CN109536366B - Nucleic acid detection micro-fluidic chip based on modified capillary and nucleic acid detection system - Google Patents
Nucleic acid detection micro-fluidic chip based on modified capillary and nucleic acid detection system Download PDFInfo
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/02—Adapting objects or devices to another
- B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
- B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
Abstract
The invention relates to a nucleic acid detection micro-fluidic chip and a nucleic acid detection system based on a modified capillary tube. The invention realizes the functions of sample cracking, nucleic acid extraction, nucleic acid amplification and nucleic acid detection, and can be deployed in non-laboratory areas such as entry and exit ports, fields and the like and detect infectious pathogens.
Description
Technical Field
The invention relates to the technical field of microfluidic nucleic acid analysis chips, in particular to a nucleic acid detection microfluidic chip based on a modified capillary and a nucleic acid detection system.
Background
In recent years, several large-scale infectious diseases such as H1N1, ebola virus, zika, and the like have been developed worldwide, and need to be detected through a nucleic acid extraction and amplification process by a specialized nucleic acid detection instrument. Generally speaking, such instruments have the disadvantages of large volume, difficult field deployment, high cost, special maintenance and the like. The microfluidic chip is widely applied to the field of biochemical analysis, such as the field of bacteria, virus and protein detection, and the like, due to the characteristics of easy integration, automation, controllable fluid and small required sample volume, and the defect that professional nucleic acid detection equipment is difficult to deploy in non-laboratory areas such as entry and exit ports, fields and the like is overcome to a certain extent.
One of the cores of the nucleic acid detection method based on the microfluidic chip is a nucleic acid extraction method, which is usually one of the following methods: boiling method, magnetic bead method and solid phase extraction method. In order to develop the on-site rapid nucleic acid detection device, research teams of the applicant have already conducted certain research on related fields and have deep research bases, for example, the integrated nucleic acid extraction real-time fluorescence PCR micro-total analysis system published in analytical chemistry by Mimi Zhao et al, the paper reports a microfluidic chip for detecting viruses in blood and a matched fluorescence quantitative detection device, but the chip reported in the paper adopts a solid phase extraction method based on a silica gel membrane to extract nucleic acid, and a sample is blood lysate, so that whole blood cannot be processed and analyzed. The patent of "apparatus and method for rapidly detecting infectious pathogens on site" filed by Liu Ying le et al describes a design of nucleic acid extraction apparatus based on the precipitation boiling method, but the method extracts a small amount of nucleic acid and has low purity, and is difficult to purify a sample with high quality requirement for nucleic acid purification such as RNA viruses. The cumonbo et al in the patent "microfluidic device for automatically extracting nucleic acid" introduced a nucleic acid extraction chip and device based on the magnetic bead method, which is not designed for the microfluidic chip, and relatively complicated in steps and difficult to be automatically integrated. The < Integrated glass microprocessor for nucleic acid amplification, loop-mediated isothermal amplification, and online detection > published by Thiago Wu Qingqing et al in Analytical chemistry reports a chip based on solid phase extraction method for nucleic acid extraction and amplification, which requires an additional nucleic acid elution step in the nucleic acid extraction process, increasing the integration difficulty of microfluidic chips. A capillary array nucleic acid in-situ amplification method based on adsorption of nucleic acid by chitosan modified glass filter paper is provided by Hui Junhou et al, which is published in Lab a Chip of < multiple sample-to-answer detection of bacteria using a peptide-activated capillary array with integrated DNA extraction, isotherma amplification and smartphone detection >, and realizes that nucleic acid can be amplified without an elution step, so that the integration difficulty is greatly reduced.
Disclosure of Invention
The invention solves the technical problems that: the defects of the prior art are overcome, and the nucleic acid detection micro-fluidic chip and the nucleic acid detection system based on the modified capillary are provided, so that the functions of sample cracking, nucleic acid extraction, nucleic acid amplification and nucleic acid detection are integrated, and the micro-fluidic chip and the nucleic acid detection system can be deployed in a non-laboratory area and can detect infectious pathogens.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the micro-fluidic chip internally comprises a modified capillary tube which is used for adsorbing target nucleic acid in a solution sample to be detected and is used as a carrier for realizing an automatic realization mode of nucleic acid adsorption, purification, amplification and detection;
the chip is of a four-layer structure comprising a substrate, a liquid path layer substrate, an elastic film and a gas path layer substrate from bottom to top; the upper surface of the liquid path layer substrate is embedded with a liquid circulation channel, a liquid mixing channel and a liquid shunting channel, and a penetrating hole is communicated with the lower surface of the liquid path layer; the lower surface of the liquid path layer is embedded with a cracking cavity and a capillary reserved cavity, and the modified capillary is embedded in the capillary reserved cavity to realize the adsorption and amplification of the nucleic acid sample; the upper surface of the gas circuit layer substrate is provided with a plurality of hollow cavities for storing detection samples, lysate or PCR reagents; the lower surface of the liquid path layer substrate is provided with a plurality of sunken air valves and penetrates through the upper surface of the liquid path layer substrate through holes, the lower surface of the liquid path layer substrate is bonded with the bottom substrate, the middle cavity part is embedded with the capillary tube, and the upper surface of the liquid path layer substrate is bonded with the lower surface of the elastic film; the upper surface and the lower surface are connected with each other through a microchannel, so that the liquid storage cavity, the cracking cavity, the capillary tube and the outlet are communicated; the lower surface of the gas circuit layer substrate is bonded with the upper surface of the elastic film to form a complete micro-fluidic chip.
The substrate is glass or a silicon wafer; the liquid path layer substrate and the gas path layer substrate are made of PDMS, PC, PMMA or COC polymer materials; the elastic membrane is made of PDMS or TPU elastic material;
in addition, according to an embodiment of the invention, the upper surface of the gas circuit layer substrate of the microfluidic chip further comprises three liquid storage cavities, the liquid storage cavities are commonly connected to a serpentine channel on the upper surface of the liquid circuit layer through a through hole penetrating through the upper surface and the lower surface of the gas circuit layer substrate, the serpentine channel is connected to a lysis cavity for lysing nucleic acid, and the lysis cavity is connected to a cavity for embedding a capillary tube.
The technical scheme adopted by the modified capillary tube is as follows: the surface of the capillary tube is enabled to carry positive charges through a surface modification technology, target nucleic acid in a solution after a sample is cracked can be enriched by the modified capillary tube, and meanwhile, the enriched nucleic acid can be spontaneously dissociated into the solution under the normal nucleic acid amplification condition;
in addition, according to an embodiment of the present invention, the modified capillary is made of any one of glass, silica or thermoplastic;
further, according to an embodiment of the present invention, the modified capillary inner wall contains a functional group or ionic compound having an amino structure at the end, including-NH2Or [ NH ]4]+A structure that adsorbs nucleic acids mainly based on hydrogen bonds and ionic bonds;
in addition, according to an embodiment of the invention, the modified capillary is fixed in the channel on the lower surface of the substrate of the microfluidic chip liquid path by interference fit, thermal bonding or plasma cleaning bonding;
in addition, according to an embodiment of the present invention, the target nucleic acid in the solution after the sample is lysed refers to a nucleic acid in a lysate environment, i.e., a lysate solution system does not need to be additionally changed;
in addition, according to an embodiment of the present invention, the normal nucleic acid amplification condition mainly means that the adsorbed nucleic acid is automatically separated from the wall of the capillary in the nucleic acid amplification reaction solution system to participate in the nucleic acid amplification reaction without additional measures, including changing the temperature, pH, and other ion concentrations in the solution.
In order to realize the automatic control of the microfluidic chip, the invention provides a matching device of the microfluidic chip, which comprises a fluid driving control system, an air valve control system, a temperature control system, an optical system, a control system and a smart phone and is used for realizing the full-automatic operation and detection process of the microfluidic chip. The method is used for realizing the full-automatic operation and detection process of the microfluidic chip and realizing the aims of sample input and result output.
The micro-fluidic chip adopts the technical scheme that: adopting a modified capillary tube embedded in a chip to adsorb and purify nucleic acid, and integrating nucleic acid amplification and detection into a microfluidic chip;
the matching device of the micro-fluidic chip adopts the following technical scheme:
the micro-fluidic chip is composed of a gas circuit layer substrate, an elastic film, a liquid circuit layer substrate, a heating cracking cavity, a first liquid storage cavity, a second liquid storage cavity, a third liquid storage cavity, a first injection pump interface, a second injection pump interface, a plurality of elastic film micro valves, a snake-shaped liquid mixing flow channel, a plurality of modified capillary reserved cavities, a first modified capillary, a second modified capillary, a third modified capillary and a fourth modified capillary;
the device comprises a fluid driving control system, an air valve control system, a temperature control system, an optical system, a control system and a smart phone, wherein the fluid driving control system and the air valve control system are connected with the upper surface of a chip air path layer substrate, the temperature control system is tightly attached to the lower surface of the chip substrate, the optical system is arranged right above the micro-fluidic chip, and the smart phone is arranged right above the optical system.
The fluid driving control system controls fluid in the microfluidic chip, the air valve control system controls the opening and closing of the elastic film micro valve, the temperature control system maintains the temperature required by nucleic acid amplification, the optical system provides excitation light with a specific wavelength and obtains emission light with the specific wavelength, the control system controls the fluid driving control system, the temperature control system and the optical system, and the image recognition function of the smart phone performs qualitative and semi-quantitative detection on a sample;
in addition, according to an embodiment of the present invention, the fluid driving control system is composed of a first syringe pump and a second syringe pump, the syringe pump is communicated with the sample inlet and outlet on the microfluidic chip, and drives the fluid by positive pressure and negative pressure;
in addition, according to one embodiment of the invention, the air valve control system is composed of an air source and a plurality of electromagnetic valves, the air source generates a positive pressure of 0.2MPa continuously and is communicated with the electromagnetic valves, and the other ends of the electromagnetic valves are respectively connected with at least one elastic film micro valve and are controlled to be opened and closed through the opening and closing of the electromagnetic valves;
in addition, according to one embodiment of the invention, the temperature control system is tightly attached to the substrate of the microfluidic chip, the temperature sensor, the Peltier, the heat sink, the heat dissipation fins and the fan are arranged inside the temperature control system, the temperature adjusting range is 0-100 ℃, the temperature adjusting precision is 0.1 ℃, the temperature rising and falling speed is 5 ℃/s, and the temperature control system is used for controlling the temperature of the capillary inside the microfluidic chip and meeting the temperature environment required by nucleic acid amplification;
in addition, according to an embodiment of the present invention, the optical system includes an excitation light source, a filter, a lens and a focusing mirror, and is mainly used for generating excitation light with a specific wavelength to irradiate the surface of the capillary of the microfluidic chip and obtaining emission light with a specific wavelength, so as to realize qualitative and semi-quantitative detection of nucleic acid amplification in the capillary;
furthermore, in accordance with an embodiment of the present invention, the control system includes a microprocessor for operating the fluid drive control system, the optical system and the temperature control system, and a wireless communication interface for communicating the microprocessor with a smart phone;
in addition, according to an embodiment of the present invention, the smartphone issues commands and obtains feedback data to the fluid drive control system, the temperature control system, and the optical system by controlling the microprocessor, detects a fluorescence signal by a camera provided in the smartphone, and performs qualitative or semi-quantitative analysis on a nucleic acid amplification result by a built-in image processing software.
In addition, according to an embodiment of the present invention, the method for analyzing the microfluidic chip includes the following steps:
(1) sample loading
A sample, lysis solution and reagent required by nucleic acid analysis are sequentially added into a first liquid storage cavity, a second liquid storage cavity and a third liquid storage cavity;
(2) mixing the sample with the lysis solution
The sample and the lysis solution are mixed under the action of a snake-shaped liquid mixing flow channel under the driving of negative pressure generated by an injection pump, and finally, the mixture is uniformly mixed and flows into a lysis cavity;
(3) sample lysis
Heating the mixed solution of the sample and the lysate in a lysis cavity through a temperature control system to release the DNA into the liquid;
(4) nucleic acid adsorption
Controlling the magnitude of the driving force, allowing the sample to pass through the modified capillary tube, and removing the lysate;
(5) elution of impurities
Injecting eluent into the first modified capillary tube, the second modified capillary tube and the third modified capillary tube through the through hole positioned above the gas circuit layer substrate by the injection pump, and then removing the eluent to obtain a nucleic acid sample;
(6) reaction liquid Loading
Loading a reaction liquid into a reaction liquid cavity above the gas circuit layer substrate through a branched flow passage to a first modified capillary, a second modified capillary, a third modified capillary and a fourth modified capillary under the action of external force driving;
(7) nucleic acid amplification
Heating a first modified capillary tube, a second modified capillary tube, a third modified capillary tube and a fourth modified capillary tube in the chip by a temperature control system, wherein the specific temperature condition required by nucleic acid amplification is determined by a selected reagent reaction system;
(8) nucleic acid detection
The modified capillary is photographed at regular time by using a smart phone to collect a fluorescence image, and the change of the fluorescence intensity of the modified capillary is recognized and detected by using built-in image recognition software, so that qualitative and semi-quantitative detection of the nucleic acid sample is realized.
The principle of the invention is as follows:
(1) the micro-fluidic chip is composed of 4 layers, a substrate, a liquid path layer substrate, an elastic film and a gas path layer substrate are respectively arranged from bottom to top, and a reagent and a sample enter and exit the cavity through a customized micro-channel and a micro-channel by the cooperation of external force drive and an elastic film valve; in addition, a modified capillary tube is embedded in the chip, nucleic acid is negatively charged in an alkaline solution (pH is greater than 7) and is adsorbed to the wall surface by the capillary tube, enrichment of a sample to be detected is realized, the capillary tube is eluted by using a weak alkaline solution, redundant impurities are removed, the nucleic acid adsorbed to the inner wall of the modified capillary tube is relatively difficult to elute due to low liquid flow rate of the inner wall surface of the tube, and a relatively pure nucleic acid sample is obtained.
(2) Based on the smart phone, the smart phone sends commands to control each system through a wireless communication means, a camera carried by the smart phone acquires a capillary fluorescence image, and the change of the capillary fluorescence intensity is detected through built-in image recognition software, so that the qualitative and semi-quantitative judgment of an amplification result is carried out.
Compared with the prior art, the invention has the following advantages:
(1) compared with the traditional nucleic acid extraction and detection method, the micro-fluidic chip and the system can realize the whole processes of nucleic acid cracking, nucleic acid adsorption, nucleic acid elution, nucleic acid amplification and detection, have simple operation steps, only need to add a sample, realize the whole process in the chip and complete the 'sample input-result output'.
(2) Compared with the traditional micro-fluidic chip for extracting nucleic acid by a magnetic bead method and a solid phase extraction method, the micro-fluidic chip containing the amino modified capillary avoids the step of eluting nucleic acid, can reduce the operation steps of nucleic acid pretreatment, and simplifies the flow;
(3) compared with the traditional microfluidic chip instrument, the invention realizes the detection of the sample based on the smart phone, reduces the manufacturing cost of the equipment and improves the portability and the flexibility of the instrument;
in summary, the invention provides a multifunctional nucleic acid analysis chip and a matching device with sample pretreatment, nucleic acid enrichment and purification, nucleic acid amplification and detection, realizes an automatic and totally enclosed microfluidic chip with sample in-and-result out, and is suitable for being applied in a scene where large-scale instruments and equipment are difficult to deploy.
Drawings
FIG. 1 is a top view of a microfluidic chip according to the present invention;
FIG. 2 is an analytic view of a microfluidic chip according to the present invention;
FIG. 3 is a schematic diagram of the structure of the device for sample injection, nucleic acid enrichment and elution of the microfluidic chip according to the present invention;
FIG. 4 is a schematic diagram of the structure of an apparatus for the amplification and detection process of the present invention.
In the figure, greek letter numbers are respectively: the system comprises a micro-fluidic chip I, a fluid driving control system II, an air valve control system III, a control system IV, a smart phone V, an optical system VI and a temperature control system VII; the Arabic numerals are respectively as follows: the gas circuit layer substrate 1, the elastic film 2, the liquid circuit layer substrate 3, the substrate 4, the cracking cavity 5, the first liquid storage cavity 61, the second liquid storage cavity 62, the third liquid storage cavity 63, the first injection pump interface 64, the second injection pump interface 65, the first elastic film micro valve 71, the second elastic film micro valve 72, the third elastic film micro valve 73, the fourth elastic film micro valve 74, the fifth elastic film micro valve 75, the sixth elastic film micro valve 76, the seventh elastic film micro valve 77, the serpentine liquid mixing channel 8, the first modified capillary reserved cavity 81, the second modified capillary reserved cavity 82, the third modified capillary reserved cavity 83, the fourth modified capillary reserved cavity 84, the first modified capillary 91, the second modified capillary 92, the third modified capillary 93, the fourth modified capillary 94, the first electromagnetic valve 101, the second electromagnetic valve 102, the third electromagnetic valve 103, the fourth electromagnetic valve 104, the device comprises an air source 11, a first injection pump 121, a second injection pump 122, a lens 13, a filter 14, an excitation light source 15, a focusing mirror 16, a heat sink 17, a heat dissipation fin 18 and a heat dissipation fan 19.
Detailed Description
The invention will be further described with reference to the following figures and examples. These examples are intended to illustrate the invention and do not limit the scope of application of the invention. Further, it should be understood that any changes and modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and those equivalents may fall within the scope of the claims attached to the present application.
As shown in fig. 1 and 2, the microfluidic chip I includes, from top to bottom, a gas circuit layer substrate 1, an elastic membrane 2, a liquid circuit layer substrate 3, and a base 4. Placing 4 capillaries into H2SO4Soaking in the solution for 1h to ensure that the inner surface of the capillary is completely covered so as to remove stains on the inner surface of the capillary; taking out the capillary tube, and washing the capillary tube for 1H by using pure water to remove H2SO4 on the inner surface of the capillary tube; finally, the capillary is placed in an alkaline compound with the tail end containing functional groups or ions with amino structures, such as polydiallyldimethylammonium chloride, chitosan or polydopamine, and the like, and the compound in the solution can react with the inner wall of the capillary, so that corresponding chemical groups are coated on the inner wall of the capillary, and the first modified capillary 91, the second modified capillary 92, the third modified capillary 93 and the fourth modified capillary 94 are obtained. And then, 4 modified capillaries are respectively transversely arranged in a first modified capillary reserved cavity 81, a second modified capillary reserved cavity 82, a third modified capillary reserved cavity 83 and a fourth modified capillary reserved cavity 84 at the bottom of the liquid path layer substrate 3. And then bonding the microfluidic chip I in sequence according to the sequence of the air circuit layer substrate 1, the elastic film 2, the liquid circuit layer substrate 3 and the substrate 4 to form the microfluidic chip I shown in the figures 1 and 2.
The air valve control system III in fig. 3 includes 4 electromagnetic valves, wherein a first electromagnetic valve 101 is connected to the first elastic film micro valve 71 in fig. 1 through an air path sealing pipeline, a second electromagnetic valve 102 is connected to the second elastic film micro valve 72 and the seventh elastic film micro valve 77, a third electromagnetic valve 103 is connected to the third elastic film micro valve 73 and the sixth elastic film micro valve 76, and a fourth electromagnetic valve 104 is connected to the fourth elastic film micro valve 74 and the fifth elastic film micro valve 75; the other end of the electromagnetic valve is connected with an air source 11 which provides air pressure with the constant size of 0.2 MPa; the control system IV controls the electromagnetic valve, and when the electromagnetic valve is opened, the air source 11 is introduced into the elastic film micro valve corresponding to the electromagnetic valve, so that the flow channel is closed.
As shown in fig. 3 and fig. 1, the fluid driving control system II in fig. 3 includes a first syringe pump 121 and a second syringe pump 122, wherein the first syringe pump 121 is connected to the first syringe pump inlet 64, and the pump is filled with deionized water with pH of 7.0; the second syringe pump 122 is connected to the second syringe pump inlet 65, with no sample and residual volume loaded inside the syringe pump.
As shown in fig. 4 and fig. 1, the microfluidic chip I in fig. 4 is placed on the temperature control system VII and is tightly connected to a heat sink 17, and the bottom of the heat sink 17 is provided with a heat dissipation fin 18 and a fan 19. The temperature of the heat sink is controlled to rise and fall through a PID algorithm built in the temperature control system, and the heat sink is matched with the heat dissipation fins 18 and the fan 19, so that the temperature required by the nucleic acid amplification reaction is maintained; an optical filter 14 in the optical system VI is right above a first modified capillary 91, a second modified capillary 92, a third modified capillary 93 and a fourth modified capillary 94 of the microfluidic chip I, and an excitation light source 15 irradiates 470nm of excitation light, and the excitation light is reflected to the first modified capillary 91, the second modified capillary 92, the third modified capillary 93 and the fourth modified capillary 94 through a focusing mirror 16 and a lens 13, and 590nm of emission light is obtained; the smart phone camera is right above the lens 13, takes a picture to collect a fluorescence signal, and analyzes fluorescence intensity through built-in image processing software.
In a second embodiment, the application process of the modified capillary-based nucleic acid detection microfluidic chip and the nucleic acid detection system according to the first embodiment is as follows:
step 1: and (4) loading a sample. And coating heat-conducting silicone grease on the bottom of the micro-fluidic chip I, and tightly attaching the micro-fluidic chip I to a temperature control system VII. Respectively placing 250 mul of blood sample to be detected and 500 mul of lysate in the first liquid storage cavity 61 and the second liquid storage cavity 62, and adding 150 mul of PCR mix into the third liquid storage cavity 63; and connecting the electromagnetic valve and the syringe pump to each elastic membrane micro valve or syringe pump interface of the microfluidic chip I through a sealing hose according to the first embodiment.
Step 2: the sample is mixed with the lysis solution. And controlling the mobile phone to control the control system IV to issue a command, and starting the air source 11, the electromagnetic valve and the injection pump. The first elastic membrane micro valve 71, the fourth elastic membrane micro valve 74 and the fifth elastic membrane micro valve 75 are closed by opening the first electromagnetic valve 101 and the fourth electromagnetic valve 104, so that a passage is formed with a liquid flow path among the first liquid storage cavity 61, the second liquid storage cavity 62 and the second injection pump inlet 65; the second injection pump 122 generates negative pressure in the channel under the stretching action to drive the sample to be decomposed and enter the cracking cavity 5, wherein the sample and the cracking cavity are spontaneously mixed in the serpentine pipeline when passing through the liquid mixing flow channel 8; when the sample fills the lysis chamber 5, the second solenoid valve 102 is opened to seal the second elastic membrane microvalve 72 and the seventh elastic membrane microvalve 77.
And step 3: and (6) cracking the sample. The temperature control system VII is controlled by the smart phone V, the heat sink 17 is heated to 95 ℃ for 10min, and the blood sample in the cracking cavity 5 is cracked and the nucleic acid to be detected is released.
And 4, step 4: and (3) adsorbing the nucleic acid. And closing the second electromagnetic valve 102, controlling the second injection pump 122 to continue stretching, allowing the cracked sample to slowly pass through the first modified capillary 91, the second modified capillary 92 and the third modified capillary 93, fixing the nucleic acid to be detected on the surface under the charge adsorption effect of the capillary surface, and finally allowing the waste liquid to enter the second injection pump 122.
And 5: and (4) eluting impurities. The first solenoid valve 101 is closed and the second solenoid valve 102 is opened, allowing communication between the passage between the first syringe pump inlet 64 and the second syringe pump inlet 65. 5ml of ionized water is driven by the first injection pump 121 to slowly pass through the first modified capillary 91, the second modified capillary 92 and the third modified capillary 93, so that residual impurities are eluted, and nucleic acid to be detected is difficult to wash away under the capillary adsorption effect; at the same time, the second syringe pump 122 is pulled, and the eluted liquid finally enters the second syringe pump 122.
Step 6: and loading the reaction solution. The first solenoid valve 101 is closed and the fourth solenoid valve 104 is opened so that the reagent chamber 63 is connected to the passage between the second syringe pump inlet 65. And (3) stretching the second injection pump 122 to enable the PCR mix to enter the first modified capillary 91, the second modified capillary 92, the third modified capillary 93 and the fourth modified capillary 94, and opening all the electromagnetic valves when 4 capillaries are filled with liquid at the same time to close all the elastic film micro valves so as to seal all the modified capillaries.
And 7: and (3) amplifying nucleic acid. Starting a temperature control system VII, controlling the amplification of nucleic acid in the sample by the temperature rise and fall of the heat sink, wherein the temperature parameters are as follows: firstly, the pre-denaturation of Taq enzyme is realized at 95 ℃ for 600 s; unwinding 95 ℃ for 15s, annealing 62 ℃ for 20s, and elongation 72 ℃ for 20s for 35 cycles.
And 8: and (3) detecting nucleic acid. An excitation light source 15 and a smart phone V are coordinated by the smart phone, a picture is taken at the extension stage of each cycle, the edge profile of the capillary is identified by internal image processing software, a fluorescence signal is obtained, a fluorescence intensity curve is obtained through a built-in algorithm, and qualitative and semi-quantitative detection of target nucleic acid is realized.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (8)
1. A nucleic acid detection micro-fluidic chip based on modified capillary is characterized in that: the micro-fluidic chip is respectively provided with a substrate, a liquid path layer substrate, an elastic film and an air path layer substrate from bottom to top; the liquid path layer substrate comprises a fluid circulation channel, a fluid mixing channel, a fluid diversion channel, a fluid converging channel, a sample cracking cavity and a modified capillary reserved cavity, and the connection sequence is as follows: the device comprises a fluid converging channel, a fluid mixing channel, a sample cracking cavity, a fluid diversion channel, a modified capillary reserved cavity and a fluid converging channel, wherein each structure is connected through a fluid flowing channel; the modified capillary is embedded in the reserved cavity of the modified capillary and used for adsorbing target nucleic acid in a solution sample to be detected and used as a carrier for realizing an automatic implementation mode of nucleic acid adsorption, purification, amplification and detection, and the technical scheme adopted by the modified capillary is as follows: the surface of the capillary tube is enabled to carry positive charges through a surface modification technology, target nucleic acid in a solution after a sample is cracked is enriched by the modified capillary tube, and meanwhile, the enriched nucleic acid is spontaneously dissociated into the solution under the normal nucleic acid amplification condition; the upper surface of the gas circuit layer substrate is provided with a plurality of convex hollow liquid storage cavities; the lower surface of the gas circuit layer substrate contains a plurality of sunken elastic film micro valves, and the gas circuit layer substrate is penetrated through the upper surface of the gas circuit layer substrate through holes; the upper surface of the liquid path layer substrate and the lower surface of the liquid path layer substrate are connected through a micro-channel and a micro-channel, so that the liquid storage cavity, the cracking cavity, the modified capillary and the outlet are communicated; and the lower surface of the gas circuit layer substrate is bonded with the upper surface of the elastic film to form a complete microfluidic chip.
2. The nucleic acid detection microfluidic chip based on the modified capillary tube of claim 1, wherein: the substrate is glass or a silicon wafer; the liquid path layer substrate and the gas path layer substrate are made of PDMS, PC, PMMA or COC polymer materials; the elastic film is made of PDMS or TPU elastic material.
3. The nucleic acid detection microfluidic chip based on the modified capillary tube of claim 1, wherein: the modified capillary is fixed in the channel on the lower surface of the liquid path substrate of the microfluidic chip through interference fit, thermal bonding or plasma cleaning bonding.
4. The nucleic acid detection microfluidic chip based on the modified capillary tube of claim 1, wherein: the modified capillary tube is made of a hard transparent material, and the hard transparent material comprises glass, silicon dioxide or thermoplastic plastics.
5. The nucleic acid detection microfluidic chip based on the modified capillary tube of claim 1, wherein:the inner wall of the modified capillary contains a compound with a functional group or ion with an amino structure at the tail end, including-NH2Or [ NH ]4]+And (5) structure.
6. A nucleic acid detection system for implementing the nucleic acid detection microfluidic chip based on the modified capillary of claim 1, wherein: the nucleic acid detection system is the microfluidic chip and a matched device, and comprises: the system comprises a micro-fluidic chip, a fluid driving control system, an air valve control system, a smart phone, an optical system and a temperature control system; the micro-fluidic chip is composed of a gas circuit layer substrate, an elastic film, a liquid circuit layer substrate, a heating cracking cavity, a first liquid storage cavity, a second liquid storage cavity, a third liquid storage cavity, a first injection pump interface, a second injection pump interface, a plurality of elastic film micro valves, a snake-shaped liquid mixing flow channel, a plurality of modified capillary reserved cavities, a first modified capillary, a second modified capillary, a third modified capillary and a fourth modified capillary; the fluid driving control system consists of a first injection pump and a second injection pump; the air valve control system consists of an air source and a plurality of electromagnetic valves; the optical system consists of a lens, a filter, an excitation light source and a focusing mirror; the temperature control system consists of a heat sink, heat dissipation fins and a heat dissipation fan; one end of each electromagnetic valve is connected with the corresponding elastic film micro valve through a gas path sealing pipeline, and the other end of each electromagnetic valve is connected with a gas source; the first injection pump is connected with an inlet of the first injection pump, and deionized water with the pH value of 7.0 is filled in the first injection pump; the second injection pump is connected with the inlet of the second injection pump, and the inside of the injection pump is not loaded with a sample and a residual volume; the micro-fluidic chip is arranged on the temperature control system and is tightly connected with the heat sink, and the bottom of the heat sink is provided with a heat radiation fin and a fan; an optical filter in the optical system is right above a capillary tube of the microfluidic chip, an excitation light source irradiates 470nm excitation light, the excitation light is reflected to the capillary tube through a focusing mirror and a lens, and 590nm emitted light is obtained; the smart phone camera is right above the lens, takes a picture to collect a fluorescence signal, and analyzes fluorescence intensity through built-in image processing software.
7. The nucleic acid detection system of the nucleic acid detection microfluidic chip based on the modified capillary tube of claim 6, wherein: the intelligent mobile phone issues commands to the fluid driving control system, the temperature control system and the optical system through the control system and obtains feedback data, the intelligent mobile phone detects fluorescence signals, and the nucleic acid amplification result is analyzed qualitatively or semi-quantitatively through built-in image processing software.
8. The nucleic acid detection system of the nucleic acid detection microfluidic chip based on the modified capillary tube of claim 6, wherein: the method comprises the following steps:
(1) sample loading
A sample, lysis solution and reagent required by nucleic acid analysis are sequentially added into a first liquid storage cavity, a second liquid storage cavity and a third liquid storage cavity;
(2) mixing the sample with the lysis solution
The sample and the lysis solution are mixed under the action of a snake-shaped liquid mixing flow channel under the driving of negative pressure generated by an injection pump, and finally, the mixture is uniformly mixed and flows into a lysis cavity;
(3) sample lysis
Heating the mixed solution of the sample and the lysate in a lysis cavity through a temperature control system to release the DNA into the liquid;
(4) nucleic acid adsorption
Controlling the magnitude of the driving force, allowing the sample to pass through the modified capillary tube, and removing the lysate;
(5) elution of impurities
Injecting eluent into the first modified capillary tube, the second modified capillary tube and the third modified capillary tube through the through hole positioned above the gas circuit layer substrate by the injection pump, and then removing the eluent to obtain a nucleic acid sample;
(6) reaction liquid Loading
Loading a reaction liquid into a reaction liquid cavity above the gas circuit layer substrate through a branched flow passage to a first modified capillary, a second modified capillary, a third modified capillary and a fourth modified capillary under the action of external force driving;
(7) nucleic acid amplification
Heating a first modified capillary tube, a second modified capillary tube, a third modified capillary tube and a fourth modified capillary tube in the chip by a temperature control system, wherein the specific temperature condition required by nucleic acid amplification is determined by a selected reagent reaction system;
(8) nucleic acid detection
The modified capillary is photographed at regular time by using a smart phone to collect a fluorescence image, and the change of the fluorescence intensity of the modified capillary is recognized and detected by using built-in image recognition software, so that qualitative and semi-quantitative detection of the nucleic acid sample is realized.
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Effective date of registration: 20231227 Address after: 244000, Room 202, 2nd Floor, Building F, Tongfang Information Port, Cuihu Second Road, Economic Development Zone, Tongling City, Anhui Province Patentee after: Anhui Zhongke Yikangda Biotechnology Co.,Ltd. Patentee after: HEFEI INSTITUTES OF PHYSICAL SCIENCE, CHINESE ACADEMY OF SCIENCES Address before: 230088 507, R & D building, Institute of technological innovation, Chinese Academy of Sciences (Hefei), northwest corner, intersection of Xiyou road and Shilian South Road, high tech Zone, Hefei City, Anhui Province Patentee before: HEFEI ZHONGKE YIKANGDA BIOMEDICAL Co.,Ltd. Patentee before: HEFEI INSTITUTES OF PHYSICAL SCIENCE, CHINESE ACADEMY OF SCIENCES |