CN102914529B - 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 fluorescent quenching system exists inner filtering effect

Technical field

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

Background technology

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

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

In measuring process, add after quencher the fluorescent reagent in biased sample that the absorption of exciting light is distributed and than pure fluorescent reagent before adding quencher, the absorption distribution of exciting light can be changed, difference on this absorption distributes, can exert an influence to fluorescence intensity equally, interference spectrum analysis result, in the time quantitatively detecting, also must be proofreaied and correct.

The impact being formed by absorption distribution and the impact of fluorescence inner filtering effect are accompanied, occur the artefact of jointly causing fluorescent quenching rate virtual height simultaneously.Current existing alignment technique is only proofreaied and correct separately for fluorescence inner filtering effect, and ignored the distribute impact that brings of the absorption simultaneously occurring with it, thereby fundamentally cannot carry out accurate correction to gained fluorescent quenching rate data, obtain real quencher rate, restrict 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 while 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 existing fluorescence inner filtering effect and fluorescent reagent to mix with quencher.Utilize the given bearing calibration of the present invention, can and absorb on fluorescence inner filtering effect the impact producing that distributes and carry out accurate correction simultaneously, thereby obtain truly reflecting the actual quencher rate of fluorescent quenching process.

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

When fluorescent quenching system of the present invention exists inner filtering effect, the feature of the precision correcting method of fluorescent quenching rate is to carry out as follows:

A, correction competition absorb the impact on spectrum:

With function I ₁(λ) represent not add the quencher fluorescence measurement spectrum that fluorescent material produces after being stimulated before, with function I ₁' (λ) represent to add the actual fluorescence Spectra of sending of fluorescent material after quencher, have:

${I_1}^{\′}\left(\mathrm{\λ}\right)=\frac{{10}^{-\mathrm{\Δ}{E}_1}(1-{10}^{-\mathrm{\Δ}{E}_2})[1-{10}^{-n\left(\mathrm{\Δ}{E}_1+\mathrm{\Δ}{E}_2\right)}]}{(1-{10}^{-n\·\mathrm{\Δ}{E}_2})[1-{10}^{-(\mathrm{\Δ}{E}_1+\mathrm{\Δ}{E}_2)}]}\×I_1\left(\mathrm{\λ}\right)---\left(1\right)$

In 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 ₁for the molar absorption coefficient of the corresponding excitation wavelength of quencher; ε ₂for the molar absorption coefficient of the corresponding excitation wavelength of fluorescent material; c ₁for the volumetric molar concentration of quencher; c ₂for the volumetric molar concentration of fluorescent material.

Order: while measuring fluorescence Spectra, width and the thickness in specimen in use pond are L, the sample in sample cell is subdivided into a series of uniform thickness elementary layers vertical with exciting light by imagination, and 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) represent to add the fluorescence measurement spectrum of biased sample after quencher, with function I ₂' (λ) represent have the actual fluorescence Spectra occurring after quenching process:

${I_2}^{\′}\left(\mathrm{\λ}\right)={10}^{\frac{n}{2}\mathrm{\Δ}{E}_1\left(\mathrm{\λ}\right)}\×I_2\left(\mathrm{\λ}\right)---\left(2\right)$

In formula (2), Δ E ₁(λ)=ε ₁(λ) c ₁Δ l is first dullness function that quencher is relevant to wavelength; ε ₁(λ) be the molar absorption coefficient function that quencher is relevant to wavelength; c ₁for the volumetric molar concentration of 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 distributing on spectrum:

1. the fluorescent reagent of choosing any one kind of them is mixed with solution, and the concentration that then successively dilution reduces solution, records concentration value and measure corresponding fluorescence Spectra; For the corresponding fluorescence Spectra of variable concentrations value, utilize respectively the uptake of Beer law calculation sample to exciting light and front 1/10th light paths of sample cell are to the absorption of exciting light and total number percent D absorbing _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 be normalized the data C obtaining _i;

3. per sample front 1/10th light paths in pond to the absorption of exciting light and total number percent D absorbing _iwith data C _i, make the two relation curve C (d);

4. for function I in step a ₁(λ) fluorescence measurement of representative spectrum, utilizes Beer law to calculate fluorescent material number percent D to the absorption of exciting light and total absorption 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 in step b ₂(λ) fluorescence measurement of representative is composed, and utilizes (3) formula to calculate fluorescent material number percent D to the absorption of exciting light and total absorption in front 1/10th light paths _j2, and determine and D according to relation curve C (d) _j2corresponding parameter value C _j2;

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

In formula (3), I ₀for incident excitating light strength, I _xfor adding the absorption of fluorescent material to exciting light after quencher;

D, calculating fluorescent quenching rate:

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

$Q=\frac{{I_{\mathrm{ls}}}^{\′}}{C_{j1}}\·\frac{C_{j2}}{{I_{2s}}^{\′}}---\left(4\right)$

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

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

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

2, bearing calibration of the present invention is simple and easy to do in practical operation, without increasing new input, utilizes Origin or other software to carry out simple mathematical operation and can thoroughly remove inner filtering effect and absorb the impact distributing.When this makes to utilize conventional equipment to carry out fluorescence quenching analysis, can not be subject to again the restriction of fluorescence inner filtering effect, not only expand the range of application of fluorescence quenching method, be conducive to reduce research cost simultaneously.

Brief description of the 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 quencher spectroscopic data result of proofreading and correct, horizontal ordinate is excitation wavelength, ordinate is the fluorescence and the ratio that adds the fluorescence after quencher adding before 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 in step c in technical solution of the present invention 1., 2., 3. carry out, measure D _iand C _irelation curve C (d).

In theory, fluorescence intensity is to sample, the absorption of exciting light to be directly proportional, but finds in experiment, after sample concentration is higher than a certain numerical value, and the phenomenon that there will be on the contrary fluorescence intensity to decline to a great extent, this is by sample, exciting light to be absorbed to the variation distributing to cause.Under perfect condition, as shown in Figure 1, in sample cell 1, sample distributes to the absorption of exciting light 2 and the fluorescence that sends is all that the uniform fluorescence 3 that now sample sends can be collected by photodetector to greatest extent uniformly, and instrument response degree is the highest.In the time that concentration increases, as shown in Figure 2, the attenuation process of the interior exciting light 5 of sample cell 4 is accelerated, near the effect that the partially absorbs grow of incident end, obviously weaken away from partially absorbing of incident end, the non-homogeneous fluorescence 6 sending for sample, the position of aiming at sample cell 4 due to photodetector in spectrometer is relatively-stationary, the variation (luminous position of fluorescence changes) that this absorption distributes will cause the fluorescence signal that enters photodetector correspondingly to reduce, instrument response degree decreases, make fluorescence intensity detected value not rise counter falling, now fluorescence intensity and sample no longer keep proportional relation to the absorption of exciting light.C (d) has reflected the response of instrument to same absorbent amount under different absorption distribution occasions, absorbs distribution parameter D if can calculate _i, just can obtain corresponding correction coefficient C according to C (d) _i, and then proofreaied and correct absorbing the impact distributing.Absorbing the impact distributing is caused and determined by the light path arrangement of instrument self, and therefore for same spectrometer, its relation curve C (d) is also changeless, and the sample that uses has nothing to do when measuring.Only need obtain relation curve C (d) by one-shot measurement, just obtain this instrument about absorbing the correction parameter distributing, in other trimming processes afterwards, no longer need this step of repetition.

2, measure respectively the absorption spectra of fluorescent reagent and quencher with absorption spectrum instrument, obtain their absorption spectra function, during for correction calculation, use.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 by fluorescence spectrophotometer ₁(λ), then utilize formula (1) to proofread and correct processing to it, 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 the absorption of exciting light and total absorption 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 by fluorescence spectrophotometer ₂(λ); Utilize formula (2) to proofread and correct processing to it, obtain curve of spectrum I ₂' (λ), and according to I ₂' (λ) calculate corresponding Fluorescence integral intensity I _2s'; For mixing material, cannot calculate simply the absorption distribution of each component with Beer law, can utilize formula (3) to calculate fluorescent material number percent D to the absorption of exciting light and total absorption 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), the granted patent referring to present inventor: denomination of invention is that bearing calibration, the patent No. of removing quencher inhalation effects in fluorescence quenching analysis are ZL200810099357.4; The absorbance of material determines that it absorbs and distributes, and selects in front 1/10th light paths the absorption of exciting light with total number percent absorbing as absorption distribution parameter D in the present embodiment _i, also can be directly in practice with the absorbance of sample as absorbing distribution parameter.

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

While not there is not inner filtering effect, fluorescent quenching rate can be by the fluorescence Spectra I that adds pure fluorescent reagent before quencher ₁(λ) and add the fluorescence Spectra I of biased sample after quencher ₂(λ) directly obtain.In the time there is inner filtering effect, if still directly use I ₁(λ) and I ₂(λ) calculate, can obtain the quencher rate of virtual height.In 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, be in order to proofread and correct absorbing the impact that produces of distributing.

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

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

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

About the mensuration that absorbs 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, dilution at double subsequently, and the sample of each concentration is carried out to fluoroscopic examination, excitation wavelength is got 355nm.The data obtained is carried out after correlation computations and normalized to the calibration curve C (d) that can be absorbed and distribute.In theory, every spectrometer has own exclusive absorption distribution correction curve.

In test, use respectively different excitation wavelengths to measure and add C ₇₀the fluorescence Spectra of forward and backward 5T, then utilizes Origin software to carry out correlation-corrected processing to experimental data, and net result is referring to Fig. 3, and in figure, dotted line is C ₇₀the relative value of absorption spectra.

In Fig. 3, round dot symbol is the quencher rate 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 is mainly 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 there is correlativity, the now impact of inner filtering effect has been corrected removal, absorbing distributes becomes main influence factor; Square symbols is that the impact of inner filtering effect and absorption distribution is combined to the quencher rate of proofreading and correct rear gained, and their size basically identical (remaining tiny difference is from the error of calculation and experimental error), 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 combining after correction, and acquired results just conforms to theory, and this has proved that in the present embodiment, bearing calibration is correct, and has good correction accuracy.

Claims (1)

1. the precision correcting method of fluorescent quenching rate when fluorescent quenching system exists inner filtering effect, described fluorescent quenching system refers to: the fluorometric investigation sample that does not generate ground-state complex after existing fluorescence inner filtering effect and fluorescent material to mix with quencher; Described bearing calibration is carried out as follows:

Step a, correction competition absorb the impact on spectrum:

With function I ₁(λ) represent not add the quencher fluorescence measurement spectrum that fluorescent material produces after being stimulated before, with function I ₁' (λ) represent to add the actual fluorescence Spectra of sending of fluorescent material after quencher, have:

${I_1}^{\text{'}}\left(\mathrm{\λ}\right)=\frac{{10}^{{\mathrm{\ΔE}}_1}(1-{10}^{{\mathrm{\ΔE}}_2})[1-{10}^{-n({\mathrm{\ΔE}}_1+{\mathrm{\ΔE}}_2)}]}{(1-{10}^{-n\·{\mathrm{\ΔE}}_2})[1-{10}^{-({\mathrm{\ΔE}}_1+{\mathrm{\ΔE}}_2)}]}\×I_1\left(\mathrm{\λ}\right)---\left(1\right)$

In 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 ₁for the molar absorption coefficient of the corresponding excitation wavelength of quencher; ε ₂for the molar absorption coefficient of the corresponding excitation wavelength of fluorescent material; c ₁for the volumetric molar concentration of quencher; c ₂for the volumetric molar concentration of fluorescent material;

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

Step b, correction absorb the impact on spectrum again:

With function I ₂(λ) represent to add the fluorescence measurement spectrum of biased sample after quencher, with function I ₂' (λ) represent have the actual fluorescence Spectra occurring after quenching process:

${I_2}^{\text{'}}\left(\mathrm{\λ}\right)={10}^{\frac{n}{2}{\mathrm{\ΔE}}_1\left(\mathrm{\λ}\right)}\×I_2\left(\mathrm{\λ}\right)---\left(2\right)$

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

It is characterized in that: after completing described step b, continue following steps c and steps d:

Step c, correction absorb the impact distributing on spectrum:

1. the fluorescent reagent of choosing any one kind of them is mixed with solution, and the concentration that then successively dilution reduces solution, records concentration value and measure corresponding fluorescence Spectra; For the corresponding fluorescence Spectra of variable concentrations value, utilize respectively the uptake of Beer law calculation sample to exciting light and front 1/10th light paths of sample cell are to the absorption of exciting light and total number percent D absorbing _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 be normalized, obtain data C _i;

3. per sample front 1/10th light paths in pond to the absorption of exciting light and total number percent D absorbing _iwith data C _i, make the two relation curve C (d);

4. for function I in step a ₁(λ) fluorescence measurement of representative spectrum, utilizes Beer law to calculate fluorescent material number percent D to the absorption of exciting light and total absorption 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 in step b ₂(λ) fluorescence measurement of representative is composed, and utilizes (3) formula to calculate fluorescent material number percent D to the absorption of exciting light and total absorption in front 1/10th light paths _j2, and determine and D according to relation curve C (d) _j2corresponding parameter value C _j2;

$I_X=I_0\·{10}^{{-\mathrm{\ΔE}}_1}\·(1-{10}^{{-\mathrm{\ΔE}}_2})\·[1-{10}^{-n({\mathrm{\ΔE}}_1+{\mathrm{\ΔE}}_2)}]/[1-{10}^{-({\mathrm{\ΔE}}_1+{\mathrm{\ΔE}}_2)}]---\left(3\right)$

In formula (3), I ₀for incident excitating light strength, I _xfor adding the absorption of fluorescent material to exciting light after quencher;

Steps d, calculating fluorescent quenching rate:

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

$Q=\frac{{I_{1s}}^{\text{'}}}{C_{j1}}\·\frac{C_{j2}}{{I_{2s}}^{\text{'}}}---\left(4\right)$

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