CN202730131U - Space oriented miniature microfluidic real-time fluorescent photo-conductive relay (PCR) work system - Google Patents
Space oriented miniature microfluidic real-time fluorescent photo-conductive relay (PCR) work system Download PDFInfo
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- CN202730131U CN202730131U CN 201220072095 CN201220072095U CN202730131U CN 202730131 U CN202730131 U CN 202730131U CN 201220072095 CN201220072095 CN 201220072095 CN 201220072095 U CN201220072095 U CN 201220072095U CN 202730131 U CN202730131 U CN 202730131U
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Abstract
A space oriented miniature microfluidic real-time fluorescent photo-conductive relay (PCR) work system belongs to the field of biology, analytical chemistry and medical detection. The work system comprises a biological chip, an injection pump and a step motor; a high-temperature denaturation area, a thermophilic extension area and a low temperature annealing area are sequentially arranged on the biological chip; a microchannel is also designed on the biological chip, sequentially passes through the three areas so as to form a reaction circulation passage, is bent in the thermophilic extension area, and then sequentially passes through the high-temperature denaturation area and the low temperature annealing area to reach the thermophilic extension area so as to form the next reaction circulation passage; a plurality of reaction circulation passages in the microchannel are sequentially connected with a micro fluorescent detection device which is arranged in a bending part of the annealing area with lower temperature in the microchannel; the inlet of the microchannel is connected with the injection pump which is connected with the step motor; and a control end of the step motor and detection signals of the micro fluorescent detection device are connected to a singlechip control system. The work system has a simple production technology, is easy to process, has high cost performance and can be disposably used.
Description
Technical field
The utility model relates to a kind of space-oriented miniature micro-fluidic real-time fluorescence PCR work system, is the pcr amplification reaction device of a kind of " function integrated morphology is miniature " type, belongs to biology, analytical chemistry and medical science detection field.
Background technology
Analytical test is frequently one of science and technology activity of human beings, innovative analysis measuring technology and corresponding innovative analysis testing tool to a great extent representing the scientific and technological level of a country, and guaranteed that the country of advanced technology is in strategic reserves and the capability of sustainable development of the knowledge and technology of association area.In the 21 century of taking as the leading factor with life science, the analytical and testing instrument of a new generation should be more microminiaturized, automatization, rapid and portability.
Micro-full analytical system (Miniaturized Total Analysis System, μ TAS) claims again chip lab (lab on a chip, LOC).The target of μ TAS is by the microminiaturization of chemical analysis apparatus and integrated, to greatest extent the function of assay laboratory is integrated on the portable equipment, or even on the chip of heart size, namely realizes " function integrated morphology is miniature " in biochemical analysis laboratory.
Along with reaching its maturity of China's manned spaceflight technology, the gradually prolongation in space mission cycle also can be more and more higher to the requirement of spacefarer's health care.Be in the microorganism serious threat spacefarer's of non-supervised state health in the spacecraft.Because Millikan's rays and the synergy that stress wait factor cause immune function of human body to reduce, Expectancy increases, and the pathogenic microorganism in the cabin can badly influence the healthy of spacefarer at this moment.Simultaneously, carry the microorganism that can under space environment, survive on the aerospacecraft, might take space station, martian surface to by the mankind or spacecraft, can cause disadvantageous effect to human research's space life form.Therefore, the real-time automatic alarm system of equipment biological hazard is very necessary on the spacecraft.
Miniature fluorescence detecting system in the biological hazard warning howler since the essence of its gordian technique to be the requirement function integrated and structure is miniature, to realize that the little full-automatic detection of lightweight volume is as target.And the little detection system of present fluorescence biosensor chip wants too that cube is little, function is many, automatization level is high.Therefore, the research of the little detection system of fluorescence in the biological hazard warning howler can be implanted on the little detection system of the fluorescence biosensor chip basis and carry out, and can revise research in the calculating under the below-G conditions to detection system, to realize " space-oriented application ".
The PCR microflow controlled biochip is a new and high technology that develops rapidly in life science in recent years, it is that the functions such as sampling, dilution, reagent adding, reaction, separation and detection are integrated in the chip, and its science and advance embody a concentrated reflection of miniature and integrated these two aspects of function of structure.Biochip technology has 4 bare bones: chip preparation, sample preparation, biochemical reaction and signal detection.Its principle is: the PCR reaction mixture enters the microchannel that is in respectively three flat-temperature zones (94 ℃, 50～60 ℃, 72 ℃) on the biochip by the flow velocity of setting under the effect of precise injection pump, sex change, renaturation and extension through PCR, thereby realize amplification cycles one time, a circulation can make the DNA total amount double.Under conditions suitable, this circulation constantly repeats, and after n the circulation amount of product D NA is increased by the 2n mode.Last testing liquid shines through excitation light source and sends fluorescence, by the photosensor collection and through exporting the electrical signal of fluorescent value after the opto-electronic conversion.
Obviously mainly there is following shortcoming in present micro-fluidic PCR work system for carry out the real-time testing of PCR fluorescence in the space:
1, temperature circulatory system adopts copper billet or aluminium block heats and fan cooling matches, and whole device energy-wasting is many, and volume is difficult to greatly dwindle, and can't realize portability;
2, fluorescence low-light spectrum detector all is directly to use traditional encapsulation phototube at present, such as photomultiplier (PMT) or charge coupled cell (CCD).Because the volume of element self is just very large, and be again that split is used, supporting light path device need to be arranged, cause the bulky of whole fluorescence detection device, may not be embedded in the biochip;
3, because the light path that needs all kinds of optics and optical fiber to form when exciting light conduction and reflected light gather is carried out optic path, not only complex structure is difficult to realize integrated, and affect the stability of real-time fluorescence detection;
4, owing to detect the metering system that does not reach the zero distance contact, can make the little detection of fluorescence be subject to non-detected object material, as forming the interference of the materials such as microchannel wall, cause measuring error;
5, the sampling control system of PCR microflow controlled biochip, temperature controlling system, fluorescence detecting system generally adopt each several part to control separately, can't carry out Real-time Feedback and integrated control to the variation of temperature in the microchannel, flow velocity and the signal between the fluoroscopic examination of liquid.
Therefore, realization is to carrying out highly integrated control between the micro-fluidic work system disparate modules of PCR, and the development volume little to can embed chip and highly sensitive be the integrated portable of realization PCR fluorescence real-time detecting system and the main direction of studying of microminiaturization to reaching the miniature fluorescence detection device that biotechnology requires.
The utility model content
In order to address the above problem well, the utility model relates to a kind of miniature micro-fluidic real-time fluorescence PCR work system, is mainly used in spatial dimension pcr amplification reaction is carried out the real-time fluorescence detection.Purpose is to realize the integrated automation control of miniature micro-fluidic real-time fluorescence PCR detection system, and the portable minisize of realizing whole work system.
This patent is to realize by the following technical solutions above-mentioned purpose:
A kind of miniature micro-fluidic real-time fluorescence PCR work system comprises biochip 1, syringe pump 2, stepper-motor 3; Be disposed with three thermostatically heating districts 5 on the described biochip 1, i.e. high-temperature denatured district, thermophilic extension area, low-temperature annealing district; The temperature in described high-temperature denatured district be 94 ℃, the temperature in low-temperature annealing district be 56 ℃, and the temperature of thermophilic extension area be 72 ℃; Also be designed with microchannel 4 on the biochip 1, microchannel 4 is successively by high-temperature denatured district, low-temperature annealing district, thermophilic extension area, consist of a reaction cycle passage, then in the bending of thermophilic extension area, arrive the thermophilic extension area through high-temperature denatured district and low-temperature annealing district successively again, consist of next reaction cycle passage; A plurality of reaction cycle passages link to each other successively in the microchannel 4; For fear of the infringement of high temperature to detection means, therefore the flex area of the annealed zone that temperature is lower in the microchannel is provided with miniature fluorescence detection device 8; The entrance of microchannel 4 links to each other with the syringe pump that is connected with stepper-motor 3; The detection signal of the control end of stepper-motor 3 and miniature fluorescence detection device 8 is connected to single-chip computer control system 10; Be provided with heat insulation air door 7 between high-temperature denatured district, low-temperature annealing district, the thermophilic extension area.
Described biochip 1 is microflow controlled biochip.
The number of described reaction cycle passage is 20 to 30.
Described single-chip computer control system 10 comprises liquid crystal indicator and keyboard input device.
Described microchannel 4 usefulness photoetching techniques or etching technique or pressure sintering, injection moulding, the little processing method of laser ablation are made at glass, silicon chip or high polymer material, or the direct cloth of kapillary is being posted on the slide glass that heats film.
Described heat insulation air door 7 is poroid.
Described miniature fluorescence detection device 8 mainly comprises the electric import and export layer 9 of excitation light source unit 11 and fluorescence detection unit 12 and proofing unit; Wherein excitation light source unit 11 is tubulose, by excitation light source 13, combined filters 14, optical microlens 16 and with whole excite the unit around the multilayer optical film 15 of the effect that intercepts fully to the high reflection of light wave in the conduit with to the outer light wave of conduit that is surrounded form; Fluorescence detection unit 12 is tubulose, forms by optical microlens 16, combined filters 18, electrooptical device 17 with the multilayer optical film 15 of the effect that intercepts fully to the high reflection of light wave in the conduit with to the outer light wave of conduit that is surrounded around the whole detecting unit;
The mode of connection of described excitation light source unit 11 is that excitation light source 13 and combined filters 14 usefulness multilayer optical films 15 are surrounded as tubulose, then adopt the formed in situ legal system to make optical microlens 16 in combined filters 14, soon ultraviolet optics curing glue drips on combined filters 14 and vertically discharges, drop onto on the combined filters 14, and from top to bottom to diffusion trickling all around, with Ultra-Violet Laser drop is shone, make its curing, form micro optical lens 16;
Described miniature fluorescence detection device 8 with the mode of connection of biochip 1 is: bottom and two side perforatings in the microchannel 4 of biochip 1, excitation light source unit 11 is placed in the base apertures, optical microlens up, fluorescence detection unit 12 place the left side, microlens is towards the right side; 4 top and right side embed another set of micro detecting device 19 again in the microchannel, and wherein excitation light source unit 11 places the top, microlens down, fluorescence detection unit 12 places the right side, microlens is towards a left side; Make optical microlens all remain the microchannel 4 of micrometer fluid towards stream;
Lower end and low-temperature annealing district that the electric import and export floor 9 of described proofing unit is positioned at biochip 1 separate, or place the groove shape passage that etches on the biochip 1, or directly place biochip 1 surface.
Device in the utility model is like this work: syringe pump 2 directly inserts the microchannel on the chip, promote the sample introduction that syringe pump is finished sample by stepper-motor 3, can give the control of the different instruction realization of stepper-motor to the microfluid flow velocity by micro-chip microprocessing systems 10 simultaneously; 4 for being full of the microchannel of microfluid to be measured, treat that fluid measured realizes the amplification of a DNA by flow through three different humidity provinces 5, microchannel, be that DNA finishes once circulation after high-temperature denatured (94 ℃), low-temperature annealing (56 ℃) and thermophilic extend (72 ℃), therefore can just can guarantee to treat that at the passage length in differing temps zone fluid measured is in the reaction times in differing temps district by setting the microchannel; 5,6 form thermostatic control system, flat-temperature zone 5 from the chip top successively the design temperature of downward three humidity provinces be respectively 94 ℃, 72 ℃ and 56 ℃, feed back to the Real Time Monitoring that microprocessing systems 10 is realized temperature after the SMD temperature sensor 6 of pasting on heating zone surface obtains the real time temperature values; Embed herein miniature fluorescence detection device 8 and treat the micrometer fluid and detect, also can treat the flow velocity of micrometer fluid simultaneously and monitor.Wherein excitation light source unit 11 can adopt conductor photodiode (LED) or semiconductor laser diode (LD) that exciting light is provided, and fluorescence detection unit 12 used electrooptical devices can be that photorectifier or silicon blue streak battery gather fluorescent signal; 9 is the electric import and export layer of miniature fluorescence detection device, and material to be detected goes out fluorescence by excitation, becomes electrical signal output after the detected unit collection; 10 is the micro-chip microprocessing systems, mainly three large modules of PCR reaction is carried out integrated control, i.e. temperature control modules, sample feeding speed measuring module and fluoroscopic examination module.
This miniature micro-fluidic real-time fluorescence PCR work system is shown in Figure 1, because pcr amplification reaction needs 20～30 amplification cycles at least, and the time of each pcr amplification circulation must be consistent.Flowing through after fluid channel to be measured enters microchannel 4 behind high-temperature denatured district, low-temperature annealing district and the thermophilic extension area is a reaction cycle, therefore need at least 30 identical reaction cycle passages, and the length of each pcr amplification circulation microchannel is the same with layout.The type of heating of temperature heating region 5 is paster type resistor to be heated film directly paste microchannel bottom and heat in this patent, its temperature variation can gather and feed back to micro-chip microprocessing systems 10 by temperature sensor 6 carries out inching, and wherein temperature sensor 6 directly sticks on paster type resistor heating film surface.For the real-time fluorescence of realizing micro-fluidic PCR reaction detects, proposing whenever to finish the fluorescent signal that amplification cycles treats fluid measured in this patent detects once, therefore the same position in each circulation microchannel is provided with miniature fluorescence detection device 8, and the every adjacent length of two real-time check points of fluorescence on the microfluidic flow direction is a circulation microchannel length.For the stability that guarantees to detect, therefore since second pcr amplification circulation, testing liquid promotes syringe pump 2 by micro-chip 10 control step motors 3 and injects the microchannel, treat that microfluid flows in the microchannel by pre-set flow velocity, when arriving check point, miniature fluorescence detection device 8 detects the actual fluorescent signal value of testing liquid, whether the front end according to fluorescent PCR reagent flows to the fluoroscopic examination point that is detecting simultaneously, the computer recording microfluid is flowed through time of this passage, just can obtain the working flow rate of the reality of microfluid in this passage and actual flow velocity and need adjustment amount, again the actual flow velocity adjustment amount be fed back to stepper-motor behind the single-chip computer control system and finish next amplification cycles thereby feed back to.
The work system that this patent proposes has following advantage:
The utility model is integrated sample feeding observing and controlling speed system, temperature controlling system and the miniature real-time fluorescence detection system of micro-fluidic real-time fluorescence PCR work system, adopt the integrated control of singlechip microprocessor, both reduced unnecessary peripherals, make more integrated micro of work system, this " function integrated morphology micro " just is being fit to the space-oriented and using, make again and in time feed back between the disparate modules and adjust, greatly shortened the work period of whole system.
Miniature fluorescence detection device 8 in the utility model is directly embedded in the microchannel, and integrated multiple non-electric key element is such as the even bundle of gathering, transmission, light of excitation light source, light, light collection, light detection etc.Owing to replaced photomultiplier PMT or the charge coupled cell CCD that can't embed chip, so that the feature size downsizing of whole device is to only having the millimeter magnitude, even can also be more further micro to micron dimension, and at the same position of each reaction cycle of microchannel check point is set, can whether flow to according to the front end of fluorescent PCR reagent the fluoroscopic examination point that is detecting the flow velocity of testing liquid is adjusted in real time.
Microchannel in the work system in the biochip, can adopt the modes such as photoetching technique, etching technique and pressure sintering, injection moulding, the little processing method of laser ablation to make at glass, silicon chip or high polymer material, also can be with the direct cloth of kapillary on the slide glass that posts the heating film, kapillary has following advantage as the used microchannel of amplified reaction and the mode such as above-mentioned: (1) manufacture craft simply is easy to processing, and the cost performance height can disposablely use; (2) capillary tube inner wall is smooth, than reduced roughness and the laminar flow effect in the passage with upper type; When (3) adopting kapillary as the microchannel, become the specified shape passage with the curing adhesive curing, owing to colloid becomes black, required lucifuge environment in the time of namely can satisfying fluoroscopic examination.Be provided with insulation hole (air door) 7 between the different warm area in the biochip, the thermal insulation effect of different warm areas is effectively improved.
Description of drawings
The miniature micro-fluidic real-time fluorescence PCR work system scantlings plan of Fig. 1;
The A-A sectional view of Fig. 2 direction shown in Figure 1;
The partial enlarged drawing of miniature fluorescence detection device in Fig. 3 passage;
Among the figure: 1 biochip; 2 syringe pumps; 3 stepper-motors; 4 microchannels; 5 thermostatically heating districts; 6 temperature sensors; 7 heat insulation air doors; 8 miniature fluorescence detection devices; The electric import and export layer of 9 proofing units; 10 single-chip computer control systems; 11 excitation light source units; 12 fluorescence detection units; 13 excitation light sources; 14,18 combined filters; 15 optical thin films; 16 optical microlenses; 17 electrooptical devices; 19 another set of miniature fluorescence detection devices; 20 chip microchannel slide glass cross sections.
Specific embodiment
Describe present embodiment in detail below in conjunction with accompanying drawing 1～3.
The structural representation of this patent as shown in Figure 1, this space-oriented miniature micro-fluidic real-time fluorescence PCR work system mainly comprises three control modules and as the biochip of carrier, be sample introduction observing and controlling speed control system, temperature controlling system and miniature fluorescence detecting system, carry out integrated control by singlechip microprocessor.The size of whole chip is approximately 80 * 60 * 10mm.The implementation content is: 2 and 3 consist of sampling device among Fig. 1, by the step motor control syringe pump liquid is injected in the microchannel 4, can set different instruction is controlled fluid to stepper-motor flow velocity by micro-chip microprocessing systems 10, microfluid to be measured is by certain flow velocity three thermostatically heating districts 5 that flow through, and its temperature variation can gather and feed back to micro-chip 10 by temperature sensor 6 carries out inching.When testing liquid arrives miniature fluorescence detection device 8 in the microchannel, finish a pcr amplification reaction circulation.At check point, miniature fluorescence detection device obtains the actual fluorescent signal value of microfluid to be measured.Proofing unit mainly comprises excitation light source unit 11 and fluorescence detection unit 12 as shown in Figure 3, excitation light source 13 general semiconductor light-emitting-diode (LED) or the semiconductor laser diodes (LD) of adopting, combined filters 14 through particular peak wavelength focuses on the tested biological microfluid by optical microlens 16, makes corresponding microfluid to be measured send fluorescence.15 is multilayer optical film, with whole excite the unit around be surrounded, form tubulose, play the effect to managing the high reflection of interior light wave and intercepting fully managing outer light wave.17 is electrooptical device, can be photodiode or silicon blue streak battery, fluorescent signal after optical microlens 16 focuses on through the combined filters 18 of particular peak wavelength after by the photodiode collection and be converted to electrical signal and output to micro-chip microprocessing systems 10 through 9.Whether the front end according to fluorescent PCR reagent flows to the fluoroscopic examination point that is detecting simultaneously, microprocessing systems can record microfluid and flow through time of this passage, therefore length/the microfluid that the obtains actual working flow rate=microchannel actual required time of this passage of flowing through, actual flow velocity needs the working flow rate of adjustment amount=Design Theory flow velocity-reality, the result illustrates that for just actual working flow rate need increase; Otherwise need to reduce; The result is zero, illustrates that the working flow rate of actual working flow rate and Theoretical Calculation design coincide, and actual flow velocity need not adjusted.Thereby realized real-time fluorescence is detected and sample introduction tests the speed and controls fast synchronization control, and combine by the micro-chip microprocessing systems with temperature controlling system and to control, thereby reduce the peripherals of chip, make more integrated micro of work system, to reach the requirement of job space.
Claims (7)
1. a miniature micro-fluidic real-time fluorescence PCR work system is characterized in that: comprise biochip (1), syringe pump (2), stepper-motor (3); Be disposed with three thermostatically heating districts (5) on the described biochip (1), i.e. high-temperature denatured district, thermophilic extension area, low-temperature annealing district; The temperature in described high-temperature denatured district be 94 ℃, the temperature in low-temperature annealing district be 56 ℃, and the temperature of thermophilic extension area be 72 ℃; Also be designed with microchannel (4) on the biochip (1), microchannel (4) is successively by high-temperature denatured district, low-temperature annealing district, thermophilic extension area, consist of a reaction cycle passage, then in the bending of thermophilic extension area, arrive the thermophilic extension area through high-temperature denatured district and low-temperature annealing district successively again, consist of next reaction cycle passage; A plurality of reaction cycle passages link to each other successively in the microchannel (4); For fear of the infringement of high temperature to detection means, therefore the flex area of the annealed zone that temperature is lower in the microchannel is provided with miniature fluorescence detection device (8); The entrance of microchannel (4) links to each other with the syringe pump that is connected with stepper-motor (3); The detection signal of the control end of stepper-motor (3) and miniature fluorescence detection device (8) is connected to single-chip computer control system (10); Be provided with heat insulation air door (7) between high-temperature denatured district, low-temperature annealing district, the thermophilic extension area.
2. a kind of miniature micro-fluidic real-time fluorescence PCR work system according to claim 1, it is characterized in that: described biochip (1) is microflow controlled biochip.
3. a kind of miniature micro-fluidic real-time fluorescence PCR work system according to claim 1, it is characterized in that: the number of described reaction cycle passage is 20 to 30.
4. a kind of miniature micro-fluidic real-time fluorescence PCR work system according to claim 1, it is characterized in that: described single-chip computer control system (10) comprises liquid crystal indicator and keyboard input device.
5. a kind of miniature micro-fluidic real-time fluorescence PCR work system according to claim 1, it is characterized in that: described microchannel (4) make at glass, silicon chip or high polymer material of photoetching technique or etching technique or pressure sintering, injection moulding, the little processing method of laser ablation, or with the direct cloth of kapillary on the slide glass that is posting the heating film.
6. a kind of miniature micro-fluidic real-time fluorescence PCR work system according to claim 1 is characterized in that: described heat insulation air door (7) is for poroid.
7. a kind of miniature micro-fluidic real-time fluorescence PCR work system according to claim 1, it is characterized in that: described miniature fluorescence detection device (8) mainly comprises the electric import and export layer (9) of excitation light source unit (11) and fluorescence detection unit (12) and proofing unit; Wherein excitation light source unit (11) is tubulose, by excitation light source (13), combined filters (14), optical microlens (16) and with whole excite the unit around the multilayer optical film (15) of the effect that intercepts fully to the high reflection of light wave in the conduit with to the outer light wave of conduit that is surrounded form; Fluorescence detection unit (12) is tubulose, forms by optical microlens (16), combined filters (18), electrooptical device (17) with the multilayer optical film (15) of the effect that intercepts fully to the high reflection of light wave in the conduit with to the outer light wave of conduit that is surrounded around the whole detecting unit;
The mode of connection of described excitation light source unit (11) is that excitation light source (13) and combined filters (14) are surrounded as tubulose with multilayer optical film (15), then adopts the formed in situ legal system to make optical microlens (16) in combined filters (14);
Fluorescence detection unit (12) is that electrooptical device (17) and combined filters (18) are surrounded as tubulose with multilayer optical film (15) equally, then makes optical microlens (16) on the spectral filter surface;
The mode of connection of described miniature fluorescence detection device (8) and biochip (1) is: bottom and two side perforatings in the microchannel (4) of biochip (1), excitation light source unit (11) is placed in the base apertures, optical microlens up, fluorescence detection unit (12) places the left side, and microlens is towards the right side; The top of (4) and right side embed another set of micro detecting device (19) again in the microchannel, and wherein excitation light source unit (11) places the top, microlens down, fluorescence detection unit (12) places the right side, microlens is towards a left side; Make optical microlens all remain the microchannel (4) of micrometer fluid towards stream;
Lower end and low-temperature annealing district that the electric import and export floor (9) of described proofing unit is positioned at biochip (1) separate, or place the groove shape passage that etches on the biochip (1), or directly place biochip (1) surface.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111020712A (en) * | 2019-11-22 | 2020-04-17 | 台州达邦贸易有限公司 | Hemp fiber rubbing device |
| CN112911105A (en) * | 2021-01-19 | 2021-06-04 | 中国计量科学研究院 | Digital PCR result reading device |
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2012
- 2012-02-29 CN CN 201220072095 patent/CN202730131U/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111020712A (en) * | 2019-11-22 | 2020-04-17 | 台州达邦贸易有限公司 | Hemp fiber rubbing device |
| CN111020712B (en) * | 2019-11-22 | 2021-03-23 | 陈子仪 | Hemp fiber rubbing device |
| CN112911105A (en) * | 2021-01-19 | 2021-06-04 | 中国计量科学研究院 | Digital PCR result reading device |
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Granted publication date: 20130213 Termination date: 20140229 |