CN102220226A - Two-path temperature control polymerase chain reactor and real-time detection device - Google Patents
Two-path temperature control polymerase chain reactor and real-time detection device Download PDFInfo
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- CN102220226A CN102220226A CN201110133178XA CN201110133178A CN102220226A CN 102220226 A CN102220226 A CN 102220226A CN 201110133178X A CN201110133178X A CN 201110133178XA CN 201110133178 A CN201110133178 A CN 201110133178A CN 102220226 A CN102220226 A CN 102220226A
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Abstract
The invention relates to a two-path temperature control polymerase chain reactor and a real-time detection device, which are applied in the field of biochemical reaction and medical detection. The two-path temperature control polymerase chain reactor comprises a base layer with a ring-shaped microfluidic channel structure, wherein the base layer is sealed with a cover board layer to form a microfluidic chip, the cover board layer is provided with a sample incoming hole and a sample outgoing hole, a microprocessor is adopted to control two semi-conductor heating and refrigerating devices, a whole microfluidic channel area forms three constant temperature areas, namely a degeneration area, an annealing area and an extension area needed by polymerase chain reaction, the semi-conductor heating and refrigerating devices are arranged under the microfluidic chip, and a platinum resistance temperature sensor manufactured by a semi-conductor process is arranged among the microfluidic chip and the two semi-conductor heating and refrigerating devices, so that temperature feedback signals are provided to the microprocessor, and closed ring control can be formed. The real-time detection system comprises a light source, a photoelectric detector and a fluorescence signal acquisition processing system. In the invention, by controlling the two semi-conductor heating and refrigerating devices, the whole microfluidic channel area forms the three constant temperature areas, and the heating and refrigerating device is simplified.
Description
Technical field
(Polymerase chain reaction PCR) realizes that on micro-fluidic chip DNA cloning and real-time fluorescence detect, and are applied to biochemical reaction and medical science detection range to the present invention relates to fluorescent polyase chain reaction.
Background technology
Microminiaturization is an important development direction in medical science and the biochemistry detection equipment, micro electro mechanical system (MEMS) technology (Micro-electro-mechanical systems, MEMS) develop into PCR be reflected at finish on the chip provide may.Have following shortcoming in the conventional fluorescent PCR instrument: the instrument volume is big, and temperature rate is slow, and dynamic temperature control, temperature accuracy are not high, the long-term temperature shock lost of life of semi-conductor heating cooler.Utilize the PCR reaction chip of MEMS technology manufacturing to be divided into three kinds now: 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, as 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, as patent 200410024703.4 etc.Little water-bath chip 200610043243.9 etc., the heating and cooling complex structure.The PCR reaction chip that utilizes the 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 present invention seeks to realize the control of three flat-temperature zones, and utilize semiconductor technology light source for processing and semi-conductor heating cooler, utilize the MEMS technique construction to simplify temperature control polymerase chain reaction and real-time detection apparatus by two-way semi-conductor heating and cooling.
For achieving the above object, the present invention takes following technical scheme:
Two-way temperature control polymerase chain reaction device and real-time detection apparatus, it comprises:
Micro-fluidic chip, this chip comprise stratum basale 1, the cover layer 2 that has annular fluid channel, and stratum basale 1 and cover layer 2 constitute micro-fluidic chip together; A sample holes 3 and a sample outlet hole 4 are arranged on the cover layer 2, and sample holes 3 and sample outlet hole 4 are positioned on the annular fluid channel; The material of stratum basale 1 is the material with good heat conductive performance; Cover layer 2 adopts light transmissive material;
Thermal cycling unit, this unit comprise semi-conductor heating cooler 5 and driving circuit, temperature sensor 6, and based on the control module of microprocessor and proportion integration differentiation algorithm; Two semi-conductor heating cooler 5 difference according to controlled temperature below annular microfluid forms sex change district, annealed zone and extension area; Temperature sensor 6 is placed between semi-conductor heating cooler 5 and the micro-fluidic chip in sex change district, the extension area, and temperature sensor 6 is put in the micro-fluidic chip below in the annealed zone; Temperature sensor 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;
Real-time fluorescence detecting unit, this unit comprise light source 9, photodetector 8 and above-mentioned control module; Light source 9 is a photodiode, is positioned between stratum basale 1 and the cover layer 2, is fixed on the stratum basale by bonding technology; Photodetector 8 is placed on the micro-fluidic chip top, over against light source 9, with optics support 7 fixed light electric explorers 8; Light source 9 and photodetector 8 vertical placements; Photodetector 8 is connected to control module.
Control module in above-mentioned thermal cycling unit and the real-time fluorescence detecting unit is connected to computer.
Described stratum basale 1 and cover layer 2 are with the bonding techniques formation micro-fluidic chip that is sealed.
Described temperature sensor 6 is a platinum sensor; Semi-conductor heating cooler 5, temperature sensor 6, light source 8, adopt semiconductor technology 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 temperature that described two semi-conductor heating cooler 5 are controlled is respectively 95 ℃ and 72 ℃, the zone at 95 ℃ semi-conductor heating cooler place forms the sex change district, the zone at 72 ℃ semi-conductor heating cooler place forms extension area, and to constitute temperature be 65 ℃ extension area because temperature descends between sex change district and the extension area.
Two-way temperature control polymerase chain reaction device and real-time detection apparatus comprise the stratum basale that has annular fluid channel structure, and stratum basale and cover layer have a sample holes and a sample outlet hole with the bonding techniques formation micro-fluidic chip that is sealed on cover layer.Adopt microprocessor digital signal processor (Digital Signal Processor, DSP) or field programmable gate array (Field-Programmable Gate Array, FPGA), make whole fluid channel zone form required three the steady temperature fields of sex change district, annealed zone, extension area of PCR reaction by control two-way semi-conductor heating and cooling.The semi-conductor heating cooler is below micro-fluidic chip, platinum resistance temperature sensor places between micro-fluidic chip and the semi-conductor heating cooler, for DSP/FPGA provides temperature feedback signal, (Proportional-integral-derivative, PID) algorithm forms closed-loop control to the proportion of utilization integral differential.Semi-conductor heating cooler and platinum resistance temperature sensor are to adopt semiconductor technology to make.The real-time fluorescence detection system comprises light source, photodetector and fluorescent signal acquisition processing system.Light source adopts photodiode, and (Light Emitting Diode LED), is positioned between stratum basale and the cover layer, is fixed on the stratum basale by bonding technology; Photodetector is placed on the micro-fluidic chip top, with support fixed light electric detection means.Light source is vertical with photodetector to be placed, and detects the fluorescent signal of PCR in real time.Fluorescent signal acquisition processing system and temperature controlling system are connected on the computer by microprocessor.
Micro-fluidic chip: utilize the manufacturing of MEMS technology to have the stratum basale of annular fluid channel, a sample holes and a sample outlet hole are arranged on the cover layer, the tilting communicating pores that designs according to principle of dynamics can prevent that sample is excessive.Stratum basale and cover layer are with the bonding techniques formation micro-fluidic chip that is sealed.Baseboard material is a thermally conductive material, and cover plate materials has the light transmissive material of good optical characteristic.Sample is from the injection port sample introduction, and sample flows in fluid channel and passes through sex change district, annealed zone, extension area successively, and sample flow one circle is promptly finished a PCR reaction.Utilize the micro-fluidic chip of MEMS technology manufacturing to realize the micro-reaction system.
Thermal cycling unit, this unit comprise semi-conductor heating cooler 5 and driving circuit, temperature sensor 6, and based on the control module of microprocessor and proportion integration differentiation algorithm.The semi-conductor heating cooler is below annular microfluidic channel.Temperature sensor is placed between semi-conductor heating cooler and the micro-fluidic chip.Semi-conductor heating cooler and platinum resistance temperature sensor are to adopt semiconductor technology to make.
Adopt microprocessor DSP/FPGA output pulse width modulating wave (Pulse-width modulating, PWM) control two-way semi-conductor heating and cooling, three platinum resistance temperature sensors provide temperature feedback signal for DSP/FPGA, 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.Denaturation temperature and the elongating temperature of two-way semi-conductor heating cooler control fluid channel respective regions constant temperature in the PCR reaction, representative temperature is not divided into 95 ℃ and 72 ℃.Can formation temperature around at two flat-temperature zones gradient fields, annealing temperature in the PCR reaction can appear in factors such as the power by control semi-conductor heating cooler, size, placement location between two flat-temperature zones, representative temperature is 65 ℃.
Real-time fluorescence proofing unit: comprise fixed support, comprise light source, photodetector and fluorescent signal acquisition processing system.The photodiode that light source adopts semiconductor technology to make is positioned between stratum basale and the cover layer, is fixed on the stratum basale by bonding technology; Photodetector is placed on the micro-fluidic chip top, with fixed support fixed light electric detection means.Light source is vertical with photodetector to be placed.Sample mobile circle in fluid channel is promptly finished amplified reaction one time, utilize the light source irradiation sample during this time, target gene combined with fluorescent group in the sample, launch the fluorescence of specific wavelength behind the absorption energy of light source, because target gene concentration and fluorescence intensity are proportional, just can realize that by fluorescence intensity real-time quantitative fluorescence detects.Photodetector is arranged in the extension area top of micro-fluidic chip.
The present invention has substantial characteristics and progress, technique effect of the present invention: compare with the PCR in real time instrument of classics, utilize MEMS fabrication techniques micro-fluidic chip, the reagent traceization; Save the heating and cooling time by 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; A highly sensitive real-time fluorescence detection system of low cost is proposed, the photodiode that light source adopts semiconductor technology to make, light source is vertical with photodetector to be placed; Microprocessor adopts digital signal processor or field programmable gate array, can be integrated on the micro-fluidic chip easily.Compare with the PCR micro-fluidic chip of having invented with the PCR in real time instrument of classics, make whole fluid channel zone form three flat-temperature zones, simplified the heating and cooling device by control two-way semi-conductor heating and cooling.
Description of drawings
The present invention is further described below in conjunction with accompanying drawing.
Fig. 1 structure vertical view of the present invention.
Fig. 2 structure side view of the present invention.
Fig. 3 is according to structure of the present invention, the figure as a result of Computer Simulation.
Fig. 4 system chart of the present invention.
Among the figure: 1, have the stratum basale of annular fluid channel, 2, cover layer, 3, sample holes, 4, sample outlet hole, 5, the semi-conductor heating cooler, 6, platinum resistance temperature sensor, 7, the optics support, 8, photodetector, 9 light sources.
Specific implementation method
As illustrated in fig. 1 and 2, the present invention includes: micro-fluidic chip, temperature controlling system and real-time fluorescence proofing unit.Being temperature controlling system below micro-fluidic chip, is the real-time fluorescence detector above micro-fluidic chip.
The micro-fluidic chip mode of connection is that the stratum basale 1 and the cover layer 2 that have annular fluid channel all are to utilize the manufacturing of MEMS technology, become one by bonding.A sample holes 3 and a sample outlet hole 4 are arranged on cover layer 2, and sample is from sample holes 3 sample introductions, and sample flows through sex change warm area, annealing warm area successively, extends warm area, and amplified reaction is finished in circulation repeatedly, discharges from sample outlet hole 4.
Be two semi-conductor heating cooler 5 below micro-fluidic chip, between micro-fluidic chip and semi-conductor heating cooler 5, platinum resistance temperature sensor 6 be set, monitor three characteristic temperatures in the PCR reaction process in real time.Adopt microprocessor DSP/FPGA output pulse width modulating wave PWM control two-way semi-conductor heating and cooling, three platinum resistance temperature sensors provide temperature feedback signal for DSP/FPGA, adopt pid algorithm to form the 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.Make fluid channel sex change district constant temperature 95 ℃ of representative temperatures, for denaturation process provides constant temperature by control sex change district semi-conductor heating cooler 5; Make fluid channel extension area constant temperature 72 ℃ of representative temperatures by control extension area semi-conductor heating cooler 5, for the extension process provides constant temperature.With two semi-conductor heating cooler 5 is center formation temperature gradient fields, sex change district and the regional temperature between the extension area in fluid channel can be lower than above two zones, can realize that by control fluid channel wherein has the temperature in one section zone to be 65 ℃ of annealing representative temperatures.As shown in Figure 3, utilize Computer Simulation software that it is carried out analogue simulation.Be example with sample 5 microlitres in this emulation, the sample parameters water replaces here, and annular fluid channel inside radius is 12.5 millimeters, and outside radius is 14 millimeters.The final condition that loads is that fluid channel sex change district constant temperature is 95 ℃ of representative temperatures, fluid channel extension area constant temperature is 72 ℃ of representative temperatures, as shown in Figure 3, heat 2.33 minutes results afterwards, between sex change district and extension area, form one section flat-temperature zone at 65 ℃, satisfy the annealed characteristic temperature, the temperature condition of whole PCR reaction is satisfied in this temperature field.
The real-time fluorescence detection system comprises light source 9, photodetector 8, optics support 7 and fluorescent signal acquisition processing system.Light source 9 adopts photodiode, is positioned between stratum basale and the cover layer, is fixed on the stratum basale by bonding technology; Photodetector 8 is placed on the micro-fluidic chip top, with optics support 7 fixed light electric detection means.Light source 9 and photodetector 8 vertical placements.Sample flows along fluid channel 1, and through sex change district, annealed zone, extension area warm area, sample flow one circle is promptly finished amplified reaction one time, and this process circulates repeatedly and just can finish repeatedly amplified reaction.Light source 9 and photodetector 8 lay respectively at the side and the top of extension area in the micro-fluidic chip, carry out a real time fluorescent quantitative and detect after this exemplary position can make sample finish once amplification.
As shown in Figure 4, fluorescent signal acquisition processing system and temperature controlling system are connected on the computer by microprocessor, the used microprocessor of heat circulating system and real-time fluorescence detection system is digital signal processor or field programmable gate array, digital signal processor or field programmable gate array are connected on the computer by serial ports or USB or bluetooth or wired or wireless modes such as Wi-Fi or internet network, and host computer procedure is controlled whole device.
Claims (6)
1. two-way temperature control polymerase chain reaction device and real-time detection apparatus, it is characterized in that: it comprises:
Micro-fluidic chip, this chip comprise stratum basale (1), the cover layer (2) that has annular fluid channel, and stratum basale (1) and cover layer (2) constitute micro-fluidic chip together; A sample holes (3) and a sample outlet hole (4) are arranged on the cover layer (2), and sample holes (3) and sample outlet hole (4) are positioned on the annular fluid channel; The material of stratum basale (1) is the material with good heat conductive performance; Cover layer (2) adopts light transmissive material;
Thermal cycling unit, this unit comprise semi-conductor heating cooler (5) and driving circuit, temperature sensor (6), and based on the control module of microprocessor proportion integration differentiation algorithm; Two semi-conductor heating cooler (5) difference according to controlled temperature below annular microfluid forms sex change district, annealed zone and extension area; Temperature sensor (6) is placed between semi-conductor heating cooler (5) and the micro-fluidic chip; Temperature sensor (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 and cooling device (5), the heating or the refrigeration temperature of control semi-conductor heating and cooling device (5);
Real-time fluorescence detecting unit, this unit comprise light source (9), photodetector (8) and above-mentioned control module; Light source (9) is a photodiode, is positioned between stratum basale (1) and the cover layer (2), is fixed on the stratum basale by bonding technology; Photodetector (8) is placed on the micro-fluidic chip top, over against light source (9), with optics support (7) fixed light electric explorer (8); Light source (9) and the vertical placement of photodetector (8); Photodetector (8) is connected to control module.
Above-mentioned control module is connected to computer.
2. temperature control according to claim 1 polymerase chain reaction and real-time detection apparatus 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. temperature control according to claim 1 polymerase chain reaction and real-time detection apparatus is characterized in that: described temperature sensor (6) is a platinum sensor; Semi-conductor heating cooler (5), temperature sensor (6), light source (8) adopt semiconductor technology to make.
4. temperature control according to claim 1 polymerase chain reaction and real-time detection apparatus 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. temperature control according to claim 1 polymerase chain reaction and real-time detection apparatus 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.
6. temperature control according to claim 1 polymerase chain reaction and real-time detection apparatus, it is characterized in that: the temperature that described two semi-conductor heating cooler (5) are controlled is respectively 95 ℃ and 72 ℃, the zone at 95 ℃ semi-conductor heating cooler place forms the sex change district, the zone at 72 ℃ semi-conductor heating cooler place forms extension area, and to constitute temperature be 65 ℃ extension area because temperature descends between sex change district and the extension area.
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| CN103777662A (en) * | 2014-01-18 | 2014-05-07 | 浙江大学 | Device and method applied in gradient temperature control |
| CN106190821A (en) * | 2016-07-01 | 2016-12-07 | 四川简因科技有限公司 | A kind of hand-held bluetooth PCR instrument being integrated with photoelectric detection system |
| CN107817850A (en) * | 2017-12-05 | 2018-03-20 | 苏州昊通仪器科技有限公司 | PCR instrument temperature control system |
| CN107991278A (en) * | 2017-12-28 | 2018-05-04 | 湖南华南光电科技股份有限公司 | Heating sampling structure based on fluorescent quenching technology trace explosive detector |
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| CN110551622A (en) * | 2019-09-18 | 2019-12-10 | 无锡百泰克生物技术有限公司 | quick PCR reaction chip and quick fluorescence quantitative detector |
| CN112779125A (en) * | 2021-02-09 | 2021-05-11 | 复旦大学 | Light-driven circulating polymerase chain reaction micro-fluidic device and application thereof |
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| CN112779125A (en) * | 2021-02-09 | 2021-05-11 | 复旦大学 | Light-driven circulating polymerase chain reaction micro-fluidic device and application thereof |
| CN113433040A (en) * | 2021-06-25 | 2021-09-24 | 国家纳米科学中心 | Modularized micro-fluidic chip for preparing nano particles and application thereof |
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