CN102220226B - Two-path temperature control polymerase chain reactor and real-time detection device - Google Patents

Abstract

Translated fromChinese

双路温控聚合酶链式反应器与实时检测装置，应用于生化反应和医学检测领域，其包括带有环形微流道结构的基底层，基底层与盖板层密封形成微流控芯片，在盖板层上有一个进样孔和一个出样孔。采用微处理器控制两个半导体加热制冷，在整个微流道区域形成聚合酶链式反应所需的变性区、退火区、延伸区三个恒定温度场。半导体加热制冷器在微流控芯片的下方，采用半导体工艺制作的铂电阻温度传感器置于微流控道片和半导体加热制冷器之间，为微处理器提供温度反馈信号，形成闭环控制。实时荧光检测系统包括光源、光电探测器和荧光信号采集处理系统。本发明通过控制两路半导体加热制冷使整个微流道区域形成三个恒温区，精简了加热制冷装置。

The double-channel temperature-controlled polymerase chain reactor and real-time detection device are used in the fields of biochemical reactions and medical detection. It includes a base layer with an annular microchannel structure, and the base layer and the cover layer are sealed to form a microfluidic chip. There is a sample inlet and a sample outlet on the cover layer. Microprocessor is used to control two semiconductor heating and cooling to form three constant temperature fields in the whole microfluidic channel area, which are denaturation zone, annealing zone and extension zone required by polymerase chain reaction. The semiconductor heating cooler is under the microfluidic chip, and the platinum resistance temperature sensor made by semiconductor technology is placed between the microfluidic chip and the semiconductor heating cooler to provide temperature feedback signals for the microprocessor to form a closed-loop control. The real-time fluorescence detection system includes a light source, a photodetector and a fluorescence signal acquisition and processing system. In the present invention, three constant temperature zones are formed in the entire microchannel region by controlling two semiconductor heating and cooling circuits, thereby simplifying the heating and cooling device.

Description

Translated fromChinese

双路温控聚合酶链式反应器与实时检测装置Dual temperature-controlled polymerase chain reactor and real-time detection device

技术领域technical field

本发明涉及荧光聚合酶链式反应(Polymerase chain reaction，PCR)在微流控芯片上实现DNA扩增及实时荧光检测，应用于生化反应和医学检测领域。The invention relates to fluorescent polymerase chain reaction (Polymerase chain reaction, PCR) on a microfluidic chip to realize DNA amplification and real-time fluorescence detection, which is applied to the fields of biochemical reactions and medical detection.

背景技术Background technique

医学与生化检测设备中微型化是一个重要发展方向，微机电系统技术(Micro-electro-mechanical systems，MEMS)的发展为PCR反应在芯片上完成提供了可能。传统荧光PCR仪中存在以下缺点：仪器体积大，升降温速率慢，动态控温，温度精度不高，半导体加热制冷器长期温度骤变寿命缩短。现在利用MEMS技术制造的PCR反应芯片分为三种：微阱式芯片、微流控芯片和微水浴芯片。微阱式芯片存在以下缺点：升降温速率慢，动态控温，温度精度不高，一般只有合金层微加热器，没有主动式的制冷器，如专利200310122607.9和200510011180.4等。微流控芯片存在以下缺点：一般只有合金层微加热器，没有主动制冷器，如专利200410024703.4等。微水浴芯片200610043243.9等，加热制冷结构复杂。利用MEMS技术制造的PCR反应芯片，解决了仪器体积大的问题，但是又出现了主动制冷、实时荧光检测在芯片上集成的难题。Miniaturization is an important development direction in medical and biochemical testing equipment. The development of micro-electro-mechanical systems (MEMS) technology provides the possibility for PCR reactions to be completed on chips. The traditional fluorescent PCR instrument has the following disadvantages: the instrument is bulky, the heating and cooling rate is slow, the temperature is dynamically controlled, the temperature accuracy is not high, and the long-term temperature sudden change life of the semiconductor heating refrigerator is shortened. There are currently three types of PCR reaction chips manufactured using MEMS technology: micro-well chips, microfluidic chips, and micro-bath chips. The micro-well chip has the following disadvantages: slow heating and cooling rate, dynamic temperature control, and low temperature accuracy. Generally, there are only alloy layer micro-heaters and no active refrigerators, such as patents 200310122607.9 and 200510011180.4. Microfluidic chips have the following disadvantages: Generally, there are only alloy layer micro-heaters and no active coolers, such as patent 200410024703.4. Micro-water bath chip 200610043243.9, etc., have complex heating and cooling structures. The PCR reaction chip manufactured by using MEMS technology solves the problem of large instrument volume, but there is also the problem of active cooling and real-time fluorescence detection integrated on the chip.

发明内容Contents of the invention

本发明目的是通过两路半导体加热制冷实现三个恒温区的控制，并且利用半导体工艺加工光源与半导体加热制冷器，利用MEMS技术构建精简温控聚合酶链式反应与实时检测装置。The purpose of the present invention is to realize the control of three constant temperature zones through two-way semiconductor heating and cooling, and use semiconductor technology to process the light source and semiconductor heating refrigerator, and use MEMS technology to build a simplified temperature-controlled polymerase chain reaction and real-time detection device.

为实现上述目的，本发明采取以下技术方案：To achieve the above object, the present invention takes the following technical solutions:

双路温控聚合酶链式反应器与实时检测装置，它包括：Two-way temperature-controlled polymerase chain reactor and real-time detection device, which includes:

微流控芯片，该芯片包括带有环形微流道的基底层1、盖板层2，基底层1和盖板层2一起构成微流控芯片；盖板层2上有一个进样孔3和一个出样孔4，进样孔3和出样孔4位于环形微流道上；基底层1的材料为具有良好导热性能的材料；盖板层2采用透光材料；A microfluidic chip, the chip includes abase layer 1 with an annular microfluidic channel, a cover layer 2, thebase layer 1 and the cover layer 2 together form a microfluidic chip; the cover layer 2 has aninjection hole 3 And asampling hole 4, thesampling hole 3 and thesampling hole 4 are located on the annular micro flow channel; the material of thebase layer 1 is a material with good thermal conductivity; the cover layer 2 adopts a light-transmitting material;

热循环单元，该单元包括半导体加热制冷器5和驱动电路、温度传感器6，以及基于微处理器与比例积分微分算法的控制模块；两个半导体加热制冷器5在环形微流体的下方根据控制温度的不同形成变性区、退火区和延伸区；变性区、延伸区中温度传感器6放置在半导体加热制冷器5和微流控芯片之间，退火区中温度传感器6放于微流控芯片下方；温度传感器6连接至控制模块，控制模块连接至驱动电路，驱动电路与半导体加热制冷器5相连，控制半导体加热制冷器5的加热或制冷温度；Thermal cycle unit, this unit comprisessemiconductor heating refrigerator 5 and drive circuit,temperature sensor 6, and based on the control module of microprocessor and proportional integral differential algorithm; Different forms of denaturation zone, annealing zone and extension zone;temperature sensor 6 in the denaturation zone and extension zone is placed between thesemiconductor heating refrigerator 5 and the microfluidic chip, and thetemperature sensor 6 in the annealing zone is placed under the microfluidic chip; Thetemperature sensor 6 is connected to the control module, the control module is connected to the driving circuit, and the driving circuit is connected to thesemiconductor heating refrigerator 5 to control the heating or cooling temperature of thesemiconductor heating refrigerator 5;

实时荧光检测单元，该单元包括光源9、光电探测器8和上述的控制模块；光源9为发光二极管，放置于基底层1和盖板层2之间，通过键合工艺固定在基底层上；光电探测器8放置在微流控芯片上方，正对光源9，用光学支架7固定光电探测器8；光源9与光电探测器8垂直放置；光电探测器8连接至控制模块。A real-time fluorescence detection unit, which includes a light source 9, aphotodetector 8 and the above-mentioned control module; the light source 9 is a light-emitting diode, placed between thebase layer 1 and the cover layer 2, and fixed on the base layer through a bonding process; Thephotodetector 8 is placed above the microfluidic chip, facing the light source 9, and thephotodetector 8 is fixed with theoptical bracket 7; the light source 9 and thephotodetector 8 are placed vertically; thephotodetector 8 is connected to the control module.

上述热循环单元和实时荧光检测单元中的控制模块连接至计算机。The control modules in the thermal cycle unit and the real-time fluorescence detection unit are connected to the computer.

所述的基底层1与盖板层2以键合技术密封在一起形成微流控芯片.Thebase layer 1 and the cover layer 2 are sealed together by bonding technology to form a microfluidic chip.

所述的温度传感器6为铂电阻传感器；半导体加热制冷器5、温度传感器6、光源8、采用半导体工艺制作、。Thetemperature sensor 6 is a platinum resistance sensor; thesemiconductor heating refrigerator 5, thetemperature sensor 6, and thelight source 8 are made by semiconductor technology.

所述的控制模块是以采用数字信号处理器DSP或现场可编程门阵列FPGA为核心的控制模块。The control module is a control module using a digital signal processor DSP or a field programmable gate array FPGA as the core.

控制整个装置的计算机接口采用串口或USB或蓝牙或Wi-Fi或internet网络有线或无线方式连接至控制模块。The computer interface that controls the entire device is connected to the control module in a wired or wireless manner using a serial port, USB, Bluetooth, Wi-Fi, or an internet network.

所述的两个半导体加热制冷器5所控制的温度分别为95℃和72℃，95℃的半导体加热制冷器所在的区域形成变性区，72℃的半导体加热制冷器所在的区域形成延伸区，变性区和延伸区之间由于温度下降构成温度为65℃的延伸区。The temperatures controlled by the twosemiconductor heating refrigerators 5 are 95°C and 72°C respectively, the area where the 95°C semiconductor heating refrigerator is located forms a denaturation zone, and the area where the 72°C semiconductor heating refrigerator is located forms an extension zone. An extension zone with a temperature of 65°C is formed between the denaturation zone and the extension zone due to the temperature drop.

双路温控聚合酶链式反应器与实时检测装置包括带有环形微流道结构的基底层，基底层与盖板层以键合技术密封在一起形成微流控芯片，在盖板层上有一个进样孔和一个出样孔。采用微处理器数字信号处理器(Digital Signal Processor，DSP)或现场可编程门阵列(Field-Programmable Gate Array，FPGA)通过控制两路半导体加热制冷，使整个微流道区域形成PCR反应所需的变性区、退火区、延伸区三个恒定温度场。半导体加热制冷器在微流控芯片的下方，铂电阻温度传感器置于微流控芯片和半导体加热制冷器之间，为DSP/FPGA提供温度反馈信号，利用比例积分微分(Proportional-integral-derivative，PID)算法形成闭环控制。半导体加热制冷器与铂电阻温度传感器是采用半导体工艺制作的。实时荧光检测系统包括光源、光电探测器和荧光信号采集处理系统。光源采用发光二极管(Light Emitting Diode，LED)，放置于基底层和盖板层之间，通过键合工艺固定在基底层上；光电探测器放置在微流控芯片上方，用支架固定光电检测装置。光源与光电探测器垂直放置，实时检测PCR的荧光信号。荧光信号采集处理系统与温度控制系统通过微处理器连接到计算机上。The two-way temperature-controlled polymerase chain reactor and real-time detection device include a base layer with a ring-shaped microchannel structure. The base layer and the cover layer are sealed together by bonding technology to form a microfluidic chip. On the cover layer There is a sample inlet and a sample outlet. Microprocessor digital signal processor (Digital Signal Processor, DSP) or field-programmable gate array (Field-Programmable Gate Array, FPGA) is used to control two-way semiconductor heating and cooling, so that the entire microchannel area forms the PCR reaction required Three constant temperature fields of denaturation zone, annealing zone and extension zone. The semiconductor heating cooler is under the microfluidic chip, and the platinum resistance temperature sensor is placed between the microfluidic chip and the semiconductor heating cooler to provide temperature feedback signals for DSP/FPGA, using proportional-integral-derivative (Proportional-integral-derivative, PID) algorithm forms a closed-loop control. The semiconductor heating refrigerator and the platinum resistance temperature sensor are made by semiconductor technology. The real-time fluorescence detection system includes a light source, a photodetector and a fluorescence signal acquisition and processing system. The light source adopts light emitting diode (Light Emitting Diode, LED), which is placed between the base layer and the cover layer, and fixed on the base layer by bonding process; the photodetector is placed above the microfluidic chip, and the photodetection device is fixed with a bracket . The light source and the photodetector are vertically placed to detect the fluorescence signal of PCR in real time. The fluorescent signal acquisition and processing system and the temperature control system are connected to the computer through a microprocessor.

微流控芯片：利用MEMS技术制造带有环形微流道的基底层，盖板层上有一个进样孔和一个出样孔，根据动力学原理设计的倾斜式贯通孔可以防止样品外溢。基底层与盖板层以键合技术密封在一起形成微流控芯片。底板材料为导热材料，盖板材料具有良好光学特性的透光材料。样品从进样口进样，样品在微流道内流动依次经过变性区、退火区、延伸区，样品流动一圈即完成一次PCR反应。利用MEMS技术制造的微流控芯片实现了微量反应体系。Microfluidic chip: MEMS technology is used to manufacture the base layer with circular microfluidic channels. There is a sample injection hole and a sample outlet hole on the cover layer. The inclined through-hole designed according to the principle of dynamics can prevent the sample from overflowing. The base layer and the cover layer are sealed together by bonding technology to form a microfluidic chip. The bottom plate material is a thermally conductive material, and the cover plate material is a light-transmitting material with good optical properties. The sample is injected from the injection port, and the sample flows through the denaturation zone, annealing zone, and extension zone in sequence in the microchannel, and a PCR reaction is completed once the sample flows in one circle. The microfluidic chip manufactured by MEMS technology realizes the micro reaction system.

热循环单元，该单元包括半导体加热制冷器5和驱动电路、温度传感器6，以及基于微处理器与比例积分微分算法的控制模块。半导体加热制冷器在环形微流体通道的下方。温度传感器放置在半导体加热制冷器和微流控芯片之间。半导体加热制冷器与铂电阻温度传感器是采用半导体工艺制作的。The thermal cycle unit includes asemiconductor heating refrigerator 5, a drive circuit, atemperature sensor 6, and a control module based on a microprocessor and a proportional-integral-differential algorithm. The semiconductor heating refrigerator is under the annular microfluidic channel. The temperature sensor is placed between the semiconductor heating refrigerator and the microfluidic chip. The semiconductor heating refrigerator and the platinum resistance temperature sensor are made by semiconductor technology.

采用微处理器DSP/FPGA输出脉宽调制波(Pulse-width modulating，PWM)控制两路半导体加热制冷，三个铂电阻温度传感器为DSP/FPGA提供温度反馈信号，采用PID算法闭环控制调节温度，使整个微流道区域形成PCR反应所需的变性区、退火区、延伸区三个恒定温度场。两路半导体加热制冷器控制微流道相应区域恒温在PCR反应中的变性温度和延伸温度，典型温度别分为95℃和72℃。在两个恒温区周围会形成温度梯度场，通过控制半导体加热制冷器的功率、尺寸、放置位置等因素，在两恒温区之间会出现PCR反应中退火温度，典型温度为65℃。Microprocessor DSP/FPGA is used to output pulse-width modulating (PWM) to control two-way semiconductor heating and cooling, three platinum resistance temperature sensors provide temperature feedback signals for DSP/FPGA, and PID algorithm is used to control and adjust the temperature in a closed loop. Make the whole microfluidic channel area form three constant temperature fields required for PCR reaction: denaturation zone, annealing zone and extension zone. The two-way semiconductor heating refrigerator controls the denaturation temperature and extension temperature of the constant temperature in the corresponding area of the microchannel in the PCR reaction. Typical temperatures are 95°C and 72°C respectively. A temperature gradient field will be formed around the two constant temperature zones. By controlling the power, size, placement and other factors of the semiconductor heating refrigerator, the annealing temperature in the PCR reaction will appear between the two constant temperature zones. The typical temperature is 65°C.

实时荧光检测装置：包括固定支架、包括光源、光电探测器和荧光信号采集处理系统。光源采用半导体工艺制作的发光二极管，放置于基底层和盖板层之间，通过键合工艺固定在基底层上；光电探测器放置在微流控芯片上方，用固定支架固定光电检测装置。光源与光电探测器垂直放置。样品在微流道内流动一圈即完成一次扩增反应，期间利用光源照射样品，样品中的靶基因结合荧光基团，吸收光源能量后发射出特定波长的荧光，由于靶基因浓度与荧光强度成比例，通过检测荧光强度就可以实现实时定量荧光检测。光电探测器位于微流控芯片中的延伸区上方。Real-time fluorescence detection device: including a fixed bracket, including a light source, a photodetector and a fluorescence signal acquisition and processing system. The light source is a light-emitting diode made by semiconductor technology, placed between the base layer and the cover layer, and fixed on the base layer through a bonding process; the photodetector is placed above the microfluidic chip, and the photodetection device is fixed with a fixed bracket. The light source is placed perpendicular to the photodetector. The sample flows a circle in the microchannel to complete an amplification reaction. During this period, the sample is irradiated with a light source. The target gene in the sample binds to the fluorescent group, absorbs the energy of the light source and emits fluorescence with a specific wavelength. Since the concentration of the target gene is proportional to the fluorescence intensity Real-time quantitative fluorescence detection can be realized by detecting the fluorescence intensity. Photodetectors are located above the extensions in the microfluidic chip.

本发明具有实质性的特点与进步，本发明的技术效果：与经典的实时PCR仪相比，利用MEMS技术制作微流控芯片，试剂微量化；通过三个恒温区的设计省去了升降温时间大大缩短了聚合酶链式反应的时间，同时延长了半导体加热制冷器的使用寿命。与已经发明的PCR微流控芯片相比，在芯片上制作半导体加热制冷器代替现有的微加热器，不仅可以加热而且可以主动制冷；提出一个低成本高灵敏度的实时荧光检测系统，光源采用半导体工艺制作的发光二极管，光源与光电探测器垂直放置；微处理器采用数字信号处理器或现场可编程门阵列，可以方便地集成在微流控芯片上。与经典的实时PCR仪和已经发明的PCR微流控芯片相比，通过控制两路半导体加热制冷使整个微流道区域形成三个恒温区，精简了加热制冷装置。The present invention has substantive features and progress. The technical effect of the present invention: Compared with the classic real-time PCR instrument, the microfluidic chip is made by using MEMS technology, and the reagents are miniaturized; the design of three constant temperature zones saves the heating and cooling The time greatly shortens the time of the polymerase chain reaction, and at the same time prolongs the service life of the semiconductor heating refrigerator. Compared with the PCR microfluidic chip that has been invented, a semiconductor heating refrigerator is made on the chip instead of the existing micro heater, which can not only heat but also actively cool; a low-cost and high-sensitivity real-time fluorescence detection system is proposed, and the light source adopts Light-emitting diodes made by semiconductor technology, the light source and photodetector are placed vertically; the microprocessor adopts digital signal processor or field programmable gate array, which can be easily integrated on the microfluidic chip. Compared with the classic real-time PCR instrument and the invented PCR microfluidic chip, the whole microfluidic area forms three constant temperature zones by controlling two semiconductor heating and cooling channels, which simplifies the heating and cooling devices.

附图说明Description of drawings

下面结合附图对本发明进一步说明。The present invention will be further described below in conjunction with the accompanying drawings.

图1本发明的结构俯视图。Fig. 1 is a top view of the structure of the present invention.

图2本发明的结构侧视图。Fig. 2 is a side view of the structure of the present invention.

图3根据本发明的结构，计算机仿真的结果图。Fig. 3 is the result diagram of computer simulation according to the structure of the present invention.

图4本发明的系统框图。Fig. 4 is a system block diagram of the present invention.

图中：1、带有环形微流道的基底层，2、盖板层，3、进样孔，4、出样孔，5、半导体加热制冷器，6、铂电阻温度传感器，7、光学支架，8、光电探测器，9光源。In the figure: 1. Base layer with annular microchannel, 2. Cover layer, 3. Injection hole, 4. Sample outlet, 5. Semiconductor heating refrigerator, 6. Platinum resistance temperature sensor, 7. Optical Support, 8, photodetector, 9 light sources.

具体实施方法Specific implementation method

如图1和2所示，本发明包括：微流控芯片、温度控制系统和实时荧光检测装置。在微流控芯片下方是温度控制系统，在微流控芯片的上方是实时荧光检测器。As shown in Figures 1 and 2, the present invention includes: a microfluidic chip, a temperature control system and a real-time fluorescence detection device. Below the microfluidic chip is a temperature control system, and above the microfluidic chip is a real-time fluorescence detector.

微流控芯片连接方式是，带有环形微流道的基底层1和盖板层2都是利用MEMS工艺制造，通过键合成为一体。在盖板层2上有一个进样孔3和一个出样孔4，样品从进样孔3进样，样品依次流过变性温区、退火温区、延伸温区，反复循环完成扩增反应，从出样孔4排出。The connection method of the microfluidic chip is that thebase layer 1 with the annular microfluidic channel and the cover layer 2 are both manufactured by MEMS technology and integrated by bonding. There is asampling hole 3 and asampling hole 4 on the cover layer 2. The sample is injected from thesampling hole 3, and the sample flows through the denaturation temperature zone, annealing temperature zone, and extension temperature zone in sequence, and the amplification reaction is completed in repeated cycles. , discharged from thesample hole 4.

在微流控芯片下方是两个半导体加热制冷器5，在微流控芯片和半导体加热制冷器5之间设置铂电阻温度传感器6，实时监测PCR反应过程中的三个特征温度。采用微处理器DSP/FPGA输出脉宽调制波PWM控制两路半导体加热制冷，三个铂电阻温度传感器为DSP/FPGA提供温度反馈信号，采用PID算法形成闭环控制调节温度，使整个微流道区域形成PCR反应所需的变性区、退火区、延伸区三个恒定温度场。通过控制变性区半导体加热制冷器5使微流道变性区恒温在典型温度95℃，为变性过程提供恒温；通过控制延伸区半导体加热制冷器5使微流道延伸区恒温在典型温度72℃，为延伸过程提供恒温。以两个半导体加热制冷器5为中心形成温度梯度场，在微流道的变性区和延伸区之间的区域温度会低于以上两个区域，通过控制可以实现微流道其中有一段区域的温度为退火典型温度65℃。如图3所示，利用计算机仿真软件对其进行仿真模拟。本仿真中以样品5微升为例，这里样品参数用水代替，环形微流道内半径为12.5毫米，外半径为14毫米。加载的边界条件为微流道变性区恒温在典型温度95℃，微流道延伸区恒温在典型温度72℃，如图3所示，加热2.33分钟之后的结果，在变性区和延伸区之间形成一段恒温区在65℃，满足退火的特征温度，此温度场满足整个PCR反应的温度条件。Below the microfluidic chip are twosemiconductor heating refrigerators 5, and a platinumresistance temperature sensor 6 is set between the microfluidic chip and thesemiconductor heating refrigerator 5 to monitor the three characteristic temperatures during the PCR reaction in real time. Microprocessor DSP/FPGA is used to output pulse width modulation wave PWM to control two-way semiconductor heating and cooling. Three platinum resistance temperature sensors provide temperature feedback signals for DSP/FPGA. Form three constant temperature fields of denaturation zone, annealing zone and extension zone required for PCR reaction. By controlling thesemiconductor heating refrigerator 5 in the denaturation area, the temperature of the denaturation area of the microchannel is kept at a typical temperature of 95°C to provide a constant temperature for the denaturation process; by controlling thesemiconductor heating refrigerator 5 in the extension area, the temperature of the extension area of the microchannel is kept at a typical temperature of 72°C. Provide constant temperature for the extension process. A temperature gradient field is formed with twosemiconductor heating refrigerators 5 as the center, and the temperature in the area between the denaturation zone and the extension zone of the micro-flow channel will be lower than the above two areas, and the temperature of a section of the micro-flow channel can be realized through control. The temperature is the typical annealing temperature of 65°C. As shown in Figure 3, use computer simulation software to simulate it. In this simulation, a sample of 5 microliters is taken as an example, where the sample parameters are replaced by water, the inner radius of the annular microchannel is 12.5 mm, and the outer radius is 14 mm. The boundary condition of loading is that the constant temperature of the denaturation zone of the microchannel is at a typical temperature of 95°C, and the constant temperature of the extension zone of the microchannel is at a typical temperature of 72°C. As shown in Figure 3, the result after heating for 2.33 minutes is between the denaturation zone and the extension zone A constant temperature zone is formed at 65°C to meet the characteristic temperature of annealing, and this temperature field meets the temperature conditions of the entire PCR reaction.

实时荧光检测系统包括光源9、光电探测器8、光学支架7和荧光信号采集处理系统。光源9采用发光二极管，放置于基底层和盖板层之间，通过键合工艺固定在基底层上；光电探测器8放置在微流控芯片上方，用光学支架7固定光电检测装置。光源9与光电探测器8垂直放置。样品沿微流道1流动，经过变性区、退火区、延伸区温区，样品流动一圈即完成一次扩增反应，此过程反复循环就可以完成多次扩增反应。光源9和光电探测器8分别位于微流控芯片中延伸区的侧方和上方，在此典型位置可以使样品完成一次扩增后进行一次实时荧光定量检测。The real-time fluorescence detection system includes a light source 9, aphotodetector 8, anoptical support 7 and a fluorescence signal acquisition and processing system. The light source 9 is a light-emitting diode, placed between the base layer and the cover layer, and fixed on the base layer through a bonding process; thephotodetector 8 is placed above the microfluidic chip, and the photodetection device is fixed by theoptical bracket 7 . The light source 9 and thephotodetector 8 are placed vertically. The sample flows along themicrochannel 1, passes through the denaturation zone, the annealing zone, and the temperature zone of the extension zone. Once the sample flows around, one amplification reaction is completed. Repeated cycles of this process can complete multiple amplification reactions. The light source 9 and thephotodetector 8 are respectively located on the side and above the extension area of the microfluidic chip. In this typical position, a real-time fluorescence quantitative detection can be performed after the sample is amplified once.

如图4所示，荧光信号采集处理系统与温度控制系统通过微处理器连接到计算机上，热循环系统和实时荧光检测系统所用的微处理器为数字信号处理器或现场可编程门阵列，数字信号处理器或现场可编程门阵列通过串口或USB或蓝牙或Wi-Fi或internet网络等有线或无线方式连接到计算机上，上位机程序控制整个装置。As shown in Figure 4, the fluorescent signal acquisition and processing system and the temperature control system are connected to the computer through a microprocessor, and the microprocessor used in the thermal cycle system and the real-time fluorescence detection system is a digital signal processor or a field programmable gate array. The signal processor or the field programmable gate array is connected to the computer through a serial port or USB or Bluetooth or Wi-Fi or internet network or other wired or wireless methods, and the host computer program controls the entire device.

Claims (4)

Translated fromChinese

1.双路温控聚合酶链式反应与实时检测装置，其特征在于：它包括：1. A two-way temperature-controlled polymerase chain reaction and real-time detection device, characterized in that it includes:微流控芯片，该芯片包括带有环形微流道的基底层（1）、盖板层（2），基底层（1）和盖板层（2）一起构成微流控芯片；盖板层（2）上有一个进样孔（3）和一个出样孔（4），进样孔（3）和出样孔（4）位于环形微流道上；基底层（1）的材料为具有良好导热性能的材料；盖板层（2）采用透光材料；A microfluidic chip, the chip includes a base layer (1) with an annular microfluidic channel, a cover layer (2), and the base layer (1) and the cover layer (2) together constitute a microfluidic chip; the cover layer (2) There is a sample inlet (3) and a sample outlet (4), the sample inlet (3) and the sample outlet (4) are located on the annular micro flow channel; the material of the base layer (1) is a good Materials with thermal conductivity; the cover layer (2) adopts light-transmitting materials;基于微处理器比例积分微分算法的控制模块，该控制模块连接至计算机；A control module based on a microprocessor proportional integral differential algorithm, which is connected to a computer;热循环单元，该单元包括半导体加热制冷器（5）和驱动电路、温度传感器（6）；两个半导体加热制冷器（5）在环形微流道的下方根据控制温度的不同形成变性区、退火区和延伸区；温度传感器（6）放置在半导体加热制冷器（5）和微流控芯片之间；温度传感器（6）连接至控制模块，控制模块连接至驱动电路，驱动电路与半导体加热制冷器（5）相连，控制半导体加热制冷器（5）的加热或制冷温度；所述的两个半导体加热制冷器（5）所控制的温度分别为95℃和72℃，95℃的半导体加热制冷器所在的区域形成变性区，72℃的半导体加热制冷器所在的区域形成延伸区，变性区和延伸区之间由于温度下降构成温度为65℃的退火区；Thermal cycle unit, which includes a semiconductor heating refrigerator (5), a drive circuit, and a temperature sensor (6); two semiconductor heating refrigerators (5) form a denaturation zone and annealing zone under the annular microchannel according to different control temperatures zone and extension zone; the temperature sensor (6) is placed between the semiconductor heating and cooling device (5) and the microfluidic chip; the temperature sensor (6) is connected to the control module, and the control module is connected to the driving circuit, and the driving circuit and the semiconductor heating and cooling connected to the semiconductor heating refrigerator (5) to control the heating or cooling temperature of the semiconductor heating refrigerator (5); the temperatures controlled by the two semiconductor heating refrigerators (5) are respectively 95°C and 72°C The area where the device is located forms a denaturation zone, the area where the 72°C semiconductor heating refrigerator is located forms an extension area, and anannealing area with a temperature of 65°C is formed between the denaturation area and the extension area due to the temperature drop;实时荧光检测单元，该单元包括光源（9）、光电探测器（8）；光源（9）为发光二极管，放置于基底层（1）和盖板层（2）之间，通过键合工艺固定在基底层上；光电探测器（8）放置在微流控芯片上方，正对光源（9），用光学支架（7）固定光电探测器（8）；光源（9）与光电探测器（8）垂直放置；光电探测器（8）连接至控制模块。A real-time fluorescence detection unit, which includes a light source (9) and a photodetector (8); the light source (9) is a light-emitting diode, placed between the base layer (1) and the cover layer (2), and fixed by a bonding process On the base layer; the photodetector (8) is placed above the microfluidic chip, facing the light source (9), and the photodetector (8) is fixed with an optical bracket (7); the light source (9) and the photodetector (8) ) placed vertically; the photodetector (8) is connected to the control module.2.根据权利要求1所述的双路温控聚合酶链式反应与实时检测装置，其特征在于：所述的基底层（1）与盖板层（2）以键合技术密封在一起形成微流控芯片。2. The two-way temperature-controlled polymerase chain reaction and real-time detection device according to claim 1, characterized in that: the base layer (1) and the cover layer (2) are sealed together by bonding technology to form Microfluidic Chip.3.根据权利要求1所述的双路温控聚合酶链式反应与实时检测装置，其特征在于：所述的控制模块是以采用数字信号处理器DSP或现场可编程门阵列FPGA为核心的控制模块。3. The two-way temperature-controlled polymerase chain reaction and real-time detection device according to claim 1, characterized in that: the control module is based on a digital signal processor DSP or a field programmable gate array FPGA. control module.4.根据权利要求1所述的双路温控聚合酶链式反应与实时检测装置，其特征在于：控制整个装置的计算机接口采用串口或USB或蓝牙或internet网络有线或无线方式连接至控制模块。4. The two-way temperature-controlled polymerase chain reaction and real-time detection device according to claim 1, characterized in that: the computer interface controlling the entire device is connected to the control module by serial port or USB or Bluetooth or internet wired or wirelessly .

Images