CN102586098B - Real-time fluorescent PCR (polymerase chain reaction) working system of space-oriented micro-volume unit - Google Patents
Real-time fluorescent PCR (polymerase chain reaction) working system of space-oriented micro-volume unit Download PDFInfo
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- CN102586098B CN102586098B CN2012100504610A CN201210050461A CN102586098B CN 102586098 B CN102586098 B CN 102586098B CN 2012100504610 A CN2012100504610 A CN 2012100504610A CN 201210050461 A CN201210050461 A CN 201210050461A CN 102586098 B CN102586098 B CN 102586098B
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Abstract
The invention which relates to a real-time fluorescent PCR working system of a space-oriented micro-volume unit belongs to the fields of the biology, the analytic chemistry and medical detection. The system comprises: a ceramic refrigeration sheet (1), an electrothermal film (2), a hard transparent plastic (3), heat conduction channels (8), a temperature sensor (9) and rubber pistons (10) which form a temperature circulation controller; LEDs (light emitting diodes) (4) as a light exciting unit, excitation light filters (5), a silicon photocell (6) as a fluorescent spectrum detector, and a detection light filter (7); and an external connection microprocessor for controlling the refrigeration of the refrigeration sheet (1) and the heating of the electrothermal film (2), a keyboard for controlling the input and the output of parameters, and a display screen. The system accords with objectives of structure miniature, function integration, light weight, small volume and fully-automatic detection. The light excitation and the fluorescent detection directly contact an object to be detected, and there are no gravity requirements, so the detection result is stable and is not interfered, thereby the system is adapted to space working requirements.
Description
Technical field
The present invention relates to the real-time fluorescence PCR work system of the long-pending unit of a kind of space-oriented microbody, mainly be applicable to the real-time fluorescence PCR work of applying in the spatial dimension under below-G conditions, belong to biology, analytical chemistry and medical science detection field.
Background technology
Along with reaching its maturity of China's manned spaceflight technology, the prolongation gradually in space mission cycle, also can be more and more higher to the requirement of spacefarer's health care.Be in the microorganism serious threat spacefarer's of non-supervised state health in spacecraft.The correlated results demonstration of the medium-term and long-term flight of US and Russia, detected 234 kinds of microorganisms in the crew module, bacterium has 40 genus, 108 kinds, wherein pathogenic bacteria has more than 20 to plant, and these pathogenic bacterias can cause the spacefarer to infect various diseases, and even affect completing smoothly of aerial mission.In long duration space flight, the growth of microorganism species and bacterial strain variation can affect performance and the working environment of plant and instrument, to the normal operation generation harm of spacecraft and plant and instrument thereof.Simultaneously, carry the microorganism that can survive on aerospacecraft under space environment, likely by the mankind or spacecraft, take space station, martian surface to, can cause disadvantageous effect to human research's space life form.For the generation that prevents infectious diseases in spaceflight, propagate and effectively treat, the microorganism in must the monitor closely spacecraft.
Polymerase chain reaction (PCR) is a kind of nucleic acid amplification technologies in in-vitro simulated natural DNA replication dna process, extend (72 ℃) by high temperature thermally denature (94 ℃ of left and right), low-temperature annealing renaturation (50～60 ℃) and thermophilic and form one-period, a circulation can make the DNA total amount double, can be 1,000,000 times of the rapid and easy amplifications of the genetic material of trace, re-use and carry out fluorescence based on FRET (fluorescence resonance energy Transfer Technology) and detect in real time,, according to fluorescence intensity and the relative information that changes of intensity, can carry out qualitative and quantitative analysis to the PCR product.
Based on the real-time fluorescence quantitative PCR instrument of above-mentioned principle, can reach to microorganism in the spacecraft of operation in spatial dimension monitoring.Real-time PCR adopts laser apparatus or fluorescence excitation light source to carry out Real-Time Monitoring, glitch-free fluorescence excitation is stablized in assurance, forming optical system by a series of lens, filter and a dichroic mirror focuses on fluorescence excitation on spectrograph, spectrograph separates fluorescence in the mode at interval according to the difference of wavelength, enter charge coupled cell (CCD camera) or photomultiplier, the sequential detection application software is collected these fluorescent signals from charge coupled cell (CCD camera) or photomultiplier, to data analysis.So far, of a great variety based on the commercial PCR instrument of above-mentioned reaction and detection principle.External brand mainly contains ABI, Roche, MJ, Bio-Rad, Stratagene, Corbtt, Cepheid etc., and rich day of domestic Hangzhou, Guangzhou reach peace gene and Shanghai Feng Ling etc.
Obviously this quasi-instrument has following shortcoming for the real-time fluorescence PCR work of carrying out the job space requirement:
1, bulky, be unfavorable for carrying.Because the volume of photomultiplier or charge coupled cell self is just very large, and be again that split is used, supporting light path system need to be arranged, cause the bulky of whole fluorescence low-light spectrum detection system, be difficult to carry out again microminiaturization.
2, use many optical fiber as optical transmission system due to optical excitation unit and light receiving unit when the transmission, unavoidably have the luminous energy loss in the coupling fiber process, make the raising of whole detection system sensitivity be severely limited.
3, because the light path that needs all kinds of optics and optical fiber to form when exciting light conduction and reflected light gather is carried out the light transmission, this has affected spectral detection system stability in actual use.
4, owing to detecting the metering system that does not reach the zero distance contact, make the low-light spectrum detect and be subject to non-detected object material,, as forming the interference of microchannel wall material, cause measuring error.
Summary of the invention
The present invention relates to the real-time fluorescence PCR work system of the long-pending unit of a kind of space-oriented microbody, be mainly used in applying under the spatial dimension below-G conditions, the real-time fluorescence detection is carried out in reaction to PCR.Purpose is temperature cycle control device, exciting light conduction device, these three of fluorescence spectrum detecting devices partly integrated, realization is applicable to the portable minisize PCR fluorescence real-time detecting system of job space requirement, reaches that function is integrated, structure is miniature, the target of the little full-automatic detection of lightweight volume.
In order to achieve the above object, the present invention takes following technical scheme:
The real-time fluorescence PCR work system of the long-pending unit of a kind of space-oriented microbody, it includes the ceramic cooling piece 1 that forms the temperature cycle control device, Electric radiant Heating Film 2, hard transparent plastic 3, passage of heat 8, temperature sensor 9, rubber piston 10; As the LED photodiode 4 of exciting light unit, exciting light spectral filter 5,, as the silicon photocell 6 of fluorescence spectrum detecting device, detect light spectral filter 7; Also include be used to controlling ceramic cooling piece 1 refrigeration the external microprocessor of Electric radiant Heating Film 2 heating, for keyboard and the display screen controlling parameter and carry out input and output.
The real-time fluorescence PCR work system of the long-pending unit of a kind of space-oriented microbody, 3 one-tenth tubulars of hard transparent plastic volume, Electric radiant Heating Film 2 is at the skin of hard transparent plastic volume 3, interior side hole at the both ends up and down of hard transparent plastic volume 3 embeds exciting light spectral filter 5, be coated with LED photodiode 4 on exciting light spectral filter 5, LED photodiode 4 is positioned at the outside of hard transparent plastic volume 3, and Electric radiant Heating Film 2 is in above-mentioned not perforate of position of opening; The inboard perforate in hard transparent plastic volume 3 ends, front and back embeds and detects light spectral filter 7, detects on light spectral filter 7 and is coated with silicon photocell 6, and silicon photocell 6 is positioned at the outside of hard transparent plastic volume 3.Passage of heat 8 is left in the outside, two ends of Electric radiant Heating Film 2.To be spliced into lateral cross section be foursquare rectangular parallelepiped to pottery cooling piece 1, and it is tangent that above-mentioned hard transparent plastic volume 3 is placed in the internal space of described rectangular parallelepiped, and between ceramic cooling piece 1 and hard transparent plastic volume 3 the falling heat-transfer elargol.Above-mentioned transparent plastics volume 3 two ends are with pistons 10 encapsulation, and embed temperature sensor 9 on the piston 10 of a side therein.
Use the silica gel adhesion between Electric radiant Heating Film 2 and hard transparent plastic volume 3.
The sheet number that is spliced into the ceramic cooling piece 1 of rectangular parallelepiped is 4.
Described piston 10 is rubber piston.
The luminous peak wavelength of photodiode 4 is 475nm; The peak wavelength of exciting light spectral filter 5 transmissions is also 475nm.
Detecting light spectral filter 7 transmission peak wavelengths is the fluorescence of 525nm.
Described ceramic cooling piece size is 5mm * 5mm, and thickness is 1mm.
Described silicon photocell and temperature sensor are of a size of longly * wide is: 1mm * 1mm.
The present invention is a non-electricity work unit of the real-time fluorescence PCR work system of the long-pending unit of microbody, integrated all non-electric key elements, but need external microprocessor, keyboard and display screen etc. during work, its working process is, pottery cooling piece 1 and hard transparent plastic volume 3 are by external microprocessor control and the feedback of temperature sensor 9, being heated to 94 ℃ makes the genetic material in the PCR reaction solution high-temperature denatured, after be cooled to 55 ℃ of low temperature renaturation of withdrawing from a secret society or underworld gang, be warming up to finally 72 ℃ and make it thermophilic extension, these 3 temperature that circulate (30～40 cycles).The light that sends from LED photodiode 4 is simultaneously filtered at spectral filter 5, the 475nm excitation light irradiation object PCR to be detected reaction solution of selecting.Material to be detected goes out fluorescence by excitation, and after detecting light spectral filter 7, the fluorescence that leaches wavelength 525nm is received by silicon photocell 6, becomes electrical signal output.
The real-time fluorescence PCR work system of the long-pending unit of microbody of the present invention compared with prior art, has following obvious advantage and beneficial effect:
The whole non-electric key element of real-time fluorescence PCR work that the present invention is integrated, temperature circulatory system, reactive system and detection system, wherein heating can reach 5mm * 5mm size with refrigeration plant, cooling piece thickness can reach 1mm, LED photodiode, silicon photocell and temperature sensor can reach 1mm * 1mm, make a whole set of work system narrow down to 5mm * 5mm size, meet structure micro, the little full-automatic target that detects of integrated, the lightweight volume of function.Optical excitation can contact with thing zero distance to be detected with fluoroscopic examination, is not subjected to the gravity requirement, makes detected result stable, and is interference-free, more adapts to the Space-Work requirement.
The present invention stands in the integrated aspect of real-time fluorescence PCR work,, if space-oriented need to be accurate to automatic sampling in the sample introduction direction, could form the integrity of using.Hand sampling of the present invention can be completed by the cosmonaut in space.
Description of drawings
The real-time fluorescence PCR work system of the long-pending unit of Fig. 1 microbody is along the longitudinal section view of fore-and-aft direction
The real-time fluorescence PCR work system of the long-pending unit of Fig. 2 microbody is along the longitudinal section view of left and right directions
The real-time fluorescence PCR work system overall schematic of the long-pending unit of Fig. 3 microbody
In figure: 1 ceramic cooling piece, 2 Electric radiant Heating Film, 3 hard transparent plastics, 4LED photodiode, 5 exciting light spectral filters, 6 silicon photocells, 7 detect light spectral filter, 8 passages of heat, 9 temperature sensors, 10 pistons.
Specific embodiment
Describe the present embodiment in detail below in conjunction with accompanying drawing 1～3.
The structural representation of the present embodiment such as Fig. 1,2 and 3.Fig. 1 is its longitudinal section view along fore-and-aft direction, can only draw ceramic cooling piece 1, Electric radiant Heating Film 2, hard transparent plastic 3 in figure, as the LED photodiode 4 of exciting light unit, exciting light spectral filter 5, passage of heat 8 and temperature sensor 9 and rubber piston 10, detecting unit can only draw a side spectral filter 7.Described ceramic cooling piece 1 every be 5mm * 5mm size, 1mm thickness, be spliced into the rectangular parallelepiped of 7mm * 5mm * 5mm size.LED photodiode 4 and exciting light spectral filter 5 form the optical excitation unit, as the luminous peak wavelength of the LED photodiode 4 of excitation light source, are wherein 475nm; The peak wavelength of exciting light spectral filter 5 transmissions is also 475nm; Silicon photocell 6, detection light spectral filter 7 form optical detecting units, and detecting light spectral filter 7 transmissive wavelength that measured matter produces is the fluorescence of 525nm, and the 525nm fluorescence that silicon photocell 6 will detect 7 transmissions of light spectral filter is converted into electrical signal; Passage of heat 8 is connected ceramic cooling piece 1 with hard transparent plastic 3, in the middle of passage of heat 8, the falling heat-transfer elargol, can make hard transparent plastic 3 internal reaction liquid better carry out temperature exchange; Temperature variation in 9 pairs of PCR reaction process of temperature sensor feeds back to external microprocessor, then is controlled the work of cooling piece 1 and Electric radiant Heating Film 2 by microprocessor; 10 is rubber piston, with whole work system sealing.
The working process of the real-time fluorescence PCR work system of the long-pending unit of whole microbody is, use needle tubing that the PCR reaction solution is injected by the piston place, start work system, by three warm area times of keyboard input temp circulation, microprocessor, according to feedback control cooling piece 1 and heating film 2 work of temperature sensor 9, carries out the PCR reaction.The light that sends from LED photodiode 4 is simultaneously filtered at spectral filter 5, the 475nm excitation light irradiation object PCR to be detected reaction solution of selecting.Material to be detected goes out fluorescence by excitation, and after detecting light spectral filter 7, the fluorescence that leaches wavelength 525nm is received by silicon photocell 6, becomes electrical signal output.Fig. 2 is the longitudinal section view of this detection system along left and right directions, can only be by ceramic cooling piece, heating film 2, hard transparent plastic 3, LED photodiode 4, exciting light spectral filter 5 and silicon photocell 6, detection light spectral filter 7 and temperature sensor 9 and a side piston 10 in figure, heat transfer path 8 fails to draw.Form the optical excitation unit by exciting light spectral filter 5 and silicon photocell 6, by detecting light spectral filter 7 and temperature sensor 9, form optical detecting unit; The purpose of using two groups of optical excitation unit and two groups of optical detecting units is further to improve detection sensitivity.The long-pending real-time fluorescence PCR work system of microbody of the present invention has following characteristics: integrated whole key elements of real-time fluorescence PCR work system, and as temperature cycle and control, excitation light source, beam splitting system, light detection etc., because the device that uses is all millimetre-sized, make the feature size downsizing of whole system to only having 5mm * 5mm * 7mm millimeter.
The long-pending real-time fluorescence PCR work system fabrication processing of microbody is as follows: at first with 3 one-tenth tubulars of hard transparent plastic volume, again Electric radiant Heating Film 2 use silica gel are sticked in its skin, embed LED photodiode 4 and exciting light spectral filter 5 in both sides, heating film up and down, front and back embed silicon photocell 6 and detect light spectral filter 7, and passage of heat 8 is left in both sides.Again, 1 four of ceramic cooling pieces are spliced into rectangular parallelepiped, the cylinder that the upper step makes is wherein built-in, and at attaching space falling heat-transfer elargol not.Last two encapsulates with piston 10, and embeds temperature sensor 9 on the bottom piston.
Claims (8)
1. the real-time fluorescence PCR work system of the long-pending unit of a space-oriented microbody, it is characterized in that: it includes the ceramic cooling piece (1) that forms the temperature cycle control device, Electric radiant Heating Film (2), hard transparent plastic (3), passage of heat (8), temperature sensor (9), rubber piston (10); As the LED photodiode (4) of exciting light unit, exciting light spectral filter (5),, as the silicon photocell (6) of fluorescence spectrum detecting device, detect light spectral filter (7); Also include be used to controlling ceramic cooling piece (1) refrigeration the external microprocessor of Electric radiant Heating Film (2) heating, for keyboard and the display screen controlling parameter and carry out input and output;
Hard transparent plastic (3) becomes tubular, Electric radiant Heating Film (2) is at the skin of hard transparent plastic (3), interior side hole at the both ends up and down of hard transparent plastic (3) embeds exciting light spectral filter (5), be coated with LED photodiode (4) on exciting light spectral filter (5), LED photodiode (4) is positioned at the outside of hard transparent plastic (3), and Electric radiant Heating Film (2) is in above-mentioned not perforate of position of opening; Before and after hard transparent plastic (3), the inboard perforate in end embeds and detects light spectral filter (7), detects on light spectral filter (7) and is coated with silicon photocell (6), and silicon photocell (6) is positioned at the outside of hard transparent plastic (3); Passage of heat (8) is left in the outside, two ends of Electric radiant Heating Film (2); To be spliced into lateral cross section be foursquare rectangular parallelepiped to pottery cooling piece (1), and it is tangent that above-mentioned hard transparent plastic (3) is placed in the internal space of described rectangular parallelepiped, and between ceramic cooling piece (1) and hard transparent plastic (3) the falling heat-transfer elargol; Above-mentioned transparent plastics (3) two ends encapsulate with piston (10), and the temperature sensor (9) of the upper embedding of the piston of a side (10) therein.
2. the real-time fluorescence PCR work system of the long-pending unit of a kind of space-oriented microbody according to claim 1, is characterized in that: use the silica gel adhesion between Electric radiant Heating Film (2) and hard transparent plastic (3).
3. a kind of space-oriented microbody according to claim 1 amasss the real-time fluorescence PCR work system of unit, and it is characterized in that: the sheet number that is spliced into the ceramic cooling piece (1) of rectangular parallelepiped is 4.
4. a kind of space-oriented microbody according to claim 1 amasss the real-time fluorescence PCR work system of unit, and it is characterized in that: described piston (10) is rubber piston.
5. a kind of space-oriented microbody according to claim 1 amasss the real-time fluorescence PCR work system of unit, and it is characterized in that: the luminous peak wavelength of photodiode (4) is 475nm; The peak wavelength of exciting light spectral filter (5) transmission is also 475nm.
6. a kind of space-oriented microbody according to claim 1 amasss the real-time fluorescence PCR work system of unit, it is characterized in that: detecting light spectral filter (7) transmission peak wavelength is the fluorescence of 525nm.
7. a kind of space-oriented microbody according to claim 1 amasss the real-time fluorescence PCR work system of unit, it is characterized in that: described ceramic cooling piece size is 5mm * 5mm, and thickness is 1mm.
8. the real-time fluorescence PCR work system of the long-pending unit of a kind of space-oriented microbody according to claim 1 is characterized in that: described silicon photocell and temperature sensor are of a size of and longly * wide are: 1mm * 1mm.
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| CN102879128B (en) * | 2012-09-26 | 2014-04-02 | 北京工业大学 | Manufacturing method special for three temperature value transmission testing device utilizing PCR(polymerase chain reaction) technology |
| CN103215184B (en) * | 2013-04-12 | 2014-07-30 | 北京理工大学 | Micro gene amplification device for miniature space experiment |
| WO2015176253A1 (en) * | 2014-05-21 | 2015-11-26 | Coyote Bioscience Co., Ltd. | Systems and methods for low power thermal cycling |
| WO2018119848A1 (en) * | 2016-12-29 | 2018-07-05 | 湖南圣湘生物科技有限公司 | Pcr fluorescence detector |
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| WO2006085948A2 (en) * | 2004-07-01 | 2006-08-17 | Cornell Research Foundation, Inc. | Real-time pcr detection of microorganisms using an integrated microfluidics platform |
| CN101255396A (en) * | 2007-02-27 | 2008-09-03 | 索尼株式会社 | Nucleic acid amplifier |
| CN101302473A (en) * | 2007-05-11 | 2008-11-12 | 索尼株式会社 | Real-time PCR system |
| CN202492522U (en) * | 2012-02-29 | 2012-10-17 | 北京工业大学 | Real-time fluorescent PCR (Polymerase Chain Reaction) working system of space-oriented micro-volume unit |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006085948A2 (en) * | 2004-07-01 | 2006-08-17 | Cornell Research Foundation, Inc. | Real-time pcr detection of microorganisms using an integrated microfluidics platform |
| CN101255396A (en) * | 2007-02-27 | 2008-09-03 | 索尼株式会社 | Nucleic acid amplifier |
| CN101302473A (en) * | 2007-05-11 | 2008-11-12 | 索尼株式会社 | Real-time PCR system |
| CN202492522U (en) * | 2012-02-29 | 2012-10-17 | 北京工业大学 | Real-time fluorescent PCR (Polymerase Chain Reaction) working system of space-oriented micro-volume unit |
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