CN101718698A - Laser-induced fluorescence analyzer with PCR-CE coupled microfluidic chip - Google Patents
Laser-induced fluorescence analyzer with PCR-CE coupled microfluidic chip Download PDFInfo
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- CN101718698A CN101718698A CN200910272874A CN200910272874A CN101718698A CN 101718698 A CN101718698 A CN 101718698A CN 200910272874 A CN200910272874 A CN 200910272874A CN 200910272874 A CN200910272874 A CN 200910272874A CN 101718698 A CN101718698 A CN 101718698A
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Abstract
The invention discloses a laser-induced fluorescence analyzer with a PCR-CE coupled microfluidic chip, relating to a biomedical detection analyzer. In the structure of the laser-induced fluorescence analyzer, a machine shell (00) is internally provided with a PCR unit (10), a CE unit (20), a laser-induced fluorescence detection unit (30) and a signal collecting and processing unit (40); after mutually connecting with t, the PCR unit (10) and the CE unit (20) are connected with each other firstly and then connected with the laser-induced fluorescence detection unit (30); and the laser-induced fluorescence detection unit (30) is connected with the signal collecting and processing unit (40). The invention integrates PCR, electrophoretic separation, and laser-induced fluorescence detection into a whole, has the advantages of small size, simple structure, easy operation, low cost, stable performance, high analysis and detection sensitivity, and short disease diagnosis period, and is suitable for disease diagnosis of genes related to diseases.
Description
Technical field
The present invention relates to a kind of biomedical detection instrument, relate in particular to a kind of PCR-CE (PCR-Capillary Electrophoresis) coupling micro-fluidic chip laser-induced fluorescence analyzer.
Background technology
The micro-total analysis unit is a brand-new field that begins to rise the beginning of the nineties in last century.The micro-total analysis unit relates to a lot of ambits, as analytical chemistry, physics, biology, medical science, pharmacy, micro-processing technology, electronics, computing machine etc., by integrated, the microminiaturization of sample preparation, separation and detection being realized the basic function of Routine Test Lab, thereby realize low cost, high flux, intellectuality, portable target.
Lab-on-chip technology is with whole breadboard function, having only on several square centimeters the microchip from process integration such as application of sample, reaction, separation, detections, at present in chemosynthesis, cellular incubation, medicament high flux screening, medical diagnosis on disease, food security, aspects such as blood analysis and environment measuring make important progress.The development of micro-fluidic chip technology makes that round pcr is achieved on the chip.Along with the development of micro-processing technology, microfluid the channel dimensions of process entered into nanoscale from micron order, make the research object of microflow control technique enter into molecular level from cell grade.Microflow control technique has become current research forward position and focus.
The detection method of micro-fluidic chip mainly contains: laser inductive fluorescence method, electrochemical process, chemoluminescence method, mass spectroscopy etc.Wherein, laser inductive fluorescence method has highly sensitive, and principle is simple, is integrated in easily on the micro-fluidic chip, becomes the main at present detection method that adopts.The optical unit of laser-Induced Fluorescence Detection mainly contains two kinds: a kind of is that the exciting light light path is vertical mutually with the fluorescence light path; Another kind is focusing altogether, and it is with microcobjective unpolarized light beam to be focused on the micro-fluidic chip raceway groove, and fluorescence excitation incides on the photomultiplier by same object lens and detects.The subject matter of the laser-Induced Fluorescence Detection technology of Chu Xianing is that the volume ratio of detecting device is bigger in the market, costs an arm and a leg, and complex structure is difficult to realize the microminiaturization of analytic unit.
Capillary electrophoresis technique has become requisite means in the analysis field since coming out.What use always in the Capillary Electrophoresis is gel electrophoresis, and amount of samples is bigger, and during operating cost, the perfusion gel is difficult, and analysis time is long, the applied voltage height.Micro flow control chip capillary electrophoresis adopts the low polymkeric substance sieving media of viscosity to serve as electrophoretic medium, and it is easier to operate.Microcurrent controlled capillary tube electrophoresis has only several centimeter length owing to separate raceway groove, and disengaging time dwindles greatly, only needs a few minutes, even tens seconds.Microcurrent controlled capillary tube electrophoresis, the agents useful for same consumption is few, and separation voltage is low, and sensitivity is higher.
At present, when carrying out the disease gene diagnosis, all be that sample extraction is come out, on the pcr amplification instrument, increase, carry out electrophoresis detection then.Detection method has: conventional capillary gel electrophoresis and microcurrent controlled capillary tube gel electrophoresis.Conventional pcr amplification instrument is because volume is big, and it is slow with cooling rate to heat up, and makes the amplification procedure length that expends time in.And the micro-fluidic chip technology makes in the enterprising performing PCR amplification of micro-fluidic chip, and proliferation time is short, and it is fast with cooling rate to heat up, and has shortened the amplification required time greatly.And the sample that amplification is come out also will spend certain sample preparation time before carrying out electrophoresis detection, makes that the whole medical diagnosis on disease cycle is longer.
Summary of the invention
Purpose of the present invention is exactly to overcome long, problem such as amount of samples is big, labor workload is big, cost is high and the cycle is long of the processing sample time that exists in existing disease gene diagnosis sample extraction, PCR sample amplification, electrophoretic separation, the whole process of laser-Induced Fluorescence Detection, and a kind of PCR-CE coupling micro-fluidic chip laser-induced fluorescence analyzer is provided.
The object of the present invention is achieved like this:
In casing, be provided with PCR unit, CE unit, laser-Induced Fluorescence Detection unit and signals collecting and processing unit;
The PCR unit be connected with the laser-Induced Fluorescence Detection unit again after the CE unit interconnects, the laser-Induced Fluorescence Detection unit is connected with processing unit with signals collecting.
Principle of work of the present invention is:
By the micro-fluidic chip technology, the PCR process and the Capillary Electrophoresis Separation Process of disease DNA sample integrate, and analysis result carries out fluorescence signal by the laser-Induced Fluorescence Detection unit and detects, and carries out the demonstration of analysis result on computers.The concentration of disease DNA sample is very low, and the PCR process is to realize that by well heater 3 different warm areas carry out amplification procedure to disease DNA, to reach the level that can detect.Realize the separation of each component in the disease DNA sample after DNA cloning is intact through Capillary Electrophoresis, analyze by result, thereby disease is diagnosed separation.
The present invention has the following advantages and good effect:
1, integrated PCR, electrophoretic separation, laser-Induced Fluorescence Detection be in one, and volume is little, simple in structure, easy operating, cost are low;
2, stable performance, analyzing and testing is highly sensitive, and the medical diagnosis on disease cycle is short.
3, be applicable to the gene clinical diagnosis of diseases related.
Description of drawings
Fig. 1 is a structured flowchart of the present invention;
Fig. 2 is structural representation of the present invention (a main pseudosection);
Fig. 3 is a light path principle figure of the present invention.
Among the figure:
The 00-casing,
The 01-support;
The 10-PCR unit,
The 11-PCR well heater, the 12-chip set, the three-dimensional mobile platform of 13-XYZ,
The integrated PCR-CE chip of 14-;
The 20-CE unit,
The 21-electrode holder, 22-controllable high-voltage out-put supply,
23-spiral translation device;
30-laser-Induced Fluorescence Detection unit,
31-semiconductor solid state laser, the 32-plane mirror, the 33-half-reflecting half mirror,
The 34-object lens, the 35-convex lens, the 36-pin hole,
The 37-optical filter, the 38-photomultiplier;
40-signals collecting and processing unit
41-signals collecting, filtering, amplification, analog to digital conversion circuit, the 42-computing machine.
Abbreviation:
1, PCR-PCR (Polymerase Chain Reaction) is a kind of Protocols in Molecular Biology, is used for amplifying specific dna fragmentation. The special dna replication dna that can regard in vitro as. The basic principle of round pcr is similar to the natural reproduction process of DNA, and its specificity depends on the Oligonucleolide primers with the complementation of target sequence two ends. PCR is made of sex change-annealing-extension three basic reactions steps: the 1. sex change of template DNA: template DNA is after being heated to 93 ℃ of left and right sides certain hours, template DNA double-stranded DNA double-stranded or that form through pcr amplification is dissociated, make it to become strand, so that it is combined with primer, for the lower whorl reaction is prepared; 2. the annealing (renaturation) of template DNA and primer: template DNA is after heat denatured becomes strand, and temperature is down to about 55 ℃, the complementary series pairing combination of primer and template DNA strand; 3. the extension of primer: dna profiling-primer bond is under the effect of TaqDNA polymerase, take dNTP as reaction raw materials, target sequence is template, press base complementrity pairing and semi-conservative replication principle, synthetic new and the semi-conservative replication chain complementation of template DNA chain, repetitive cycling sex change--fire--three processes of extending just can obtain more " semi-conservative replication chain ", and this new chain can become again the template of circulation next time. Whenever finish a circulation and need 2～4 minutes, just can genes of interest amplification be expanded amplify millions of times in 2～3 hours.
2, CE-Capillary Electrophoresis (Capillary electrophoresis) is a kind of separate analytical technique. Have fast, efficient, high sensitivity, easily quantitatively, the advantages such as favorable reproducibility and automation, be widely used in the compartment analysis research of little molecule, small ion, polypeptide and protein. It demonstrates again huge potentiality aspect separate nucleic acid. The basic device of Capillary Electrophoresis is one and is full of the capillary of electrophoretic buffer and enters capillary from an end capillaceous by " pressure " or " electromigration " with two bottle micro-examples that link to each other with the capillary two ends. During electrophoresis, two electrodes that are connected with high voltage source soak respectively in the buffer solution of people's capillary two ends bottle. Sample court and self electrically charged opposite polarity electrode direction swimming. Each component because of its molecular size, the difference migration rate difference of the character such as electrically charged number, isoelectric point, move to successively near the photodetector of capillary output, detect, record absorbance, and on screen take transit time as abscissa, absorbance is that ordinate is dynamically recorded each component intuitively with the form of absworption peak.
Embodiment
Describe in detail below in conjunction with drawings and Examples:
One, overall
As Fig. 1, the present invention is provided with PCR unit 10, CE unit 20, laser-Induced Fluorescence Detection unit 30 and signals collecting and processing unit 40 in casing 00;
Two, functional block
0, casing 00
As Fig. 2, in casing 00, be provided with support 01, use for fixing other functional block.
1, the PCR unit 10
As Fig. 2, PCR unit 10 comprises PCR well heater 11, chip set 12, the three-dimensional mobile platform 13 of XYZ and integrated PCR-CE chip 14;
The support 01 at middle part is provided with the three-dimensional mobile platform 13 of XYZ in the casing 00, is connected with chip set 12 at the left of the three-dimensional mobile platform 13 of XYZ, is respectively arranged with integrated PCR-CE chip 14 and PCR well heater 11 at the upper and lower of chip set 12;
Described PCR well heater 11 is a kind of electric heaters of being made up of 1～3 heat block (heating material commonly used);
Described chip set 12 is a kind of for the metal frame that connects and support usefulness;
The three-dimensional mobile platform 13 of described XYZ is a kind of outsourcing pieces commonly used;
Described integrated PCR-CE chip 14 is a kind of micro-fluidic chips of design voluntarily, partly is made up of PCR part and CE; PCR partly is a PCR reaction tank or a PCR reaction channel; The CE part is made up of sample feeding passage and sample separation passage, is right-angled intersection or double T and links to each other; The sample feeding passage of the reaction tank of PCR part or reaction channel and CE part is interconnected.
This integrated PCR-CE chip 14 is to utilize classical photoetching development and wet etching method to produce micron-sized passage on glass, utilize drill bit to get out liquid storage tank in relevant position on glass, again with the flat glass of an other same material at high temperature bonding obtain; This integrated PCR-CE chip 14 also can be by the electroformed mould general laws organic polymer polymethylmethacrylate heating and pressurizing on the electroforming mould to be prepared from.
2, the CE unit 20
As Fig. 2, CE unit 20 comprises electrode holder 21, controllable high-voltage out-put supply 22 and spiral translation device 23;
Described electrode holder 21 is pcb boards of a kind of 2-4 of including platinum electrode;
Described controllable high-voltage out-put supply 22 is a kind of 0～6000V adjustable high voltage power supply;
Described spiral translation device 23 has the listing product;
3, the laser-Induced Fluorescence Detection unit 30
As Fig. 2,3, laser-Induced Fluorescence Detection unit 30 comprises solid state laser 31, plane mirror 32, half-reflecting half mirror 33, object lens 34, convex lens 35, pin hole 36, optical filter 37 and photomultiplier 38;
Its light path is: the laser that solid state laser 31 produces enters half-reflecting half mirror 33 after plane mirror 32 reflections; Its reflected light promptly produces fluorescence through object lens 34 behind integrated PCR-CE chip 14, fluorescence is gone into photoelectricity multiplier tube 38 by object lens 34, half-reflecting half mirror 33, convex lens 35, pin hole 36 and optical filter 37 are laggard again successively, detects fluorescence signal (simulating signal).
4, signals collecting and processing unit 40
As Fig. 2, signals collecting and processing unit 40 comprise preceding latter linked signals collecting, filtering, amplification, analog to digital conversion circuit 41 and computing machine 42;
Signals collecting, filtering, amplification, analog to digital conversion circuit 41 are a kind of circuit commonly used, realize collection, filtering, amplification and the analog to digital conversion of fluorescence signal, obtain fluorescence signal (digital signal);
Three, the operation steps of this analysis instrument is as follows:
The integrated PCR-CE chip 14 that 1. will add sample solution and buffering solution is positioned on the chip set 12, adjustable screw translation device 23 moves down electrode holder 21, thereby the electrode on the electrode holder 21 is inserted respectively in the liquid storage tank of integrated PCR-CE chip 14 correspondences; Use the three-dimensional mobile platform 13 of XYZ to adjust the position of integrated PCR-CE chip 14, make the sense channel of integrated PCR-CE chip 14 and laser-Induced Fluorescence Detection light path at grade.
2. open semiconductor solid state laser 31, regulate the three-dimensional mobile platform 13 of XYZ again, the focus point of laser beam is detected on the check point of raceway groove at integrated PCR-CE chip 14, build the lid of shell 00.
3. open PCR well heater 11, treat to open photomultiplier 38 after pcr amplification finishes, opening signal collection, filtering, amplification, analog to digital conversion circuit 41 are opened controllable high-voltage out-put supply 22, regulate the operating voltage of photomultiplier 38, select suitable detection sensitivity.
4. regulate controllable high-voltage out-put supply 22, on integrated PCR-CE chip 14, carry out sample introduction, the detachment process of sample simultaneously synchronously; In this process, the demonstration of the detection of fluorescence signal, the collection of data and processing, fluorescence pattern all can be carried out simultaneously.
5. after treating that the sample analysis process finishes, integrated PCR-CE chip 14 is taken out, clean up with the liquid storage tank and the raceway groove of deionized water with integrated PCR-CE chip 14.
Claims (5)
1.PCR-CE coupling micro-fluidic chip laser-induced fluorescence analyzer is characterized in that:
In casing (00), be provided with PCR unit (10), CE unit (20), laser-Induced Fluorescence Detection unit (30) and signals collecting and processing unit (40);
PCR unit (10) be connected with laser-Induced Fluorescence Detection unit (30) again after CE unit (20) interconnect, laser-Induced Fluorescence Detection unit (30) are connected with processing unit (40) with signals collecting.
2. by the described laser-induced fluorescence analyzer of claim 1, it is characterized in that:
Described PCR unit (10) comprises PCR well heater (11), chip set (12), the three-dimensional mobile platform (13) of XYZ and integrated PCR-CE chip (14);
The support (01) at middle part is provided with the three-dimensional mobile platform (13) of XYZ in the casing (00), left at the three-dimensional mobile platform of XYZ (13) is connected with chip set (12), is respectively arranged with integrated PCR-CE chip (14) and PCR well heater (11) at the upper and lower of chip set (12);
Described PCR well heater (11) is a kind of electric heater of being made up of 1～3 heat block;
Described chip set (12) is a kind of for the metal frame that connects and support usefulness;
The three-dimensional mobile platform of described XYZ (13) is a kind of outsourcing piece commonly used;
Described integrated PCR-CE chip (14) is a kind of micro-fluidic chip, partly is made up of PCR part and CE; PCR partly is a PCR reaction tank or a PCR reaction channel; The CE part is made up of sample feeding passage and sample separation passage, is right-angled intersection or double T and links to each other; The sample feeding passage of the reaction tank of PCR part or reaction channel and CE part is interconnected.
3. by the described laser-induced fluorescence analyzer of claim 1, it is characterized in that:
Described CE unit (20) comprises electrode holder (21), controllable high-voltage out-put supply (22) and spiral translation device (23);
Described electrode holder (21) is the pcb board of a kind of 2-4 of including platinum electrode;
Described controllable high-voltage out-put supply (22) is a kind of 0～6000V adjustable high voltage power supply;
Described spiral translation device (23) has the listing product;
Spiral translation device (23) is fixed on the chip set (12), and electrode holder (21) links to each other with spiral translation device (23), by adjustable screw translation device (23) electrode holder (21) is moved up and down; Electrode holder (21) is connected with controllable high-voltage out-put supply (22) by lead, powers to platinum electrode.
4. by the described laser-induced fluorescence analyzer of claim 1, it is characterized in that:
Described laser-Induced Fluorescence Detection unit (30) comprises solid state laser (31), plane mirror (32), half-reflecting half mirror (33), object lens (34), convex lens (35), pin hole (36), optical filter (37) and photomultiplier (38);
Its light path is: the laser that solid state laser (31) produces enters half-reflecting half mirror (33) after plane mirror (32) reflection; Its reflected light promptly produces fluorescence through object lens (34) behind integrated PCR-CE chip (14), fluorescence is gone into photoelectricity multiplier tube (38) by object lens (34), half-reflecting half mirror (33), convex lens (35), pin hole (36) and optical filter (37) are laggard again successively, detects fluorescence signal.
5. by the described laser-induced fluorescence analyzer of claim 1, it is characterized in that:
Signals collecting and processing unit (40) comprise preceding latter linked signals collecting, filtering, amplification, analog to digital conversion circuit (41) and computing machine (42).
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| CN2009102728741A CN101718698B (en) | 2009-11-20 | 2009-11-20 | Laser-induced fluorescence analyzer with PCR-CE coupled microfluidic chip |
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| CN101718698B CN101718698B (en) | 2011-08-17 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103063646A (en) * | 2013-01-14 | 2013-04-24 | 武汉大学 | Dual-purpose laser-induced fluorescence detecting system |
| CN103194383A (en) * | 2013-04-09 | 2013-07-10 | 湖北民族学院 | Chip level PCR-LVCE integrated system |
| CN104007091A (en) * | 2013-02-26 | 2014-08-27 | 中国科学院天津工业生物技术研究所 | High-throughput detection system for microbe based on droplet microfluidic chip |
| CN106906137A (en) * | 2017-03-19 | 2017-06-30 | 北京化工大学 | A kind of high-throughput nucleic acid analysis device based on micro-fluidic chip |
| CN107338189A (en) * | 2011-05-04 | 2017-11-10 | 卢米耐克斯公司 | Apparatus and method for for integrated sample preparation, reaction and detection |
| CN107807114A (en) * | 2017-09-12 | 2018-03-16 | 华南师范大学 | Gene based on functionalization capillary is quick, quantified system analysis and method |
| CN111610173A (en) * | 2020-05-27 | 2020-09-01 | 中国水利水电科学研究院 | Three-dimensional fluid concentration field calibration device and calibration method |
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2009
- 2009-11-20 CN CN2009102728741A patent/CN101718698B/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107338189A (en) * | 2011-05-04 | 2017-11-10 | 卢米耐克斯公司 | Apparatus and method for for integrated sample preparation, reaction and detection |
| CN103063646A (en) * | 2013-01-14 | 2013-04-24 | 武汉大学 | Dual-purpose laser-induced fluorescence detecting system |
| CN103063646B (en) * | 2013-01-14 | 2015-04-29 | 武汉大学 | Dual-purpose laser-induced fluorescence detecting system |
| CN104007091A (en) * | 2013-02-26 | 2014-08-27 | 中国科学院天津工业生物技术研究所 | High-throughput detection system for microbe based on droplet microfluidic chip |
| CN103194383A (en) * | 2013-04-09 | 2013-07-10 | 湖北民族学院 | Chip level PCR-LVCE integrated system |
| CN103194383B (en) * | 2013-04-09 | 2014-09-03 | 湖北民族学院 | Chip level PCR-LVCE integrated system |
| CN106906137A (en) * | 2017-03-19 | 2017-06-30 | 北京化工大学 | A kind of high-throughput nucleic acid analysis device based on micro-fluidic chip |
| CN106906137B (en) * | 2017-03-19 | 2019-02-12 | 北京化工大学 | A kind of high-throughput nucleic acid analysis device based on micro-fluidic chip |
| CN107807114A (en) * | 2017-09-12 | 2018-03-16 | 华南师范大学 | Gene based on functionalization capillary is quick, quantified system analysis and method |
| CN111610173A (en) * | 2020-05-27 | 2020-09-01 | 中国水利水电科学研究院 | Three-dimensional fluid concentration field calibration device and calibration method |
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