CN102586098B - Real-time fluorescent PCR (polymerase chain reaction) working system of space-oriented micro-volume unit - Google Patents

Abstract

A real-time fluorescent PCR working system for space-oriented micro-volume units, which belongs to the fields of biology, analytical chemistry and medical detection; it includes a ceramic cooling plate (1) and an electric heating film (2) forming a temperature cycle control device, which are hard and transparent Plastic (3), heat conduction channel (8), temperature sensor (9), rubber piston (10); LED light-emitting diode (4) as excitation light unit, excitation light filter (5), as fluorescence spectrum detection device A silicon photocell (6), a detection light filter (7); also includes an external microprocessor for controlling the cooling of the ceramic refrigeration sheet (1), heating of the electric heating film (2), a keyboard for inputting and outputting control parameters, and display screen. This system meets the goals of structure miniaturization, function integration, light weight and small size fully automatic detection. Optical excitation and fluorescence detection are in zero-distance contact with the object to be detected, and are not required by gravity, so that the detection results are stable and free from interference, and are more suitable for space work requirements.

Description

A real-time fluorescent PCR working system for space-oriented micro-volume unit

technical field

The invention relates to a space-oriented real-time fluorescent PCR working system for micro-volume units, which is mainly suitable for the real-time fluorescent PCR work applied in the space range under the condition of weightlessness, and belongs to the fields of biology, analytical chemistry and medical detection.

Background technique

With the maturity of my country's manned spaceflight technology and the gradual extension of the space mission cycle, the requirements for astronauts' health protection will become higher and higher. The unmonitored microorganisms in the spacecraft seriously threaten the health of astronauts. The relevant results of the US-Russia mid-to-long-term flight show that 234 kinds of microorganisms were detected in the crew cabin. There are 40 genera and 108 species of bacteria, including more than 20 kinds of pathogenic bacteria. These pathogenic bacteria can cause astronauts to be infected with various diseases. , and even affect the successful completion of the mission. During long-term spaceflight, the growth of microbial flora and the variation of strains will affect the performance of instruments and equipment and the working environment conditions, and cause harm to the normal operation of spacecraft and its instruments and equipment. At the same time, the spacecraft carry microorganisms that can survive in the space environment, which may be brought to the space station or the surface of Mars by humans or spacecraft, which will adversely affect human research on life forms in space. In order to prevent the occurrence, spread and effective treatment of infectious diseases in space flight, the microorganisms in the spacecraft must be closely monitored.

Polymerase chain reaction (PCR) is a nucleic acid amplification technique that simulates the natural DNA replication process in vitro, which consists of high-temperature thermal denaturation (about 94°C), low-temperature annealing and renaturation (50-60°C) and suitable temperature extension (72°C). ℃) to form a cycle, one cycle can double the total amount of DNA, and the trace amount of genetic material can be rapidly and easily amplified by a million times, and then used for fluorescence real-time detection based on FRET (fluorescence resonance energy transfer technology), According to the information of the fluorescence intensity and the relative change of the intensity, qualitative and quantitative analysis can be performed on the PCR products.

The real-time fluorescent quantitative PCR instrument based on the above principles can achieve the monitoring of microorganisms in spacecraft operating within the space range. The real-time quantitative PCR instrument uses a laser or a fluorescent excitation light source for real-time monitoring to ensure stable and non-interfering fluorescence excitation. The optical system consists of a series of lenses, filters and a dichroic mirror to focus the excited fluorescence onto the spectrometer. Fluorescence is separated by wavelength into a charge-coupled device (CCD camera) or photomultiplier tube, from which the sequence detection application software collects these fluorescent signals and analyzes the data. So far, there are various types of commercialized PCR instruments based on the above-mentioned reaction and detection principles. Foreign brands mainly include ABI, Roche, MJ, Bio-Rad, Stratagene, Corbtt, Cepheid, etc., and domestic brands such as Hangzhou Bioer, Guangzhou Daan Gene and Shanghai Fengling.

Obviously, this type of instrument has the following disadvantages for the real-time fluorescent PCR work required by space operations:

1. It is bulky and not easy to carry. Since the photomultiplier tube or the charge-coupled device itself is very large, and it is used separately, a supporting optical system is required, which makes the entire fluorescent microspectral detection system bulky and difficult to miniaturize.

2. Since the optical excitation unit and the light receiving unit use many optical fibers as the optical transmission system during transmission, it is inevitable that there will be loss of optical energy during the fiber coupling process, which severely limits the improvement of the sensitivity of the entire detection system.

3. Since the optical path composed of various optical devices and optical fibers is required for light transmission during excitation light transmission and reflected light collection, this affects the stability of the spectral detection system in actual use.

4. Since the detection has not achieved the zero-distance contact measurement method, the microspectral detection is interfered by non-detection objects, such as the substances that make up the microchannel wall, resulting in measurement errors.

Contents of the invention

The invention relates to a space-oriented real-time fluorescent PCR working system for micro-volume units, which is mainly used for real-time fluorescent detection of PCR reactions under the condition of weightlessness in the space range. The purpose is to integrate the three parts of the temperature cycle control device, the excitation light transmission device, and the fluorescence spectrum detection device to realize a portable miniaturized PCR fluorescence real-time detection system suitable for space operation requirements, to achieve functional integration, structure miniaturization, light weight and small size Automatic detection of targets.

In order to achieve the above object, the present invention takes the following technical solutions:

A real-time fluorescent PCR working system for space-oriented micro-volume units, which includes a ceramic cooling chip 1 forming a temperature cycle control device, an electric heating film 2, a hard transparent plastic 3, a heat conduction channel 8, a temperature sensor 9, and a rubber piston 10 ; LED light-emitting diode 4 as an excitation light unit, an excitation light filter 5, a silicon photocell 6 as a fluorescence spectrum detection device, and a detection light filter 7; it also includes a ceramic cooling chip 1 for controlling cooling, and an electric heating film 2 for heating External microprocessor, keyboard and display for input and output of control parameters.

A real-time fluorescent PCR working system for space-oriented micro-volume units, the hard transparent plastic roll 3 is in a cylindrical shape, the electric heating film 2 is on the outer layer of the hard transparent plastic roll 3, and the upper and lower ends of the hard transparent plastic roll 3 The inner opening of the part is embedded with an excitation light filter 5, and the excitation light filter 5 is covered with an LED light emitting diode 4, and the LED light emitting diode 4 is located on the outside of the hard transparent plastic roll 3, and the electrothermal film 2 is at the position of the above opening. No holes are opened; the inner side of the front and rear ends of the hard transparent plastic roll 3 is perforated to embed the detection light filter 7 , the detection light filter 7 is covered with a silicon photocell 6 , and the silicon photocell 6 is located on the outside of the hard transparent plastic roll 3 . Heat conduction channels 8 are left outside the two ends of the electrothermal film 2 . The ceramic refrigeration sheet 1 is spliced into a rectangular parallelepiped with a square cross section, and the above-mentioned hard transparent plastic roll 3 is placed in the inner space of the rectangular parallelepiped, which is tangent and filled between the ceramic refrigeration sheet 1 and the hard transparent plastic roll 3. Thermally conductive silver glue. Both ends of the above-mentioned transparent plastic roll 3 are encapsulated with a piston 10, and a temperature sensor 9 is embedded on the piston 10 at one side.

Use silica gel to adhere between the electrothermal film 2 and the hard transparent plastic roll 3 .

The number of the ceramic refrigeration sheets 1 spliced into a rectangular parallelepiped is 4 pieces.

The piston 10 is a rubber piston.

The peak wavelength of light emitted by the light emitting diode 4 is 475nm; the peak wavelength transmitted by the excitation light filter 5 is also 475nm.

The detection light filter 7 transmits fluorescence with a wavelength of 525nm.

The size of the ceramic refrigeration sheet is 5mm×5mm, and the thickness is 1mm.

The dimensions of the silicon photocell and the temperature sensor are: length×width: 1mm×1mm.

The present invention is a non-electrical working unit of the real-time fluorescent PCR working system of micro volume unit, which integrates all non-electrical elements, but needs external microprocessor, keyboard and display screen, etc. when working, and its working process is as follows: ceramic refrigeration plate 1 And the hard transparent plastic roll 3 is controlled by an external microprocessor and the feedback of the temperature sensor 9, heated to 94°C to denature the genetic material in the PCR reaction solution at high temperature, then cooled to 55°C for low-temperature refolding, and finally raised to 72°C ℃ to make it stretch at a suitable temperature, and cycle these three temperatures (30 to 40 cycles). At the same time, the light emitted from the LED light-emitting diode 4 is filtered at the filter 5, and the selected 475nm excitation light irradiates the PCR reaction solution of the object to be detected. The substance to be detected emits fluorescence by the excitation light, and after passing through the detection optical filter 7, the fluorescence with a wavelength of 525nm filtered out is received by the silicon photocell 6 and becomes an electrical signal output.

Compared with the prior art, the micro-volume unit real-time fluorescent PCR working system of the present invention has the following obvious advantages and beneficial effects:

The invention integrates all non-electrical elements of real-time fluorescent PCR work, temperature circulation system, reaction system and detection system, wherein the heating and cooling device can reach a size of 5mm×5mm, the thickness of the cooling sheet can reach 1mm, LED light-emitting diodes, silicon photocells and The temperature sensor can reach 1mm×1mm, which reduces the whole working system to 5mm×5mm, which meets the goal of structure miniaturization, function integration, light weight and small size, and fully automatic detection. Optical excitation and fluorescence detection can be in zero-distance contact with the object to be detected, without the requirement of gravity, so that the detection results are stable, free from interference, and more suitable for space work requirements.

In terms of real-time fluorescent PCR work integration, the present invention needs to be accurate in the direction of sample injection to automatically inject samples if it is space-oriented, so as to constitute the completeness of use. The manual sample injection of the present invention can be completed by astronauts in space.

Description of drawings

Fig. 1 Radial cross-sectional view of the real-time fluorescent PCR working system in the microvolume unit along the front-to-back direction

Fig. 2 Radial cross-sectional view of the real-time fluorescent PCR working system in the microvolume unit along the left and right directions

Figure 3 The overall schematic diagram of the real-time fluorescent PCR working system in micro volume units

In the figure: 1 ceramic cooling plate, 2 electric heating film, 3 hard transparent plastic, 4 LED light emitting diode, 5 excitation light filter, 6 silicon photocell, 7 detection light filter, 8 heat conduction channel, 9 temperature sensor, 10 piston .

specific embodiment

This embodiment will be described in detail below in conjunction with accompanying drawings 1 to 3 .

Schematic diagrams of the structure of this embodiment are shown in Figures 1, 2 and 3. Fig. 1 is its radial cross-sectional view along the front and rear directions, in which only ceramic cooling plate 1, electrothermal film 2, hard transparent plastic 3, LED light-emitting diode 4 as excitation light unit, and excitation light filter 5 can be drawn in the figure , heat conduction channel 8, temperature sensor 9 and rubber piston 10, the detection unit can only draw one side filter 7. Each of the ceramic cooling chips 1 is 5mm×5mm in size and 1mm in thickness, and is spliced into a cuboid with a size of 7mm×5mm×5mm. The LED light-emitting diode 4 and the excitation light filter 5 form a light excitation unit, wherein the peak wavelength of the light emitted by the LED light-emitting diode 4 as the excitation light source is 475nm; the peak wavelength transmitted by the excitation light filter 5 is also 475nm; the silicon photocell 6 1. The detection light filter 7 forms a light detection unit, the detection light filter 7 can transmit the fluorescence with a wavelength of 525nm produced by the measured substance, and the silicon photocell 6 converts the 525nm fluorescence transmitted by the detection light filter 7 into an electrical signal; The heat conduction channel 8 connects the ceramic cooling chip 1 with the hard transparent plastic 3, and the middle of the heat conduction channel 8 is filled with heat conduction silver glue, which can make the reaction solution in the hard transparent plastic 3 perform better temperature exchange; The temperature change is fed back to the external microprocessor, and then the microprocessor controls the work of the cooling plate 1 and the electric heating film 2; 10 is a rubber piston, which closes the entire working system.

The working process of the real-time fluorescent PCR working system of the whole micro-volume unit is to inject the PCR reaction solution from the piston using a needle tube, start the working system, input the time of the three temperature zones of the temperature cycle through the keyboard, and the microprocessor will control the temperature according to the temperature sensor 9. Feedback controls the work of the cooling sheet 1 and the heating film 2 to carry out the PCR reaction. At the same time, the light emitted from the LED light-emitting diode 4 is filtered at the filter 5, and the selected 475nm excitation light irradiates the PCR reaction solution of the object to be detected. The substance to be detected emits fluorescence by the excitation light, and after passing through the detection optical filter 7, the fluorescence with a wavelength of 525nm filtered out is received by the silicon photocell 6 and becomes an electrical signal output. Fig. 2 is a radial cross-sectional view of the detection system along the left and right directions. In the figure, only a ceramic cooling plate, a heating film 2, a hard transparent plastic 3, an LED light-emitting diode 4, an excitation light filter 5 and a silicon photocell 6, Detection optical filter 7 and temperature sensor 9 and one side piston 10, heat transfer channel 8 can't be drawn. The light excitation unit is composed of the excitation light filter 5 and the silicon photocell 6, and the light detection unit is composed of the detection light filter 7 and the temperature sensor 9; the purpose of using two groups of light excitation units and two groups of light detection units is to further improve the Detection sensitivity. The micro-volume real-time fluorescent PCR working system of the present invention has the following characteristics: it integrates all the elements of the real-time fluorescent PCR working system, such as temperature cycle and control, excitation light source, spectroscopic system, light detection and so on. Since the devices used are all millimeter-scale, the feature size of the entire system is reduced to only 5mm x 5mm x 7mm.

The production process of the micro-volume real-time fluorescent PCR working system is as follows: First, roll 3 hard transparent plastic into a cylindrical shape, then glue the electric heating film 2 to its outer layer with silica gel, and embed LED light-emitting diodes 4 and excitation on the upper and lower sides of the heating film. The optical filter 5 is embedded with a silicon photocell 6 and a detection optical filter 7 at the front and back, and heat conduction channels 8 are left on both sides. Again, splice the four pieces of the ceramic cooling plate 1 into a rectangular cuboid, put the cylinder made in the previous step into it, and fill the uncontacted space with thermal silver glue. The last two ends are packaged with a piston 10, and a temperature sensor 9 is embedded on the bottom piston.

Claims (8)

1. A space-oriented micro-volume unit real-time fluorescent PCR working system, characterized in that: it includes a ceramic refrigeration chip (1) forming a temperature cycle control device, an electric heating film (2), and a hard transparent plastic (3) , heat conduction channel (8), temperature sensor (9), rubber piston (10); LED light-emitting diode (4) as excitation light unit, excitation light filter (5), silicon photocell (6) as fluorescence spectrum detection device ), the detection optical filter (7); also includes an external microprocessor for controlling the cooling of the ceramic refrigeration sheet (1), heating of the electric heating film (2), a keyboard and a display screen for input and output of control parameters; The hard transparent plastic (3) is in the shape of a cylinder, the electric heating film (2) is on the outer layer of the hard transparent plastic (3), and the excitation light filter is embedded in the inner side of the upper and lower ends of the hard transparent plastic (3) sheet (5), the excitation light filter (5) is covered with LED light-emitting diodes (4), the LED light-emitting diodes (4) are located on the outside of the hard transparent plastic (3), and the electric heating film (2) is in the above-mentioned opening There are no openings at the position; the front and rear ends of the hard transparent plastic (3) have holes embedded in the detection light filter (7), and the detection light filter (7) is covered with silicon photocells (6), silicon photocells (6) Located on the outside of the hard transparent plastic (3); heat conduction channels (8) are left on the outside of both ends of the electric heating film (2); the ceramic cooling plate (1) is spliced into a rectangular parallelepiped with a square cross section, and the above hard transparent plastic ( 3) The internal space placed in the cuboid is tangent, and the thermal conductive silver glue is filled between the ceramic cooling plate (1) and the hard transparent plastic (3); the two ends of the transparent plastic (3) are connected by pistons (10 ) package, and a temperature sensor (9) is embedded on the piston (10) on one side. 2. A space-oriented real-time fluorescent PCR working system for micro-volume units according to claim 1, characterized in that: the electrothermal film (2) and the hard transparent plastic (3) are bonded with silica gel. 3. A real-time fluorescent PCR working system for space-oriented micro-volume units according to claim 1, characterized in that the number of ceramic refrigeration sheets (1) spliced into a cuboid is 4 pieces. 4. A space-oriented real-time fluorescent PCR working system for micro-volume units according to claim 1, characterized in that: the piston (10) is a rubber piston. 5. A space-oriented real-time fluorescent PCR working system for micro-volume units according to claim 1, characterized in that: the peak wavelength of the light emitted by the light-emitting diode (4) is 475nm; the excitation light filter (5) transmits The peak wavelength is also 475nm. 6. A real-time fluorescent PCR working system for space-oriented micro-volume units according to claim 1, characterized in that the detection optical filter (7) transmits fluorescence with a wavelength of 525nm. 7. A space-oriented real-time fluorescence PCR working system for micro-volume units according to claim 1, characterized in that: the size of the ceramic refrigeration plate is 5mm×5mm, and the thickness is 1mm. 8 . A real-time fluorescent PCR working system for space-oriented micro-volume units according to claim 1 , characterized in that the dimensions of the silicon photoelectric cell and the temperature sensor are: length×width: 1mm×1mm.

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