CN102411024B - Capillary array electrophoresis detection method based on spatial correction and spectral correction - Google Patents
Capillary array electrophoresis detection method based on spatial correction and spectral correction Download PDFInfo
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- CN102411024B CN102411024B CN201110160549.3A CN201110160549A CN102411024B CN 102411024 B CN102411024 B CN 102411024B CN 201110160549 A CN201110160549 A CN 201110160549A CN 102411024 B CN102411024 B CN 102411024B
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Abstract
The invention provides a capillary array electrophoresis detection method based on spatial correction and spectral correction, and the method comprises the following steps: (1) utilizing a Raman spectrum to position imaging positions of all capillaries in a capillary array on a charge coupled element; (2) performing spectrum unscrambling on multi-color fluorescent dyes in each capillary through the spatial correction; and (3) performing analysis on DNA (deoxyribonucleic acid) fragments of electrophoresis of the capillary array. In the method, the Raman spectrum of laser excitation capillary quartz or solvent water in the capillaries is adopted for realizing spectral imaging, thereby realizing the position positioning and parallelism adjustment of the capillaries on a CCD (charge coupled device); a fluorescent dye combined matrix is adopted for realizing simultaneous detection of the multi-color fluorescent dyes in the single capillary and the pixel combination way is adopted for improving the stability of a distribution matrix, reducing the amount of calculation and improving the detection stability; and a device for implementing the method is simple in structure and easy to operate.
Description
Technical field
The present invention relates to a kind of capillary array electrophoresis detection method, belong to high flux trace analysis field.
Background technology
DNA sequencing is the important technology in modern molecular biosciences research, and electrophoretic techniques is requisite technological means in DNA sequencing.Because kapillary has good heat dissipation, allow to add at kapillary two ends the high voltage to 30kV, more than the longitudinal electric field intensity of separation capillary can reach 400V/cm, thereby lock out operation can complete in a short period of time, reach very high separation efficiency (theoretical cam curve reach 400000/m more than, be up to the 107/m order of magnitude).Because internal diameter capillaceous very little (general < 100 μ m), to internal diameter 50 μ m, the kapillary that length is 50cm, its volume less than 1 μ L, sampling volume is in nL level, sample concentration can be lower than 10～4mol/L.It is desired efficient that capillary electrophoresis technique has reached instrumental analysis, and fast, amount of samples waits the most excellent feature of fundamental sum less.In addition, capillary electrophoresis technique also has easy robotization, easy and simple to handle, holds the advantages such as agent consumes less, and environmental pollution is little.First capillary electrophoresis technique application concentrates on amino acid, carbohydrate, on the compartment analysis of the biomolecule such as nucleic acid and protein, along with the development of technique and perfect, its apply gradually to medical and health, food chemistry, the fields such as environment are permeated.Capillary electrophoresis technique is also applied to the high speed order-checking of DNA, the efficient separation of protein, carbohydrate analysis, cell analysis, chiral resolution, the mensuration of physicochemical constant, production engineering control etc.
In order to improve the flux of capillary detection, capillary array is widely used in biochemical analysis as high flux electrophoresis analysis means.Carry out the qualitative and quantitative analysis of trace reagent at field widespread use capillary arrays such as medical treatment, chemical, bio-pharmaceuticals, forensic analysises.Capillary array can be realized the analysis of reagent in batches simultaneously.But because intercapillary position relationship exists uncertainly, while making capillary array carry out the qualitative and quantitative analysis of trace reagent, there is error.In addition for detecting when multicolor fluorescence dyestuff in single kapillary, because the fluorescent band of fluorescent marker is wider, cause overlapping very greatly between different fluorescence spectrums, be difficult to them well to distinguish by common light splitting, this has also caused the very large error of capillary electrophoresis analysis.Still do not eliminate at present the mature technology of these errors.
Summary of the invention
In view of this, in order to overcome the deficiencies in the prior art, the invention provides one and can accurately correct each kapillary position can accurately identify the specific capillary array electrophoresis detection method of each fluorescent marker in capillary array, greatly improve the degree of accuracy of capillary detection.
The invention provides a kind of capillary array electrophoresis detection method based on free-air correction and spectrum correction, described method comprises step: (1) utilizes the image space of each kapillary on charge coupled cell in Raman spectrum locating capillary array, (2) by spectrum correction, the multicolor fluorescence dyestuff in every capillary is carried out to spectrum unscrambling, (3) are analyzed the DNA segment of capillary array electrophoresis.
Further, described method step (1) comprising: Raman light is sent in the capillary bundle of A. laser beam irradiation, B. be imaged on charge coupled cell CCD by Raman image instrument upper, the spectrogram that C. analyzes on charge coupled cell CCD positions each kapillary in capillary bundle.
Further, Raman light is sent in the aqueous solvent scattering in described laser beam irradiation capillary bundle, and laser beam used is double laser beam, and the wavelength of described double laser beam is respectively 488nm and 514.5nm.
Further, in described method step C., take pixel count as transverse axis, spectral intensity is that the longitudinal axis obtains free-air correction spectrogram, identifies the effective width obtaining between every capillary center and every capillary by peak.
Further, described method step (2), use laser beam irradiation kapillary, the fluorescent dye generation fluorescence spectrum that is excited in kapillary, described fluorescence spectrum is imaged on charge coupled cell area array CCD through imaging device, by n kind fluorescent marker used imaging on described area array CCD respectively, obtain the spectral distribution of n kind fluorescence on described CCD, it is that fluorescence on the each pixel of m × p area array CCD is recorded by force that every kind of fluorescence is dropped on to pixel, obtain the matrix P of m × n, matrix P is carried out to normalizing and obtain matrix Q=(Q
ij)
m × n, by Q matrix to fluorescence used imaging on CCD combination carry out spectrum spectrum unscrambling, wherein: n, m, p are natural number.
Further, described fluorescence spectrum is imaged on line array CCD through imaging device.
Further, described method step (3) is to upper each the frame spectroscopic data obtaining of described charge coupled cell CCD, by free-air correction, distinguish the spectroscopic data that every capillary is corresponding, to the spectroscopic data in each capillary, obtain spectroscopic data and the independently position distribution of every kind of fluorescence of every kind of dyestuff by spectrum correction matrix spectrum unscrambling, continuous multiple frames spectroscopic data is processed to the DNA segment information that obtains.
The realization of the inventive method divides three steps.The first step realizes the location recognition of capillary array by free-air correction; Second step is realized the multicolor fluorescence dyestuff in a capillary is carried out to spectrum unscrambling by spectrum correction; The 3rd step is carried out DNA segment analysis.
1. free-air correction
The object of free-air correction is in order to provide the image space of every capillary on CCD.Adopt water scattering laser generation Raman scattering in gel, the position of every capillary is determined in the imaging of measurement scattered light on CCD.Impinge upon on kapillary with laser, laser, by the water scattering in capillary gel, sends Raman light, and by getting optical slits, grating beam splitting, is imaged on CCD upper after lens focus, and the spectrogram of analyzing on CCD determines that capillary position distributes.
Free-air correction adopts double laser beam, is respectively 488nm and 514.5nm.The feature Raman peaks of water is positioned at 3400cm
-1place.For 488nm (20491.8cm
-1), the raman characteristic peak of water scattering is positioned at 17091.8cm
-1, corresponding wavelength is 585nm.For 514.5nm (19436.3cm
-1), the raman characteristic peak of water scattering is positioned at 16036.3cm
-1, corresponding wavelength is 623.6nm.Its spectrum is mainly distributed in 2800cm
-1to 3800cm
-1between wave number.
488nm and 514.5nm laser are projected on data acquisition CCD and can obtain Fig. 3 by the feature Raman peaks of water scattering.Blue light (left side) is to gather 488nm laser by the Raman light intensity of water scattering, what ruddiness (right side) was corresponding is to gather 514.5nm laser by the Raman fluorescence intensity of water scattering, the corresponding capillary in each peak, adopt adjacent capillary space to gather the feature of the Raman light that different wave length laser is scattered, be that odd number kapillary gathers the Raman light that 488nm laser instrument excites, even numbers kapillary gathers the Raman light that 514.5nm laser instrument excites, and vice versa.From figure, we can see, 488nm and 514.5nm laser can be separated just by the Raman peaks of water scattering, and within being just in time distributed in CCD sensing range: 500～660nm.This has met the demand that two groups of data are proofreaied and correct that gathers in free-air correction just.Fig. 4 has provided the frame spectrogram on free-air correction CCD, can obviously see corresponding two bright spots (Raman peak values) on every capillary from Fig. 4.
Fig. 4 is carried out to the summation of transverse axis data as spectral intensity, and take longitudinal axis pixel as horizontal ordinate, mapping provides free-air correction spectrogram as shown in Figure 5.Can provide every capillary center (as shown in Figure 6) and effective width (one or two pixel is got in general left and right) by the identification of simple peak.
2, spectrum correction
For realizing the specificity analyses to detection signal, must proofread and correct processing, the most general technical method is to set up pigment combinations fluorescence signal matrix, the correction of signal intensity during for data analysis.The intensity signal of fluorescence is not had to too harsh requirement, as long as obtain fluorescence, position and time signal just can obtain desired experimental result accurately.But the fluorescent band of general fluorescent marker is wider, and this causes the overlapping composition between different fluorescence spectrums very large, be difficult to them well to distinguish by common light splitting.If adopted, spectrum is opened up to obtain to the very wide method with the higher resolution characteristic of acquisition, can make fluorescence intensity degradation, detection sensitivity and signal to noise ratio (S/N ratio) just become a difficult problem.For the singularity of this class detection method, we are carrying out will carrying out spectrum correction to used known fluorescent dye before fluorescence detection, to obtain every kind of mark fluorescent spectrum used imaging separately on CCD of laser excitation, obtain the distribution matrix of fluorescence spectrum according to imaging, to just carrying out spectrum unscrambling to obtain desired fluorogram to the fluorescence Spectra of surveying with normalizing matrix after matrix normalizing.
Fluorescence spectrum band is wider and intensity is very weak, is difficult to allow fluorescence imaging well distinguish with area array CCD, if spectrum is opened up very widely, to obtain compared with high resolution capacity, glimmering light intensity can seriously weaken, and the sensitivity of signal and signal to noise ratio (S/N ratio) can decline.If spectral evolution is narrow, fluorescence is more intense, and Overlapping of fluorescence spectra will aggravate, even cannot interpretation spectrum peak position, and spectrum unscrambling becomes a difficult problem.In order to take into account above two aspects, general instrument has all adopted photomultiplier to replace CCD.This will adopt multiple photomultipliers simultaneously, make beam splitting system become very complicated.For two of fluorescence signal sensitivity and spectrum spectrum unscramblings difficult problems for restriction mutually, the present invention adopts LASER Excited Fluorescence spectrum, and the method that fluorescence spectrum carries out spectrum correction is carried out to ccd signal processing.Adopt the method without the too many broadening degree of paying close attention to spectrum, as long as fluorescence spectrum shape used not exclusively overlaps, intensity distributions difference, just can obtain by spectrum correction the relative intensity of various fluorescence, upper CCD all photoreceptor signals are all effectively used simultaneously, thereby increase the sensitivity of CCD, improve the signal to noise ratio (S/N ratio) of fluorescence signal.
The step of fluorescence signal on area array CCD being carried out to spectrum unscrambling with spectrum correction is as follows.
First we will to know that fluorescent marker used has several, be set as n kind.We allow the imaging on area array CCD respectively of n kind mark fluorescent, obtain the spectral distribution of this n kind fluorescence on CCD.Area array CCD pixel is m × p.The fluorescence that every kind of fluorescence is dropped on the each pixel of CCD is recorded by force, obtains the matrix P of a m × n.The fluorescence intensity difference being excited due to every kind of label is very large, for the convenience that the later stage is carried out signal processing, matrix P is carried out to normalizing and obtain matrix Q=(Q
ij)
m × n.
By Q matrix, we just can the combination of the imaging on CCD carry out spectrum spectrum unscrambling to fluorescence used.The fluorescent intensity of adopting from each pixel of CCD as certain moment is distributed as m × 1 matrix B, and the relative intensity of every kind of fluorescence is a n × 1 matrix X: QX=B.Carry out matrixing and obtain X=(Q
tq)
-1(Q
tb).Can learn from above formula, when we have obtained after spectrum correction matrix Q, drop on the intensity distributions matrix B of each pixel on area array CCD as long as obtain any time fluorescence, the relative intensity that just can obtain various fluorescence by spectrum correction distributes, thereby obtains the spectral information of fluorescence used.In experiment below, can find out, even if the Overlapping of fluorescence spectra on CCD is very serious, even cannot interpretation go out peak position, also can obtain by this method good fluorogram.
3, DNA fragmentation analysis
While carrying out DNA segment analysis, to obtaining each the frame spectroscopic data on CCD, first by free-air correction, distinguish the spectroscopic data that every capillary is corresponding.To the spectroscopic data in each capillary, obtain the spectroscopic data of every kind of dyestuff by spectrum correction matrix spectrum unscrambling.Originally distribute after spuious spectrum light splitting and obtain independently position distribution of every kind of fluorescence, the corresponding spectral wavelength of distributing position.Continuous multiple frames spectroscopic data is processed to the DNA segment information that obtains.
Beneficial effect of the present invention
(1) adopt the Raman spectrum of laser excitation quartz or water to realize light spectrum image-forming;
(2) can adopt single or multi-laser to excite;
(3) realize position location and the depth of parallelism adjustment of kapillary on CCD;
(4) when adopting fluorochrome combinations matrix to realize in single kapillary multicolor fluorescence dyestuff, detect;
(5) can reclaim fluorescent dye and be dispersed in the efficient light on all pixels on line array CCD, spectrum utilization factor is high;
(6) can adopt the mode of pixel combination to improve the stability of distribution matrix, reduce calculated amount, improve the stability detecting;
(7) can apply with line array CCD and detect the fluoroscopic examination that realizes single capillary, also can be applied to the fluorescence spectrophotometer processing that realizes capillary array on face CCD;
(8) method implement device is simple in structure, easy to operate.
Accompanying drawing explanation
Fig. 1 is that free-air correction method realizes schematic diagram;
Fig. 2 is the Raman spectrogram of water: the feature Raman peaks that is water scattering;
Fig. 3 is that the Raman spectrogram of water: 488nm and 514.5nm laser are projected on data acquisition CCD by the feature Raman peaks of water scattering;
Fig. 4 is the image of capillary array Raman light on CCD;
Fig. 5 is locus positioning analysis schematic diagram: transverse axis is number of picture elements, and the longitudinal axis is spectral intensity mapping;
Data are analyzed in Fig. 6 space orientation;
Fig. 7 is that spectrum correction method realizes schematic diagram;
Wherein: 1-laser beam, 2-capillary array, 3-single capillary, 4-Raman light, 5-Raman spectrum imaging CCD, 6-fluorescent dye.
Embodiment
Now by reference to the accompanying drawings and embodiment the inventive method is described in further detail.
The realization of the inventive method divides three steps: the first step realizes the location recognition of capillary array by free-air correction; Second step is realized the multicolor fluorescence dyestuff in a capillary is carried out to spectrum unscrambling by spectrum correction; The 3rd step is carried out DNA segment analysis.
With reference to figure 1, impinge upon the detection window place of capillary bundle 2 by laser beam 1, laser is by the water scattering in solution in the quartz of single capillary 3 or pipe, send Raman light 4, by getting optical slits, grating beam splitting, is imaged on after lens focus on CCD 5, and the spectrogram of analyzing on CCD 5 carries out location, position to capillary bundle.
With reference to figure 7, in the time of the detection window of fluorescent dye 6 by kapillary 3, excited by laser beam 1, fluorescence spectrum is imaged on line array CCD 5 through imaging device.
1, free-air correction
The object of free-air correction is in order to provide the image space of every capillary on CCD.Adopt water scattering laser generation Raman scattering in gel, the position of every capillary is determined in the imaging of measurement scattered light on CCD.Impinge upon on kapillary with laser, laser, by the water scattering in capillary gel, sends Raman light, and by getting optical slits, grating beam splitting, is imaged on CCD upper after lens focus, and the spectrogram of analyzing on CCD determines that capillary position distributes.
2, spectrum correction
The step of fluorescence signal on area array CCD being carried out to spectrum unscrambling with spectrum correction is as follows.
First we will to know that fluorescent marker used has several, be set as n kind.We allow the imaging on area array CCD respectively of n kind mark fluorescent, obtain the spectral distribution of this n kind fluorescence on CCD.For convenient data processing, we are divided into several fritters (if every 14 pixel × 3 pixels are) whole area array CCD, are labeled as bin, suppose and altogether use m bin, general m > 4n.Here explain and why the pixel on CCD will be done to bin processing: if the step of fluorescence signal on area array CCD being carried out to spectrum unscrambling with spectrum correction is as follows.
First we will to know that fluorescent marker used has several, be set as n kind.We allow the imaging on area array CCD respectively of n kind mark fluorescent, obtain the spectral distribution of this n kind fluorescence on CCD.For convenient data processing, we are divided into several fritters (if every 14 pixel × 3 pixels are) whole area array CCD, are labeled as bin, suppose and altogether use m bin, general m > 4n.Here explain and why the pixel on CCD will be done to bin processing: if
Take the pixel on CCD as measuring unit, the matrix meeting stability of setting up is very poor, and a little less than antijamming capability, small disturbance just may bring unpredictable results; If what bin selected certainly is too large, the stability of matrix can be fine, but spectrophotometric result can be poor.Therefore be typically chosen in 20bin left and right, and weigh the antijamming capability of matrix by the conditional number of transition matrix.The fluorescence that every kind of fluorescence is dropped on the each bin of CCD is recorded by force, obtains the matrix P of a m × n.The fluorescence intensity difference being excited due to every kind of label is very large, for the convenience that the later stage is carried out signal processing, matrix P is carried out to normalizing and obtain matrix Q=(Q
ij)
m × n.
By Q matrix, we just can the combination of the imaging on CCD carry out spectrum spectrum unscrambling to fluorescence used.The fluorescent intensity of adopting from each bin of CCD as certain moment is distributed as m × 1 matrix B, and the relative intensity of every kind of fluorescence is a n × 1 matrix X: QX=B.Carry out matrixing and obtain X=(Q
tq)
-1(Q
tb).Can learn from above formula, when we have obtained after spectrum correction matrix Q, drop on the intensity distributions matrix B of each bin on area array CCD as long as obtain any time fluorescence, the relative intensity that just can obtain various fluorescence by spectrum correction distributes, thereby obtains the spectral information of fluorescence used.In experiment below, can find out, even if the Overlapping of fluorescence spectra on CCD is very serious, even cannot interpretation go out peak position, also can obtain by this method good fluorogram.
3, DNA fragmentation analysis
While carrying out DNA segment analysis, to obtaining each the frame spectroscopic data on CCD, first by free-air correction, distinguish the spectroscopic data that every capillary is corresponding.To the spectroscopic data in each capillary, obtain the spectroscopic data of every kind of dyestuff by spectrum correction matrix spectrum unscrambling.Originally distribute after spuious spectrum light splitting and obtain independently position distribution of every kind of fluorescence, the corresponding spectral wavelength of distributing position.Continuous multiple frames spectroscopic data is processed to the DNA segment information that obtains.
Although some preferred embodiment of inventing by reference, invention is described, but those of ordinary skill in the art should be appreciated that and can make various changes to it in the form and details, and do not depart from the spirit and scope of the present invention that appended claims limits.
Claims (2)
1. the capillary array electrophoresis detection method based on free-air correction and spectrum correction, it is characterized in that, described method comprises step: (1) utilizes the image space of each kapillary on charge coupled cell area array CCD in Raman spectrum locating capillary array, (2) by spectrum correction, the n kind fluorescent marker in every capillary is carried out to spectrum unscrambling, (3) DNA segment of capillary array electrophoresis is analyzed
Described method step (1) comprising: A. laser beam irradiation capillary bundle, laser is by the aqueous solvent scattering in kapillary, send Raman light, B. be imaged on charge coupled cell area array CCD by Raman image instrument, C. the spectrogram of analyzing on charge coupled cell area array CCD positions each kapillary in capillary bundle
Laser beam used is double laser beam, the wavelength of described double laser beam is respectively 488nm and 514.5nm, adjacent capillary space is gathered to the Raman light that different wave length laser is scattered, be that odd number kapillary gathers the Raman light that 488nm laser beam excites, even numbers kapillary gathers the Raman light that 514.5nm laser beam excites, or even numbers kapillary gathers the Raman light that 488nm laser beam excites, odd number kapillary gathers the Raman light that 514.5nm laser beam excites
In described method step C., take pixel count as transverse axis, carry out the summation of transverse axis data and obtain free-air correction spectrogram as spectral intensity, identify the effective width that obtains every capillary center and every capillary by peak,
Described method step (2), use laser beam irradiation kapillary, the fluorescent marker generation fluorescence spectrum that is excited in kapillary, described fluorescence spectrum is imaged on charge coupled cell area array CCD through imaging device, by n kind fluorescent marker used imaging on described charge coupled cell area array CCD respectively, obtain the spectral distribution of n kind fluorescence on described charge coupled cell area array CCD, it is that fluorescence intensity in the each pixel of m × p charge coupled cell area array CCD is recorded that every kind of fluorescence is dropped on to pixel, obtain the matrix P of m × n, matrix P is carried out to normalizing and obtain matrix Q=(Q
ij)
m × nwherein: n, m, p are natural number, by Q matrix to fluorescence used imaging on charge coupled cell area array CCD combination carry out spectrum spectrum unscrambling: the fluorescent intensity that certain moment gathers from each pixel of charge coupled cell area array CCD is distributed as m × 1 matrix B, the relative intensity of every kind of fluorescence is a n × 1 matrix X, QX=B, carries out matrixing and obtains X=(Q
tq)
-1(Q
tb), thus obtain the spectral information of fluorescence used,
Described method step (3) is to each the frame spectroscopic data obtaining on described charge coupled cell area array CCD, by free-air correction, distinguish the spectroscopic data that every capillary is corresponding, to the spectroscopic data in each capillary, obtain spectroscopic data and the independently position distribution of every kind of fluorescence of every kind of fluorescent marker by spectrum correction matrix spectrum unscrambling, continuous multiple frames spectroscopic data is processed to the DNA segment information that obtains.
2. according to the capillary array electrophoresis detection method based on free-air correction and spectrum correction claimed in claim 1, it is characterized in that, described area array CCD is replaced with to line array CCD.
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| RU2017131051A (en) * | 2015-02-06 | 2019-03-06 | Лайф Текнолоджиз Корпорейшн | SYSTEM AND METHODS FOR CALIBRATING BINDING DYES |
| CN105572093A (en) * | 2016-01-27 | 2016-05-11 | 公安部第一研究所 | Multichannel capillary array performance testing device and method |
| CN106840007A (en) * | 2017-04-07 | 2017-06-13 | 赵�怡 | A kind of spacescan system and method for combination adjustable laser range finding probe array and intelligent terminal |
| CN107976478A (en) * | 2017-11-21 | 2018-05-01 | 南京溯远基因科技有限公司 | A kind of more dyestuff collection method for nucleic acid analysis and its application based on Capillary Electrophoresis |
| EP3844491A1 (en) * | 2018-08-31 | 2021-07-07 | Life Technologies Corporation | Methods and apparatus for extended dynamic range from single exposures in capillary electrophoresis |
| JP7022670B2 (en) * | 2018-09-10 | 2022-02-18 | 株式会社日立ハイテク | Spectrum calibration device and spectrum calibration method |
| US20220229013A1 (en) * | 2019-06-06 | 2022-07-21 | Hitachi High-Tech Corporation | Multicapillary electrophoresis device, and sample analysis method |
| CN113358609B (en) * | 2021-03-15 | 2024-06-21 | 公安部第一研究所 | Method for dynamically correcting DNA fluorescence spectrum by using multicolor fluorescence internal standard |
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