CN112126587B - Nucleic acid detection chip device, nucleic acid detection chip and preparation method thereof - Google Patents
Nucleic acid detection chip device, nucleic acid detection chip and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of nucleic acid detection, and discloses a preparation method of a nucleic acid detection chip. Also discloses the structure of the nucleic acid detection chip and the nucleic acid detection chip device. The invention can realize nucleic acid detection with high sensitivity, low cost, easy operation and high accuracy, and can realize nucleic acid detection of various pathogens such as coronavirus such as SARS and influenza virus.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a nucleic acid detection chip device, a nucleic acid detection chip and a preparation method thereof.
Background
According to the recommendation of the laboratory technical guide for the detection of pneumonia caused by infection with novel coronavirus (sixth edition), the conventional method for detecting novel coronavirus (SARS-CoV-2) is RT-qPCR technology. The application of RT-qPCR technology firstly needs to purify virus nucleic acid, detection personnel need to directly contact exposed high-concentration virus particles and genome nucleic acid for a long time, the risk of infection is high, and laboratory environment with biological safety level more than 2 is needed, so that the detection speed of patients is limited, and the detection requirement of new outbreak infectious disease pathogens cannot be met. On the other hand, the purified nucleic acid is reverse transcribed into cDNA and then amplified by the quantitative fluorescence PCR technique, and thus a false positive signal is very likely to be generated. Meanwhile, because a fluorescent quantitative PCR instrument is required, the usability of virus nucleic acid detection is limited, and real-time rapid high-throughput detection cannot be realized; and the RT-qPCR requires operators to have certain molecular biology basis, and because of the huge infectivity of the new coronavirus, the detection needs to be carried out in a strictly partitioned PCR laboratory with certain protection level, thereby bringing huge difficulty to the detection of a large amount of samples due to epidemic outbreak. In addition, since nucleic acid diagnosis has a certain degree of false negative, it is necessary to effectively separate new coronavirus from influenza virus and other similar diseases so as to prevent cross infection of patients.
Nucleic acid detection is widely used for detection of biological pathogen infection, especially viral infection. Because the nucleic acid content in the sample to be detected is extremely low, amplification is usually required to obtain sufficient nucleic acid to be detected, and finally, a fluorescence, color development or potential signal is used as a report system. The current nucleic acid amplification technologies include two major types of nucleic acid amplification technologies, such as temperature-variable amplification technologies, e.g., polymerase Chain Reaction (PCR), real-time quantitative PCR (qPCR), nested PCR, reverse transcription real-time quantitative PCR (RT-qptc), etc., and Recombinase Polymerase Amplification (RPA), loop-mediated isothermal amplification (LAMP), etc. The new coronavirus is a positive-strand RNA virus, and virus ssRNA is firstly reversely transcribed into cDNA, and then temperature-variable or isothermal amplification can be carried out. Wherein, the detection sensitivity of the real-time quantitative PCR technology can reach single copy pathogen genome, but an expensive qPCR instrument is needed; while other isothermal amplification techniques have a simple amplification apparatus, they have poor detection accuracy and are prone to cause false positives due to non-specific amplification.
On the other hand, CRISPR proteins such as Cas12, which recognize DNA sequences, are applied to nucleic acid detection. Professor Zhang Fengchi of the 2016 Massachusetts institute of technology and technology discovered that Cas13a (C2C 2) protein can cleave irrelevant ssRNA in addition to target RNA, and Zhang Fengcou developed a CRISPR/Cas13 a-based nucleic acid detection method, SHERLOCK. In 2018, the action mechanism of Cas12a is researched by the Zhao national Screen/King gold team, the Cas12a can cut any ssDNA after the ternary complex of Cas12a, crRNA and dsDNA is formed, and a nucleic acid detection technology HOLMES is developed. At the same time, researchers at Berkeley, california university, have also developed nucleic acid detection methods of similar principles. These methods recognize nucleic acid sequences other than primers as soon as possible, but recognition and reading of more nucleic acid sequences is greatly limited by the difficulty of simultaneous measurement of multiple grnas. Although the CRISPR/Cas-based method can realize high-sensitivity detection of single-copy pathogen genomes through isothermal amplification and other technologies, the detection concentration limit of the method is 10 < -12 > Mol/L to 10 < -13 > Mol/L, the detection sensitivity is greatly limited, a microfluidic chip and other technologies are required to integrate the recognition capability of a plurality of target sites, the sensitivity is improved, and false negative results are reduced.
The micro-fluidic Chip is a technology for researching the accurate control and analysis of biochemical microfluid in a micron-scale channel, an experimental platform of the micro-fluidic Chip is called a Lab-On-a-Chip (Lab On a Chip), the functions of a biological laboratory and a chemical laboratory can be miniaturized to a Chip with a plurality of square centimeters, the micro-fluidic Chip has the characteristics of trace, low cost, high flux, integration, miniaturization and the like, and has unique advantages which cannot be compared with a conventional macro system in the research and application processes. A series of effective new research methods and new technologies, including pretreatment of microorganism samples, separation, culture, drug screening, single cell analysis, rapid detection and the like, have appeared by combining microfluidic technology with microbiology. For example, scientists from the poland scientific institute have analyzed the effects of single and mixed antibiotics on bacterial growth using an automated microfluidic technique, enabling rapid detection of MIC (minimal inhibition Concentration), and rapid screening of effective combinations of antibiotics; in recent years, microfluidic technology has been developed rapidly, and a new research idea is provided for single cell analysis of bacteria, so that rapid, high-sensitivity and high-resolution analysis of drug-resistant bacteria is possible. However, in the related research for drug-resistant bacteria, due to the complexity of the microbial type and the drug-resistant phenotype, the technology of microfluidic chips suitable for detection of clinical general type is still blank at present.
The existing gold standard for detecting the new coronavirus is an RT-qPCR method, and the technical scheme of the RT-qPCR method generally comprises the steps of firstly performing reverse transcription on the genome of the new coronavirus to form cDNA, then designing a specific primer to amplify a specific section on the cDNA, and detecting the copy number of initial template DNA by using a fluorescent signal generated by enzyme digestion of a fluorescent-labeled probe by polymerase (Taqman method) or a fluorescent signal generated by doping SYBR Green dye into double-stranded DNA generated by amplification (SYBR Green method).
For the technique of using Taqman probe, if the design of the probe is not good enough, the annealing effect of the probe and the target DNA is not good or the probe can not anneal, thereby causing false negative in detection; in the SYBR Green method, if non-specific amplification occurs or the system is contaminated with other DNA or auto-amplification occurs in the system, false positive results. These defects are inherent in the technical scheme and cannot be eliminated by a method such as system optimization, so that the conventional RT-qPCR method is a nucleic acid detection gold standard, but has the problem of false positive or false negative which cannot be eliminated.
Another currently widely used protocol for the detection of new corona is immunoassay. Due to the immune function, the new coronavirus will attack the human immune system after invading the human body, and the specific antibody will be generated in the body of the patient. However, since the production of antibodies by the human body is often delayed, immediate detection of the virus cannot be performed at the first time, thereby delaying the optimal isolation time and possibly causing unintended transmission. It is therefore important that the initial infection of the virus be detected.
Since immunization requires a certain time to produce antibodies, the inevitable drawbacks of viral antibody detection cannot be detected at the first time.
Because the new coronavirus has extremely strong infectivity and is easy to be changed, the detection of the new coronavirus faces a plurality of problems at present: the detection needs sampling in a professional site, processing samples in a closed environment, using a qPCR instrument and being operated by professional personnel, and has the advantages of high false positive and false negative, low detection flux, incapability of simultaneously carrying out strain typing and the like.
Disclosure of Invention
The present invention is intended to solve the above problems, and an object of the present invention is to provide a nucleic acid detection chip device, a nucleic acid detection chip, and a method for producing the same, which can achieve nucleic acid detection with high sensitivity, low cost, easy operation, and high accuracy, and can achieve detection of various pathogenic nucleic acids such as coronavirus such as SARS, and influenza virus.
The technical scheme adopted by the invention is as follows:
a preparation method of a nucleic acid detection chip is characterized by comprising the following steps:
(1) Forming a modified PDMS block on the modified chip template, and forming a modified chip groove on the surface of the modified PDMS block;
(2) Performing amination modification on the glass sheet, covering one surface of a modification chip groove on the modification PDMS block on the aminated glass sheet, and forming a plurality of parallel modification chip channels by the modification chip groove and the glass sheet;
(3) Sequentially filling streptavidin solution into a channel of the modified chip, incubating, filling biotin-labeled gRNA, incubating, filling Cas protein, and incubating;
(4) Removing the PMDS block on the glass sheet to form a modified chip;
(5) Forming a reaction PDMS chip on the reaction chip template, forming a valve PDMS chip on the valve core chip template, forming a plurality of parallel reaction chip grooves on the reaction PDMS layer, wherein the valve PDMS block is formed with a plurality of parallel valve core sheet grooves, and the end parts of the plurality of valve core sheet grooves are communicated;
(6) Taking down the reaction PDMS chip and the valve PDMS block, sealing the reaction PDM chip to one surface of a valve core sheet groove of the valve PDMS block to form packaging PDMS, sealing the valve core sheet groove to form a valve core sheet channel, wherein the reaction chip groove is vertical to the valve core sheet channel;
(7) And sealing the packaging PDMS with the glass sheet to form the nucleic acid detection chip, wherein the reaction chip groove is matched with the glass sheet to form a reaction chip channel, the reaction chip channel is vertical to the modification chip channel, and the valve core sheet channel is positioned in the interval of the modification chip channel.
Further, in the step (1), the PDMS prepolymer and the cross-linking agent are proportionally taken, stirred, mixed and poured onto the modified chip template, the thickness of the PDMS prepolymer is 3-10 mm, the PDMS block is formed after the PDMS prepolymer and the cross-linking agent are thermally dried, solidified and cooled, the PDMS block is taken down from the modified chip template, holes are formed in the surface of the PDMS block, and the modified PDMS block is communicated with the modified chip groove.
Further, in the step (2), the process of performing amination modification on the glass sheet is as follows: and (3) soaking the glass sheet in a toluene solution containing APTES with a certain concentration for amination modification, and taking out and drying the glass sheet after the amination modification is finished.
Further, in the step (3), the streptavidin solution incubation is 4 ℃ overnight incubation, after completion, the modified chip channel is washed with PBS solution, and the biotin-labeled gRNA perfusion incubation and LbCas12a perfusion incubation are 37 ℃ incubation for 30 minutes.
Further, in the step (5), the process of forming the reactive PDMS layer on the reactive chip template is as follows: uniformly throwing PDMS on the reaction chip template, and baking and curing to form a reaction PDMS layer; the process of forming the valve PDMS block on the valve chip template comprises the following steps: and pouring PDMS (polydimethylsiloxane) on the valve chip template, wherein the thickness of the PDMS is 3-10 mm, and performing heat curing to form a valve PDMS block, wherein the reaction chip channel and the valve chip channel are completed by arranging bulges on the reaction chip template and the valve chip template.
Further, the width of the modified chip channel is 50-1000 microns, the width of the reaction chip channel is 50-1000 microns, and the width of the valve chip channel is 50-200 microns.
Further, in the forming process of the modified PDMS chip, the reaction PDMS chip and the valve PDMS chip, soaking and drying are carried out for 80 ℃ for half an hour, and then cooling is carried out to room temperature to form the PDMS chip.
Furthermore, the nucleic acid detection chip is punched and communicated to holes at two ends of the reaction chip channel to form a sample inlet and a sample outlet, and communicated to the hole of the valve chip channel to form an inflation inlet.
A nucleic acid detection chip prepared by the preparation method of the nucleic acid detection chip.
A nucleic acid detection chip device comprises the nucleic acid detection chip and is characterized by further comprising a chip support frame, wherein the nucleic acid detection chip is fixed on the chip support frame, a microscopic imaging window is formed in the chip support frame, a sample liquid storage tank and a pneumatic control port are arranged on one side of the chip support frame, the sample liquid storage tank is connected to a sample inlet of a reaction chip channel, and the pneumatic control port is connected to an inflation port of a valve chip channel.
The invention has the beneficial effects that:
(1) Detection equipment and a kit aiming at detection of high-risk and strong infectious pathogens and independent of a high-grade biosafety laboratory;
(2) The nucleic acid detection with high sensitivity, low cost, easy operation and high accuracy is realized;
(3) The detection of various pathogenic nucleic acids such as coronavirus such as SARS and influenza virus is realized, and the rapid response to the burst virus infection is realized;
(4) The platform support is provided for the future detection of other types of new outbreak infectious disease pathogens;
(5) The invention utilizes the super-sensitive single-stranded DNA cutting capability of Cas12a to exert the advantages of high throughput and less sample consumption of the microfluidic microarray.
Drawings
FIG. 1 is a flow chart showing the production of a nucleic acid detecting chip according to the present invention;
FIG. 2 is a schematic diagram showing the structure of a nucleic acid detecting chip;
FIG. 3 is a schematic diagram showing the structure of a nucleic acid detecting chip apparatus;
FIG. 4 is a graph showing the effect of RT-RPA amplification on 100 copies per microliter of template;
FIG. 5 is a graph showing the effect of fluorescence in the channels corresponding to the sample and gRNA.
Detailed Description
The following will describe in detail specific embodiments of the nucleic acid detecting chip device, the nucleic acid detecting chip and the method for manufacturing the same according to the present invention with reference to the accompanying drawings.
The preparation method of the nucleic acid detection chip is divided into three parts, namely manufacturing a modification chip, a reaction chip channel and a valve chip channel.
Referring to fig. 1, a modified chip was fabricated as 10: taking the prepolymer and the cross-linking agent according to the proportion of 1, fully stirring and mixing the prepolymer and the cross-linking agent, then pouring the prepolymer and the cross-linking agent on a modified chip template, vacuumizing the modified chip template to remove bubbles, curing the modified chip template at 80 ℃ for half an hour, taking the modified chip template out of the modified chip template to cool the modified chip template to room temperature to form a modified PDMS block, taking the modified PDMS block out of the modified chip template, cutting the modified chip template along the structure, forming a modified chip through groove on the modified chip template by the modified PDMS block, and punching the modified PDMS block to communicate the modified chip groove.
And ultrasonically cleaning the perforated modified PDMS block and the glass sheet for 10-20 minutes by using absolute ethyl alcohol, ultrasonically cleaning the glass sheet for 10-20 minutes by using ultrapure water, and drying the glass sheet by using nitrogen. Cleaning the glass sheet by using a plasma machine for 1-10 minutes, then soaking the glass sheet in a toluene solution containing APTES with certain concentration for 2 hours for amination modification, taking out the glass sheet, and drying the glass sheet by using nitrogen. And covering the modified PDMS block on the aminated glass sheet, and forming a modified chip channel by the modified chip groove and the glass sheet.
And (3) pouring a streptavidin solution with a certain concentration into the channel of the modified chip through the hole on the modified PDMS block, and incubating overnight at 4 ℃. Washing the modified chip channel with PBS solution, then infusing biotin-labeled gRNA into the modified chip channel, incubating for about 30 minutes at 37 ℃, then infusing Cas protein into the modified chip channel, incubating for about 30 minutes at 37 ℃, washing out unbound Cas1 protein, and drying with nitrogen for later use.
And finally, removing the upper modified PDMS block to finish manufacturing the modified chip.
Manufacturing a reaction chip channel and a valve chip channel: adding about 5 ml of PDMS on the reaction chip template, uniformly throwing the PDMS by a spin coater, drying the PDMS for 30 minutes at 80 ℃, and taking out the PDMS. Because the reaction chip template is provided with the bulges, the reaction chip groove is formed on the reaction PDMS layer which is taken out after the preparation is finished.
And pouring PDMS (polydimethylsiloxane) on the valve chip template, wherein the thickness of the PDMS is about 3-10 mm, removing bubbles in vacuum, curing at 80 ℃ for half an hour, taking out, and cooling to room temperature to form a valve PDMS block.
Referring to fig. 2, since the valve core piece template is designed with a protrusion, a valve core piece groove is formed on the valve PDMS block 3 after the valve core piece template is manufactured and taken out, and the end parts of the valve core piece groove are connected. And taking the valve PDMS block 3 out of the valve template, dividing along the structure, cleaning the valve PDMS block 3 together with the reaction PDMS layer by using a plasma machine for 1-10 minutes, and then butting and sealing the reaction PDMS layer and the valve PDMS block 3, wherein the reaction chip groove is vertical to the valve core sheet groove. And forming a valve chip channel 2 at the sealing position, curing at 80 ℃ for about 30 minutes, taking out, carefully cutting and taking off along the edge of the valve PDMS block 3, carrying out plasma treatment, and sealing with the modified glass sheet 4 to form a reaction chip channel 1, wherein the reaction chip channel 1 is vertical to the modified chip channel 5, and the valve chip channel 2 is positioned in the interval of the modified chip channel 5.
The above process completes the manufacturing process of the nucleic acid detection chip. The width of the modified chip channel 5 is 50-1000 microns, the width of the reaction chip channel 1 is 50-1000 microns, and the width of the valve chip channel 2 is 50-200 microns.
And finally, punching a hole on the nucleic acid detection chip, communicating the hole to the two ends of the reaction chip channel 1 to form a sample inlet 6 and a sample outlet 7, communicating the hole to the valve chip channel 2 to form an inflation inlet 8.
Referring to fig. 3, the nucleic acid detecting chip 9 is mounted on the chip support frame 10 to form a nucleic acid detecting chip device, the chip support frame 10 is provided with a microscopic imaging window 11, one side of the chip support frame 10 is provided with a sample liquid reservoir 12 and an air pressure control port 13, the sample liquid reservoir 12 is connected to the sample inlet 6 of the reaction chip channel, and the air pressure control port 13 is connected to the air charging port 8 of the valve chip channel. The sample liquid storage tank injects a sample into the reaction chip channel from the sample inlet, the sample is respectively shunted to each modification strip through the reaction chip channel, at the moment, high-pressure gas is filled into the valve chip channel, and the reaction chip channel is divided into a plurality of sections of independent reaction areas after the valve chip channel expands. For example, a reaction solution is prepared, wherein the reaction solution contains 1 × Cas protein reaction buffer solution NEBuffer 2.1, an amplification product and single-stranded DNA (reporter) with a length of 5nt-30nt respectively marked with a fluorescent group and a quenching group, the reaction solution is poured into a channel of a reaction chip, high-pressure gas is immediately injected into the channel of the valve chip, so that part of the channel of the reaction chip is deformed and clings to lower glass, and series flow of the reaction solution is blocked.
The pretreated sample is transferred into a sample storage pool, and is driven to enter a chip sample inlet, and in the process of flowing through a reaction chip channel in the chip, the sample is in contact reaction with immobilized guide RNA and Cas12a fixed on a glass substrate at the lower layer of the chip, so that the purpose of nucleic acid detection is realized. In order to avoid potential cross-contamination between guide RNA reaction sites, after the reaction chip channels are filled with sample solution, the membrane chip channels are pressurized and expanded, so that each reaction site is divided into independent reaction chambers, the liquid exchange between the sites is blocked, and the excess sample solution is discharged from the sample outlet. After the reaction is finished, the fluorescence signal of each reaction site can be observed through a microscopic imaging window under a microscope, and the qualitative detection of a specific nucleic acid sequence is realized.
See fig. 4, to verify the feasibility of on-glass modification of gRNA and LbCas12a and camera capture fluorescence signal analysis. Three different targets are designed on the novel crown S gene, wherein one target is a D614G site which is widely popular in Europe and America. First we successfully performed single-target and triple-target RPA amplifications using the RPA kit.
Referring to attached figure 5, the amplification primer pair is intP1-1+ intP1-2, intP2-1+ intP2-2, D614G-1+ D614G-2. Corresponding dsDNA and reporter are injected into the channels of the reaction chip for respectively modifying the crRNA1, the crRNA1 and the crRNA3, fluorescence is generated in the channels, and no fluorescence signal is generated in the channels only by injecting water into the channels.
The invention realizes 4 major breakthroughs in nucleic acid detection:
1. safety: can prevent secondary infection. A set of closed detection system is developed, the whole process of extraction, purification, amplification and detection of the virus nucleic acid is closed in a closed system, and the exposure of operators in the detection process is effectively avoided.
2. Is convenient and fast: the microfluid one-stop operation realizes the miniaturization of the instrument. An integrated closed microfluidic chip is developed, and the extraction and amplification of viral nucleic acid are closed in a closed system, so that the exposure of operators in the detection process is effectively avoided; the automatic, quick and safe detection of pathogenic microorganisms and viruses on a miniaturized instrument platform is realized, so that the aim of quickly screening suspected virus carriers on site is fulfilled.
3. Accurate and sensitive: based on the innovative detection principle of the CRISPR/Cas12a system. The multiple Cas12a precise recognition sequence technology is introduced, so that the reading of multi-segment sequence information of pathogen genome is realized, and false positive caused by nucleic acid amplification is reduced; meanwhile, the multi-target simultaneous detection can monitor the evolution and mutation of the virus more, reduce the false negative of the detection, and can classify pathogenic microorganisms and viruses by combining the design of multiple targets.
4. And (3) fast reading: the detection equipment adopts a rapid fluorescence photographing technology, the microarray chip realizes high-flux detection, and the miniaturized automatic focusing module optimizes the low-power lens, simplifies the light path and has more sensitive detection; a locus signal detection algorithm, which optimizes a single molecule fluorescence detection algorithm; the portable and modularized detection light path is a totally-enclosed integration, and the computer is an external control interface.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A preparation method of a nucleic acid detection chip is characterized in that: the method comprises the following steps:
(1) Forming a modified PDMS block on the modified chip template, and forming a modified chip groove on the surface of the modified PDMS block;
(2) Performing amination modification on the glass sheet, covering one surface of a modification chip groove on the modification PDMS block on the aminated glass sheet, and forming a plurality of parallel modification chip channels by the modification chip groove and the glass sheet;
(3) Sequentially filling streptavidin solution into a channel of the modified chip, incubating, filling biotin-labeled gRNA, incubating, filling Cas protein, and incubating;
(4) Removing the PMDS block on the glass sheet to form a modified chip;
(5) Forming a reaction PDMS chip on the reaction chip template, forming a valve PDMS chip on the valve core chip template, forming a plurality of parallel reaction chip grooves on the reaction PDMS layer, wherein the valve PDMS block is formed with a plurality of parallel valve core sheet grooves, and the end parts of the plurality of valve core sheet grooves are communicated;
(6) Taking down the reaction PDMS chip and the valve PDMS block, sealing the reaction PDM chip to one surface of a valve core sheet groove of the valve PDMS block to form packaging PDMS, sealing the valve core sheet groove to form a valve core sheet channel, wherein the reaction chip groove is vertical to the valve core sheet channel;
(7) And sealing the packaging PDMS with the glass sheet to form the nucleic acid detection chip, wherein the reaction chip groove is matched with the glass sheet to form a reaction chip channel, the reaction chip channel is vertical to the modification chip channel, and the valve core sheet channel is positioned in the interval of the modification chip channel.
2. The method for preparing a nucleic acid detecting chip according to claim 1, wherein: in the step (1), the PDMS prepolymer and the cross-linking agent are proportionally taken, stirred, mixed and poured onto the modified chip template, the thickness of the modified chip template is 3-10 mm, the modified PDMS block is formed after the thermal drying, solidification and cooling, the modified PDMS block is taken down from the modified chip template, holes are formed in the surface of the modified PDMS block, and the modified PDMS block is communicated with the modified chip groove.
3. The method for preparing a nucleic acid detecting chip according to claim 2, wherein: in the step (2), the process of performing amination modification on the glass sheet is as follows: and (3) soaking the glass sheet in a toluene solution containing APTES with a certain concentration for amination modification, and taking out and drying the glass sheet after the amination modification is finished.
4. The method for preparing a nucleic acid detecting chip according to claim 3, wherein: in the step (3), the streptavidin solution incubation is 4 ℃ overnight incubation, after completion, the modified chip channel is washed with a PBS solution, and the biotin-labeled gRNA incubation and LbCas12a incubation are perfused for 30 minutes at 37 ℃.
5. The method for preparing a nucleic acid detecting chip according to claim 4, wherein: in the step (5), the process of forming the reactive PDMS layer on the reactive chip template is: uniformly throwing PDMS on the reaction chip template, and baking and curing to form a reaction PDMS layer; the process of forming the valve PDMS block on the valve chip template comprises the following steps: and pouring PDMS (polydimethylsiloxane) on the valve chip template, wherein the thickness of the PDMS is 3-10 mm, and performing heat curing to form a valve PDMS block, wherein the reaction chip channel and the valve chip channel are completed by arranging bulges on the reaction chip template and the valve chip template.
6. The method for preparing a nucleic acid detecting chip according to claim 4, wherein: the width of the modifying chip channel is 50-1000 microns, the width of the reaction chip channel is 50-1000 microns, and the width of the valve chip channel is 50-200 microns.
7. The method for preparing a nucleic acid detecting chip according to claim 4, wherein: and in the forming process of the modified PDMS chip, the reaction PDMS chip and the valve PDMS chip, soaking and drying for half an hour at 80 ℃ and then cooling to room temperature to form the PDMS chip.
8. The method for preparing a nucleic acid detecting chip according to claim 4, wherein: the nucleic acid detection chip is punched and communicated to holes at two ends of a reaction chip channel to form a sample inlet and a sample outlet, and communicated to holes of a valve chip channel to form an inflation inlet.
9. A nucleic acid detecting chip produced by the method for producing a nucleic acid detecting chip according to any one of claims 1 to 8.
10. A nucleic acid detecting chip device comprising the nucleic acid detecting chip according to claim 9, characterized in that: the nucleic acid detection chip is fixed on the chip support frame, a microscopic imaging window is formed on the chip support frame, a sample liquid storage tank and an air pressure control port are arranged on one side of the chip support frame, the sample liquid storage tank is connected to a sample inlet of the reaction chip channel, and the air pressure control port is connected to an inflation port of the valve chip channel.
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| CN113311160A (en) * | 2021-06-17 | 2021-08-27 | 山东科讯生物芯片技术有限公司 | Micro-fluidic biochip for rapidly detecting SARS-CoV-2 antigen and IgG/IgM antibody |
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