CN108169301A - A kind of high-throughput quantification detects the closed experimental system setup of nucleic acid reaction - Google Patents

Abstract

The invention relates to a closed experimental system device for high-throughput quantitative detection of nucleic acid reactions. The sections are respectively provided with pressure relief structures. Compared with the prior art, the high-throughput nucleic acid reaction airtight biochip of the present invention is simple in structure, good in sealing performance, and low in cost, and can effectively solve the problem of aerosol pollution caused in the process of adding samples; moreover, the biochip can make The entire detection process can be completed in a unified sealing system, so as to ensure the integrity of the sample, and isolate potential pathogens from the operator to achieve rapid detection, high sensitivity, and high precision detection.

Description

A closed experimental system device for high-throughput quantitative detection of nucleic acid reactions

technical field

The invention relates to the technical field of bioelectrochemical detection, in particular to a closed experimental system device for high-throughput quantitative detection of nucleic acid reactions.

Background technique

In recent years, with the development of society and the advancement of biomedical technology, the number of various tests performed each year has increased significantly, and the requirements for on-site rapid detection and processing speed have become higher and higher. On-site operations require high sensitivity, simple and fast The need is also becoming more urgent. Among the diagnostic techniques for the detection of pathogenic bacteria, genetic bacterial identification is the most sensitive, specific, and accurate, and for these reasons, is accepted as the diagnosis of the definitive pathogen.

Real-time polymerase chain reaction (qPCR) has been widely used for the identification and quantification of pathogenic bacteria. However, conventional PCR techniques require thermal cycling steps of denaturation, annealing, and elongation, along with a laborious sample pretreatment step and bulky, expensive analytical instruments. These disadvantages make traditional PCR methods unsuitable for point-of-care testing (POCT). In recent years, isothermal nucleic acid amplification techniques have developed rapidly, including loop-mediated isothermal amplification (lamp), nucleic acid sequence amplification (NASBA), spin-dependent amplification (HDA), recombinant polymerase amplification (RPA) and rolling circle amplification ( RCA) has been developed. Among them, RPA is a highly rapid and sensitive amplification target DNA sequence at a relatively low temperature (39°C), and then the polymerase initiates the synthesis of complementary DNA strands. During the RPA reaction, the amplification of targeted genes can be monitored by targeted detection of real-time probes for 8 different types of RPA to detect viruses or pathogenic bacteria, which greatly simplifies the entire gene analysis process.

At present, various microfluidic devices for POCT pathogenic viruses or bacteria have been proposed, which have the advantages of low sample consumption, rapid heat transfer, and complete integration. However, these microfluidic devices generally require external pumps or valve actuators and complicated The tubing system used to control reagent flow and its reusability was not satisfactory. In particular, due to the isothermal amplification detection method, it is difficult to achieve quantitative detection with general technical means. How to realize the quantitative detection under the isothermal amplification system has always puzzled the majority of scientific researchers. Under the microfluidic system, how to make the controlled liquid infusion flow slowly and orderly is also a technical problem.

Contents of the invention

The technical problem to be solved by the present invention is to provide a closed experimental system device with good sealing performance and simple operation for high-throughput quantitative detection of nucleic acid reaction in view of the prior art, which effectively solves the aerosol problem that plagues the isothermal amplification system. At the same time, the quantitative detection of isothermal amplification technology is realized by electrochemical technology.

The technical solution adopted by the present invention to solve the above technical problems is: a high-throughput quantitative detection nucleic acid reaction airtight experimental system device, including a sheet body, which is characterized in that the sheet body is provided with sequentially connected liquid inlet sections, detection The reaction section, the liquid waste section, the liquid inlet section and the detection reaction section are respectively provided with pressure relief structures.

Preferably, the sheet body has a hollow structure, and the hollow structure constitutes the above-mentioned liquid inlet section, detection reaction section and waste liquid section respectively; the above-mentioned sheet body is a layer structure, which includes a first layer, a second layer and a third layer , and formed by stacking the first layer, the second layer and the third layer up and down, the first layer is provided with a first hollow pattern, the second layer is provided with a second hollow pattern, the first hollow pattern and The second hollow pattern is stacked up and down to form the above hollow structure, and the third layer is provided with a control component capable of controlling the above pressure relief structure.

Preferably, the liquid inlet section includes a liquid inlet, a liquid inlet pool, and a liquid inlet pipeline, the reaction section includes a liquid separation pipeline, an infusion pipeline, and a detection reaction pool, and the waste liquid section includes a waste liquid pool; The above-mentioned liquid inlet is communicated with the liquid inlet pool, and the liquid inlet pool is communicated with one end of the liquid inlet pipeline, and one end of the liquid distribution pipeline is communicated with the other end of the liquid inlet pipeline, and the other end is communicated with the waste liquid pool. There is a one-to-one correspondence between the pipelines and the detection reaction pools. One end of each infusion pipeline communicates with the liquid separation pipeline, and the other end communicates with the detection reaction pools. Through the above design, a complete and orderly reaction system can be formed in the hollow structure of the sheet body, so that the entire detection process can be successfully completed in the above reaction system, and each infusion pipeline can be used as the detection point of the light source detector.

Preferably, distribution pools corresponding to the above-mentioned detection reaction pools are formed on the liquid distribution pipeline, and each infusion pipeline communicates with the liquid separation pipeline through the corresponding distribution pools. The centrifugal forces are respectively smaller than the centrifugal forces at the corresponding detection reaction cells. In this way, according to the principle of fluid mechanics, the sample in the distribution pool can smoothly flow into the corresponding detection reaction pool under the action of centrifugal force.

As a preference, under the centrifugation state, the centrifugal force at each of the detection reaction pools increases evenly from the place close to the liquid inlet pool to the place far away from the liquid inlet pool, so that the samples in the liquid inlet pool can reach each detection reaction pool smoothly, ensuring The testing process went smoothly.

As a preference, the pressure relief structure in the detection reaction section corresponds to the detection reaction pool one by one, each of the pressure relief structures includes a pressure relief pool and a pressure relief hole opened on the pressure relief pool, and each pressure relief pool is connected to the pressure relief pool respectively. The corresponding detection reaction pools are connected through pressure relief pipelines. By setting a pressure relief structure in the detection reaction section, when the air pressure in the detection reaction section increases during the infusion process or the detection process, the pressure can be released through the pressure relief structure, thereby ensuring the stability of the reaction system and ensuring smooth detection conduct.

Preferably, the pressure relief structure of the liquid inlet section is one, and the pressure relief structure includes a pressure relief pool and a pressure relief hole opened on the pressure relief pool, the pressure relief pool corresponds to the above liquid inlet pool and passes through The pressure line is connected.

Preferably, the control assembly includes valves corresponding to each pressure relief hole, and each valve can close the corresponding pressure relief hole, which not only makes the structure of the pressure relief structure simple, but also realizes its sensitivity to pressure control sex.

Preferably, the third layer is provided with a membrane body, and the membrane body forms a hollow arc section corresponding to each pressure relief hole, and the area surrounded by each hollow arc section forms the above-mentioned valve, and one end of each valve is connected to the membrane body, and the other One end is free and can move relative to the pressure relief hole under the actuation of air pressure. Through the above design, the sensitivity of the pressure relief structure to pressure control can be further ensured.

Preferably, each of the detection reaction cells is respectively provided with a detection three-electrode circuit, and the interfaces of each detection three-electrode circuit are respectively exposed outside the chip. The invention detects samples by setting a detection three-electrode circuit and using cyclic voltammetry, so that the detection data is intuitive and the detection result is accurate.

Compared with the prior art, the present invention has the advantages that the device of the closed experimental system for high-throughput quantitative detection of nucleic acid reaction in the present invention has simple structure, good airtightness, and low cost, and can effectively solve the aerosol pollution caused in the process of adding samples At the same time, it can solve the problem of quantitative detection under the isothermal amplification system; or use the microfluidic technology to detect the cells in the shunt infusion through this system. Moreover, the experimental system includes a liquid inlet section, a detection reaction section, and a waste liquid section, so that the entire detection process can be completed in a unified sealed system, thereby ensuring the integrity of the sample and keeping potential pathogens away from the operator. Isolation, to achieve rapid detection and high sensitivity, high precision detection. The high-throughput quantitative detection nucleic acid reaction airtight experimental system device, a single detection mode, can be applied to corresponding processes such as microfluidic detection of cells.

Description of drawings

FIG. 1 is a schematic diagram of the three-dimensional structure of a closed experimental system device for high-throughput quantitative detection of nucleic acid reactions in an embodiment of the present invention;

Fig. 2 is the perspective view of the high-throughput quantitative detection nucleic acid reaction airtight experimental system device in the embodiment of the present invention;

3 is an exploded view of the structure of the closed experimental system device for high-throughput quantitative detection of nucleic acid reactions in the embodiment of the present invention;

Fig. 4 is a schematic structural diagram of another direction of Fig. 3;

Fig. 5 is the perspective view of part A in Fig. 4;

Fig. 6 is a structural exploded view of part A in Fig. 4;

Fig. 7 is a schematic structural diagram of another direction of Fig. 6;

Fig. 8 is a schematic diagram of the use state (centrifugal state) of the closed experimental system for high-throughput quantitative detection of nucleic acid reactions in the embodiment of the present invention.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

As shown in Figures 1 to 7, a closed experimental system device for high-throughput quantitative detection of nucleic acid reactions includes a sheet body 1, and the sheet body 1 is provided with a hollow structure 2, and the hollow structure 2 includes sequentially connected liquid inlet sections 21 , a detection reaction section 22 and a liquid waste section 23 , the liquid inlet section 21 and the detection reaction section 22 are respectively provided with a pressure relief structure 24 . The above-mentioned sheet body 1 is a layer structure, which includes a first layer 3, a second layer 4, and a third layer 5, and is formed by stacking the first layer 3, the second layer 4, and the third layer 5 up and down. One layer 3 is provided with a first hollow pattern 201, and the second layer 4 is provided with a second hollow pattern 202. The first hollow pattern 201 and the second hollow pattern 202 are stacked up and down to form the above hollow structure 2, and the above third A control assembly capable of controlling the above-mentioned pressure relief structure 24 is arranged on the layer 5 .

Further, the above-mentioned liquid inlet section 21 includes a liquid inlet 211, a liquid inlet pool 212, and a liquid inlet pipeline 213. The above-mentioned detection reaction section 22 includes a liquid distribution pipeline 221, an infusion pipeline 222, and a detection reaction pool 223. The above-mentioned waste liquid section 23 includes a waste liquid reservoir 231. The above liquid inlet 211 is included in the sheet body 1 and communicates with the liquid inlet pool 212, the liquid inlet pool 212 communicates with one end of the liquid inlet pipeline 213, and one end of the liquid distribution pipeline 221 communicates with the other end of the liquid inlet pipeline 213 , the other end communicates with the waste liquid pool 231, the above-mentioned infusion pipeline 222 corresponds to the detection reaction pool 223 one by one and is respectively multiple, and one end of each infusion pipeline 222 communicates with the liquid separation pipeline 221 respectively, and the other end communicates with the detection reaction pool 223 respectively. The reaction pool 223 is connected. Through the above design, a complete and orderly reaction system can be formed in the hollow structure 2 of the sheet body 1, so that the entire detection process can be successfully completed in the above reaction system. The pipelines in the present invention can adopt microfluidic fine pipelines made by engraving technology, or capillary pipelines made by moulds.

In order to make the sample entering the liquid separation pipeline 221 enter each detection reaction pool 223 more smoothly, a distribution pool 221a corresponding to the above-mentioned detection reaction pool 223 is formed on the liquid separation line 221, and each infusion pipeline 222 is respectively The corresponding distribution pools 221 a communicate with the liquid separation pipeline 221 , and in a centrifugal state, the centrifugal force at each distribution pool 221 a is smaller than the centrifugal force at the corresponding detection reaction pool 223 . In this way, according to the principle of fluid mechanics, the sample in the distribution pool 221 a can smoothly flow into the corresponding detection reaction pool 223 under the action of centrifugal force. In addition, under the centrifugal state, the centrifugal force at each of the above-mentioned detection reaction pools 223 increases evenly from the place close to the liquid inlet pool 212 to the place far away from the liquid inlet pool 212, so that the samples in the liquid inlet pool 212 can reach each detection reaction pool 223 smoothly. In order to ensure the smooth progress of the testing process. As shown in Figure 8.

Further, the pressure relief structure 24 includes a pressure relief pool 241 and a pressure relief hole 242 opened on the pressure relief pool 241 . Wherein, there are a plurality of pressure relief structures 24 on the detection reaction section 22 and one-to-one correspondence with the detection reaction pools 223 , and each pressure relief pool 241 communicates with the corresponding detection reaction pool 223 through a pressure relief pipeline 243 . There is one pressure relief structure 24 of the liquid inlet section 21 , and the pressure relief pool 241 corresponds to the liquid inlet pool 212 and communicates with it through a pressure relief pipeline 243 . By setting the pressure relief structure 24 on the liquid inlet section 21 and the detection reaction section 22 respectively, when the air pressure of the system increases during the detection process, the pressure relief structure 24 can be used to release the pressure, thereby ensuring the stability of the reaction system, thereby ensuring The detection went smoothly.

The above-mentioned control assembly can be realized in various ways. In this embodiment, preferably, the above-mentioned control assembly includes a valve 72 corresponding to each pressure relief hole 242 one-to-one, and each valve 72 can close the corresponding pressure relief hole 242 . Further, specifically, the third layer 5 is provided with a membrane body 7, the membrane body 7 forms a hollow arc section 71 corresponding to each pressure relief hole 242, and the area surrounded by each hollow arc section 71 forms the above-mentioned valve 72, and each valve 72 One end is connected to the membrane body 7, and the other end is a free end that can move relative to the pressure relief hole 242 driven by air pressure.

In this embodiment, the sample is detected by cyclic voltammetry, that is, a detection three-electrode circuit 6 is respectively arranged in each of the above-mentioned detection reaction cells 223, and the interface 61 of each detection three-electrode circuit 6 is respectively exposed outside the sheet body 1 Each interface 61 communicates with a multi-channel probe (not shown) of the detection instrument, so as to realize the detection of the sample.

In the above-mentioned three-electrode system, the measured system is composed of a research electrode, a reference electrode and an auxiliary electrode. Among them, the research electrode is also called the working electrode or the test electrode, that is, the MH electrode. The electrode process that occurs on the electrode is our research object; the reference electrode is used to measure the potential of the research electrode, and the electrode should have a known The electrode potential must be constant and stable, and no polarization shall occur during the measurement process; the auxiliary electrode is also called the counter electrode, which is only used to pass current to realize the polarization of the research electrode, and the surface area of the electrode should be larger than that of the research electrode. In this example, dyes are used as electrochemical signal markers to detect DNA or cells to be detected. When cyclic voltammetry is used to apply a cyclic voltage, the current generated by the oxidation-reduction reaction between the electrode and the dye is studied: when the sample contains The more the object to be tested, the more dyes will be embedded in the DNA or cell chain of the object to be tested, and the less dyes will be in contact with the electrodes, the more the researched current will drop, and the faster the current will drop. In this way, the quantitative detection of the substance to be detected in the sample can be realized by measuring the current signal.

During detection, the high-throughput quantitative detection nucleic acid reaction closed experimental system device of the present invention is placed on the multi-channel distribution plate on the centrifuge, and the high-throughput quantitative detection nucleic acid reaction closed experimental system device is placed in the detection tank, There are metal tentacles one-to-one corresponding to the interfaces 61 of the detection three-electrode circuits 6 of the above-mentioned detection reaction pools 223 in the detection groove, and the interface 61 of the detection three-electrode circuits 6 of the corresponding detection reaction pools 223 is to be connected to each metal antennae. After connection, it is placed in a thermostat, and a three-electrode circuit is drawn out of the detection tank, and the three-electrode circuit communicates with the multi-channel probe of the detection instrument. Samples and dyes are preset through the liquid inlet 211 , the device is turned on, and the samples enter into each detection reaction pool 223 under the action of centrifugal force through rotation. When the sample contains more substances to be tested, the more dyes are embedded in the DNA or cell chains of the samples to be tested, and the less dyes are in contact with the electrodes, the more the researched current drops, so that the current drops The speed is also faster, so that the quantitative detection of the substance to be detected in the sample can be realized by measuring the current signal.

It can be seen from the above that the present invention uses biochemical sensors as detection devices, utilizes the principles of fluid mechanics, and uses microfluidic means to complete high-throughput reactions, thereby realizing multiple high-throughput microfluidic biological experimental detection.

Claims (10)

1. A high-throughput quantitative detection nucleic acid reaction airtight experimental system device, including a sheet body, is characterized in that, the sheet body is provided with a liquid inlet section, a detection reaction section, and a waste liquid section that are sequentially connected, and the above-mentioned liquid inlet section Pressure relief structures are respectively arranged on the detection reaction section and the detection reaction section. 2. The high-throughput quantitative detection nucleic acid reaction airtight experimental system device as claimed in claim 1, characterized in that, there is a hollow structure in the sheet body, and the hollow structure respectively constitutes the above-mentioned liquid inlet section, detection reaction section and waste liquid part; The above-mentioned sheet body is a layer structure, which includes a first layer, a second layer and a third layer, and is formed by stacking the first layer, the second layer and the third layer up and down, and the first layer is provided with a first A hollow pattern, the second layer is provided with a second hollow pattern, the first hollow pattern and the second hollow pattern are stacked up and down to form the above hollow structure, the third layer is provided with a control component capable of controlling the above pressure relief structure . 3. The high-throughput quantitative detection nucleic acid reaction closed experimental system device according to claim 2, wherein the liquid inlet section includes a liquid inlet, a liquid inlet pool and a liquid inlet pipeline, and the detection reaction section includes The liquid separation pipeline, the infusion pipeline and the detection reaction pool, the waste liquid section includes a waste liquid pool; The above-mentioned liquid inlet is connected with the liquid inlet pool, and the liquid inlet pool is connected with one end of the liquid inlet pipeline, and one end of the liquid distribution pipeline is connected with the other end of the liquid inlet pipeline, and the other end of the liquid separation pipeline is connected with the waste liquid The above-mentioned infusion pipelines correspond to the detection reaction pools one by one and there are multiple ones respectively. One end of each infusion pipeline communicates with the liquid separation pipeline, and the other end communicates with the detection reaction pools respectively. 4. The high-throughput quantitative detection nucleic acid reaction airtight experimental system device as claimed in claim 3, is characterized in that, a distribution pool corresponding to the above-mentioned detection reaction pool is formed on the liquid separation line, and each infusion line is respectively The corresponding distribution pools communicate with the liquid separation pipeline, and in the centrifugal state, the centrifugal force at each distribution pool is smaller than the centrifugal force at the corresponding detection reaction pool. 5. The high-throughput quantitative detection nucleic acid reaction closed experimental system device as claimed in claim 4, characterized in that, under the centrifugal state, the centrifugal force at each of the detection reaction pools is from near the inlet pool to away from the inlet pool Increment evenly. 6. The high-throughput quantitative detection nucleic acid reaction airtight experimental system device as claimed in claim 3, is characterized in that, the pressure relief structure on the detection reaction section is multiple and corresponds to the detection reaction pool one by one, each of the The pressure relief structures all include a pressure relief pool and a pressure relief hole opened on the pressure relief pool, and each pressure relief pool is connected with the corresponding detection reaction pool through a pressure relief pipeline. 7. The high-throughput quantitative detection nucleic acid reaction closed experimental system device as claimed in claim 3, wherein the pressure relief structure of the liquid inlet section is one, and the pressure relief structure includes a pressure relief pool and is set in the The pressure relief hole on the pressure relief pool corresponds to the above-mentioned liquid inlet pool and communicates with the pressure relief pipeline. 8. The high-throughput quantitative detection nucleic acid reaction airtight experimental system device according to claim 6 or 7, wherein the control assembly includes valves corresponding to each pressure relief hole, and each valve can be closed correspondingly pressure relief hole. 9. The high-throughput quantitative detection nucleic acid reaction airtight experimental system device as claimed in claim 8, characterized in that, the third layer is provided with a membrane body, and the membrane body forms a hollowed-out arc section corresponding to each pressure relief hole , the area surrounded by each hollow arc section forms the valve, one end of each valve is connected to the membrane body, and the other end is a free end that can move relative to the pressure relief hole under the pressure of air pressure. 10. The high-throughput quantitative detection nucleic acid reaction airtight experimental system device according to any one of claims 3 to 5, wherein each detection reaction pool is respectively provided with a detection three-electrode circuit, and each detection three-electrode circuit The interfaces of the electrode circuits are respectively exposed outside the chip.