CN104946505A - Microfluidic chip for realizing PCR (polymerase chain reaction) and virus rapid detection device for real-time PCR - Google Patents
Microfluidic chip for realizing PCR (polymerase chain reaction) and virus rapid detection device for real-time PCR Download PDFInfo
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Abstract
The invention provides a microfluidic chip for realizing PCR (polymerase chain reaction). The microfluidic chip comprises a reaction layer, wherein a sample tank is arranged in the reaction layer; multiple groups of reaction zones are arranged around the sample tank; each group of the reaction zones at least comprises a reaction tank; each reaction tank is connected with the sample tank by virtue of a flow passage; and the sample tank is connected with the outside by virtue of a sample inlet hole. Meanwhile, the invention also provides a virus rapid detection device comprising the microfluidic chip for real-time PCR. The microfluidic chip is small in used sample dosage and high in reliability to save the use cost; and moreover, different detection reagents can be added into the microfluidic chip according to different test needs to achieve standardization and normalization. Meanwhile, the detection device not only is simple to operate and convenient to use, but also is convenient to carry.
Description
Technical field
The present invention relates to micro fluidic chip technical field, be especially a kind ofly applied to the micro-fluidic chip realizing PCR of life science and the viral device for fast detecting of PCR in real time.
Background technology
Since nineteen ninety proposes micro-total analysis system concept, microfluidic chip technology opens wide development space in fields such as chemistry, life science, environmental science and Food sciences.Microfluidic chip technology is based on microfluidic control technology, guide analytical instrument to miniaturization, integrated, automatization, high-throughput fast quantitative analysis future development, for the real-time detection of biochemical environmental samples, on-the site analysis provide a kind of possible strategy, it has wide market outlook in medical diagnosis and the epidemic monitoring of agricultural breeding system.
Polymerase chain reaction (Polymerase Chain Reaction, PCR) also known as cell-free molecular cloning or the directed enzymatic amplification technique of the external primer of specific DNA sequences, it is a kind of method of external enzyme' s catalysis specific DNA fragment, reacted by a few step such as high-temperature denatured, low-temperature annealing and thermophilic extension and form one-period, circulation is carried out, make target DNA be able to rapid amplification, there is high specificity, highly sensitive, easy and simple to handle, the feature such as save time.It not only can be used for the fundamental researchs such as gene isolation, clone and nucleic acid sequence analysis, also can be used for the diagnosis of disease, animal virus and epidemic situation rapid detection or any place having DNA, RNA.In clinical medicine and animal epidemics Monitoring systems, PCR is used to differentiate genetic diseases and rapid detection virus, courses of infection.By traditional method, detect virus, germ infection need pathogen culture could be identified several weeks, and adopt achievement of the present invention, can judge in human body and zooblast (such as blood cell), whether to there is virus fast, the DNA of germ and making a definite diagnosis.
PCR in real time (real-time PCR), belongs to the one of quantitative PCR (quantitative-PCR), in certain hour DNA amount of amplification based on carry out the quantitative analysis of DNA.Real-Time PCR quantitation uses fluorochrome, has two kinds of methods at present.A kind of is in DNA, insert special fluorochrome; A kind of fluorescent probe that another kind of use can combine with few nucleotide sequence specific in amplification DNA sequence dna.PCR in real time combines with reverse transcription PCR (reverse-transcription PCR), can find out the gene of specified time, cell, in-house special expression with the RNA of trace.The combination of these two kinds of RT-PCR is referred to as " quantitative RT-PCR (quantitative RT-PCR) ".Because this technology not only achieves the leap of PCR from qualitative to quantitative, and compared with Standard PCR, there is specificity stronger, effective solution PCR pollution, level of automation high.Real-time quantitative PCR refers to the amount immediately being measured specific product during PCR exponential amplification by the change of monitoring fluorescent signal power continuously, and infers the original bulk of goal gene accordingly, does not need to take out PCR primer and is separated.Current real-time quantitative PCR, as an extremely effective experimental technique, has been widely used in the every field of molecular biology research.
At present, the reaction series of strata of real-time quantitative PCR often adopt AND DEWATERING FOR ORIFICE STRUCTURE as the carrier of sample, so not only make the heat of reaction zone heating and cooling absorption and release excessive, thus cause reaction zone warming and cooling rate slower; And the sample reagent volume that the sample carriers of AND DEWATERING FOR ORIFICE STRUCTURE needs at least will, in 10 microlitre ranks, be unfavorable for reducing use cost; Meanwhile, in fields such as such as environmental monitoring, the anti-biochemistry of spot inspection quarantine and battlefield, need portable fast analyser, and the large complicated operation of viral device for fast detecting volume of the PCR in real time that prior art provides is not suitable for operation in the wild.
Summary of the invention
For solving the problem existing for above-mentioned prior art, the object of the present invention is to provide the micro-fluidic chip realizing PCR that a kind of sample dosage uses less, reliability is high.
To achieve these goals, an object of the present invention is to provide a kind of micro-fluidic chip realizing PCR, comprise responding layer, sample slot is provided with in described responding layer, many group reactions district is provided with around described sample slot, every group reaction district at least comprises a reactive tank, and each reactive tank is connected with sample slot by runner, and described sample slot is connected with the external world by sample holes.
Preferably, described micro-fluidic chip also comprises the pneumatic control layer be covered on above described responding layer, and described pneumatic control layer comprises multiple control valve and the gas pipeline for connection control valve, and described control valve is placed in the top of runner.
Preferably, described responding layer comprises substrate and is covered on the thin film layer on substrate, and described sample slot, sample holes, reactive tank and runner are located at the side towards substrate on thin film layer, and described pneumatic control layer is covered on the top of thin film layer.
Preferably, described thin film layer adopts PDMS material to make.
Preferably, described micro-fluidic chip also comprises the water filling layer be made up of gas permeable material be located at above described pneumatic control layer.
Preferably, be coated with sealing coat in the reactive tank on described thin film layer and runner, described sealing coat adopts gas impermeable material.
Preferably, described pneumatic control layer adopts resilient material to make, and the thickness of described pneumatic control layer is 20 ~ 50 μm.
Preferably, described responding layer comprises base plate and is covered on the cover plate on base plate, described sample slot, reactive tank and runner are located at the side towards cover plate on base plate, and described sample holes is located at the position relative to sample slot on cover plate, and described base plate and cover plate all adopt plastic material to make.
Another object of the present invention is to provide a kind of viral device for fast detecting of PCR in real time, comprising:
Micro-fluidic chip as above;
Actuating unit: for providing rotary power for described micro-fluidic chip;
Temperature control unit: for controlling and monitoring the temperature of reaction zone;
Acquisition of signal unit: for the signal in detection reaction district, and be transferred to data processing and control unit processes;
Data processing and control unit: realize the collection to temperature control unit and acquisition of signal cell data, process, storage and display, control the motion of described actuating unit simultaneously.
Preferably, described temperature control unit comprises is located at multiple semiconductor temperature-control sheet around reaction zone and multiple temperature sensor respectively, and described semiconductor temperature-control sheet is for controlling the temperature of reaction zone, and described temperature sensor is used for the temperature in detection reaction district; Described acquisition of signal unit comprises excitation light source, optical delivery unit and photodetector; Described excitation light source is used for sending exciting light and irradiates reaction zone, makes the sample in reaction zone send fluorescence; Optical delivery unit is for transmitting the fluorescence of exciting light and collection sample; Optical signal for detecting fluorescent signal, and is converted to electric signal transmission and processes to data processing and control unit by described photodetector.
Beneficial effect:
The micro-fluidic chip realizing PCR provided by the invention, the sample dosage not only used is little, reliability is high, thus has saved use cost; And, can need according to different tests to add different detection reagent, thus reach stdn and standardization.Meanwhile, present invention also offers the viral device for fast detecting of the PCR in real time comprising this micro-fluidic chip, this proofing unit is not only simple to operate, easy to use, and is easy to carry.
Accompanying drawing explanation
The structural representation realizing the micro-fluidic chip of PCR that Fig. 1 provides for the embodiment of the present invention 1, wherein, Fig. 1 a is the structural representation of substrate in responding layer; Fig. 1 b is the structural representation of thin film layer in responding layer; Fig. 1 c is the structural representation of pneumatic control layer; Fig. 1 d is the structural representation of water filling layer.
The schematic diagram of the viral device for fast detecting of the PCR in real time that Fig. 2 embodiment of the present invention provides.
The structural representation realizing the micro-fluidic chip of PCR that Fig. 3 provides for the embodiment of the present invention 2, wherein, Fig. 3 a is the structural representation of substrate in responding layer; The structural representation of thin film layer in Fig. 3 b responding layer; Fig. 3 c is the structural representation of pneumatic control layer.
The structural representation realizing the micro-fluidic chip of PCR that Fig. 4 provides for the embodiment of the present invention 3, wherein, Fig. 4 a is the structural representation of base plate in responding layer; The structural representation of Fig. 3 b responding layer cover plate.
Embodiment
As previously mentioned, the object of the present invention is to provide a kind of micro-fluidic chip realizing PCR, comprise responding layer, sample slot is provided with in described responding layer, many group reactions district is provided with around described sample slot, every group reaction district at least comprises a reactive tank, and each reactive tank is connected with sample slot by runner, and described sample slot is connected with the external world by sample holes.Meanwhile, another object of the present invention is to provide a kind of viral device for fast detecting comprising the PCR in real time of this micro-fluidic chip.
In order to set forth technical characterstic of the present invention and structure better, below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.
Embodiment 1
Consult Fig. 1, the micro-fluidic chip of what the present embodiment provided realize PCR comprises responding layer and is located at the pneumatic control layer 20 above responding layer.Wherein, consult Fig. 1 a and Fig. 1 b, the thin film layer 120 that responding layer comprises substrate 110 and is covered on substrate 110.Wherein, substrate 110 is make as rigid transparent materials such as glass, and thin film layer 120 adopts resilient material to make.In one preferably embodiment, thin film layer 120 adopts polydimethylsiloxane (polydimethylsiloxane, PDMS) material to make, and it has the advantages such as good optical clear, good adhesivity, unreactiveness.The reaction zone 121 thin film layer 120 being provided with the sample slot 123 for holding sample towards the side of substrate 110 and being located at around sample slot 123, wherein, sample is entered in sample slot 123 by the sample holes 124 be located in sample slot 123.In the present embodiment, responding layer comprises 12 group reaction districts 121, and wherein every group reaction district 121 comprises three reactive tanks 1211, is connected between each reactive tank 1211 and sample slot 123 by runner 122.Sample slot 123 comprises polysaccharase, enzyme antibody, base pair and the reaction reagent for the primer designed by certain virus or bacterium etc. for placing, preferably, reactive tank 1211 in same group reaction district 121 places identical reflection reagent, and in reactive tank between different group reaction district 121 1211, place different reflection reagent, the accuracy of detection so not only can be improved by reperformance test, simultaneously, in one-time detection test, various detection can also be carried out to sample, thus improve the range of application of this micro-fluidic chip.
Consult Fig. 1 c, pneumatic control layer 20 comprises multiple control valve 210 and the gas pipeline 220 for connection control valve 210, and wherein, pneumatic control layer 20 is covered on the top of the thin film layer 120 of responding layer, and control valve 210 is located at the top of runner 122.Key-course 20 adopts resilient material to make, and preferably, it adopts PDMS material to make, and its thickness is 20 ~ 50 μm.Gas pipeline 220 is provided with an inlet mouth 221, and this inlet mouth 221 is connected with gas under pressure.When from inlet mouth 221 insufflation gas, under the influence of air pressure, control valve 210 produces deformation, thus the thin film layer 120 in responding layer is fitted on substrate 110 well, and then closes runner 122, stops sample to enter reaction zone 121.
Further, the evaporation of moisture in reaction zone 121 during in order to reduce to test, thus improve the reliability of test, this micro-fluidic chip also comprises the water filling layer 30 be located at above pneumatic control layer 20.This water filling layer 30 adopts gas permeable material to make, and preferably, it adopts PMDS material to make.Consult Fig. 1 d, the water filling layer 30 that the present embodiment provides totally in comb shape, but is not limited to this.Whole water filling layer 30 is a cavity configuration, which is provided with water filling port 310, in use, distilled water is injected into from inlet 310 in the cavity of water filling layer 30, moisture in water filling layer 30 is evaporated and is entered into the top of responding layer by PMDS material, under making reaction zone 121 be in saturated vapor pressure atmosphere, thus reach the object of the moisture evaporation in inhibited reaction district 121.
Consult Fig. 2, based on same inventive concept, the present invention also provides a kind of viral device for fast detecting of PCR in real time, comprising: micro-fluidic chip 1 as above; Actuating unit 4: for providing rotary power for described micro-fluidic chip 1; Temperature control unit 5: for controlling and monitoring the temperature of reaction zone 121; Acquisition of signal unit 6: for the signal in detection reaction district, and be transferred to data processing and control unit processes.Data processing and control unit 7: realize the collection to temperature control unit 5 and acquisition of signal unit 6 data, process, storage and display, control the motion of actuating unit 4 simultaneously.
Wherein, temperature control unit 5 comprises and is placed in semiconductor temperature-control sheet 51 around each reaction zone 121 and temperature sensor 52 respectively.Preferably, the below of each reaction zone 121 is provided with semiconductor temperature control chip 51, and each semiconductor temperature-control sheet 51 is controlled by data processing and control unit 7 respectively; The surrounding of each reactive tank 1211 is equipped with a temperature sensor 52, and each temperature sensor 52 is controlled by data processing and control unit 7 respectively.
Acquisition of signal unit 6 comprises excitation light source 61, optical delivery unit 62 and photodetector 63.Wherein, excitation light source 61, for sending exciting light and irradiating reaction zone 121, makes the sample in it send fluorescence; Optical delivery unit 62 is for transmitting the fluorescence of exciting light and collection sample; Optical signal for detecting fluorescent signal, and is converted to electric signal transmission and processes to data processing and control unit 7 by photodetector 63.
In use, sample is joined sample slot 123 from sample holes 124 after distilled water diluting, under the control of data processing and control unit 7, actuating unit 4 drives micro-fluidic chip to rotate, sample is made to enter in reactive tank 1211 by runner 122, now, the sample total in whole reaction zone 121 tens receive be raised to hundreds of receive rise scope between.After sample enters into reactive tank 1211, close actuating unit 4, inflate simultaneously to key-course 20, until runner 122 is closed by control valve 210, now reaction zone 121 becomes a reaction system closed; Meanwhile, distilled water is injected into water filling layer 30, thus prevents the moisture in reaction zone 121 from evaporating.Open temp control unit 5, semiconductor temperature-control sheet 51 heats up to each reaction zone 121 or lowers the temperature under the control of data processing and control unit 7; Meanwhile, the temperature of each reactive tank 1211 monitored by temperature sensor 52 under the control of data processing and control unit 7.Now, each reaction zone can have different temperature of reaction according to the difference of reaction reagent.After reacting completely, data processing and control unit 7 control signal probe unit 6 work, and process the fluorescent signal collected, last Output rusults.
The micro-fluidic chip that the present embodiment provides, can add the reaction reagent that many groups are different, thus meet various different demand, and then reach stdn and standardization in each reaction zone 121.And the sample dosage that this micro-fluidic chip uses is little, and reliability is high, thus has saved use cost.Meanwhile, this proofing unit simple to operate, easy to use, be easy to carry.
Embodiment 2
Consult Fig. 3, the micro-fluidic chip of what the present embodiment provided realize PCR comprises responding layer, be located at pneumatic control layer 20 above responding layer, wherein, responding layer has structure identical in embodiment 1 with pneumatic key-course 20.As different from Example 1, thin film layer 120 in the responding layer that the present embodiment provides adopts PMDS material to make, it is coated with the sealing coat that employing is made as gas impermeable materials such as polyphenylene ethyls in reactive tank 1211 and runner 122, and this sealing coat is for stoping the evaporation of moisture in reaction zone 121.
The micro-fluidic chip that the present embodiment provides, replaces water filling layer by the sealing coat be located in reactive tank 1211 and runner 122, thus the evaporation of moisture in inhibited reaction district 121, not only structure is simple, easy to use, but also reduces cost.
Embodiment 3
Consult Fig. 4, the micro-fluidic chip of what the present embodiment provided realize PCR comprises responding layer, and wherein, the cover plate 140 that responding layer comprises base plate 130 and is covered on base plate 130, described base plate 130 and cover plate 140 all adopt plastic material to make.The reaction zone 121 that base plate 130 is provided with the sample slot 123 for holding sample towards the side of cover plate 140 and is located at around sample slot 123, wherein, sample is entered in sample slot 123 by the sample holes 124 be located at relative to the position of sample slot 123 on cover plate 140.In the present embodiment, responding layer comprises 12 group reaction districts 121, and wherein every group reaction district 121 comprises three reactive tanks 1211, is connected between each reactive tank 1211 and sample slot 123 by runner 122.Preferably, the position of reaction zone 121 is higher than sample slot 123, and make sample be easy to flow into reaction zone 121 in sample slot 123 like this, on the contrary, the sample in reaction zone 121 is difficult to flow back into sample slot 123, thus eliminates the use of control valve.
The micro-fluidic chip that the present embodiment provides is the simplest embodiment of one of the present invention, because base plate 130 adopts plastic material, therefore sample slot, reactive tank and runner can be formed on base plate 130 straight through the mode of injection moulding, thus simplify preparation flow; And, because plastics itself do not have ventilation property, thus eliminate the design suppressing moisture evaporation, simplify preparation flow further; Meanwhile, plastics itself have that cost is low, the simple advantage of injection moulding, therefore can also reduce preparation cost.
In sum, the micro-fluidic chip realizing PCR provided by the invention, the sample dosage not only used is little, reliability is high, thus has saved use cost; And, can need according to different tests to add different detection reagent, thus reach stdn and standardization.Meanwhile, present invention also offers the viral device for fast detecting of the PCR in real time comprising this micro-fluidic chip, this proofing unit is not only simple to operate, easy to use, and is easy to carry.
It should be noted that, in this article, the such as relational terms of first and second grades and so on is only used for an entity or operation to separate with another entity or operational zone, and not necessarily requires or imply the relation that there is any this reality between these entities or operation or sequentially.And, term " comprises ", " comprising " or its any other variant are intended to contain comprising of nonexcludability, thus make to comprise the process of a series of key element, method, article or equipment and not only comprise those key elements, but also comprise other key elements clearly do not listed, or also comprise by the intrinsic key element of this process, method, article or equipment.When not more restrictions, the key element limited by statement " comprising ... ", and be not precluded within process, method, article or the equipment comprising described key element and also there is other identical element.
Although the present invention is described in detail with reference to its exemplary embodiment and shows, but will be understood by those skilled in the art that, when not departing from the spirit and scope of the present invention be defined by the claims, the various changes of form and details can be carried out to it.
Claims (10)
1. one kind realizes the micro-fluidic chip of PCR, it is characterized in that, comprise responding layer, sample slot (123) is provided with in described responding layer, described sample slot (123) is provided with many group reactions district (121) around, every group reaction district (121) at least comprises a reactive tank (1211), and each reactive tank (1211) is connected with sample slot (123) by runner (122), and described sample slot (123) is connected with the external world by sample holes (124).
2. micro-fluidic chip according to claim 1, it is characterized in that, described micro-fluidic chip also comprises the pneumatic control layer (20) be covered on above described responding layer, described pneumatic control layer (20) comprises multiple control valve (210) and the gas pipeline (220) for connection control valve (210), and described control valve (210) is placed in the top of runner (122).
3. micro-fluidic chip according to claim 2, it is characterized in that, described responding layer comprises substrate (110) and is covered on the thin film layer (120) on substrate (110), described sample slot (123), sample holes (124), reactive tank (123) and runner (122) are located at the side towards substrate (110) on thin film layer (120), and described pneumatic control layer (20) is covered on the top of thin film layer (120).
4. micro-fluidic chip according to claim 3, is characterized in that, described thin film layer (120) adopts PDMS material to make.
5. micro-fluidic chip according to claim 4, is characterized in that, described micro-fluidic chip also comprises the water filling layer (30) be made up of gas permeable material being located at described pneumatic control layer (20) top.
6. micro-fluidic chip according to claim 4, is characterized in that, is coated with sealing coat in the reactive tank (1211) on described thin film layer (120) and runner (122), and described sealing coat adopts gas impermeable material.
7. micro-fluidic chip according to claim 3, is characterized in that, described pneumatic control layer (20) adopts resilient material to make, and the thickness of described pneumatic control layer is 20 ~ 50 μm.
8. micro-fluidic chip according to claim 1, it is characterized in that, described responding layer comprises base plate (130) and is covered on the cover plate (140) on base plate (130), described sample slot (123), reactive tank (1211) and runner (122) are located at the side towards cover plate (124) on base plate (130), described sample holes (124) is located at the position relative to sample slot (123) on cover plate (140), and described base plate (123) and cover plate (124) all adopt plastic material to make.
9. a viral device for fast detecting for PCR in real time, is characterized in that, comprising:
The arbitrary described micro-fluidic chip (1) of claim 1-8;
Actuating unit (4): for providing rotary power for described micro-fluidic chip (1);
Temperature control unit (5): for controlling and monitoring the temperature of reaction zone (121);
Acquisition of signal unit (6): for the signal in detection reaction district (121), and be transferred to data processing and control unit (7) processes;
Data processing and control unit (7): realize the collection to temperature control unit (5) and acquisition of signal unit (6) data, process, storage and display, control the motion of described actuating unit (4) simultaneously.
10. viral device for fast detecting according to claim 9, it is characterized in that, described temperature control unit (5) comprises the multiple semiconductor temperature-control sheets (51) and multiple temperature sensor (52) of being located at reaction zone (121) surrounding respectively, described semiconductor temperature-control sheet (51) is for controlling the temperature of reaction zone (121), and described temperature sensor (52) is for the temperature of detection reaction district (121); Described acquisition of signal unit (6) comprises excitation light source (61), optical delivery unit (62) and photodetector (63); Described excitation light source (61), for sending exciting light and irradiating reaction zone (121), makes the sample in reaction zone (121) send fluorescence; Optical delivery unit (62) is for transmitting the fluorescence of exciting light and collection sample; Optical signal for detecting fluorescent signal, and is converted to electric signal transmission and processes to data processing and control unit (7) by described photodetector (63).
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| CN108855254A (en) * | 2018-04-11 | 2018-11-23 | 上海理工大学 | A kind of protein concentration detection chip based on microflow control technique |
| CN109735444A (en) * | 2018-03-08 | 2019-05-10 | 中国科学院上海微系统与信息技术研究所 | A kind of micro chamber multicolor fluorescence digital pcr chip and application |
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