CN102220226A - Two-path temperature control polymerase chain reactor and real-time detection device - Google Patents

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

Dual temperature-controlled polymerase chain reactor and real-time detection device

technical field

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.

Background technique

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.

Contents of the invention

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.

To achieve the above object, the present invention takes the following technical solutions:

Two-way temperature-controlled polymerase chain reactor and real-time detection device, which includes:

A microfluidic chip, the chip includes a base layer 1 with an annular microfluidic channel, a cover layer 2, the base layer 1 and the cover layer 2 together form a microfluidic chip; the cover layer 2 has an injection hole 3 And a sampling hole 4, the sampling hole 3 and the sampling hole 4 are located on the annular micro flow channel; the material of the base layer 1 is a material with good thermal conductivity; the cover layer 2 adopts a light-transmitting material;

Thermal cycle unit, this unit comprises semiconductor 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 the semiconductor heating refrigerator 5 and the microfluidic chip, and the temperature sensor 6 in the annealing zone is placed under the microfluidic chip; The temperature sensor 6 is connected to the control module, the control module is connected to the driving circuit, and the driving circuit is connected to the semiconductor heating refrigerator 5 to control the heating or cooling temperature of the semiconductor heating refrigerator 5;

A real-time fluorescence detection unit, which includes a light source 9, a photodetector 8 and the above-mentioned control module; the light source 9 is a light-emitting diode, placed between the base layer 1 and the cover layer 2, and fixed on the base layer through a bonding process; The photodetector 8 is placed above the microfluidic chip, facing the light source 9, and the photodetector 8 is fixed with the optical bracket 7; the light source 9 and the photodetector 8 are placed vertically; the photodetector 8 is connected to the control module.

The control modules in the thermal cycle unit and the real-time fluorescence detection unit are connected to the computer.

The base layer 1 and the cover layer 2 are sealed together by bonding technology to form a microfluidic chip.

The temperature sensor 6 is a platinum resistance sensor; the semiconductor heating refrigerator 5, the temperature sensor 6, and the light source 8 are made by semiconductor technology.

The control module is a control module using a digital signal processor DSP or a field programmable gate array FPGA as the core.

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.

The temperatures controlled by the two semiconductor 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.

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.

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.

The thermal cycle unit includes a semiconductor heating refrigerator 5, a drive circuit, a temperature 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.

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.

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.

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.

Description of drawings

The present invention will be further described below in conjunction with the accompanying drawings.

Fig. 1 is a top view of the structure of the present invention.

Fig. 2 is a side view of the structure of the present invention.

Fig. 3 is the result diagram of computer simulation according to the structure of the present invention.

Fig. 4 is a system block diagram of the present invention.

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.

Specific implementation method

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.

The connection method of the microfluidic chip is that the base layer 1 with the annular microfluidic channel and the cover layer 2 are both manufactured by MEMS technology and integrated by bonding. There is a sampling hole 3 and a sampling hole 4 on the cover layer 2. The sample is injected from the sampling 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 the sample hole 4.

Below the microfluidic chip are two semiconductor heating refrigerators 5, and a platinum resistance temperature sensor 6 is set between the microfluidic chip and the semiconductor 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 the semiconductor 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 the semiconductor 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 two semiconductor 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 conditions for loading are constant temperature in the microchannel denaturation zone at a typical temperature of 95°C, and constant temperature in the microchannel extension zone 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.

The real-time fluorescence detection system includes a light source 9, a photodetector 8, an optical 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; the photodetector 8 is placed above the microfluidic chip, and the photodetection device is fixed by the optical bracket 7 . The light source 9 and the photodetector 8 are placed vertically. The sample flows along the microchannel 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 the photodetector 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.

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 (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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