CN102286358A - Microfluidic control chip for realizing PCR (Polymerase Chain Reaction) and real-time PCR virus quick detection device - Google Patents
Microfluidic control chip for realizing PCR (Polymerase Chain Reaction) and real-time PCR virus quick detection device Download PDFInfo
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Abstract
The invention relates to a microfluidic control chip which is formed by bonding and sealing PDMS (Polydimethylsiloxane) material integrated with a micro valve, and comprises an upper layer of air control passage, a middle layer of PDMS membrane and a lower layer of microfluidic passage. A real-time PCR virus quick detection device comprises the microfluidic control chip, a temperature control unit, a signal detection unit and a data processing and control unit. The invention designs the microfluidic control chip which is integrated with the micro valve, is in a three-layer PDMS structure, and is used for virus detection and the real-time fluorescent PCR detection device. Compared with the traditional method, the invention has the advantages of short time, less sample usage, quick detection speed, simpleness and convenience in operation, integration and the like.
Description
Technical field
The present invention relates to a kind of realization polymerase chain reaction (Polymerase Chain Reaction, the viral device for fast detecting of micro-fluidic chip PCR) and PCR in real time.
Background technology
Since nineteen ninety proposed the micro-total analysis system notion, the micro-fluidic chip technology had been opened up wide development space in fields such as chemistry, life science, environmental science and Food sciences.The micro-fluidic chip technology is based on the microfluidic control technology, the guiding analytical instrument develops to miniaturization, integrated, automatization, high-throughput fast quantitative analysis direction, for real-time detection, the on-the site analysis of biochemical environmental sample provides a kind of possible strategy, it has vast market prospect aspect medical diagnosis and the epidemic monitoring of agricultural breeding system.
Polymerase chain reaction (Polymease Chain Reaction, PCR) claim the external primer of cell-free molecular cloning or specific DNA sequences directed enzymatic amplification technique again, it is a kind of method of the synthetic specific DNA fragment of external enzymatic, form one-period by several steps reactions such as high-temperature denatured, low-temperature annealing and thermophilic extension, circulation is carried out, make target DNA be able to rapid amplification, have high specificity, highly sensitive, easy and simple to handle, characteristics such as save time.It not only can be used for fundamental researchs such as gene isolation, clone and nucleic acid sequence analysis, also can be used for diagnosis, animal virus and the epidemic situation rapid detection of disease or the place of any DNA of having, RNA.In clinical medicine and animal epidemics Monitoring systems, PCR is used to differentiate genetic diseases and rapid detection virus, courses of infection.With traditional method, detect the infection of virus, germ and need could identify pathogen culture several weeks, and adopt achievement of the present invention, can judge whether there is the DNA of virus, germ in human body and the zooblast (such as blood cell) and makes a definite diagnosis fast.
PCR in real time (real-time PCR) belongs to a kind of of quantitative PCR (Q-PCR), is the quantitative analysis that DNA is carried out on the basis with the amount of amplification of DNA in the certain hour.PCR in real time is quantitatively used fluorochrome, and two kinds of methods are arranged at present.A kind of is to insert special fluorochrome in DNA; A kind of fluorescent probe that another kind of use can combine with specific few nucleotide sequence in the amplification dna sequence dna.Real time PCR combines with 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 realized the leap of PCR from qualitative to quantitative, and compares with conventional PCR, have specificity stronger, effectively solve characteristics such as PCR pollution, level of automation height.Real-time quantitative PCR is meant during the amplification of PCR index measures the amount of specificity product immediately by the variation of continuous monitoring fluorescent signal power, and infers the original bulk of goal gene in view of the above, does not need to take out the PCR product and separates.Real-time quantitative PCR has been widely used in the every field of molecular biology research as an extremely effective experimental technique at present.
The TaqMan probe is to add a specific fluorescent probe when pcr amplification when adding a pair of primer, and this probe is an oligonucleotide, and two ends are mark report fluorophor and a cancellation fluorophor respectively.When probe was complete, the reporter group fluorescent signal emitted was absorbed by quenching group; During pcr amplification, 5 '-3 ' 5 prime excision enzyme activity of Taq enzyme is cut degraded with the probe enzyme, the report fluorophor is separated with the cancellation fluorophor, thereby fluorescence detecting system can receive fluorescent signal, it is DNA chain of every amplification, just there is a fluorescence molecule to form, can realizes that the accumulation of fluorescent signal and PCR product form fully synchronously.
Traditional pcr amplification instrument exists shortcomings such as thermal capacity is big, heating and cooling speed is slow, sample consumption height.Usually finish an amplification procedure and need spend several hours, and big for the demand of reactant.Along with the development of MEMS and micro-fluidic chip technology, have that thermal capacity is low, amplification rate fast, the sample consumption is little, cost of manufacture is low, can abandon and detect in real time advantage such as amplified production based on the viral device for fast detecting of micro-fluidic chip and PCR in real time.
Summary of the invention
The technology of the present invention is dealt with problems: overcome the deficiencies in the prior art, the micro-fluidic chip of a kind of PCR of realization and the viral device for fast detecting of PCR in real time are provided, can under micro-fluidic environment, carry out quick, the accurately detection of small sample amount, reduce the PCR reagent dosage and shorten temperature required round-robin time of pcr amplification of realization, and pcr amplification product is detected in real time.
Technical solution of the present invention is as follows: a kind of micro-fluidic chip of realizing PCR, it is characterized in that: described micro-fluidic chip is by integrated three strata dimethyl siloxane (Polydimethylsiloxane of little valve, PDMS) sealing-in of material bonding forms, the gas control channel that comprises the upper strata, the PDMS film in middle layer, the microfluidic channel of lower floor; Wherein:
Top tank air control channel is made up of four little valves, wherein two little valves lay respectively at reaction chamber corresponding upper gas control channel in two ends, two little valves are parallel with a little valve of an end in addition, described three parallel Micropumps of little valves composition; Each little valve is formed by valve seat and little valve gas channels, vertical linking to each other between valve seat and the little valve gas channels; When ambient atmos enters little valve gas channels by valve seat after, the PDMS film in extruding middle layer, the PDMS film projection in middle layer is stopped up lower floor's microfluidic channel, pressure by pilot-gas, can control the opening and closing of four little valves, three parallel little valve opening and closing successively in the Micropump can drive the fluid in lower floor's microfluidic channel;
The microfluidic channel of lower floor comprises two sample injection channels, little hybrid channel, PCR reaction chamber and waste liquid exhaust channel; Described two sample injection channels are used for injecting sample and PCR reaction solution respectively; Described little hybrid channel is used for sample and PCR reaction solution that the injection channel adds are carried out thorough mixing, is beneficial to carry out the PCR reaction; Described PCR reaction chamber is used for storing the PCR reaction solution, finish PCR reaction after, described waste liquid exhaust channel is used for discharging the waste liquid after the PCR reaction finishes.
During experiment, PCR reaction reagent and DNA sample to be amplified are injected from two sample injection channels respectively, under the control of data processing and control unit, three parallel little valve opening and closing successively in the top tank air control channel, drive PCR reaction solution and DNA sample to be amplified and enter little hybrid channel by two sample injection channels respectively, in little hybrid channel, carry out thorough mixing, flow into the PCR reaction chamber, two little valves that close two ends this moment, make the PCR reaction solution under the control of temperature control unit, finish the PCR reaction, after reaction is finished, open two little valves, open Micropump, reaction solution is discharged from the waste liquid exhaust channel.
A kind of viral device for fast detecting of PCR in real time comprises micro-fluidic chip, temperature control unit, acquisition of signal unit and data processing and control unit, wherein:
Described temperature control unit is made up of semiconductor temperature-control sheet and temperature sensor, the semiconductor temperature-control sheet is used for changing the temperature of PCR reaction chamber, temperature sensor is used to detect the temperature of PCR reaction chamber and is electrical signal with temperature transition, by being stored in electrical signal in data processing and the control unit and the mutual relationship between the temperature in advance, determine the temperature of PCR reaction chamber and the semiconductor temperature-control sheet is carried out feedback control, can realize high-precision temperature control;
Described acquisition of signal unit is made up of excitation light source, optical delivery unit and photodetector; The fluorescence dye or the fluorescent probe of PCR reaction solution in the excitation light irradiation PCR reaction chamber that excitation light source sends make fluorescence dye or the fluorescent probe generation fluorescence that is stimulated; The optical delivery unit is used for transmitting exciting light and collects the fluorescence of sample; Described photodetector is used for surveying fluorescent signal, and optical signal is converted to electrical signal is transferred to data processing and control unit is handled;
Described data processing and control unit are realized collection, processing, storage and the demonstration to temperature control unit and acquisition of signal cell data; Data processing and control unit are at first given an order to temperature control unit, and the semiconductor temperature-control sheet is heated the PCR reaction reagent in the PCR reaction chamber, and temperature sensor is measured the temperature of the PCR reaction reagent in the PCR reaction chamber in real time; When reaching set(ting)value, data processing and control unit are given an order and are stopped heating, thereby realize high-precision temperature control; After a pcr amplification circulation is finished, data processing and control unit are given an order to the acquisition of signal unit, open excitation light source, irradiation PCR reaction chamber, the fluorescence that produces is transferred to photodetector, converts through photodetector that electrical signal is sent to data processing and control unit is handled and preserved to.
As long as can send the exciting light of specific wavelength among the present invention, excitation light source can be light sources such as xenon lamp or photodiode.The optical delivery unit can be freeboard or optical waveguides, as optical fiber, adopts full Optical Fiber Transmission structure can reduce the detection system volume greatly, is easy to integrated.Photodetector can be photomultiplier, photorectifier, charge coupled device (Charge-coupled Device, CCD) etc.
The present invention's advantage compared with prior art is: the invention has designed the micro-fluidic chip and the real-time fluorescence PCR proofing unit of three layers of PDMS structure that are used for the virus detection of integrated little valve.The present invention compares with traditional method, has advantages such as sample injection time is short, amount of samples is few, detection speed is fast, easy and simple to handle and integrated.
Description of drawings
Fig. 1 proofing unit block diagram of the present invention, 1 micro-fluidic chip, 2 temperature control units, 3 acquisition of signal unit, 4 data processing and control unit;
Fig. 2 is three layers of PDMS microfluidic chip structure figure of integrated little valve of the present invention, the 11st, top tank air control channel, the 12nd, the PDMS film in middle layer, the 13rd, lower floor's microfluidic channel, the 111st, valve seat, the 112nd, little valve gas channels, 131, the 132nd, the sample injection channel, the 133rd, little hybrid channel, the 134th, PCR reaction chamber, the 135th, waste liquid exhaust channel;
Fig. 3 is three layers of PDMS microfluidic chip structure figure of integrated little valve of the present invention, the 141,142,143, the 144th, and little valve, three parallel little valves 142,143,144 are formed a Micropump 15;
Fig. 4 is for the little opening of valves among the present invention and close synoptic diagram, 11 upper strata gas circuit key-courses, 12 middle layer PDMS films, 13 microfluidic channel;
Fig. 5 is acquisition of signal modular construction figure, 31 excitation light sources, 32 photodetectors, 33 optical delivery unit, 1 micro-fluidic chip;
Fig. 6 is little opening of valves among the present invention and closed photo;
Fig. 7 is FITC fluorescence spectrum figure.
Embodiment
Following example is made the present invention and being further specified, but not thereby limiting the invention, supports the technological innovation and the exploitation of multiple application system.
Shown in Fig. 2,3, the micro-fluidic chip 1 among the present invention is formed by the PDMS material bonding sealing-in of integrated little valve, comprises the gas control channel 11 on upper strata, the PDMS film 12 in middle layer, the microfluidic channel 13 of lower floor; PDMS film 12 thickness in middle layer are 80-120um, are 100um among this embodiment of the present invention.
Top tank air control channel 11 is made up of four little valves 141,142,143,144, wherein two little valves 141,142 lay respectively at reaction chamber 134 corresponding upper gas control channels 11 in two ends, in addition one of two little valves 143,144 and an end little valve 142 is parallel, Micropump 15 of three parallel little valves 142,143,144 compositions.Each little valve is formed by valve seat 111, little valve gas channels 112, vertical linking to each other between valve seat 111 and the little valve gas channels 112.After ambient atmos enters little valve gas channels 112 by valve seat 111, the PDMS film 12 in extruding middle layer, PDMS film 12 projections in middle layer are stopped up lower floor's microfluidic channel 13, pressure by pilot-gas, can control the opening and closing of four little valves 141,142,143,144, three parallel little valve 142,143,144 opening and closing successively in the Micropump 15 can drive the fluid in lower floor's microfluidic channel 13.
The microfluidic channel 13 of lower floor comprises that two sample injection channels 131,132, little hybrid channel 133, PCR reaction chamber 134 and 135, two sample injection channels of waste liquid exhaust channel 131,132 are used for injecting sample and PCR reagent respectively.PCR reaction chamber 134 is used for storing the PCR reaction solution.Waste liquid exhaust channel 35 is used for discharging the waste liquid after the PCR reaction finishes.
During experiment, with PCR reaction reagent and DNA sample to be amplified respectively from sample injection channel 131,132 inject chip, under the control of data processing and control unit 4, little valve 142 in the top tank air control channel 11,143,144 opening and closing successively, drive PCR reaction kit and DNA sample to be amplified and enter little hybrid channel 133, in little hybrid channel 133, carry out thorough mixing, flow into PCR reaction chamber 134, close little valve 141 this moment, 142, make the PCR reaction solution under the control of temperature control unit 2, finish the PCR reaction, after reaction is finished, open little valve 141,142, open Micropump 15, reaction solution is discharged from waste liquid exhaust channel 135.
As shown in Figure 4, the little valve on the micro-fluidic chip 1 comprises upper strata gas circuit key-course 11, middle layer PDMS film 12, the microfluidic channel 13 of lower floor.When gas enters gas circuit key-course 11, middle layer PDMS film 12 pressurizeds block the microfluidic channel 13 of lower floor to lower process, and continuing increases air pressure, can cut off the microfluidic channel 13 of lower floor fully, and this moment, little valve was in closing condition.
As shown in Figure 1, micro-fluidic chip and real-time fluorescence PCR virus device for fast detecting is made of micro-fluidic chip 1, temperature control unit 2, acquisition of signal unit 3 and data processing and control unit 4.
As shown in Figure 5, detecting signal unit is by excitation light source 31, photodetector 32, optical delivery unit 33 is formed, the light that excitation light source sends shines PCR reaction chamber 134 in the micro-fluidic chip 1 by the optical delivery unit, and the fluorescence of generation is sent to photodetector 32 by optical delivery unit 33 and detects.
Microfluidic channel width 100um, degree of depth 75um, gas control channel width 200um, degree of depth 75um, scope: width and depth ratio get final product less than 20.
Working process of the present invention: PCR reaction reagent and sample to be amplified are respectively from micro-fluidic chip inlet 131,132 inject microfluidic channel, data processing and control unit 4 are given an order Micropump 15 are started working, under the control of data processing and control unit 4, little valve 142 in the top tank air control channel 11,143,144 opening and closing successively, drive PCR reaction kit and DNA sample to be amplified and enter little hybrid channel 133, in little hybrid channel 133, carry out thorough mixing, flow into PCR reaction chamber 134, data processing and control unit 4 were given an order and were closed little valve 141 this moment, 142, temperature control unit 2 is started working, the 21 energising heating of semiconductor temperature-control sheet, at first the PCR reaction solution is heated to 95 ℃ of sex change, after keeping certain hour, reduce the temperature to 55 ℃ of annealing by the received current direction that changes semiconductor temperature-control sheet 21, and then be heated to 72 ℃ and extend, the segmental once amplification of target dna is finished in such circulation, whole PCR process need is finished about 30～35 circulations, after reaction was finished, reaction solution was discharged from waste liquid exhaust channel 135.After each PCR circulation is finished, data processing and control unit 4 are given an order and are made 3 work of acquisition of signal unit, open excitation light source 31, exciting light shines pcr amplification product by optical delivery unit 32, the fluorescence that produces is transferred to photodetector 33 by optical delivery unit 32, photodetector 33 is converted to electrical signal with optical signal, and electrical signal is sent to data processing and control unit 4 carries out data processing, storage and demonstration.
The making of 1: three layer of PDMS micro-fluidic chip of embodiment
Micro-fluidic chip is to be formed by upper strata control channel 11, intermediary PDMS thin film layer 12 and the 13 reversible sealing-in of lower floor's microfluidic channel layer.PDMS film thickness 100um, the width of microfluidic channel and the degree of depth are respectively 100um and 75um, and the width and the degree of depth of gas circuit control channel are respectively 200um, 75um.Micro-fluidic chip microscopically in the clean room is aimed at sealing-in, and guarantees little valve 141,142,143,144 and Micropump 15 works better.The opening and closing of forming three little valves 142,143,144 of Micropumps 15 by 4 controls of data processing and control unit realize driving the function of microfluid, Fig. 6 is little opening of valves and the closed photo of taking at microscopically, microscopically was taken when the left side one width of cloth figure was little opening of valves upper strata gas channels and lower floor's microfluidic channel photo, the right is little valve microscopically is taken when closing upper strata gas channels and lower floor's microfluidic channel photo, and visible this moment, the upper strata passage was closed little valve to lower process.
Embodiment 2: fluorescein isothiocyanate in the micro-fluidic chip passage (Fluoresceinisothiocyanate, FITC) detection of fluorescent signal
To be added with fluorescein isothiocyanate (Fluoresceinisothiocyanate, FITC) testing sample is from sample inlet 131,132 inject micro-fluidic chip 1, three little valves 142 in data processing and the control unit 4 control Micropumps 15,143,144 successively closure or openness drive sample flow and enter micro mixer 133, after in micro mixer 133, carrying out thorough mixing, sample enters PCR reaction chamber 134, data processing and the control unit 4 opening signal probe unit 3 of giving an order, excitation light source 31 in the acquisition of signal unit 3 sends exciting light, by optical delivery unit 32 irradiation PCR reaction chambers 134, under the excitation light irradiation of 480nm, FITC will produce the fluorescence that 520nm is a centre wavelength, fluorescence still is transferred to photodetector 33 by the collection of optical delivery unit and surveys, fluorescence spectrum as shown in Figure 7, as seen the fluorescent emission peak value of FITC is near 520nm, and the result proves that signal probe unit 3 can successfully detect the fluorescence in the PCR reaction chamber.
Claims (4)
1. micro-fluidic chip of realizing PCR, it is characterized in that: described micro-fluidic chip (1) is formed by the PDMS material bonding sealing-in of integrated little valve, the gas control channel (11) that comprises the upper strata, the PDMS film (12) in middle layer, the microfluidic channel of lower floor (13); Wherein:
Top tank air control channel (11) is made up of four little valves (141,142,143,144), wherein two little valves (141,142) lay respectively at reaction chamber (134) corresponding upper gas control channel (11) in two ends, two little valves (143,144) are parallel with a little valve (142) of an end in addition, and described three parallel little valves (142,143,144) are formed a Micropump (15); Each little valve is formed by valve seat (111), little valve gas channels (112), vertical linking to each other between valve seat (111) and the little valve gas channels (112); After ambient atmos enters little valve gas channels (112) by valve seat (111), the PDMS film (12) in extruding middle layer, PDMS film (12) projection in middle layer is stopped up lower floor's microfluidic channel (13), pressure by pilot-gas, can control the opening and closing of four little valves (141,142,143,144), three parallel little valves (142,143,144) opening and closing successively in the Micropump (15) can drive the fluid in lower floor's microfluidic channel (13);
The microfluidic channel of lower floor (13) comprises two sample injection channels (131,132), little hybrid channel (133), PCR reaction chamber (134) and waste liquid exhaust channel (135); Described two sample injection channels (131,132) are used for injecting sample and PCR reaction solution respectively; Described little hybrid channel (133) is used for sample and PCR reaction solution that the injection channel adds are carried out thorough mixing, is beneficial to carry out the PCR reaction; Described PCR reaction chamber (134) is used for storing the PCR reaction solution, finish PCR reaction after, described waste liquid exhaust channel (35) is used for discharging the waste liquid after the PCR reaction finishes.
2. the micro-fluidic chip of realization PCR according to claim 1, it is characterized in that: PDMS film (12) thickness in described middle layer is 80-120um.
3. the viral device for fast detecting of a PCR in real time is characterized in that comprising: the described micro-fluidic chip of claim 1 (1), temperature control unit (2), acquisition of signal unit (3) and data processing and control unit (4), wherein:
Described temperature control unit (2) is made up of semiconductor temperature-control sheet (21) and temperature sensor (22), semiconductor temperature-control sheet (21) is used for changing the temperature of PCR reaction chamber (134), temperature sensor (22) is used to detect the temperature of PCR reaction chamber (134) and is electrical signal with temperature transition, by being stored in electrical signal in data processing and the control unit (4) and the mutual relationship between the temperature in advance, determine the temperature of PCR reaction chamber (134) and semiconductor temperature-control sheet (21) is carried out feedback control, can realize high-precision temperature control;
Described acquisition of signal unit (3) is made up of excitation light source (31), optical delivery unit (32) and photodetector (33); Fluorescence dye or fluorescent probe in the excitation light irradiation PCR reaction chamber (134) that excitation light source (31) sends in the PCR reaction solution make fluorescence dye or the fluorescent probe generation fluorescence that is stimulated; Optical delivery unit (32) is used for transmitting exciting light and collects the fluorescence of sample; Described photodetector (33) is used for surveying fluorescent signal, and optical signal is converted to electrical signal is transferred to data processing and control unit (4) is handled;
Described data processing and control unit (4) are realized collection, processing, storage and the demonstration to temperature control unit and acquisition of signal cell data; Data processing and control unit (4) are at first given an order to temperature control unit (2), semiconductor temperature-control sheet (21) is heated the PCR reaction solution in the PCR reaction chamber (134), and temperature sensor (22) is measured the temperature of the PCR reaction solution in the PCR reaction chamber (134) in real time; When reaching set(ting)value, data processing and control unit (4) are given an order and are stopped heating, thereby realize high-precision temperature control; After a pcr amplification circulation is finished, data processing and control unit (4) are given an order to acquisition of signal unit (3), open excitation light source (31), irradiation PCR reaction chamber (134), the fluorescence that produces is transferred to photodetector (33), converts through photodetector (33) that electrical signal is sent to data processing and control unit (4) is handled and preserved to.
4. according to the viral device for fast detecting of the described PCR in real time of claim 3, it is characterized in that: described photodetector (33) is photomultiplier, photorectifier or ccd detector.
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