distributed on one side of two mounting grooves 8 on the gland 1, and are in a group of two silica gel plugs, and each group corresponds to a gland sample inlet 101 and a gland gas outlet 102 of one mounting groove 8 respectively. One sealing mechanism 2 can seal two gland sample inlets 101 and two gland gas outlets 102 corresponding to two chips 5, namely two mounting grooves 8. The material of the sealing plug 202 may also be an elastic substrate of other adhesive layers.

Referring to fig. 1 and 2, two iron sheets 301 and 302 are symmetrically embedded in a gland 1 through an injection molding process, and the two iron sheets 301 and 302 are symmetrically arranged at both sides of the gland 1. In this embodiment, four mounting grooves 8 are distributed in the middle of the gland 1 in a shape like a Chinese character 'tian', and the iron sheets 301 and 302 are distributed on the outer sides of the four mounting grooves 8 on the gland 1. The two iron sheets 301 and 302 are used for generating attraction with a magnet on an external device, and the chip 5 in the four mounting grooves 8 between the iron sheets 301 and 302 is pressed by the attraction, so that the elastic adhesive tape 6 between the chip 5 and the mounting grooves 8 is squeezed. The elastic buffering effect of the elastic adhesive tape 6 is exerted by squeezing the elastic adhesive tape 6, and the sealing performance between the chip 5 and the mounting groove 8 is increased, so that the sealing performance of the whole detection process is improved.

Specifically, under the action of the attraction force between the magnet and the iron sheets 301 and 302, the elastic tape 6 is used to realize the elastic buffering between the glass sheet and the mounting groove 8, so as to further improve the sealing performance between the sample inlet 504 of the chip 5 and the liquid inlet micro-hole 103 of the pressing cover, and between the gas outlet 505 of the chip 5 and the liquid outlet micro-hole 104 of the pressing cover. In addition, the adopted foam adhesive tape has high temperature resistance, so that the reaction liquid can not leak into the air to cause aerosol pollution in the rapid temperature rise and fall process. In the extrusion process, the deviation caused by the height difference of the chips 5 in the mounting groove 8 can be adjusted by utilizing the buffer action of the elastic adhesive tape 6, so that each chip can obtain good sealing performance. In other embodiments, the number and distribution of the iron sheets 5 may also be adjusted according to the number and distribution of the mounting slots 8, for example, four iron sheets may be symmetrically placed two by two outside the mounting slots 8, or may also be distributed between the mounting slots 8.

As shown in fig. 10 and 11, four positioning holes 4 are symmetrically arranged at four corners of the gland 1 during the injection molding process. Four positioning holes 4 are used to position the cover 1 and the chip 5 on an external device. The heat conducting sheet 508 is connected with the external heat conducting seat 7, the heat conducting seat 7 is connected with a heat source, the heat conducting sheet 508 (connected with the heat conducting seat 7) on the chip 5 is accurately positioned with the external heat source through the four positioning holes 4, the heat of the external heat can be transferred to the heat conducting sheet 508 through the heat conducting seat 7, and the heat conducting sheet 508 transfers the heat to the second material layer 507, namely a silicon wafer layer. Therefore, the temperature rise and the temperature fall of the amplification cavity 501 and the temperature rise and the temperature fall of the heat transfer seat 7 can be effectively kept at the same frequency, the average temperature rise and the average temperature fall can be realized to be more than 15 ℃/s, and the time consumption of 40 PCR amplification cycles in the past market is reduced to 5min from 60 min. The amplification time is shortened, the detection efficiency is further improved, and the detection cost is reduced.

As shown in fig. 9, two hand-held structures 110 are symmetrically disposed on two sides of the gland 1, so that an operator can use the microfluidic chip conveniently, and the use process of nucleic acid amplification detection is safer and more convenient.

The structure of the microfluidic chip provided by this embodiment is as follows:

two symmetrically distributed handheld structures 110, two symmetrically distributed sealing mechanisms 2, two symmetrically distributed iron sheets 301 and 302, four symmetrically distributed positioning holes 4 and four symmetrically distributed mounting grooves 8 are formed on the plastic gland 1. Each mounting groove 8 is internally assembled with one chip 5, and four chips 5 are assembled. A layer of elastic adhesive tape 6 is arranged between each chip 5 and the mounting groove 8, and the chips 5 are fixed in the mounting grooves 8 through the elastic adhesive tapes 6.

Each chip 5 is internally provided with one amplification cavity 501, and the microfluidic chip provided by the embodiment has four chips 5, namely four amplification cavities 501, so that four samples can be detected simultaneously. Taking a chip 5 as an example, the chip 5 is formed by sequentially stacking, connecting and compounding three layers of materials, wherein the first layer is a glass sheet, the second layer is a silicon sheet, and the third layer is a heat-conducting sheet 508; a sample inlet 504 and a gas outlet 505 are formed in the glass sheet, and an amplification cavity 501, a liquid inlet micro-channel 502 and a liquid outlet micro-channel 503 are formed in the silicon sheet. A gland sample inlet 101, a gland liquid inlet micropore 103, a gland gas outlet 102 and a gland liquid outlet micropore 104 are formed on the mounting groove 8. The elastic adhesive tape 6 is internally provided with a liquid inlet 601 and a liquid outlet 602.

The gland sample inlet 101, the gland liquid inlet micropore 103 (the outside is covered with a liquid inlet hole 601), the sample inlet hole 504, the liquid inlet microchannel 502 and the amplification cavity 501 are communicated in sequence. The gland gas outlet 102, the gland liquid outlet micropore 104 (the outside is covered with a liquid outlet 602), the gas outlet 505, the liquid outlet microchannel 503 and the amplification cavity 501 are communicated in sequence. Each sealing mechanism 2 is correspondingly provided with four silica gel plugs, each two silica gel plugs form a group, and each group corresponds to one gland sample inlet 101 and one gland gas outlet 102. In the using process, the gland sample inlet 101 and the gland gas outlet 102 are sealed by the sealing plug 202 on the sealing mechanism 2, so as to form a totally-closed PCR nucleic acid amplification detection environment.

The working principle of the embodiment is as follows: the sample enters the gland liquid inlet micro hole 103 from the gland sample inlet 101 on the gland 1, enters the sample inlet hole 504 of the chip 5 from the gland liquid inlet micro hole 103 (the space between the gland liquid inlet micro hole 103 and the sample inlet hole 504 is sealed by a foam tape provided with a liquid inlet hole 601), flows into the liquid inlet micro channel 502 of the chip 5 from the sample inlet hole 504, and then flows into the coiled pipe amplification cavity 501 from the liquid inlet micro channel 502. The gas originally retained in the coiled pipe amplification cavity 501 is discharged from the liquid outlet micro-channel 503 and the gas outlet 505 of the chip 5 to the gland liquid outlet micro-hole 104, and finally discharged from the gland gas outlet 102. At this time, the silica gel plugs on the sealing plug 202 block the gland sample inlet 101 and the gland gas outlet 102, thereby completing the sealing of the chip 5, forming a fully closed PCR reaction environment, and starting the reaction. The heat conductive sheet 508 is heated by the heat transfer base 7, and the temperature of the amplification chamber 501 is cyclically increased and decreased. The position corresponding to the chip 5 in the mounting groove 8 is the fluorescence detection area of the external device, and after the amplification reaction of the sample is completed in the coiled pipe amplification cavity 501 of the chip 5, the external device can be used for performing fluorescence detection on the sample.

The above embodiment describes a case of assembling four silicon-based chips 5 by one gland 1, but the technical scheme protected by the present invention is not limited thereto, the design of the microfluidic chip provided by the present invention has great amplification, and the silicon-based chips 5 can be selectively assembled, and can realize 1 to 4 sample detections. In other embodiments, more amplification chambers 501, for example, four serpentine amplification chambers 501, may be disposed in the silicon wafer of each chip 5, so that amplification detection of 1 to 16 samples can be simultaneously achieved. Even more samples can be tested, i.e. the silicon wafer and gland 1 designs are diversified.

Most of the existing microfluidic chip consumables are single materials, such as pure glass materials or pure PC materials. In one embodiment of the invention, the consumables of the microfluidic chip use various materials such as PC plastic, elastic foam and cotton rubber materials, glass, si materials, silica gel, iron sheets and the like, the target microfluidic chip is prepared by utilizing a multi-material combination mode, and the full-closed PCR amplification environment is realized. According to the closed chip consumable combined by the silicon-based material and the high polymer material, the extracted nucleic acid or the non-extracted nucleic acid is added into the amplification cavity 501 of the chip 5 for amplification and detection, so that closed-tube nucleic acid amplification detection is realized. And the microfluidic chip is prepared by adopting a wafer low-cost etching process and injection molding production gland, the production cost of the product is low, and the product is suitable for large-scale application and popularization, and the product of the invention is produced in batch.

In one embodiment of the invention, the silica gel sealing plugs are used for sealing the gland sample inlet 101 and the gland gas outlet 102, the elastic buffering performance of the foam adhesive tape is used for sealing the gap between the gland 1 (the mounting groove 8) and the chip 5, and the iron sheet and the magnet assembled on the PCR equipment are embedded in the gland 1 to generate suction force to extrude the foam adhesive tape between the chip 5 and the gland 1, so that the sealing performance of the cavity of the chip 5 is realized. The microfluidic chip provided by the invention can realize a totally-enclosed PCR amplification environment without independently arranging a cover plate on the gland 1, so that the reaction liquid cannot leak into the air to cause aerosol pollution in the rapid temperature rise and fall process. The structure for realizing PCR amplification environment sealing enables the interior of the chip 5 to be completely isolated from the external environment, so that the chip is free from external pollution and wide in applicable scene.

In one embodiment of the present invention, the positioning hole 4 is used to position the heat-conducting plate 508 and the heat-transfer base 7 in contact with the heat-conducting plate 508, and is positioned at the heat source of the external device. The heat-conductive sheet 508 is in direct contact with the silicon wafer in which the serpentine amplification chamber 501 is formed, and covers the serpentine amplification chamber 501 in the silicon wafer. The rapid semiconductor thermal technology of the device can be combined to provide a rapid temperature rise and fall heat source, and then the heat conduction is carried out on the amplification cavity 501 of the chip 5 through the copper sheet heat conduction seat 7 with high heat conductivity. Because the amplification cavity 501 is a three-dimensional silicon material coiled pipe, the temperature rising and reducing efficiency of the PCR reagent can almost have the same frequency as the temperature rising and reducing efficiency of the heat transfer seat 7, the heat conduction efficiency is improved, forty thermal cycle amplification can be completed within five minutes, the rapid temperature rising and reducing can be realized, the consumed time of 40 PCR amplification cycles in the past market can be reduced to 5min, and the consumed time of PCR detection is greatly shortened. Can utilize the elastic buffer design of bubble cotton sticky tape and conducting strip 508, thereby double buffering makes the silicon chip can effectual contact to the copper sheet heat transfer seat 7 on the PCR equipment and does not produce the air cavity, and heat transfer effect is better.

The above description of the embodiments is only intended to facilitate the understanding of the method and the core idea of the present invention. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

Claims (14)

1. A closed micro-fluidic chip for nucleic acid amplification detection is characterized in that: comprises a gland and at least one chip; the first surface of the gland is provided with at least one mounting groove, and the second surface is provided with at least one gland sample inlet and a gland gas outlet corresponding to each mounting groove; at least one amplification part is formed inside the chip, and the chip is installed in the installation groove; an elastic adhesive tape is arranged at the bottom of the mounting groove and fixes the chip in the mounting groove; at least two micro-cavity holes are formed in the elastic adhesive tape, and the at least two micro-cavity holes seal the communication position of the gland sample inlet and the amplification part and the communication position of the gland gas outlet and the amplification part; the second face of gland is provided with at least one sealing mechanism, sealing mechanism is used for sealing the gland inlet with the gland gas outlet.

2. The closed microfluidic chip for nucleic acid amplification detection according to claim 1, wherein: the first surface of the gland is provided with four mounting grooves, and the second surface is provided with one gland sample inlet and one gland gas outlet corresponding to each mounting groove; one chip is installed in each installation groove, and an amplification part is formed in each chip.

3. The closed microfluidic chip for nucleic acid amplification detection according to claim 1 or 2, wherein: the first face of gland is provided with at least one iron sheet, iron sheet and the magnet interact on the external equipment produce suction.

4. The closed microfluidic chip for nucleic acid amplification detection according to claim 3, wherein: the iron sheet that the first face of gland set up totally two, two the iron sheet symmetry sets up the both sides of gland the periphery of mounting groove.

5. The closed microfluidic chip for nucleic acid amplification detection according to claim 1 or 2, wherein: the sealing mechanism comprises a fixing structure fixed on the gland and a plurality of sealing plugs connected with the fixing structure, and the sealing plugs are used for sealing the gland sample inlet and the gland gas outlet.

6. The enclosed microfluidic chip for nucleic acid amplification detection according to claim 5, wherein: the sealing plug is made of an elastic base material of silica gel or a glue coating layer.

7. The closed microfluidic chip for nucleic acid amplification detection according to claim 1, wherein: the elastic adhesive tape is a high-temperature-resistant foam adhesive tape.

8. The closed microfluidic chip for nucleic acid amplification detection according to claim 1, wherein: the chip comprises a first material layer and a second material layer, wherein the first material layer is positioned between the elastic adhesive tape and the second material layer; the first material layer is a sample inflow and outflow circulation layer, and the second material layer is an amplification layer.

9. The closed microfluidic chip for nucleic acid amplification detection according to claim 8, wherein: the chip further comprises a heat conducting sheet, the heat conducting sheet is located on the surface, on one side, of the second material layer, and the surface is far away from the first material layer, and the heat conducting sheet is connected with an external heat source.

10. The enclosed microfluidic chip for nucleic acid amplification detection according to claim 8, wherein: at least one sample inlet hole and at least one gas outlet hole are formed in the first material layer, and at least one liquid inlet micro-channel, an amplification cavity and a liquid outlet micro-channel are formed in the second material layer; the gland sample inlet, the sample inlet hole, the liquid inlet micro-channel and the amplification cavity are communicated in sequence, and the gland gas outlet, the gas outlet hole, the liquid outlet micro-channel and the amplification cavity are communicated in sequence.

11. The enclosed microfluidic chip for nucleic acid amplification detection according to claim 10, wherein: the amplification cavity is a coiled pipe amplification cavity.

12. The closed microfluidic chip for nucleic acid amplification detection according to any one of claims 8 to 11, wherein: the first material layer is a glass sheet, and the second material layer is a silicon wafer.

13. The closed microfluidic chip for nucleic acid amplification detection according to claim 1 or 2, wherein: the pressing cover is provided with at least one positioning hole, and the pressing cover is positioned on external equipment through the positioning hole.

14. The closed microfluidic chip for nucleic acid amplification detection according to claim 1 or 2, wherein: and hand-held structures are symmetrically arranged on two sides of the gland.

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