CN1900715A - Standard chip for biological PCR micro path fluorescent detection and its preparing method - Google Patents
Standard chip for biological PCR micro path fluorescent detection and its preparing method Download PDFInfo
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- CN1900715A CN1900715A CN 200610088818 CN200610088818A CN1900715A CN 1900715 A CN1900715 A CN 1900715A CN 200610088818 CN200610088818 CN 200610088818 CN 200610088818 A CN200610088818 A CN 200610088818A CN 1900715 A CN1900715 A CN 1900715A
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- 238000001514 detection method Methods 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims description 18
- 239000011521 glass Substances 0.000 claims abstract description 50
- 238000004088 simulation Methods 0.000 claims abstract description 28
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 14
- 230000003321 amplification Effects 0.000 claims abstract description 10
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 8
- 239000000463 material Substances 0.000 claims abstract description 5
- 238000006243 chemical reaction Methods 0.000 claims description 18
- 238000012408 PCR amplification Methods 0.000 claims description 8
- 238000010222 PCR analysis Methods 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 229920003023 plastic Polymers 0.000 claims description 6
- 239000004033 plastic Substances 0.000 claims description 6
- 238000003753 real-time PCR Methods 0.000 claims description 6
- 230000000630 rising effect Effects 0.000 claims description 4
- 238000001228 spectrum Methods 0.000 claims description 4
- 238000005459 micromachining Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 3
- 238000003672 processing method Methods 0.000 claims description 3
- 238000002189 fluorescence spectrum Methods 0.000 abstract description 14
- 238000007731 hot pressing Methods 0.000 abstract 1
- 230000010076 replication Effects 0.000 abstract 1
- 238000004458 analytical method Methods 0.000 description 10
- 238000005516 engineering process Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000012742 biochemical analysis Methods 0.000 description 3
- 238000012921 fluorescence analysis Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 2
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 2
- 239000004926 polymethyl methacrylate Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 235000015170 shellfish Nutrition 0.000 description 2
- 230000003595 spectral effect Effects 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000011953 bioanalysis Methods 0.000 description 1
- 230000029918 bioluminescence Effects 0.000 description 1
- 238000005415 bioluminescence Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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Abstract
The chip includes upper piece of organic glass, and lower piece of organic glass. N pieces of independent simulation micro channel are setup on the said lower piece of organic glass. Being setup on upper piece of organic glass, micropores are corresponding to two ends of the said micro channels, and micropores are sealed. Using hot pressing linkage seal upper piece of organic glass and lower piece of organic glass permanently. Fluorescent materials with incremental fluorescence intensities are injected to N pieces of independent simulation micro channel in sequence. The selected fluorescence intensities can reflect changing trend of amplification curve of number of replications for characteristic gene of the biologic PCR fluorescent reagent. The invention solves issue of working calibration of detection device for PCR fluorescence spectrum.
Description
Technical field
The present invention relates to the metering comparison device of a kind of biochemical micro-total analysis system chip PCR (PCR) fluorescence microchannel spectral detection system, be mainly used in the calibration of microchannel fluorescence spectrum pick-up unit, belong to biology and medical science detection range.
Background technology
Along with micro-total analysis system (Miniaturized Total AnalysisSystems at the beginning of the nineties in last century, μ-TAS) notion is proposed first, in recent years, all kinds of bioanalysiss on the micro-total analysis system conceptual foundation and chemical analysis chip (comprising: the research fast development micro-fluidic biological pcr chip), the micro-total analysis system technology has become the most important research and development direction of innovation formula analytical and testing instrument and technology, is subjected to the concern of countries in the world deeply.
(μ-TAS) is an analysis experiment lab system of realizing the integrated and structure micromation of the function of whole analytic processes on the basis of microminiaturization to so-called micro-total analysis system.Generally, it is whole breadboard function, be the many processes and the step of biochemical analysis, comprise sampling, dilute, add reagent, reaction, separation, detection etc. and be integrated on the as far as possible little operating platform that present this micromation operating platform is the chip (structure miniature size to tens square millimeters of development) about 100 square millimeters.μ-TAS has outstanding features such as detection speed is fast, sample dosage is few, flux height, is widely used in the research of all kinds of biochemical analysis chips.In μ-TAS, chip volume is very little, and its sample size is a micro updating only, and the detection cell volume is very little, and finishes in the level in second more than the analyzing and testing, so require very high to the sensitivity and the response speed of its detection architecture.Detection method is the key factor of the final detection limit of decision μ-TAS, is one of core technology of μ-TAS.In the μ that grew up in recent years-TAS detection method, the fluorescence spectrum detection method becomes most popular detection technique in μ-TAS research field gradually because its selectivity is good, the trace qualitative and quantitative analysis is highly sensitive and characteristics such as non-destructive detection.
The biological PCR fluorescence spectrum detection method of biochemical analysis chip, actual is that fluorescent material in the microchannel on the chip is carried out spectral detection.There are some different with the fluorescence spectrum detection technique in the chemical analysis field: at first, small and the signal of the amount of detected object sends a little little, thereby the detected fluorescence spectrum weak output signal that causes, need use the microchannel fluorescence spectrum pick-up unit of high detection sensitivity; Secondly, also do not have the standard substance in a kind of picture chemical analysis field at present, can be used for calibrating biological PCR fluorescence microchannel spectrum detection device.
Summary of the invention
A kind of biological PCR fluorescence microchannel comparison examination criteria chip production method and device thereof are provided among the present invention, and this chip can be used for calibrating biological PCR fluorescence microchannel spectrum detection device.
In order to achieve the above object, the utility model has taked to take off technical scheme.A kind of standard chips that is used for the biological PCR micro fluoroscopic examination, this chip mainly includes an organic glass 3, following sheet organic glass 4, wherein, 4 are provided with the individual microchannel 2 of independently simulating of N on following sheet organic glass, 3 corresponding simulation two ends, microchannel are respectively arranged with micropore 1 and seal this hole on last organic glass, the method permanent seal of last slice organic glass 3 and the 4 employing thermocompression bondings of following sheet organic glass; Be marked with the fluorescent material that fluorescence intensity increases progressively N simulation successively in the microchannel, selected fluorescence intensity can reflect the variation tendency of copy number amplification curve of the specific gene of this kind biological PCR fluorescent reagent.
The span of described N is 4～40.
Described last slice organic glass 3, the thickness of sheet organic glass 4 is 1mm down.
A kind of preparation method who is used for the standard chips of biological PCR micro fluoroscopic examination recited above, this method is carried out according to the following steps:
1) two organic glass up and down that choosing size is identical, on excimer laser micromachining system, use the identical manufacture craft in microchannel of directly writing micro-processing method, adopt and preparing in the biological PCR analysis chip, microchannel 2 is independently simulated in N of 4 processing on following sheet organic glass, and width, the degree of depth, the surfaceness of simulation microchannel 2 are identical with the microchannel in the biological PCR analysis chip;
2) on the last organic glass 3, simulate corresponding position, 2 two ends, microchannel and all be processed with micropore 1 with each bar on the following sheet organic glass 4, micropore 1 is connected with the two ends of simulation microchannel 2, and adopting the thermocompression bonding mode again is one with two organic glass bondings;
3) the low platform area of the fluorescence signal on the copy number amplification curve of the specific gene of biological PCR fluorescent reagent, fluorescence signal lifting thresholding (C
TValue), fluorescence signal rising area, the high platform area of fluorescence signal choose the point of the different fluorescence intensities of representing in the pcr amplification process respectively, selected N point can reflect this plots changes;
4) use ripe commercial real-time fluorescent quantitative PCR detector, in the plastics reaction tube, a kind of biological PCR fluorescence reaction thing is carried out in the process of pcr amplification, when pcr amplification temperature cycles number of times proceeds to the cycle index of first selected some correspondence, biological PCR fluorescence reaction thing in one of them plastics reaction tube used the ripe commercial real-time fluorescent quantitative PCR detector from this take out, be injected into down by micropore 1 in first simulation microchannel 2 of sheet organic glass 4, seal the micropore 1 at these 2 two ends, simulation microchannel then;
5), prepare the as above PCR fluorescence reaction thing of institute's reconnaissance correspondence more successively, and be injected into successively in the corresponding simulation microchannel 2, and seal the micropore 1 at its two ends according to said method.
Design concept of the present invention is: the microchannel of the biological PCR fluorescence analysis chip of microchannel flow control working method is the microcarrier container of analyte in the analysis chip.In the present invention, the identical manufacture craft in microchannel in making the employing of simulation microchannel and preparing biological PCR fluorescence analysis chip, and all kinds of parameters (as: width of microchannel, the degree of depth and surfaceness etc.) of simulation microchannel are the same with the parameter of microchannel in the biological PCR fluorescence analysis chip.Therefore, when biological PCR fluorescence spectrum pick-up unit is calibrated, can keep consistency.Simulate the homoreactant that injects different fluorescence intensities in the microchannel successively for N among the present invention, selected fluorescence intensity is to determine as follows.Be exactly when biological PCR reacts, the fluorescence intensity when the shellfish number of specific gene increases with the specific gene copy number increases according to certain rules.The PCR fluorescent quantitative detection device is exactly by the detection to the fluorescence intensity that increases with the specific gene copy number, comes shellfish number to specific gene to carry out detection by quantitative.The rule of the copy number amplification curve of biological PCR specific gene as shown in Figure 3, general ordinate be and the consistent fluorescence intensity of specific gene amplification copy number, horizontal ordinate is the biological PCR temperature cycles number of times that increases in the biological round pcr.In the copy number amplification curve of biological PCR specific gene, generally be divided into: fluorescence signal hangs down platform area, fluorescence signal lifting thresholding (C
TValue), the fluorescence signal rising area, the high platform area of fluorescence signal in this four " zone ", is chosen N the point that can reflect this plots changes.The homoreactant of the different fluorescence intensities that this N point is corresponding is injected into respectively in N the simulation microchannel successively, so this N PCR fluorescence reaction thing of simulating in the microchannel can reflect the trend of the bioluminescence change curve that this kind fluorescent PCR increases.Calibrate PVR fluorescence spectrum pick-up unit with this.
Use the present invention as follows: to use PCR microchannel fluorescence spectrum pick-up unit that a certain microchannel of the calibration chip among the present invention is detected to the calibration steps of PCR microchannel fluorescence spectrum pick-up unit, again the fluorescence intensity in testing result and this passage is compared, if these two groups comparison fluorescence intensity levels occur inequality, we just are as the criterion with the fluorescence intensity of the expression in the calibration chip, and PCR microchannel fluorescence spectrum pick-up unit is calibrated.This device can only be calibrated the identical PCR microchannel fluorescence spectrum pick-up unit of reactant in the simulation microchannel in fluorescence reaction thing and this device in its microchannel.
Through experimental verification, the present invention has solved the problem of the work calibration of PCR fluorescence spectrum pick-up unit in chip use and micro integrated research.
Description of drawings
Fig. 1 has six structural drawing of the present invention of independently simulating the microchannel
Fig. 2 sectional view of the present invention
The copy number amplification curve of Fig. 3 biological PCR specific gene
Among the figure: 1, micropore, 2, the simulation microchannel, 3, last slice organic glass, 4, sheet organic glass down.
Embodiment
Below in conjunction with Fig. 1～Fig. 3 embodiments of the invention are described.This chip mainly includes a PMMA organic glass 3, following sheet PMMA organic glass 4, and last slice organic glass 3, the thickness of sheet organic glass 4 is 1mm down.Wherein, 4 are provided with six and independently simulate microchannel 2 on following sheet organic glass, corresponding simulation two ends, microchannel are processed with micropore 1 respectively and seal this micropore 1 on last organic glass 3, the method permanent seal of last slice organic glass 3 and the 4 employing thermocompression bondings of following sheet organic glass; Be marked with the fluorescent material that fluorescence intensity increases progressively six simulations successively in the microchannels 2, selected fluorescence intensity can reflect the variation tendency of copy number amplification curve of the specific gene of this kind biological PCR fluorescent reagent.
Above described chip production method, it is characterized in that this method is carried out according to the following steps:
1) two organic glass up and down that choosing size is identical, on excimer laser micromachining system, use the identical manufacture craft in microchannel of directly writing micro-processing method, adopt and preparing in the biological PCR analysis chip, microchannel 2 is independently simulated in N of 4 processing on following sheet organic glass, and width, the degree of depth, the surfaceness of simulation microchannel 2 are identical with the microchannel in the biological PCR analysis chip.
2) on the last organic glass, simulate corresponding position, 2 two ends, microchannel and all be processed with micropore 1 with each bar on the following sheet organic glass, micropore 1 is connected with the two ends of simulation microchannel 2, and adopting the thermocompression bonding mode again is one with two organic glass bondings.
3) the low platform area of the fluorescence signal on the copy number amplification curve of the specific gene of biological PCR fluorescent reagent is chosen 2 points, chooses fluorescence signal lifting thresholding (C
TValue) be a point, choose 2 points in the fluorescence signal rising area, choose 1 point in the high platform area of fluorescence signal, 6 selected points can reflect this plots changes.
4) use ripe commercial real-time fluorescent quantitative PCR detector, in the plastics reaction tube, a kind of biological PCR fluorescence reaction thing is carried out in the process of pcr amplification, when pcr amplification temperature cycles number of times proceeds to the cycle index of first selected some correspondence, biological PCR fluorescence reaction thing in one of them plastics reaction tube used the ripe commercial real-time fluorescent quantitative PCR detector from this take out, be injected into down by micropore 1 in first simulation microchannel 2 of sheet organic glass, seal the micropore 1 at these 2 two ends, simulation microchannel then.
5), prepare the as above PCR fluorescence reaction thing of institute's reconnaissance correspondence more successively, and be injected into successively in the corresponding simulation microchannel, and seal the micropore 1 at its two ends according to said method.
Claims (4)
1, a kind of standard chips that is used for the biological PCR micro fluoroscopic examination, it is characterized in that: this chip mainly includes an organic glass (3), following sheet organic glass (4), wherein, 4 are provided with the individual microchannel (2) of independently simulating of N on following sheet organic glass, go up corresponding simulation two ends, microchannel at last organic glass (3) and be respectively arranged with micropore (1) and seal this hole, the method permanent seal of last slice organic glass (3) and following sheet organic glass (4) employing thermocompression bonding; Be marked with the fluorescent material that fluorescence intensity increases progressively N simulation successively in the microchannel, selected fluorescence intensity can reflect the variation tendency of copy number amplification curve of the specific gene of this kind biological PCR fluorescent reagent.
2, the microchannel chip that is used for biological PCR fluorescence microchannel spectrum detection device comparison standard according to claim 1, it is characterized in that: the span of described N is 4～40.
3, the microchannel chip that is used for biological PCR fluorescence microchannel spectrum detection device comparison standard according to claim 1 is characterized in that: described last slice organic glass (3), the thickness of sheet organic glass (4) is 1mm down.
4, a kind of preparation method who is used for the standard chips of biological PCR micro fluoroscopic examination described in the claim 1 is characterized in that this method is carried out according to the following steps:
1) two organic glass up and down that choosing size is identical, on excimer laser micromachining system, use the identical manufacture craft in microchannel of directly writing micro-processing method, adopt and preparing in the biological PCR analysis chip, microchannel (2) is independently simulated in N of (4) processing on following sheet organic glass, and width, the degree of depth, the surfaceness of simulation microchannel (2) are identical with the microchannel in the biological PCR analysis chip;
2) go up at last organic glass (3), simulate microchannel with each bar on the following sheet organic glass (4)
(2) corresponding position, two ends all is processed with micropore (1), and micropore (1) is connected with the two ends of simulation microchannel (2), and adopting the thermocompression bonding mode again is one with two organic glass bondings;
3) the low platform area of the fluorescence signal on the copy number amplification curve of the specific gene of biological PCR fluorescent reagent, fluorescence signal lifting thresholding (C
TValue), fluorescence signal rising area, the high platform area of fluorescence signal choose the point of the different fluorescence intensities of representing in the pcr amplification process respectively, selected N point can reflect this plots changes:
4) use ripe commercial real-time fluorescent quantitative PCR detector, in the plastics reaction tube, a kind of biological PCR fluorescence reaction thing is carried out in the process of pcr amplification, when pcr amplification temperature cycles number of times proceeds to the cycle index of first selected some correspondence, biological PCR fluorescence reaction thing in one of them plastics reaction tube used the ripe commercial real-time fluorescent quantitative PCR detector from this take out, be injected into down by micropore (1) in first simulation microchannel (2) of sheet organic glass (4), seal the micropore (1) at these two ends, simulation microchannel (2) then;
5), prepare the as above PCR fluorescence reaction thing of institute's reconnaissance correspondence more successively, and be injected into successively in the corresponding simulation microchannel (2), and seal the micropore (1) at its two ends according to said method.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2006100888189A CN100533144C (en) | 2006-07-19 | 2006-07-19 | Standard chip for biological PCR micro path fluorescent detection and its preparing method |
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2006100888189A CN100533144C (en) | 2006-07-19 | 2006-07-19 | Standard chip for biological PCR micro path fluorescent detection and its preparing method |
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| CN1900715A true CN1900715A (en) | 2007-01-24 |
| CN100533144C CN100533144C (en) | 2009-08-26 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277294A (en) * | 2011-08-03 | 2011-12-14 | 浙江大学 | High-density array chip device used for digital nucleic acid amplification application of device |
| CN104076031A (en) * | 2013-03-28 | 2014-10-01 | 优志旺电机株式会社 | Test piece for point inspection and analyzing device |
| CN104880440A (en) * | 2015-05-11 | 2015-09-02 | 深圳市天吉新创科技有限公司 | Standard color card, production method of standard color card and biological analysis detection set |
| CN110146477A (en) * | 2019-05-31 | 2019-08-20 | 成都博奥晶芯生物科技有限公司 | A kind of preparation method and its calibration method of concentration gradient calibration chip |
| CN111545257A (en) * | 2020-04-23 | 2020-08-18 | 成都博奥独立医学实验室有限公司 | Multifunctional micro-fluidic chip and preparation and application thereof |
-
2006
- 2006-07-19 CN CNB2006100888189A patent/CN100533144C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277294A (en) * | 2011-08-03 | 2011-12-14 | 浙江大学 | High-density array chip device used for digital nucleic acid amplification application of device |
| CN104076031A (en) * | 2013-03-28 | 2014-10-01 | 优志旺电机株式会社 | Test piece for point inspection and analyzing device |
| CN104880440A (en) * | 2015-05-11 | 2015-09-02 | 深圳市天吉新创科技有限公司 | Standard color card, production method of standard color card and biological analysis detection set |
| CN110146477A (en) * | 2019-05-31 | 2019-08-20 | 成都博奥晶芯生物科技有限公司 | A kind of preparation method and its calibration method of concentration gradient calibration chip |
| CN110146477B (en) * | 2019-05-31 | 2023-08-04 | 成都博奥晶芯生物科技有限公司 | Preparation method of concentration gradient calibration chip and calibration method thereof |
| CN111545257A (en) * | 2020-04-23 | 2020-08-18 | 成都博奥独立医学实验室有限公司 | Multifunctional micro-fluidic chip and preparation and application thereof |
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| Publication number | Publication date |
|---|---|
| CN100533144C (en) | 2009-08-26 |
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