CN102604824A - Space-oriented mini micro-fluidic real-time fluorescent PCR (Plymerase Chain Reaction) working system - Google Patents
Space-oriented mini micro-fluidic real-time fluorescent PCR (Plymerase Chain Reaction) working system Download PDFInfo
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Abstract
The invention discloses a space-oriented mini micro-fluidic real-time fluorescent PCR (Plymerase Chain Reaction) working system, belonging to the fields of biology, analytical chemistry and medicine detection and comprising a biological chip, an injection pump and a step motor, wherein a high temperature degeneration zone, a thermophilic extension zone and a low temperature annealing zone are arranged on the biological chip in sequence; a micro-channel is arranged on the biological chip, the micro-channel passes through three zones in sequence so as to form a reaction circulation channel, and is bent in the thermophilic extension zone, and reaches the thermophilic extension zone through the high temperature degeneration zone and the low temperature annealing zone in sequence so as to form the next reaction circulation channel; multiple reaction circulation channels in the micro-channel are connected in sequence, a bending zone of an annealing zone with lower temperature in the micro-channel is provided with a mini fluorescence detection device; the inlet of the micro-channel is connected with the injection pump with the step motor; and the control end of the step motor and a detection signal of the fluorescence detection device are connected to a single-chip microcomputer control system. The system, provided by the invention, has the advantages of simple production process, easy processing, high cost performance and disposable use.
Description
Technical field
The present invention 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 range.
Background technology
Analytical test is one of the most frequent human science and technology activity; Innovative analysis measuring technology and corresponding innovative analysis testing tool have been represented the scientific and technological level of a country to a great extent, and have guaranteed strategic reserve and the capability of sustainable development of the country of advanced technology at the knowledge and technology of association area.In the 21 century of taking as the leading factor with life science, analytical and testing instrument microminiaturization more, robotization, the rapid and portability of a new generation.
Micro-full analytical system (Miniaturized Total Analysis System, μ TAS) claim again chip lab (lab on a chip, LOC).The target of μ TAS is through the microminiaturization of chemical analysis apparatus and integrated, is integrated into the function of assay laboratory on the portable equipment to greatest extent, or even on the chip of heart size, promptly realizes biochemical analysis breadboard " function integrated morphology is miniature ".
Along with reaching its maturity of China's manned spaceflight technology, the prolongation gradually in space mission cycle also can be increasingly high to the requirement of spacefarer's health care.Be in the mikrobe serious threat spacefarer's of non-monitor 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 mikrobe that can under space environment, survive on the aerospacecraft, might take space station, martian surface to, can cause disadvantageous effect human research's space life form through the mankind or spacecraft.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 is because that the essence of its gordian technique is the requirement function is integrated miniature with structure, to realize that the little full-automatic detection of volume in light weight is a 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 carry out Study on Correction to the calculating of detection system under below-G conditions, to realize " space-oriented application ".
The PCR microflow controlled biochip is a new and high technology that in life science, develops rapidly in recent years; It is with sampling, dilutes, adds functions such as reagent, reaction, separation and detection and be integrated in the chip, and it is scientific to embody a concentrated reflection of miniature and integrated these two aspects of function of structure with advance.Biochip technology has 4 bare bones: chip preparation, specimen preparation, biochemical reaction and signal detection.Its principle is: the PCR reaction mixture is getting into the microchannel that is in three flat-temperature zones (94 ℃, 50～60 ℃, 72 ℃) on the biochip respectively 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 vacuum photocell at present, like PM (PMT) or charge coupled cell (CCD).Because the volume of element self is just very big, and be again that split is used, need supporting light path device, cause the bulky of whole fluorescence detection device, possibly not be embedded in the biochip;
3, because the light path that when exciting light conduction and reflected light are gathered, needs all kinds of optics and optical fiber to form is carried out optic path, not only complex structure is difficult to realize integrated, and influence 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 receive non-detected object material,, cause measuring error as forming the interference of materials such as microchannel wall;
5, the sampling control system of PCR microflow controlled biochip, temperature controlling system, fluorescence detecting system generally adopt the each several part independent control, can't the flow velocity and the signal between the fluoroscopic examination of variation of temperature, liquid in the microchannel be fed back and integrated control in real time.
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 and microminiaturized main direction of studying of realization PCR fluorescence real-time detecting system to the miniature fluorescence detection device that can reach the biotechnology requirement.
Summary of the invention
In order to address the above problem well, the present invention relates to a kind of miniature micro-fluidic real-time fluorescence PCR work system, be 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 minisizeization that realizes whole work system.
This patent is to adopt following technical scheme to realize 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, promptly high-temperature denatured district, thermophilic extension area, low-temperature annealing district; The temperature in said 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 through high-temperature denatured district, low-temperature annealing district, thermophilic extension area; Constitute a reaction cycle passage; In the bending of thermophilic extension area, arrive the thermophilic extension area through high-temperature denatured district and low-temperature annealing district successively more then, constitute 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 inlet 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 a 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 entry device.
Described microchannel 4 usefulness photoetching techniques or etching technique or pressure sintering, injection moulding, the little processing method of laser ablation are made on 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 comprises that mainly the excitation light source unit 11 and the electricity of fluorescence detection unit 12 and proofing unit export into layer 9; Wherein excitation light source unit 11 is a tubulose, forms by excitation light source 13, combined filters 14, optical microlens 16 with the whole 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 unitary that excites; Fluorescence detection unit 12 is a 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; On combined filters 14, adopt the formed in situ legal system to make optical microlens 16 then, the glue of ultraviolet optics curing soon drips on combined filters 14 and vertically discharges, and drops 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 micrometer fluidic microchannel 4 towards stream;
The electricity of described proofing unit is exported the lower end and the low-temperature annealing district that are positioned at biochip 1 into layer 9 and is separated, or places the groove shape passage that etches on the biochip 1, or directly places biochip 1 surface.
Device among the present invention is work like this: syringe pump 2 directly inserts the microchannel on the chip; Promote the sample introduction that syringe pump is accomplished sample by stepper-motor 3, can give the different instruction of stepper-motor through micro-chip microprocessing systems 10 simultaneously and realize control the microfluid flow velocity; 4 for being full of the microchannel of microfluid to be measured; Treat that fluid measured realizes the amplification of a DNA through flow through three different temperature districts 5, microchannel; Be that DNA accomplishes once circulation after high-temperature denatured (94 ℃), low-temperature annealing (56 ℃) and thermophilic extend (72 ℃), therefore can just can guarantee to treat the reaction times of fluid measured at the passage length in differing temps zone through setting the microchannel in the differing temps district; 5,6 form thermostatic control system; Flat-temperature zone 5 is respectively 94 ℃, 72 ℃ and 56 ℃ from the design temperature of downward successively three humidity provinces, chip top, feeds back to the real-time monitoring that microprocessing systems 10 is realized temperature after the SMD TP of pasting on the surface, heating zone 6 obtains the real time temperature value; Embed miniature fluorescence detection device 8 herein and treat the micrometer fluid and detect, also can treat the micrometer flow rate of 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 are gathered fluorescent signal; 9 export into layer for the electricity of miniature fluorescence detection device, and material to be detected goes out fluorescence by excitation, become electrical signal output after the unit to be detected collection; 10 is the micro-chip microprocessing systems, mainly three big 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 each pcr amplification round-robin time must be consistent.Behind flow through behind the entering microchannel 4, fluid channel to be measured high-temperature denatured district, low-temperature annealing district and thermophilic extension area is a reaction cycle; Therefore need 30 identical reaction cycle passages at least, and the length of each pcr amplification circulation microchannel is the same with layout.The type of heating of temperature heating region 5 is SMD resistive heating film directly to be pasted the bottom, microchannel heat in this patent; Its temperature variation can be gathered and feed back to micro-chip microprocessing systems 10 and carried out inching through TP 6, and wherein TP 6 directly sticks on SMD resistive heating film surface.For the real-time fluorescence of realizing micro-fluidic PCR reaction detects; Proposing amplification cycles of every completion in this patent treats the fluorescent signal of fluid measured and 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 injection microchannels by micro-chip 10 control step motors 3; 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, and the working flow rate and the actual flow velocity that just can obtain the reality of microfluid in this passage need adjustment amount, again the actual flow velocity adjustment amount is fed back to stepper-motor behind the single-chip computer control system and accomplishes next amplification cycles thereby feed back to.
The work system that this patent proposed has following advantage:
The present invention is integrated the sample feeding observing and controlling speed system of micro-fluidic real-time fluorescence PCR work system, temperature controlling system and miniature real-time fluorescence detection system; Adopt the integrated control of singlechip microprocessor; Both reduced unnecessary peripherals, and made work system integrated microization more, 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 whole system operation cycle.
Miniature fluorescence detection device 8 among the present invention is directly embedded in the microchannel, and integrated multiple non-electric key element is like the even bundle of gathering, transmission, light of excitation light source, light, light collection, light detection etc.Owing to replaced the PM PMT or the charge coupled cell CCD that can't embed chip; Make the characteristic dimension of whole device narrow down to and have only the millimeter magnitude; Even can also be more further micro to micron dimension; And in the microchannel same position of each reaction cycle is provided with check point, can whether flow to the fluoroscopic examination point that is detecting according to the front end of fluorescent PCR reagent the flow velocity of testing liquid is adjusted in real time.
Microchannel in the work system in the biochip; Can adopt modes such as photoetching technique, etching technique and pressure sintering, injection moulding, the little processing method of laser ablation on glass, silicon chip or high polymer material, to make; Also can be on the slide glass that posts the heating film with the direct cloth of kapillary; Kapillary has following advantage as used microchannel of amplified reaction and 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, become the specified shape passage with the curing adhesive curing as the microchannel, owing to colloid becomes black, required lucifuge environment in the time of promptly 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 TPs; 7 heat insulation air doors; 8 miniature fluorescence detection devices; The electricity of 9 proofing units is exported into layer; 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
Specify present embodiment below in conjunction with accompanying drawing 1～3.
The structural representation of this patent is 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 system, temperature controlling system and miniature fluorescence detecting system, carry out integrated control by singlechip microprocessor.The size of entire chip is approximately 80 * 60 * 10mm.The practical implementation content is: 2 and 3 constitute sampling device among Fig. 1; By the step motor control syringe pump liquid is injected in the microchannel 4; Can set different instruction through micro-chip microprocessing systems 10 and control flow rate of fluid to stepper-motor; Microfluid to be measured is by certain flow velocity three thermostatically heating districts 5 that flow through, and its temperature variation can be gathered and feed back to micro-chip 10 and carried out inching through TP 6.When testing liquid arrives the miniature fluorescence detection device 8 in the microchannel, accomplishes a pcr amplification reaction and circulate.At check point, miniature fluorescence detection device obtains the actual fluorescent signal value of microfluid to be measured.Proofing unit as shown in Figure 3 mainly comprises excitation light source unit 11 and fluorescence detection unit 12; Excitation light source 13 general semiconductor light-emitting-diode (LED) or the semiconductor laser diodes (LD) of adopting; After optical microlens 16 focuses on the tested biological microfluid, make corresponding microfluid to be measured send fluorescence through the combined filters 14 of particular peak wavelength.15 is multilayer optical film, and whole exciting is surrounded around unitary, forms tubulose, plays to the high reflection of light wave in managing with to managing the effect that outer light wave intercepts fully.17 is electrooptical device; Can be photodiode or silicon blue streak battery, fluorescent signal after optical microlens 16 focuses on through combined filters 18 backs of particular peak wavelength by the photodiode collection and convert electrical signal into 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 write down microfluid and flow through time of this passage; Therefore length/the microfluid that the obtains actual working flow rate=one 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, and the result explains that for just actual working flow rate need increase; Otherwise need to reduce; The result is zero, explains that the working flow rate of actual working flow rate and Theoretical Calculation design coincide, and actual flow velocity need not adjusted.Thereby having realized real-time fluorescence detected to test the speed with sample introduction 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 work system integrated microization more, 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), promptly high-temperature denatured district, thermophilic extension area, low-temperature annealing district; The temperature in said 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 through high-temperature denatured district, low-temperature annealing district, thermophilic extension area; Constitute a reaction cycle passage; In the bending of thermophilic extension area, arrive the thermophilic extension area through high-temperature denatured district and low-temperature annealing district successively more then, constitute 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 inlet 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 a 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 entry 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 on glass, silicon chip or high polymer material with 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 is characterized in that: described miniature fluorescence detection device (8) mainly comprise the electricity of excitation light source unit (11) and fluorescence detection unit (12) and proofing unit export into the layer (9); Wherein excitation light source unit (11) is a tubulose, forms by excitation light source (13), combined filters (14), optical microlens (16) with the whole 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 unitary that excites; Fluorescence detection unit (12) is a 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); Going up in combined filters (14) then adopts the formed in situ legal system to make optical microlens (16); Being about to ultraviolet optics curing glue drips from the upward vertical release of combined filters (14); Drop onto on the combined filters (14), and, drop is shone with Ultra-Violet Laser from top to bottom to diffusion trickling all around; Make its curing, form micro optical lens (16);
Fluorescence detection unit (12) is that electrooptical device (17) and combined filters (18) are surrounded as tubulose with multilayer optical film (15) equally, makes optical microlens (16) on the spectral filter surface then;
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) place the left side, 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 micrometer fluidic microchannel (4) towards stream;
The electricity of described proofing unit is exported the lower end and the low-temperature annealing district that are positioned at biochip (1) into layer (9) and is separated, or places the groove shape passage that etches on the biochip (1), or directly places biochip (1) surface.
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| CN111346685A (en) * | 2020-03-10 | 2020-06-30 | 中国科学院苏州生物医学工程技术研究所 | Device and method capable of realizing rapid temperature control |
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