CN202099269U - Polymerase chain reactor and real-time micro optics detection device - Google Patents
Polymerase chain reactor and real-time micro optics detection device Download PDFInfo
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- CN202099269U CN202099269U CN2011201659588U CN201120165958U CN202099269U CN 202099269 U CN202099269 U CN 202099269U CN 2011201659588 U CN2011201659588 U CN 2011201659588U CN 201120165958 U CN201120165958 U CN 201120165958U CN 202099269 U CN202099269 U CN 202099269U
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Abstract
The utility model discloses a polymerase chain reactor and real-time micro optics detection device, which relates to the field of biochemical reaction and medical detection and comprises a substrate layer with a spiral type micro-channel structure, wherein the substrate layer and a cover plate layer are sealed. Semiconductor heating refrigerators are arranged under a micro-flow control chip, the three semiconductor heating refrigerators are controlled by a microprocessor, a degeneration region, an annealing region and an extension region required for a polymerase chain reaction are formed in the whole micro-channel region. Three platinum resistor temperature sensors are arranged between the micro-flow control channel chip and the semiconductor heating refrigerators to form closed loop temperature control. A real-time fluorescence detecting system comprises a light source, a photoelectric detector and a fluorescence signal acquiring and processing system. A line array light-emitting diode is used as the light source, a photomultiplier is used as the photoelectric detector, and the line array light-emitting diode and the photomultiplier are fixed at the light source and the photoelectric detector and are positioned above the extension region of the micro-flow control chip through an optics bracket. In the utility model, temperature rising and reducing time is omitted, the time for the polymerase chain reaction is shortened, and the service life of the semiconductor heating refrigerators is prolonged.
Description
Technical field
(Polymerase Chain Reaction PCR) realizes that on micro-fluidic chip DNA cloning and real-time fluorescence detect to the present invention relates to the polymerase chain reaction.Be applied to biochemical reaction and medical science detection range.
Background technology
Microminiaturization is an important development direction in medical science and the biochemistry detection equipment, and (Micro-electro-mechanical systems, the PCR that develops into MEMS) is reflected on the chip and accomplishes that provide maybe micro electro mechanical system (MEMS) technology.Have following shortcoming in the conventional fluorescent PCR appearance: the instrument volume is big, and temperature rate is slow, and the dynamic temperature control precision is not high, the long-term temperature shock lost of life of semi-conductor heating cooler.The PCR reaction chip that utilizes the MEMS technology to make now is divided into three kinds: little trap formula chip, micro-fluidic chip and little water-bath chip.There is following shortcoming in little trap formula chip: temperature rate is slow, and dynamic temperature control, temperature accuracy are not high, generally has only the alloy layer micro-heater, does not have active refrigerator, like patent 200310122607.9 and 200510011180.4 etc.There is following shortcoming in micro-fluidic chip: generally have only the alloy layer micro-heater, do not have the active refrigeration device, like patent 200410024703.4 etc.Little water-bath chip 200610043243.9 etc., the heating and cooling complex structure.The PCR reaction chip that utilizes MEMS technology to make has solved the bulky problem of instrument, but has occurred active refrigeration again, real-time fluorescence detects a difficult problem integrated on chip.
Summary of the invention
The object of the invention provides a kind of miniature polymerase chain reaction device and real-time micro-optic proofing unit that can carry out fluoroscopic examination simultaneously in the reaction of the enterprising performing PCR of micro-fluidic chip, and it has accuracy of temperature control height, characteristics that stability is strong.
For realizing above-mentioned purpose, the present invention takes following technical scheme: polymerase chain reaction device and real-time micro-optic proofing unit, and it comprises:
Micro-fluidic chip, it comprises stratum basale 1, the cover layer 2 that has the volution fluid channel, stratum basale 1 forms micro-fluidic chip with cover layer 2; On cover layer 2, there are two holes to be respectively sample holes and sample outlet hole, lay respectively at the starting point and the terminal point of fluid channel; The pneumatic pump 10 that provides pneumatic power to make sample flow links to each other with sample holes 3; The material of stratum basale 1 is the good material of heat conductivility; Cover layer 2 adopts light transmissive material;
The thermal cycling unit, it comprises semi-conductor heating cooler 5 and driving circuit, TP 6, and based on the control module of microprocessor and PID algorithm; 3 semi-conductor heating cooler 5 form sex change district, annealed zone and extension area along sample in microfluidic channel successively below the volution microfluidic channel; TP 6 is placed between semi-conductor heating cooler 5 and the micro-fluidic chip; TP 6 is connected to control module, and control module is connected to driving circuit, and driving circuit links to each other with semi-conductor heating cooler 5, the heating or the refrigeration temperature of control semi-conductor heating cooler 5;
The real-time fluorescence detecting unit, this system comprises light source 7, photodetector 9 and above-mentioned control module; Light source 7 adopts the linear array photodiodes, and photodetector 9 adopts PMs, described linear array photodiode and PM secured in parallel, and become the angle of spending greater than 10 with the volution fluid channel; Light source linear array photodiode and PM are positioned at micro-fluidic chip extension area top; The fluorescence of fluid channel is pooled the cylindrical mirror of straight line light and accepts straight line light with its knockdown micro-optical device of spherical lens that focuses on a bit, place the top of fluid channel, the emergent light of spherical lens is received by photodetector 9; Photodetector 9 is connected to control module;
Control module in above-mentioned thermal cycling unit and the real-time fluorescence detecting unit is connected to computingmachine.
Described stratum basale 1 and cover layer 2 are with the bonding techniques formation micro-fluidic chip that is sealed.
Described TP 6 is a platinum sensor; Semi-conductor heating cooler 5, TP 6, light source 8 adopt semiconductor technology to make.
Described control module is to be the control core module to adopt digital signal processor DSP or on-site programmable gate array FPGA.
The computer interface of controlling whole device adopts serial ports or USB or bluetooth or Wi-Fi or the wired or wireless mode of internet network to be connected to control module.
The representative temperature in said sex change district is 95 ℃, and the representative temperature of annealed zone is 65 ℃, and the representative temperature of extension area is 72 ℃.
Sample pneumatic pump behind the injection port sample introduction provides pneumatic power to make sample flow to the injection port air inlet.Sample flows in fluid channel, passes through sex change district, annealed zone, extension area successively, and sample flow one circle is promptly accomplished a PCR reaction.The micro-fluidic chip that utilizes the MEMS technology to make has been realized the micro-reaction system.
DSP/FPGA output pulse width modulating wave (Pulse-width modulating is adopted in described temperature control; PWM) control three block semiconductor heating cooler; Three platinum resistance temperature sensors are that DSP/FPGA provides temperature feedback signal; Adopt pid algorithm closed-loop control attemperation, make whole fluid channel zone form required three the steady temperature fields of sex change district, annealed zone, extension area of PCR reaction.Three semi-conductor heating cooler leave certain gap each other, utilize air heat insulation.
Light source linear array LED and PM are positioned at micro-fluidic chip extension area top in the real-time fluorescence detection system; Make cylindrical mirror and the knockdown micro-optical device of spherical lens; Place the top of reaction channel; The fluorescence of each reaction channel receives with PM because the cylindrical mirror effect of converging becoming straight line focuses on a bit through the line that spherical lens will converge again again.This process circulates repeatedly and can accomplish the amplification that repeatedly reaction realizes DNA, carries out real-time fluorescence simultaneously and detects.The light source irradiation sample, the target gene combined with fluorescent group in the sample absorbs the fluorescence of launching specific wavelength behind the energy of light source, because target gene concentration is proportional with fluorescence intensity, just can realize the real-time quantitative fluorescence detection through fluorescence intensity.
The present invention has substantial characteristics and progress, technique effect of the present invention: compare with the PCR in real time appearance of classics, utilize MEMS fabrication techniques micro-fluidic chip, the reagent traceization; Save the heating and cooling time through the design of three flat-temperature zones and shortened the time of polymerase chain reaction greatly, prolonged the work-ing life of semi-conductor heating cooler simultaneously.Compare with the PCR micro-fluidic chip of having invented, on chip, make the semi-conductor heating cooler and replace existing micro-heater, not only can heat but also can active refrigeration; Invent a highly sensitive real-time fluorescence detection system of low cost; Adopt and make cylindrical mirror and the knockdown micro-optical device of spherical lens; The fluorescent signal of the PCR of all passages is converged in the high-performance photoelectricity multiplier tube, realize that real-time quantitative fluorescence detects.
Description of drawings
Below in conjunction with accompanying drawing patent of the present invention is further specified.
Fig. 1 structure vertical view of the present invention.
Fig. 2 structure side-looking of the present invention side.
Fig. 3 is the A-A sectional view of Fig. 1.
Fig. 4 system chart of the present invention.
Among the figure 1, have a stratum basale of volution fluid channel, 2, cover layer, 3, sample holes, 4, sample outlet hole, 5, the semi-conductor heating cooler, 6, TP, 7, light source, 8, micro-optical device, 9, photodetector, 10, pneumatic pump.
Embodiment
As illustrated in fig. 1 and 2, the present invention's miniature polymerase chain reaction device and real-time micro-optic proofing unit comprise micro-fluidic chip, pneumatic pump, temperature controlling system and fluorescence detection device.Being temperature controlling system below fluid channel, is fluorescence detection device above fluid channel, and pneumatic pump is not done concrete regulation as long as realize its POF.
The micro-fluidic chip mode of connection utilizes the manufacturing of MEMS technology to have the stratum basale 1 and cover layer 2 of volution fluid channel, and both become micro-fluidic chip through bonding.On cover layer 2, there are two communicating poress to be respectively sample holes 3, sample outlet hole 4; Sample holes 3, sample outlet hole 4 are corresponding with the starting point and the terminal point of fluid channel respectively; Sample is from sample holes 3 sample introductions; Pneumatic pump 10 is from sample holes 3 air inlets behind the sample introduction, and sample flows through the air pressure mode, and unnecessary gas is discharged from sample outlet hole 4.The flow velocity of sample can be controlled through the air input of pneumatic pump 10, and then the real time position of sample can be known according to fluid channel size and sample flow rate.Concrete regulation is not done in the position of pneumatic pump 10, regulates as the case may be, as long as realize its function.
Be three semi-conductor heating cooler 5 below micro-fluidic chip, it covers whole fluid channel zone, between three semi-conductor heating cooler 5, leaves certain gap, utilizes air heat insulation.Make fluid channel sex change district constant temperature 95 ℃ of representative temperatures, for denaturation process provides constant temperature through control sex change district semi-conductor heating cooler 5; Make fluid channel annealed zone constant temperature 65 ℃ of representative temperatures, for annealing process provides constant temperature through control annealed zone semi-conductor heating cooler 5; Make fluid channel extension area constant temperature 72 ℃ of representative temperatures through control extension area semi-conductor heating cooler 5, for the extension process provides constant temperature.Laying temperature transmitter 6 between micro-fluidic chip and the semi-conductor heating and cooling sheet 5.1 minute sex change stage, annealing stage 30 seconds, the extension stage is then decided according to the dna fragmentation length that will increase, and generally needs 1 minute.Sample is accomplished one time the polymerase chain reaction through sex change, annealing and extension, and the polymerase chain reaction process circulates repeatedly and realizes the amplification of DNA.Before the amplified reaction, need preparatory sex change in 10 minutes usually.
It above micro-fluidic chip the real-time fluorescence proofing unit; The linear array photodiode that light source 7 adopts semiconductor technology to make, photodetector 9 adopts PM, linear array photodiode and PM secured in parallel; And angled with spiral fluid channel, greater than 10 degree.Linear array photodiode and PM are positioned at micro-fluidic chip extension area top, and sample is a mobile circle in fluid channel, has promptly flow through sex change district, annealed zone, extension area respectively, accomplishes one time the polymerase chain reaction, and array light source throws light on once; Make cylindrical mirror and the knockdown micro-optical device 8 of spherical lens; Place each circle top of reaction channel; The fluorescence of each reaction channel is because the cylindrical mirror effect of converging becoming straight line; The line that spherical lens will converge focuses on a bit again, receives with PM, first order fluorescence is carried out in reaction survey; The polymerase chain reaction process circulates repeatedly and realizes the amplification of DNA, carries out real-time fluorescence simultaneously and detects.As shown in Figure 4; The photodetector 9 of fluorescent signal acquisition processing system is connected on the computingmachine through control module with the TP 6 of temperature controlling system; The microprocessor of control module is digital signal processor or field programmable gate array; Digital signal processor or field programmable gate array are connected on the computingmachine host computer procedure control total system through serial ports or USB or bluetooth or wired or wireless modes such as Wi-Fi or internet network.
Claims (5)
1. polymerase chain reaction device and real-time micro-optic proofing unit, it is characterized in that: it comprises:
Micro-fluidic chip, it comprises stratum basale (1), the cover layer (2) that has the volution fluid channel, stratum basale (1) forms micro-fluidic chip with cover layer (2); On cover layer (2), there are two holes to be respectively sample holes and sample outlet hole, lay respectively at the starting point and the terminal point of fluid channel; The pneumatic pump (10) that provides pneumatic power to make sample flow links to each other with sample holes (3); The material of stratum basale (1) is the good material of heat conductivility; Cover layer (2) adopts light transmissive material;
The thermal cycling unit, it comprises semi-conductor heating cooler (5) and driving circuit, TP (6), and based on the control module of microprocessor and PID algorithm; 3 semi-conductor heating cooler (5) form sex change district, annealed zone and extension area along sample in microfluidic channel successively below the volution microfluidic channel; TP (6) is placed between semi-conductor heating cooler (5) and the micro-fluidic chip; TP (6) is connected to control module, and control module is connected to driving circuit, and (5 link to each other driving circuit, the heating or the refrigeration temperature of control semi-conductor heating cooler (5) with the semi-conductor heating cooler; Three semi-conductor heating cooler leave the space each other;
The real-time fluorescence detecting unit, this system comprises light source (7), photodetector (9) and above-mentioned control module; Light source (7) adopts the linear array photodiode, and photodetector (9) adopts PM, described linear array photodiode and PM secured in parallel, and become the angle of spending greater than (10) with the volution fluid channel; Light source linear array photodiode and PM are positioned at micro-fluidic chip extension area top; The fluorescence of fluid channel is pooled the cylindrical mirror of straight line light and accepts straight line light with its knockdown micro-optical device of spherical lens that focuses on a bit, place the top of fluid channel, the emergent light of spherical lens is received by photodetector (9); Photodetector (9) is connected to control module;
Above-mentioned control module is connected to computingmachine.
2. polymerase chain reaction according to claim 1 device and real-time micro-optic proofing unit is characterized in that: described stratum basale (1) and cover layer (2) are with the bonding techniques formation micro-fluidic chip that is sealed.
3. polymerase chain reaction according to claim 1 device and real-time micro-optic proofing unit is characterized in that: described TP (6) is a platinum sensor; Semi-conductor heating cooler (5), TP (6), light source (7) adopt semiconductor technology to make.
4. polymerase chain reaction according to claim 1 device and real-time micro-optic proofing unit is characterized in that: described control module is to be the control core module to adopt digital signal processor DSP or on-site programmable gate array FPGA.
5. polymerase chain reaction according to claim 1 device and real-time micro-optic proofing unit is characterized in that: the computer interface of controlling whole device adopts serial ports or USB or bluetooth or Wi-Fi or the wired or wireless mode of internet network to be connected to control module.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011201659588U CN202099269U (en) | 2011-05-23 | 2011-05-23 | Polymerase chain reactor and real-time micro optics detection device |
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| CN2011201659588U CN202099269U (en) | 2011-05-23 | 2011-05-23 | Polymerase chain reactor and real-time micro optics detection device |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105092480A (en) * | 2014-05-23 | 2015-11-25 | 中国科学院物理研究所 | Biochip used for OIRD detection method, and detection method thereof |
| CN107505251A (en) * | 2017-07-26 | 2017-12-22 | 西安理工大学 | Flow cytometer based on microflow control technique and linear array detector |
| CN107591380A (en) * | 2017-08-03 | 2018-01-16 | 电子科技大学 | A kind of fluid channel radiator of integrated temperature sensor |
| CN109072159A (en) * | 2016-05-18 | 2018-12-21 | 日本板硝子株式会社 | The control method of reaction treating device and reaction treating device |
| CN113574161A (en) * | 2019-03-15 | 2021-10-29 | 国立研究开发法人产业技术综合研究所 | Nucleic acid amplification method |
-
2011
- 2011-05-23 CN CN2011201659588U patent/CN202099269U/en not_active Expired - Lifetime
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105092480A (en) * | 2014-05-23 | 2015-11-25 | 中国科学院物理研究所 | Biochip used for OIRD detection method, and detection method thereof |
| CN105092480B (en) * | 2014-05-23 | 2017-08-01 | 中国科学院物理研究所 | A kind of biochip and its detection method for OIRD detection methods |
| CN109072159A (en) * | 2016-05-18 | 2018-12-21 | 日本板硝子株式会社 | The control method of reaction treating device and reaction treating device |
| CN109072159B (en) * | 2016-05-18 | 2022-05-10 | 日本板硝子株式会社 | Reaction processing apparatus and method for controlling reaction processing apparatus |
| US11465150B2 (en) | 2016-05-18 | 2022-10-11 | Nippon Sheet Glass Company, Limited | Reaction treatment device and method for controlling reaction treatment device |
| CN107505251A (en) * | 2017-07-26 | 2017-12-22 | 西安理工大学 | Flow cytometer based on microflow control technique and linear array detector |
| CN107591380A (en) * | 2017-08-03 | 2018-01-16 | 电子科技大学 | A kind of fluid channel radiator of integrated temperature sensor |
| CN107591380B (en) * | 2017-08-03 | 2019-08-13 | 电子科技大学 | A kind of fluid channel radiator of integrated temperature sensor |
| CN113574161A (en) * | 2019-03-15 | 2021-10-29 | 国立研究开发法人产业技术综合研究所 | Nucleic acid amplification method |
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| C14 | Grant of patent or utility model | ||
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