CN109916871A - A kind of FRET efficiency method for quantitative measuring applied to Apoptosis detection - Google Patents
A kind of FRET efficiency method for quantitative measuring applied to Apoptosis detection Download PDFInfo
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- CN109916871A CN109916871A CN201910264449.1A CN201910264449A CN109916871A CN 109916871 A CN109916871 A CN 109916871A CN 201910264449 A CN201910264449 A CN 201910264449A CN 109916871 A CN109916871 A CN 109916871A
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Abstract
The present invention relates to a kind of FRET efficiency method for quantitative measuring applied to Apoptosis detection.The present invention has the characteristics that the concentration ratio 1:1 of donor fluorophore and acceptor fluorophore for Apoptosis detection FRET probe, correction system fluorescence crosstalk is not needed, it does not need measuring system sensitization and transforming factor is quenched yet, it only needs Plasmid series known to 1 parameter as reference, the Quantitative Monitoring of Apoptosis time-space process can be fast implemented.The present invention is not high to the configuration requirement of instrument, at low cost, has many advantages, such as to can be realized FRET apparent efficiency dynamic, quantitative measurment.
Description
Technical field
The present invention relates to the method for quantitative measuring of fluorescence resonance energy transfer (FRET) apparent efficiency, and in particular to Yi Zhongying
FRET efficiency method for quantitative measuring for Apoptosis detection.
Background technique
Fluorescence resonance energy transfer (FRET) technology is all widely used in biophysics, biochemistry.In conjunction with
Fluorescence microscope can be accurately positioned biomolecule and carry out real-time monitoring, such as protein-egg to its molecular dynamics behavior
FRET caused by interaction, protein-DNA interaction, proteinase activity, protein conformation variation of white matter etc. is apparently imitated
Rate variation.
Quick, the accurate detection of Apoptosis has important value in biology, medical domain, and now some studies pointed out that Guang day eggs
White enzyme (caspases) plays a crucial role in apoptosis process.Currently, mainly passing through for the FRET probe of apoptosis detection
The activity for detecting the Caspases such as caspase-2, caspase-3, caspase-8, caspase-9 carries out the space-time spy of apoptosis
Journal of Sex Research.Apoptosis detects FRET probe by linking fluorophor on the specific substrate of caspases, passes through analysis of fluorescence
The process of FRET efficiency change research apoptosis between group.This kind of probe has outstanding feature: donor fluorophore in probe
With the concentration ratio 1:1 of acceptor fluorophore.
Report that FRET apparent efficiency measurement method can be not only used for Apoptosis at present, it can also be used to protein interaction
Deng the research of other intracellular molecules events.But when for apoptosis process analysis, these method and steps are excessively cumbersome, institute
Need equipment cost higher.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of FRET efficiency quantitative measurments applied to Apoptosis detection
Method, it is only necessary to which Plasmid series known to 1 parameter can fast implement the quantitative prison of Apoptosis time-space process as reference
It surveys.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of FRET efficiency method for quantitative measuring applied to Apoptosis detection, comprising the following steps:
Step S1: it selects a kind of for acceptor density ratioWith FRET apparent efficiency EApp stringKnown Plasmid series are as ginseng
According to plasmid；
Step S2: donor excitation is generated referring to plasmid using donor exciter filter, and is filtered by donor emission
Mating plate and CCD obtain cell fluorescent images, measure fluorescence intensity ID string；
Step S3: receptor excitation is generated referring to plasmid using receptor exciter filter, and is filtered by acceptor emission
Mating plate and CCD obtain cell fluorescent images, measure fluorescence intensity IA string；
Step S4: donor excitation apoptosis is generated using donor exciter filter and detects FRET probe, and passes through confession
Body emits optical filter and CCD obtains cell fluorescent images, measures fluorescence intensity ID apoptosis；
Step S5: using receptor exciter filter generate receptor excitation apoptosis detect FRET probe, and by by
Body emits optical filter and CCD obtains cell fluorescent images, measures fluorescence intensity IA apoptosis；
Step S6: building apoptosis detects FRET probe apparent efficiency EApp apoptosisComputation model, and obtained according to step S1-S5
Apoptosis detection FRET probe apparent efficiency E is calculated in dataApp apoptosis。
Further, it is described referring to supplied in plasmid, acceptor fluorophore and apoptosis detection FRET probe in supply, acceptor fluorescence
Group is consistent.
Further, it is supplied in the apoptosis detection FRET probe, acceptor density ratio is 1:1
Further, two groups of independent filter sets, including donor filter set and receptor filter is arranged in the measurement process
Mating plate group；The donor filter set includes: 1 piece of donor exciter filter, 1 piece of donor dichroic sheet, 1 piece of donor emission optical filtering
Piece, for measuring ID stringAnd ID apoptosis；The receptor filter set includes: 1 piece of receptor exciter filter, 1 piece of receptor dichroic sheet, 1 piece
Acceptor emission filter, and receptor exciting light produced by receptor exciter filter can not excited donor fluorophor, for surveying
Measure IA stringAnd IA apoptosis。
Further, the step S6 specifically:
Step S61: according to
It calculates
In formula: ID stringTo be C referring to concentration in plasmidD stringThe fluorescence intensity that is emitted by donor filter set of donor；EApp string
For the FRET apparent efficiency referring to plasmid；IA stringTo be C referring to concentration in plasmidA stringReceptor emitted by receptor filter set
Fluorescence intensity；For incident intensity；texDFor donor exciter filter transmittance function；CD stringIt is dense referring to donor in plasmid
Degree；For the extinction coefficient referring to donor in plasmid；QDFor the quantum yield referring to donor in plasmid；tDMDFor donor dichroic sheet
Transmittance function；temDFor donor emission optical filter transmittance function；SDIt is CCD to the receptance function of donor-emitted light；texA
For receptor exciter filter transmittance function；CA stringFor referring to acceptor density in plasmid；For the delustring system referring to receptor in plasmid
Number；QAFor the quantum yield referring to receptor in plasmid；tDMAFor the transmittance function of receptor dichroic sheet；temAFor acceptor emission optical filtering
Piece transmittance function；SAIt is CCD to the receptance function of acceptor emission light；
Step S62: according to Apoptosis detect FRET probe in supply, acceptor density ratio be 1:1, and donor fluorescent albumen,
Acceptor fluorescent protein is identical as referring to plasmid, and measuring instrument and filter set are also consistent, obtains:
Then:
Compared with the prior art, the invention has the following beneficial effects:
The present invention only needs a kind to refer to plasmid and 2 groups of independent filter sets, and is not needing correction system fluorescence crosstalk
The Quantitative Monitoring of Apoptosis time-space process is realized in the case where transforming factor (G-factor) is quenched with measuring system sensitization.Have
It is not high to the configuration requirement of instrument, it is at low cost, have many advantages, such as to can be realized FRET apparent efficiency dynamic, quantitative measurment
Detailed description of the invention
Fig. 1 is test result schematic diagram in one embodiment of the invention.
Specific embodiment
The present invention will be further described with reference to the accompanying drawings and embodiments.
Please refer to Fig. 1, the present embodiment in order to more preferably examine the validity of measurement method, using it is a kind of supply, acceptor density ratio
The plasmid C32V simulation apoptosis of 1:1 detects FRET probe, and measures the FRET apparent efficiency of the plasmid；
1, analogue probe to be measured
C32V is for acceptor density ratio 1:1
2, referring to Plasmid series
VCV cerulean--venus Plasmid series are for acceptor density ratio 1:2, FRET apparent efficiency EApp string=69.3 ±
1.0% (being measured using FLIM method)
3, measuring instrument and filter set
Microscope: Olympus IX73
CCD: shore pine (HAMAMATSU) ORCA-Flash4.0 LT C1140-42U CMOS
Excitation light source: metal halid lamp Olympus U-HGLGPS
Object lens: 1.3 oil of Olympus 40X/NA
Donor filter set (Chroma): exciter filter 435/20, dichroic sheet 455LP, transmitting optical filter 480/30
Receptor filter set (Chroma): exciter filter 495/20, dichroic sheet 515LP, transmitting optical filter 540/30
4, cell culture and transfection
HeLa cell is placed on the newborn bovine serum that 90%DMEM culture medium is added 10% and is trained containing 37 DEG C of 5% carbon dioxide
It supports and is cultivated in case.It is cells trypsinised, it goes in 15mm Tissue Culture Dish, after culture 24 hours, when cell is grown to
When 70-90%, with in-vitro transfection reagent TurbofectTMPlasmid wink is transferred to HeLa cell.
5, measurement process
11 cells transfected referring to plasmid VCV are chosen, I in fluorescent image is obtainedD stringAnd IA string, and calculateTake it
Average value.
| Cell serial number | IA string/ID string |
| 1 | 8.153665973 |
| 2 | 7.594195923 |
| 3 | 7.301798161 |
| 4 | 6.954468208 |
| 5 | 8.09711851 |
| 6 | 8.307591258 |
| 7 | 8.195907037 |
| 8 | 7.734814357 |
| 9 | 7.643352486 |
| 10 | 7.667624668 |
| 11 | 7.860605342 |
| Average value | 7.773740175 |
The cell for choosing 10 transfection analogue probe C32V to be measured, obtains I in fluorescent imageD is to be measuredAnd IA is to be measured, and by previous step
Obtained in rapidAverage value brings formula into, calculates EApp is to be measured.The calculation formula of C32V plasmid apparent efficiency:
Because plasmid C32V plasmid to be measured for acceptor density ratio be 1:1, with reference to Plasmid series for acceptor density ratio be 1:
2, with reference to the apparent efficiency E of plasmid VCVApp string=69.3%, so formula can be further simplified are as follows:
| Cell serial number | EApp is to be measured |
| 1 | 0.313270564 |
| 2 | 0.328022443 |
| 3 | 0.319582091 |
| 4 | 0.279902152 |
| 5 | 0.331665889 |
| 6 | 0.331999346 |
| 7 | 0.32238694 |
| 8 | 0.298031987 |
| 9 | 0.334395811 |
| 10 | 0.327791721 |
| Average value | 31.8704894 ± 1.6% |
6, measurement result is analyzed
Use FLIM method measurement C32V plasmid apparent efficiency be 31.2%, the present invention measurement C32V plasmid apparent effect
Rate is 31.8704894 ± 1.6%, it was demonstrated that the accuracy of the invention measured.And the standard deviation of measurement result of the present invention is
1.6%, it was demonstrated that the stability of the invention measured.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
Claims (5)
1. a kind of FRET efficiency method for quantitative measuring applied to Apoptosis detection, which comprises the following steps:
Step S1: it selects a kind of for acceptor density ratioWith FRET apparent efficiency EApp stringKnown Plasmid series are used as referring to matter
Grain；
Step S2: donor excitation is generated referring to plasmid using donor exciter filter, and passes through donor emission optical filter
Cell fluorescent images are obtained with CCD, measure fluorescence intensity ID string；
Step S3: receptor excitation is generated referring to plasmid using receptor exciter filter, and passes through acceptor emission filter
Cell fluorescent images are obtained with CCD, measure fluorescence intensity IA string；
Step S4: donor excitation apoptosis is generated using donor exciter filter and detects FRET probe, and is sent out by donor
It penetrates optical filter and CCD obtains cell fluorescent images, measure fluorescence intensity ID apoptosis；
Step S5: receptor excitation apoptosis is generated using receptor exciter filter and detects FRET probe, and is sent out by receptor
It penetrates optical filter and CCD obtains cell fluorescent images, measure fluorescence intensity IA apoptosis；
Step S6: building apoptosis detects FRET probe apparent efficiency EApp apoptosisComputation model, and data are obtained according to step S1-S5
Apoptosis detection FRET probe apparent efficiency E is calculatedApp apoptosis。
2. a kind of FRET efficiency method for quantitative measuring applied to Apoptosis detection according to claim 1, feature
Be: it is described referring to supplied in plasmid, acceptor fluorophore and apoptosis detection FRET probe in supply, acceptor fluorophore it is consistent.
3. a kind of FRET efficiency method for quantitative measuring applied to Apoptosis detection according to claim 1, feature
It is: is supplied in the apoptosis detection FRET probe, acceptor density ratio is 1:1.
4. a kind of FRET efficiency method for quantitative measuring applied to Apoptosis detection according to claim 1, feature
Be: two groups of independent filter sets, including donor filter set and receptor filter set are arranged in the measurement process；The confession
Body filter set includes: 1 piece of donor exciter filter, 1 piece of donor dichroic sheet, 1 piece of donor emission optical filter, for measuring ID string
And ID apoptosis；The receptor filter set includes: 1 piece of receptor exciter filter, 1 piece of receptor dichroic sheet, 1 piece of acceptor emission optical filtering
Piece, and receptor exciting light produced by receptor exciter filter can not excited donor fluorophor, for measuring IA stringAnd IA apoptosis。
5. the FRET efficiency method for quantitative measuring according to claim 3 applied to Apoptosis detection, it is characterised in that:
The step S6 specifically:
Step S61: according to
It calculates
In formula: ID stringTo be C referring to concentration in plasmidD stringThe fluorescence intensity that is emitted by donor filter set of donor；EApp stringFor ginseng
According to the FRET apparent efficiency of plasmid；IA stringTo be C referring to concentration in plasmidA stringThe fluorescence that is emitted by receptor filter set of receptor
Intensity；For incident intensity；texDFor donor exciter filter transmittance function；CD stringFor referring to donor concentrations in plasmid；For the extinction coefficient referring to donor in plasmid；QDFor the quantum yield referring to donor in plasmid；tD perfume (or spice) DFor the saturating of donor dichroic sheet
Cross rate function；tE perfume (or spice) DFor donor emission optical filter transmittance function；SDIt is CCD to the receptance function of donor-emitted light；texAFor by
Body exciter filter transmittance function；CA stringFor referring to acceptor density in plasmid；For the extinction coefficient referring to receptor in plasmid；
QAFor the quantum yield referring to receptor in plasmid；tD perfume (or spice) AFor the transmittance function of receptor dichroic sheet；tE perfume (or spice) AFor acceptor emission filter
Transmittance function；SAIt is CCD to the receptance function of acceptor emission light；
Step S62: detected in FRET probe according to Apoptosis supply, acceptor density ratio is 1:1, and donor fluorescent albumen, receptor
Fluorescin is identical as referring to plasmid, and measuring instrument and filter set are also consistent, obtains:
Then:
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Citations (4)
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|---|---|---|---|---|
| WO2001066723A2 (en) * | 2000-03-10 | 2001-09-13 | Mcgill University | Hetero-oligomeric g protein-coupled receptors as drug target |
| CN102636465A (en) * | 2011-10-26 | 2012-08-15 | 华南师范大学 | FRET (Fluorescence Resonance Energy Transfer) efficiency quantitative detecting method based on partial acceptor photo-bleaching and donor-acceptor alternate excitation |
| CN106290268A (en) * | 2016-07-12 | 2017-01-04 | 华南师范大学 | A kind of method utilizing single series connection donor-acceptor structure measurement receptor donor ratio of extinction coefficient |
| CN108732147A (en) * | 2018-04-23 | 2018-11-02 | 南京邮电大学 | The method for detecting apoptosis process based on FRET effects |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001066723A2 (en) * | 2000-03-10 | 2001-09-13 | Mcgill University | Hetero-oligomeric g protein-coupled receptors as drug target |
| CN102636465A (en) * | 2011-10-26 | 2012-08-15 | 华南师范大学 | FRET (Fluorescence Resonance Energy Transfer) efficiency quantitative detecting method based on partial acceptor photo-bleaching and donor-acceptor alternate excitation |
| CN106290268A (en) * | 2016-07-12 | 2017-01-04 | 华南师范大学 | A kind of method utilizing single series connection donor-acceptor structure measurement receptor donor ratio of extinction coefficient |
| CN108732147A (en) * | 2018-04-23 | 2018-11-02 | 南京邮电大学 | The method for detecting apoptosis process based on FRET effects |
Non-Patent Citations (2)
| Title |
|---|
| MARKUS REHM等: "Single-cell Fluorescence Resonance Energy Transfer Analysis Demonstrates That Caspase Activation during Apoptosis Is a Rapid Process ROLE OF CASPASE-3", 《JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| 张建伟等: "荧光共振能量转移( FRET) 的定量检测及其应用", 《华南师范大学学报（自然科学版）》 * |
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