CN102914529A - Accurate correction method of fluorescence quenching rate in inner filtering efficiency process of fluorescence quenching system - Google Patents

Abstract

The invention discloses an accurate correction method of a fluorescence quenching rate in an inner filtering efficiency process of a fluorescence quenching system, and the method is characterized by comprising the following steps of: firstly, correcting influences of competitive absorption on a light spectrum; then, correcting the influences of absorption on the light spectrum; and correcting the influences of absorption distribution on the light spectrum. The method disclosed by the invention can be used for carrying out combined correction on the influences caused by a fluorescence inner filtering efficiency and absorption distribution, so as to ensure that the light spectrum of the quenching system is accurately corrected; and the fluorescence quenching rate in the quenching process is really reflected.

Description

The precision correcting method of fluorescent quenching rate when there is inner filtering effect in the fluorescent quenching system

Technical field

The present invention relates to fluorescent spectroscopy, particularly in the situation that has the fluorescence inner filtering effect, the spectroscopic analysis methods in the fluorescent quenching test.

Background technology

Fluorometry is the method that the fluorescence Spectra according to material is carried out (comprising the parameters such as intensity, shape) identification and assay, wherein fluorescence quenching method can be used for analyzing that some autofluorescence is difficult detects but have the content of the material of quencher characteristic, can be used for also in the analysis of fluorescence quenching process between two kinds of materials that the energy of (being between fluorescent material and the quencher) shifts or electronics shifts.

For the fluorescent quenching system, when having comparatively seriously crossover between the absorption spectra of fluorescent material and quencher and the corresponding fluorescence Spectra, fluoroscopic examination will be subject to the interference of fluorescence inner filtering effect (IFE) inevitably.Inner filtering effect can be divided into competition by its mechanism of action and absorb (primary IFE) and absorb (secondary IFE) two processes again, can directly affect intensity and the spectrum shape of fluorescence Spectra, and the parameters such as the intensity of spectrum, shape are the fundamental basis of fluorescence analysis, therefore, if the impact of inner filtering effect is not proofreaied and correct, just can't obtain correct fluorescence analysis result.

In the measuring process, add behind the quencher fluorescent reagent in the biased sample to exciting Optical Absorption to distribute before add quencher pure fluorescent reagent to exciting the Optical Absorption distribution to change, difference on this absorption distributes, can exert an influence to fluorescence intensity equally, the interference spectrum analysis result, when quantitatively detecting, also must be proofreaied and correct.

The impact that forms by absorb distributing and the impact of fluorescence inner filtering effect is accompanied, simultaneously appearance, the artefact of jointly causing fluorescent quenching rate virtual height.Present existing alignment technique is only proofreaied and correct separately for the fluorescence inner filtering effect, and ignored the impact that the absorption distributions that occurs simultaneously with it brings, thereby fundamentally can't carry out accurate correction to gained fluorescent quenching rate data, obtain real quencher rate, restricted the application of fluorescence quenching method.

Summary of the invention

The present invention is for avoiding the existing weak point of above-mentioned prior art, the precision correcting method of fluorescent quenching rate when providing a kind of fluorescent quenching system to have inner filtering effect, described fluorescent quenching system refers to: the fluorometric investigation sample that does not generate ground-state complex after having fluorescence inner filtering effect and fluorescent reagent and quencher mixing.Utilize the given bearing calibration of the present invention, can carry out simultaneously accurate correction to the impact of fluorescence inner filtering effect and the generation of absorption distributions, thereby obtain truly to reflect the actual quencher rate of fluorescent quenching process.

Technical solution problem of the present invention adopts following technical scheme:

The characteristics of the precision correcting method of fluorescent quenching rate were to carry out as follows when there was inner filtering effect in fluorescent quenching system of the present invention:

A, correction competition absorb the impact on spectrum:

With function I ₁(λ) expression does not add before the quencher fluorescent material rear fluorescence measurement spectrum that produces that is stimulated, with function I ₁' (λ) expression adds the actual fluorescence Spectra of sending of fluorescent material behind the quencher, then have:

${{\mathrm{<figure-callout\; id="1"\; label="I"\; filenames="HDA00002358356100011.png,HDA00002358356100013.png"\; state="\{\{state\}\}">I</figure-callout>}}_1}^{\&prime;}\left(\mathrm{\&lambda;}\right)=\frac{{10}^{-\mathrm{\&Delta;}{E}_1}(1-{10}^{-\mathrm{\&Delta;}{E}_2})[1-{10}^{-n\left(\mathrm{\&Delta;}{E}_1+\mathrm{\&Delta;}{E}_2\right)}]}{(1-{10}^{-n\&CenterDot;\mathrm{\&Delta;}{E}_2})[1-{10}^{-(\mathrm{\&Delta;}{E}_1+\mathrm{\&Delta;}{E}_2)}]}\&times;I_1\left(\mathrm{\&lambda;}\right)---\left(1\right)$

In the formula (1), Δ E ₁=ε ₁c ₁Δ l is first dullness of the corresponding excitation wavelength of quencher; Δ E ₂=ε ₂c ₂Δ l is first dullness of the corresponding excitation wavelength of fluorescent material; Δ l is the thickness of sample segmentation elementary layer; N is the number of plies of sample segmentation elementary layer; ε ₁Molar absorption coefficient for the corresponding excitation wavelength of quencher; ε ₂Molar absorption coefficient for the corresponding excitation wavelength of fluorescent material; c ₁Volumetric molar concentration for quencher; c ₂Volumetric molar concentration for fluorescent material.

Order: width and the thickness in specimen in use pond are L when measuring fluorescence Spectra, and imagination is subdivided into a series of uniform thickness elementary layers vertical with exciting light with the sample in the sample cell, and then Δ l, n, the triangular pass of L are: n * Δ l=L;

B, correction absorb the impact on spectrum again:

With function I ₂(λ) the fluorescence measurement spectrum of biased sample behind the expression adding quencher is with function I ₂' (λ) represent then have the actual fluorescence Spectra that occurs after quenching process:

${I_2}^{\&prime;}\left(\mathrm{\&lambda;}\right)={10}^{\frac{\mathrm{<figure-callout\; id="2"\; label="n"\; filenames="HDA00002358356100011.png"\; state="\{\{state\}\}">n</figure-callout>}}{2}\mathrm{\&Delta;}{E}_1\left(\mathrm{\&lambda;}\right)}\&times;I_2\left(\mathrm{\&lambda;}\right)---\left(2\right)$

In the formula (2), Δ E ₁(λ)=ε ₁(λ) c ₁Δ l is the quencher first dullness function relevant with wavelength; ε ₁(λ) be the quencher molar absorption coefficient function relevant with wavelength; c ₁Volumetric molar concentration for quencher; Δ l is the thickness of sample segmentation elementary layer; N is the number of plies of sample segmentation elementary layer.

C, correction absorb the impact that distributes on spectrum:

1. the fluorescent reagent of choosing any one kind of them is mixed with solution, then dilutes one by one the concentration that reduces solution, and the record concentration value is also measured corresponding fluorescence Spectra; For the corresponding fluorescence Spectra of variable concentrations value, utilize respectively the Beer law calculation sample to the uptake of exciting light

And front 1/10th light paths of sample cell are to exciting Optical Absorption and total number percent D that absorbs _i, and calculate corresponding Fluorescence integral intensity S _i

2. calculate Fluorescence integral intensity S _iWith the uptake of sample to exciting light

Ratio, and carry out normalized, the data C that obtains _i

3. per sample front 1/10th light paths in pond to exciting Optical Absorption and total number percent D that absorbs _iWith data C _i, make the two relation curve C (d);

4. for function I among the step a ₁(λ) fluorescence measurement of representative spectrum utilizes Beer law to calculate fluorescent material number percent D to exciting Optical Absorption and always absorbing in front 1/10th light paths _J1, and determine and D according to relation curve C (d) _J1Corresponding parameter value C _J1

5. for function I among the step b ₂(λ) fluorescence measurement of representative spectrum utilizes (3) formula to calculate fluorescent material number percent D to exciting Optical Absorption and always absorbing in front 1/10th light paths _J2, and determine and D according to relation curve C (d) _J2Corresponding parameter value C _J2

$I_X={\mathrm{<figure-callout\; id="0"\; label="I"\; filenames="HDA00002358356100013.png"\; state="\{\{state\}\}">I</figure-callout>}}_0\&CenterDot;{10}^{-\mathrm{\&Delta;}{\mathrm{<figure-callout\; id="1"\; label="E"\; filenames="HDA00002358356100011.png,HDA00002358356100013.png"\; state="\{\{state\}\}">E</figure-callout>}}_1}\&CenterDot;(1-{10}^{-\mathrm{\&Delta;}{E}_2})\&CenterDot;[1-{10}^{-n(\mathrm{\&Delta;}{E}_1+\mathrm{\&Delta;}{E}_2)}]/[1-{10}^{-(\mathrm{\&Delta;}{E}_1+\mathrm{\&Delta;}{E}_2)}]---\left(3\right)$

In the formula (3), I ₀Be incident excitating light strength, I _XFor fluorescent material after adding quencher to exciting Optical Absorption;

D, calculating fluorescent quenching rate:

Difference computing function I ₁' (λ) and I ₂' (λ) Fluorescence integral intensity, be designated as I _1s' and I _2s', then calculate quencher rate Q by (4) formula:

$Q=\frac{{I_{\mathrm{ls}}}^{\&prime;}}{{\mathrm{<figure-callout\; id="1"\; label="C\; j"\; filenames="HDA00002358356100011.png,HDA00002358356100013.png"\; state="\{\{state\}\}">C</figure-callout>}}_j}\&CenterDot;\frac{C_{j2}}{{I_{2s}}^{\&prime;}}---\left(4\right)$

The script of fluorescent quenching rate is defined as: fluorescent quenching rate=(fluorescence after the original fluorescence of fluorescent material-adding quencher)/original fluorescence of fluorescent material, when considering to use the Stern-Volmer equation, can directly calculate more the original fluorescence of fluorescent material and the ratio that adds quencher fluorescence afterwards, formula (4) is namely derived by rear a kind of form, result of calculation has been eliminated the fluorescence inner filtering effect and absorbed the impact that distributes, and is corresponding with actual quenching process.

Compared with the prior art, beneficial effect of the present invention is embodied in:

1, when having comparatively seriously crossover between the absorption spectra of fluorescent material and quencher and the corresponding fluorescence Spectra, will be subject to inevitably the interference of fluorescence inner filtering effect when using conventional equipment to carry out fluoroscopic examination, form the artefact of quencher rate virtual height.Existing alignment technique is only proofreaied and correct separately for the fluorescence inner filtering effect, has but ignored the impact that simultaneous, fluorescent material with it causes the difference that excites the Optical Absorption degree of depth, thereby can't really realize the accurate correction of spectrum.Bearing calibration of the present invention is united correction simultaneously on the impact of fluorescence inner filtering effect and absorption distribution, guarantees the spectrum of described Quenching System is realized accurate correction, obtains the true fluorescent quenching rate that reflects quenching process.

2, bearing calibration of the present invention is simple and easy to do in practical operation, and need not increases new input, utilizes Origin or other software to carry out simple mathematical operation and can thoroughly remove inner filtering effect and absorb the impact that distributes.This has not only enlarged the range of application of fluorescence quenching method so that when utilizing conventional equipment to carry out fluorescence quenching analysis, can not be subject to the restriction of fluorescence inner filtering effect again, is conducive to simultaneously reduce research cost.

Description of drawings

Fig. 1 is the desirable distribution schematic diagram that absorbs of the exciting light of corresponding low absorbance;

Fig. 2 is the imperfect absorption distribution schematic diagram of the exciting light of corresponding high absorbance;

Fig. 3 is for utilizing the present invention to five polythiophenes (5T) and fullerene (C ₇₀) the result that proofreaies and correct of quencher spectroscopic data, horizontal ordinate is excitation wavelength, ordinate is for adding the fluorescence and the ratio that adds the fluorescence behind the quencher before the quencher;

Number in the figure: 1 sample cell; 2 exciting lights; The uniform fluorescence that 3 samples send; 4 sample cells; 5 exciting lights; The non-homogeneous fluorescence that 6 samples send.

Embodiment

The inventive method is carried out according to the following procedure:

1, according to 1., 2., 3. carrying out among the step c in the technical solution of the present invention, measures D _iAnd C _iRelation curve C (d).

In theory, fluorescence intensity and sample be to exciting Optical Absorption to be directly proportional, but find in the experiment that after sample concentration is higher than a certain numerical value, the phenomenon that fluorescence intensity declines to a great extent can occur on the contrary, this is by sample exciting light to be absorbed the variation that distributes to cause.Under the perfect condition, as shown in Figure 1, sample all is uniformly to the absorption distribution of exciting light 2 and the fluorescence that sends thereof in the sample cell 1, and the uniform fluorescence 3 that this moment, sample sent can be collected by photodetector to greatest extent, and the instrument response degree is the highest.When concentration increases, as shown in Figure 2, the attenuation process of sample cell 4 interior exciting lights 5 is accelerated, the effect that partially absorbs grow near the incident end, obviously weaken away from partially absorbing then of incident end, the non-homogeneous fluorescence 6 that sends for sample, because the position of photodetector aligning sample cell 4 is relatively-stationary in the spectrometer, the fluorescence signal that the variation that this absorption distributes (luminous position of fluorescence changes) will cause entering photodetector correspondingly reduces, the instrument response degree decreases, so that the fluorescence intensity detected value does not rise counter falling, this moment, fluorescence intensity and sample were to exciting Optical Absorption no longer to keep proportional relation.C (d) has reflected that instrument absorbs distribution parameter D to the response of same absorbent amount if can calculate under the different absorption distribution occasions _i, just can obtain corresponding correction coefficient C according to C (d) _i, and then the impact that absorb to distribute proofreaied and correct.The impact that absorb to distribute is that the light path arrangement by instrument self causes and determines that therefore for same spectrometer, its relation curve C (d) also is changeless, and employed sample has nothing to do when measuring.Only need to obtain relation curve C (d) by one-shot measurement, just obtained this instrument about absorbing the correction parameter that distributes, no longer need this step of repetition in other trimming processes afterwards.

2, measure respectively the absorption spectra of fluorescent reagent and quencher with absorption spectrum instrument, obtain their absorption spectra function, use during for correction calculation.If the absorption spectra function of fluorescent reagent and quencher is known, can save this step.

3, measure the fluorescence Spectra I of pure fluorescent reagent with fluorescence spectrophotometer ₁(λ), then utilize formula (1) that it is proofreaied and correct processing, obtain curve of spectrum I ₁' (λ), and according to I ₁' (λ) calculate corresponding Fluorescence integral intensity I _1s'; Calculate fluorescent material number percent D to exciting Optical Absorption and always absorbing in front 1/10th light paths according to Beer law _J1, determined and D by relation curve C (d) _J1Corresponding parameter value C _J1

4, measure the fluorescence Spectra I of fluorescent reagent and quencher biased sample with fluorescence spectrophotometer ₂(λ); Utilize formula (2) that it is proofreaied and correct processing, obtain curve of spectrum I ₂' (λ), and according to I ₂' (λ) calculate corresponding Fluorescence integral intensity I _2s'; For mixing material, can't calculate simply the absorption distribution of each component with Beer law, can utilize formula (3) to calculate fluorescent material number percent D to exciting Optical Absorption and always absorbing in front 1/10th light paths _J2, and determine and D according to relation curve C (d) _J2Corresponding parameter value C _J2

About implication and the derivation of formula (1), formula (2) and formula (3), referring to the present application people's granted patent: denomination of invention is that bearing calibration, the patent No. of removing the quencher inhalation effects in the fluorescence quenching analysis are ZL200810099357.4; The absorbance of material determines that it absorb to distribute, and selects in the present embodiment in front 1/10th light paths exciting Optical Absorption and total number percent that absorbs as absorption distribution parameter D _i, also can be directly in the practice with the absorbance of sample as absorbing distribution parameter.

5, calculate according to formula (4), obtain the fluorescent quenching rate Q behind the accurate correction.

When not having inner filtering effect, the fluorescent quenching rate can be by the fluorescence Spectra I of pure fluorescent reagent before the adding quencher ₁(λ) and add the fluorescence Spectra I of biased sample behind the quencher ₂(λ) directly obtain.When having inner filtering effect, if still directly use I ₁(λ) and I ₂(λ) calculate, can obtain the quencher rate of virtual height.In the formula (4), use respectively I _1s' and I _2s' alternative I ₁(λ) and I ₂(λ), realized the correction to inner filtering effect, and C _J1And C _J2Introducing, then be in order to proofread and correct absorbing the impact that distributions produces.

Embodiment: five polythiophenes (5T) and fullerene (C ₇₀) the accurate correction of fluorescent quenching spectrum process

The absorption spectra of 5T and fluorescence Spectra and C ₇₀Absorption spectra between all have largely crossover, its mixed liquor does not generate ground-state complex, the photic electronics of the part of the rear 5T that is stimulated will be to C ₇₀Shift, form Quenching of fluorescence.Solvent adopts o-dichlorobenzene, and it is transparent in the ultraviolet/visible light scope, nothing absorbs.

Absorption spectra is measured the UV-2550 uv-visible absorption spectroscopy instrument that adopts Shimadzu company; The F-4500 fluorescence spectrophotometer of Hitachi, Ltd is then used in the detection of fluorescence Spectra, is equipped with water bath with thermostatic control; Experiment is finished under 25 ℃ of conditions.

About absorbing the mensuration of distribution correction curve C (d), select three polythiophene 3T as fluorescent reagent, being mixed with initial concentration with o-dichlorobenzene as solvent is 2 * 10 ^-4The solution of mol/l dilutes subsequently at double, and the sample of each concentration is carried out fluoroscopic examination, and excitation wavelength is got 355nm.After the data obtained carried out correlation computations and normalized, the calibration curve C (d) that can be absorbed and distribute.In theory, every spectrometer has own exclusive absorption distribution correction curve.

Use respectively different excitation wavelengths to measure adding C in the test ₇₀Then the fluorescence Spectra of forward and backward 5T is utilized Origin software that experimental data is carried out correlation-corrected and is processed, and net result is referring to Fig. 3, and dotted line is C among the figure ₇₀The relative value of absorption spectra.

Among Fig. 3, the quencher rate of round dot symbol for directly calculating without any correction, very obvious, their varying in size under different excitation wavelength conditions, its profile and C ₇₀Absorption spectra between present strong positive correlation, this mainly is caused by the impact of inner filtering effect; The quencher rate of triangle representative gained after inner filtering effect is proofreaied and correct separately can find out that their size is not identical yet, still with C ₇₀Absorption spectra have correlativity, this moment, the impact of inner filtering effect was corrected removal, absorb distributing becomes main influence factor; Square symbols then is on inner filtering effect and absorbs the quencher rate that the rear gained of correction is united in the impact that distributes that their big or small basically identical (remaining tiny difference is from the error of calculation and experimental error) is with excitation wavelength and C ₇₀Absorption spectra all irrelevant.

According to the description of Stern-Volmer equation, the size and the excitation wavelength that shift corresponding fluorescent quenching rate with photic electronics are irrelevant.Above embodiment shows to only have through after uniting correction, and acquired results just conforms to theory, and this has proved that bearing calibration is correct in the present embodiment, and has good correction accuracy.

Claims (1)

1. the precision correcting method of fluorescent quenching rate when there is inner filtering effect in the fluorescent quenching system, described fluorescent quenching system refers to: the fluorometric investigation sample that does not generate ground-state complex after having fluorescence inner filtering effect and fluorescent material and quencher mixing; It is characterized in that described bearing calibration carries out as follows:

A, correction competition absorb the impact on spectrum:

With function I ₁(λ) expression does not add before the quencher fluorescent material rear fluorescence measurement spectrum that produces that is stimulated, with function I ₁' (λ) expression adds the actual fluorescence Spectra of sending of fluorescent material behind the quencher, then have:

${I_1}^{\&prime;}\left(\mathrm{\&lambda;}\right)=\frac{{10}^{-\mathrm{\&Delta;}{E}_1}(1-{10}^{-\mathrm{\&Delta;}{E}_2})[1-{10}^{-n\left(\mathrm{\&Delta;}{E}_1+\mathrm{\&Delta;}{E}_2\right)}]}{(1-{10}^{-n\&CenterDot;\mathrm{\&Delta;}{E}_2})[1-{10}^{-(\mathrm{\&Delta;}{E}_1+\mathrm{\&Delta;}{E}_2)}]}\&times;I_1\left(\mathrm{\&lambda;}\right)---\left(1\right)$

In the formula (1), Δ E ₁=ε ₁c ₁Δ l is first dullness of the corresponding excitation wavelength of quencher; Δ E ₂=ε ₂c ₂Δ l is first dullness of the corresponding excitation wavelength of fluorescent material; Δ l is the thickness of sample segmentation elementary layer; N is the number of plies of sample segmentation elementary layer; ε ₁Molar absorption coefficient for the corresponding excitation wavelength of quencher; ε ₂Molar absorption coefficient for the corresponding excitation wavelength of fluorescent material; c ₁Volumetric molar concentration for quencher; c ₂Volumetric molar concentration for fluorescent material;

Order: width and the thickness in specimen in use pond are L when measuring fluorescence Spectra, and imagination is subdivided into a series of uniform thickness elementary layers vertical with exciting light with the sample in the sample cell, and then Δ l, n, the triangular pass of L are: n * Δ l=L;

B, correction absorb the impact on spectrum again:

With function I ₂(λ) the fluorescence measurement spectrum of biased sample behind the expression adding quencher is with function I ₂' (λ) represent then have the actual fluorescence Spectra that occurs after quenching process:

${I_2}^{\&prime;}\left(\mathrm{\&lambda;}\right)={10}^{\frac{n}{2}\mathrm{\&Delta;}{E}_1\left(\mathrm{\&lambda;}\right)}\&times;I_2\left(\mathrm{\&lambda;}\right)---\left(2\right)$

In the formula (2), Δ E ₁(λ)=ε ₁(λ) c ₁Δ l is the quencher first dullness function relevant with wavelength; ε ₁(λ) be the quencher molar absorption coefficient function relevant with wavelength; c ₁Volumetric molar concentration for quencher; Δ l is the thickness of sample segmentation elementary layer; N is the number of plies of sample segmentation elementary layer;

C, correction absorb the impact that distributes on spectrum:

1. the fluorescent reagent of choosing any one kind of them is mixed with solution, then dilutes one by one the concentration that reduces solution, and the record concentration value is also measured corresponding fluorescence Spectra; For the corresponding fluorescence Spectra of variable concentrations value, utilize respectively the Beer law calculation sample to the uptake of exciting light

And front 1/10th light paths of sample cell are to exciting Optical Absorption and total number percent D that absorbs _i, and calculate corresponding Fluorescence integral intensity S _i

2. calculate Fluorescence integral intensity S _iWith the uptake of sample to exciting light Ratio, and carry out normalized, obtain data C _i

3. per sample front 1/10th light paths in pond to exciting Optical Absorption and total number percent D that absorbs _iWith data C _i, make the two relation curve C (d);

4. for function I among the step a ₁(λ) fluorescence measurement of representative spectrum utilizes Beer law to calculate fluorescent material number percent D to exciting Optical Absorption and always absorbing in front 1/10th light paths _J1, and determine and D according to relation curve C (d) _J1Corresponding parameter value C _J1

5. for function I among the step b ₂(λ) fluorescence measurement of representative spectrum utilizes (3) formula to calculate fluorescent material number percent D to exciting Optical Absorption and always absorbing in front 1/10th light paths _J2, and determine and D according to relation curve C (d) _J2Corresponding parameter value C _J2

$I_X=I_0\&CenterDot;{10}^{-\mathrm{\&Delta;}{E}_1}\&CenterDot;(1-{10}^{-\mathrm{\&Delta;}{E}_2})\&CenterDot;[1-{10}^{-n(\mathrm{\&Delta;}{E}_1+\mathrm{\&Delta;}{E}_2)}]/[1-{10}^{-(\mathrm{\&Delta;}{E}_1+\mathrm{\&Delta;}{E}_2)}]---\left(3\right)$

In the formula (3), I ₀Be incident excitating light strength, I _XFor fluorescent material after adding quencher to exciting Optical Absorption;

D, calculating fluorescent quenching rate:

Difference computing function I ₁' (λ) and I ₂' (λ) Fluorescence integral intensity, be designated as I _1s' and I _2s', then calculate quencher rate Q by (4) formula:

$Q=\frac{{I_{\mathrm{ls}}}^{\&prime;}}{C_{j1}}\&CenterDot;\frac{C_{j2}}{{I_{2s}}^{\&prime;}}---\left(4\right)$

The Q value of being calculated gained by formula (4) is exactly through the fluorescent quenching rate after the accurate correction processing, and these data have been eliminated the fluorescence inner filtering effect and absorbed the impact that distributes, and are corresponding with actual quenching process.

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