CN101644708A - Fluorescence polarization enhancing immune capillary electrophoresis analysis - Google Patents
Fluorescence polarization enhancing immune capillary electrophoresis analysis Download PDFInfo
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- CN101644708A CN101644708A CN200910092316A CN200910092316A CN101644708A CN 101644708 A CN101644708 A CN 101644708A CN 200910092316 A CN200910092316 A CN 200910092316A CN 200910092316 A CN200910092316 A CN 200910092316A CN 101644708 A CN101644708 A CN 101644708A
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- fluorescent dye
- polarization
- capillary electrophoresis
- spectrogram
- dna
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Abstract
The invention relates to a fluorescence polarization enhancing immune capillary electrophoresis analysis technique and the application thereof in the biology sample detection. Fluorescent dye with small molecular weight has fast rotational speed and nearly can depolarize when being simulated by polarized light; when the fluorescent dye is bond with macromolecule matters usch DNA or protein and thelike, fluorescence polarization obviously increases. Based on characteristics, the invention utilizes vertical polarized lights and horizontal polarized light deduction technique (I<v>-I<h>) technique to eliminate the interference of falling-off fluorescent dye on target compound in the capillary electrophoresis detection and the further separation is not needed. The invention has the advantagesof simple and fast operation and like, and can be used for immune capillary electrophoresis-laser induced fluorescence accuracy determination and quantitative analysis in aspects such as DNA adduction, DNA methylating level and biology macromolecule interaction.
Description
Technical field
The present invention relates to the Biological Detection technical field, specifically is that fluorescence polarization is combined with the immune capillary electrophoretic techniques, improves the immune capillary electrophoresis analysis level, can be applicable to the analysis of various biomolecules.
Background technology
The immune capillary electrophoretic techniques is a kind of analytical approach that grows up in conjunction with the height selectivity of the efficient isolation technics fast of Capillary Electrophoresis and immunoassay, be widely used in the interaction of research biomacromolecule (protein-protein, DNA-protein, Ag-Ab), aspects such as dna adduct detection.Can reach the characteristics such as high sensitivity of single molecules level in conjunction with the laser-Induced Fluorescence Detection method, this method has become one of important technology of biological sample separation detection, has advantages such as amount of samples is few, detection speed is fast, sensitivity height.But because there is certain instability in fluorescent dye with combining of biomacromolecule, when carrying out immune capillary electrophoresis-laser-Induced Fluorescence Detection, thereby fluorescent dye hydrolysis obscission impact analysis result may occur, need further to separate removal.
Fluorescence polarization technology is a kind of important analysis means, can reflect fluorescently-labeled rotation difference in nano-seconds, has higher using value aspect the research intermolecular interaction.Usually, fluorescent material is because its molecular weight is little, and rotational speed is fast, and the fluorescence that produces when excited by polarized light almost can depolarization; When fluorescent material is attached to macromolecular substances, as DNA, protein etc., its rotation is limited and speed is slack-off, and fluorescence polarization enlarges markedly.The polarized light of launching when being subjected to the plane polarization optical excitation according to fluorescence molecule changes, and judges the binding mode between target molecule.
The present invention combines two kinds of analytical technologies of fluorescence polarization and immune capillary electrophoresis, the method that subtracts button by polarization is eliminated the interference that the hydrolysis from the biomacromolecule of mark of fluorescent dye in the immune capillary electrophoresis detection comes off and produces, need not further separation, have advantage such as fast easy and simple to handle, can be applied to the immune capillary electrophoresis analysis of multiple biological sample.
Summary of the invention
The immune capillary electrophoresis analysis technology that the purpose of this invention is to provide a kind of fluorescence polarization enhancement mode.
With orthogonal polarized light fluorescence excitation dyestuff, can detect emission light, i.e. orthogonal polarized light (I in the plane of polarization of vertical and level
v) and horizontal polarization light (I
h).Generally speaking, the fluorescent dye that molecular weight is little, its rotational speed is fast, and when excited by polarized light, the fluorescence response of orthogonal polarized light and horizontal polarization light is roughly the same, almost can depolarization; And rotational speed is slack-off after being attached to macromolecular substances such as DNA and protein when fluorescent dye, and fluorescence polarization enlarges markedly.
Based on this characteristic, the present invention utilizes orthogonal polarized light and horizontal polarization light to subtract button (I
v-I
h) technology gets rid of in the immune capillary electrophoresis detection interference to the target immune complex of the fluorescent dye that comes off from the fluorescence labeling biomacromolecule.Because fluorescent dye I
v=I
h, I
v-I
hApproach zero, polarization almost can be removed its interference after subtracting button fully.Be marked on the fluorescent dye on the biomacromolecule, have bigger polarization value, the fluorescent dye that therefore is marked on the biomacromolecule carries out still partly existing after polarization subtracts button.Therefore be merely able to detect the fluorescent dye signal that is marked on the biomacromolecule after immune capillary electrophoresis-laser-induced fluorescence (LIF) polarization subtracts button, need not the interference that further separation can be got rid of the fluorescent dye that comes off.
The present invention has overcome the interference of the fluorescent dye that comes off in traditional immune capillary electrophoresis-laser-Induced Fluorescence Detection method, with the combination with it of online fluorescence polarization technology, subtract the button method by polarization and need not further separation, in chromatographic work station, directly realize the coming off deduction of fluorescent dye, easy and simple to handle, can be used for the immune capillary electrophoresis-laser-induced fluorescence (LIF) Accurate Analysis of multiple biological samples such as dna adduct, dna methylation level and interaction of biomacromolecules.
Description of drawings
Fig. 1 is that online fluorescence polarization technology binary channels detects synoptic diagram;
Fig. 2 is that the immuno-CE-LIFP of antibody complex detects electrophoretogram;
Fig. 3 detects the whole methylation level of m λ DNA of 3 μ g/mL for immuno-CE-LIFP;
Fig. 4 detects the whole methylation level of m λ DNA of 0.01 μ g/mL for immuno-CE-LIFP.
Embodiment
Specific implementation method of the present invention is summarized as follows:
When carrying out immunoassay, fluorescent dye splits away off from the biomacromolecule of mark easily, influence the detection of target compound, and online fluorescence polarization technology can be got rid of the interference of the fluorescent dye that comes off, need not further separation, realize detection the target compound with this.
In immune capillary electrophoresis-laser-induced fluorescence (LIF) Polarization Detection (immuno-CE-LIFP) process, the fluorescent dye horizontal polarization light (I that is excited and produces after the optical excitation as shown in Figure 1
h) and orthogonal polarized light (I
v), detect by different photomultipliers respectively, through the signal converter light signal is converted into electric signal postscript input computer, utilize chromatographic work station to carry out data processing.In chromatographic work station, carry out the button that subtracts of signal between horizontal polarization light and the orthogonal polarized light, obtain horizontal polarization light (I
h) and orthogonal polarized light (I
v) signal subtracts the new electrophoresis spectrogram behind the button.
Simple fluorescent dye is because the little I of molecular weight
v-I
hApproach zero, almost disappear in the new electrophoresis spectrogram after subtracting button.And be marked on fluorescent dye on the biomacromolecule, and it is limited that molecular weight increases rotational speed, and its polarization value increases, so be marked on fluorescent dye on the biomacromolecule at orthogonal polarized light (I
v) and horizontal polarization light (I
h) signal subtracts behind the button still that part exists.Only underlined fluorescent dye signal on biomacromolecule in the new electrophoresis spectrogram after polarization subtracts button, the signal of the fluorescent dye that comes off disturbs and is then almost deducted fully, therefore qualitative the and quantitative test that can determine the target compound.
The immuno-CE-LIFP of embodiment 1, fluorescent-labeled antibody compound detects
The F of the anti-IgG1 of Alexa Fluor 546 fluorescently-labeled Fc specificitys (ab ') fragment can with non-covalent combination of 5-methylcytosine monoclonal antibody of IgG1 hypotype, form antibody complex, this antibody complex is carried out immuno-CE-LIFP detects.Accompanying drawing 2 shows, spectrum Fig. 1 and 2 is respectively the antagonist compound and carries out immuno-CE-LIFP horizontal polarization light (I when analyzing
h) and orthogonal polarized light (I
v) the electrophoresis spectrogram of signal.In chromatographic work station, horizontal polarization photoelectrophoresis spectrogram is made as background, in orthogonal polarized light electrophoresis spectrogram, carries out background deduction, by subtract button (I in linear polarization
v-I
h) acquisition spectrogram 3.The electrophoresis spectrogram is analyzed, and fluorescently-labeled two anti-can't separating with anti-two anti-compounds, overlapping is a peak (peak 1).The peak 2 of analytical spectra Fig. 1 and 2, discovery peak 2 is made up of a and b two parts, and wherein b is the acromion at peak 2.Polarization subtracts button postpeak 2a and still has 8% signal, and peak 2b almost disappears, and proves that peak 2b is the signal peak of fluorescent dye of coming off.The part existence of peak 2a may be to be caused by the albumen of the unknown-dyestuff combination, has certain polarization and therefore can not deduct fully.The result proves that the polarization that the fluorescent dye that comes off is almost equal to zero can subtract the button elimination by polarization.
Utilize the 5-methylcytosine monoclonal antibody anti-as one, the F of the anti-IgG1 of Alexa Fluor 546 fluorescently-labeled Fc specificitys (ab ') fragment for the present invention adopt two anti-, after the DNA reaction to be measured with antibody and 3 μ g/mL, can form the dna antibody immune complex, according to the immune complex growing amount whole methylation level among the λ DNA (m λ DNA) that handles of determining to methylate.Immuno-CE-LIFP analyzes DNA to be measured and antibody response potpourri, and spectrum Fig. 1 and 2 is respectively the reaction mixture compound is carried out immuno-CE-LIFP horizontal polarization light (I when analyzing in the accompanying drawing 3
h) and orthogonal polarized light (I
v) the electrophoresis spectrogram of signal, as described in the example 1 by subtract button (I in linear polarization
v-I
h) acquisition spectrogram 3.Spectrogram 3 shows that there are a and two peaks of b in peak 2, by deducting in linear polarization, peak 2a and 2b still exist, and by the degree of separation of deducting the two increase (at least 2 times), show that with example 1 comparing result peak 2b for getting rid of target dna-antibody mediated immunity compound that the fluorescent dye that comes off disturbs, carries out integration to DNA-antibody mediated immunity compound peak area and can carry out accurate quantification to whole methylation level among the DNA to be measured.
M λ DNA and one anti-(the 5-methylcytosine monoclonal antibody) and two of 0.01 μ g/mL are resisted (F of the anti-IgG1 of Alexa Fluor 546 fluorescently-labeled Fc specificitys (ab ') fragment) reaction, immuno-CE-LIFP detects the whole methylation level of DNA to be measured.Spectrogram 1 is buckled (I for the immuno-CE-LIFP of antibody complex subtracts in linear polarization in the accompanying drawing 4
v-I
h) spectrogram, spectrogram 2 subtracts button (I for the immuno-CE-LIFP of DNA to be measured and antibody response potpourri in linear polarization
v-I
h) spectrogram.Spectrogram 1 shows that peak 2b disappears by subtract the button back in linear polarization, and fluorescent dye disturbs in order to come off; Spectrogram 2 shows that peak 2b still exist by subtracting button in linear polarization, is the eliminating target dna-antibody mediated immunity compound that fluorescent dye disturbs that comes off.When DNA concentration to be measured only is 0.01 μ g/mL by subtract the button technology in linear polarization, still can Accurate Analysis DNA-antibody mediated immunity compound, the immune capillary electrophoresis analysis level obviously improves.
Claims (6)
1, a kind of fluorescence polarization immune capillary electrophoresis analysis technique.
2, based on the described method of claim 1, it is characterized by: the present invention combines fluorescence polarization technology with the immune capillary electrophoresis method, and the immune capillary electrophoresis analysis level is significantly improved.
3, based on the described method of claim 1, it is characterized by: photomultiplier detects the horizontal polarization light (I that fluorescent dye is excited and afterwards produces respectively
h) and orthogonal polarized light (I
v), after conversion of signals, utilize chromatographic work station to carry out data processing.In chromatographic work station, the electrophoresis spectrogram that will derive from orthogonal polarized light (or horizontal polarization light) is made as the background spectrogram, carries out the background correction operation in the electrophoresis spectrogram that comes from horizontal polarization light (or orthogonal polarized light), obtains orthogonal polarized light (I
v) and horizontal polarization light (I
h) signal subtracts the new electrophoresis spectrogram behind the button.
4, based on the described method of claim 6, it is characterized by: because fluorescent dye I
v=I
h, I
v-I
hApproach zero, after polarization subtracts button, almost can complete obiteration in new electrophoresis spectrogram.Fluorochrome label is on biomacromolecule, and its polarization value increases, and is marked on the fluorescent dye still part existence in new electrophoresis spectrogram on the biomacromolecule behind the polarization deduction.
5, based on the described method of claim 1-4, it is characterized by: reaction mixture is carried out immune capillary electrophoresis-laser-induced fluorescence (LIF) analysis, only underlined fluorescent dye signal on biomacromolecule in the new electrophoresis spectrogram after polarization subtracts button, the signal of fluorescent dye of coming off disturbs then almost to be deducted fully, therefore need not further separation, the qualitative and quantitative test that can determine the target compound.
6, based on the described method of claim 1, it is characterized by: the present invention can be used for the accurately qualitative and quantitative test of immune capillary electrophoresis-laser-induced fluorescence (LIF) of many biological samples such as dna adduct, dna methylation level and interaction of biomacromolecules.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020001416A1 (en) * | 2018-06-26 | 2020-01-02 | 深圳市圣必智科技开发有限公司 | Method for determining dna methylation level at specific site in biological sample and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020001416A1 (en) * | 2018-06-26 | 2020-01-02 | 深圳市圣必智科技开发有限公司 | Method for determining dna methylation level at specific site in biological sample and application thereof |
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Application publication date: 20100210 |