JP4909744B2 - Capillary electrophoresis apparatus and electrophoresis method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a capillary electrophoresis apparatus in which simultaneity can be ensured between sensitivity and data acquisition to decrease false signals while spectral calibration data acquisition is eliminated in each capillary exchange. <P>SOLUTION: In the capillary electrophoresis apparatus, a capillary position shift is detected in each capillary exchange by detecting a capillary position. A capillary position measuring light source is provided in the capillary electrophoresis apparatus. The capillary is irradiated with light emitted from the capillary position measuring light source, and a capillary image is detected with a two-dimensional detector, whereby a position deviation of the capillary is determined. On the basis of the position deviation of the capillary, a data acquisition area is set in the two-dimensional detector, or a reference fluorescent light spectrum determined from the capillary at the standard position is corrected. <P>COPYRIGHT: (C)2007,JPO&amp;INPIT

Description

  The present invention relates to an electrophoresis apparatus for separating and analyzing nucleic acids, proteins, and the like by electrophoresis, and more particularly to a capillary electrophoresis apparatus.

  In a capillary electrophoresis apparatus, a laser light source is generally used as an excitation light source. However, in recent years, it has been proposed to use a light emitting diode (LED) as an excitation light source for the purpose of reducing the price of a capillary electrophoresis apparatus. Patent Document 1 discloses an electrophoresis apparatus using a light emitting diode (LED).

Patent Documents 2 and 3 propose an electrophoresis apparatus that irradiates excitation light in the direction of the capillary axis, rather than a method of irradiating laser light perpendicular to the capillary axis. The excitation light propagates in the capillary and excites the detection target substance moving in the capillary regardless of its position.
Since a wide excitation region, that is, a detection region is obtained, the sensitivity of the apparatus can be increased. The detection light from the linear light emitting part is dispersed by a diffraction grating and detected by a two-dimensional detector.

Patent Documents 4 and 5 describe examples of conventional methods for acquiring wavelength calibration data.
Non-Patent Document 1 discloses a method using a plurality of filters instead of a diffraction grating.

US2003 / 0178312-A1 JP 5-52810 A Japanese Patent No. 2833119 US 6,821,402 US 6,863,791 Sea Cornell et al .; Biotechnics, Vol. 5, 648 (1987) (C. Connell et.al .; BioTechniques 5, 342 (1987))

  As a result of intensive studies by the present inventor, the following problems have been found.

  In the techniques disclosed in Patent Documents 2 and 3, since the capillary itself serves as an excitation part and a detection part, there arises a problem that the detection position shifts due to replacement of the capillary. Therefore, it is necessary to acquire wavelength calibration data every time the capillary is replaced. If the wavelength calibration data is inaccurate, a pseudo signal due to a wavelength shift is generated in the analysis process. When the wavelength shift increases, the pseudo signal also increases, and the reliability of the analysis result decreases.

  In addition, the wavelength calibration data acquisition methods disclosed in Patent Documents 4 and 5 require a known sample to be actually electrophoresed, which is very troublesome for the operator.

  Further, in the method disclosed in Non-Patent Document 1, since a plurality of filters are rotated, it is difficult to ensure the sensitivity and the synchronization of data acquisition.

  That is, in the method using a diffraction grating, it is possible to ensure the sensitivity and the synchronization of data acquisition, but it is necessary to acquire the wavelength calibration data every time the capillary is replaced. On the other hand, in the method using a plurality of filters, it is not necessary to acquire wavelength calibration data for each capillary exchange, but it is not possible to ensure the sensitivity and the synchronization of data acquisition.

  An object of the present invention is to provide a capillary electrophoresis apparatus that makes it unnecessary to acquire wavelength calibration data every time a capillary is exchanged, secures the sensitivity and synchronization of data acquisition, and reduces pseudo signals. .

  The present invention relates to a capillary electrophoresis apparatus that detects the position of a capillary every time the capillary is replaced and detects a displacement of the capillary.

  According to the present invention, the capillary electrophoresis apparatus is provided with a light source for capillary position measurement. The capillary position is irradiated with light from a capillary position measurement light source, and a capillary image is detected by a two-dimensional detector, thereby obtaining a capillary position deviation.

  Based on the deviation of the capillary position, a data acquisition region in the two-dimensional detector is set, or the reference fluorescence spectrum obtained from the capillary at the reference position is corrected.

  According to the present invention, it is not necessary to acquire wavelength calibration data every time a capillary is exchanged, and it is possible to reduce the pseudo signal without impairing the signal intensity and the simultaneity of signal acquisition.

  The above and other novel features and benefits of the present invention will be described below with reference to the drawings. However, the drawings are for explanation only, and do not limit the scope of the present invention.

  As shown in FIG. 1A, the capillary electrophoresis apparatus of this example includes a capillary array 101, an irradiation optical unit 121, a detection optical unit 131, an autosampler unit 141, an electrophoresis medium filling unit 150, a power supply unit 110, and a temperature control unit. 161. The capillary electrophoresis apparatus of this example uses a plurality of capillaries filled with an electrophoresis medium, introduces a sample into each capillary, and separates and analyzes sample components in the sample. For example, a plurality of samples having a DNA-containing sample are analyzed at the same time to analyze the base sequence.

  The capillary array 101 is composed of a plurality of capillaries 102. In the example of FIG. 1, the capillaries are composed of two capillaries 102, but may be one, four, eight, or the like. The capillary 102 is a quartz thin tube having an inner diameter of several tens to several hundreds of microns and an outer diameter of several hundreds of microns. The surface of the capillary 102 is coated with polyimide to improve the strength. A fluorine coating may be used in the portion where light is propagated.

  The capillary array 101 is a detachable replacement member, and is replaced with a new one when the quality deteriorates due to a predetermined number of analyzes and the separation performance decreases. When the measurement method is changed and the length of the capillary 102 needs to be changed, the capillary array 101 is replaced with a different length. Thereby, the length of the capillary 102 can be arbitrarily adjusted.

  The capillary array 101 includes an irradiation unit 103 irradiated with excitation light, a sample introduction end 105 for introducing a sample, and a capillary head 108 formed by bundling a plurality of capillaries. The sample introduction end 105 is held by the sample introduction unit 104.

  As shown in FIG. 1B, a hollow electrode 106 is inserted at the tip of the capillary 102, and the tip protrudes slightly from the hollow electrode 106. The hollow electrode 106 is, for example, a stainless steel pipe. The capillary head 108 is connected to the electrophoresis medium filling unit 150.

  The irradiation optical unit 121 includes a light source 123, a first condenser lens 124, an irradiation filter 125, and a second condenser lens 126.

  In the irradiation unit 103, the capillary 102 is supported on the glass substrate 107. Excitation light from the irradiation optical unit 121 is applied to the capillary 102. The light source 123 generates excitation light 122 that irradiates the irradiation unit 103 of the capillary array 101. A laser light source is usually used as the light source 123, but in this example, a light emitting diode (LED) is used.

The first condenser lens 124 condenses the excitation light 122 from the light source 123. The irradiation filter 125 cuts unnecessary wavelength components from the excitation light 122. The second condenser lens 126 condenses the excitation light 122. The light collected by the second condenser lens 126 is applied to the capillary 102. According to this example, the excitation light is irradiated along the capillary axis or inclined at a predetermined angle with respect to the capillary axis.

  A sample component separated by electrophoresis in the capillary 102 is irradiated with excitation light 122. The phosphor labeled on the sample component emits light having a different wavelength for each sample component. This light is detected by the detection optical unit 131.

  The detection optical unit 131 includes a first camera lens 132, a detection filter 133, a diffraction grating 134, a second camera lens 135, and a two-dimensional detector 136. Details of the detection optical unit 131 will be described later with reference to FIG.

  The autosampler unit 141 conveys the sample container 143, the buffer container 144, the cleaning container 145, and the waste liquid container 146 directly under the sample introduction unit 104.

  The sample container 143 is a container that holds a plurality of trace samples, and is transported immediately below the sample introduction unit 104 when the sample is introduced. The sample is, for example, a solution containing a large number of nucleic acids having an appropriate length (size) in which four types of nucleoside base molecules are fluorescently labeled.

  For example, the sample container 143 is configured such that a septa, which is a resin sheet, is placed on a sample plate having 24 rows and 16 columns of wells capable of holding a sample of several tens of μL, and is sandwiched between the holder and a clip. ing. The septa usually have a sealed through hole at a position corresponding to the well to prevent evaporation of the sample in the well. When the sample is introduced, the sample introduction end 105 can come into contact with the sample through the through hole. Moreover, a protective film can be stuck on the upper surface of the septa to prevent sample evaporation.

  The buffer container 144 is a container that holds a buffer solution that immerses the sample introduction end 105, and is transported directly below the sample introduction unit 104 during electrophoresis analysis. Further, during the standby of the apparatus, it is transported directly under the sample introduction unit 104 as in the case of electrophoresis analysis, and the sample introduction end 105 is immersed in a buffer solution to prevent the electrophoresis medium in the capillary 102 from drying.

  The cleaning container 145 is a container that holds a cleaning liquid for cleaning the sample introduction end 105, and is transported immediately below the sample introduction unit 104 when the electrophoresis medium is filled, during preliminary migration, and after sample introduction. By immersing the sample introduction end 105 in the cleaning liquid in the cleaning container, the sample introduction end 105 can be cleaned and contamination can be avoided.

  The waste liquid container 146 is a container that holds a used electrophoresis medium, and is transported immediately below the sample introduction unit 104 when the electrophoresis medium is filled. When the electrophoresis medium is filled, the used electrophoresis medium discharged from the sample introduction end 105 is received.

  The electrophoresis medium filling unit 150 includes a polymer filling block 151, a syringe 152, a tube 153, a solenoid valve 154, and an anode buffer container 112, and automatically fills the capillary 102 with a new electrophoresis medium before starting analysis.

  The polymer filling block 151 has a polymer flow path 155. The polymer flow path 155 communicates with a syringe 152 filled with a migration medium and a tube 153 provided with an electromagnetic valve 154. The other end of the tube 153 is immersed in a buffer solution held in the anode buffer container 112. The capillary head 108 is attached to the polymer filling block 151 while maintaining pressure and air tightness.

  The power supply unit 110 includes a high-voltage power supply 111 that generates a high voltage of around 15 kV. The negative electrode of the high voltage power supply 111 is connected to the hollow electrode 106, and the positive electrode is grounded via an ammeter 114. One end of the anode electrode 113 is immersed in a buffer solution in the anode buffer container 112, and the other end is grounded.

  A method for filling the capillary 102 with the electrophoresis medium using the electrophoresis medium filling unit 150 will be briefly described. A waste liquid container 146 is disposed immediately below the sample introduction unit 104, the electromagnetic valve 154 is closed, and the plunger of the syringe 152 is pushed. As a result, the electrophoresis medium in the syringe 152 flows into the capillary 102 from the capillary head 108 via the polymer channel 155. The used electrophoresis medium in the capillary 102 is discharged from the sample introduction end 105 and received in the waste liquid container 146.

  A method for introducing a sample into the capillary 102 will be briefly described. The inside of the capillary 102, the polymer channel 155, and the tube 153 is filled with the electrophoresis medium. The sample container 143 is disposed immediately below the sample introduction unit 104, the sample introduction end 105 is immersed in the sample held in the well of the sample container 143, and the electromagnetic valve 154 is opened. Thus, the hollow electrode 106, the sample in the sample container 143, the migration path in the capillary 102, the polymer flow path 155 of the polymer filling block 151, the tube 153, and the anode buffer container 112 are provided between the positive electrode and the negative electrode of the high-voltage power supply 111. Current path composed of the buffer solution and the anode electrode 113 is formed. Then, a pulse voltage is applied to this energization path with the hollow electrode 106 as a negative potential and the anode electrode 113 as a positive potential. As a result, a negatively charged sample component, such as DNA, present in the well is introduced from the sample introduction end 105 into the migration path. The sample introduction method is not limited to electrophoresis, and the sample may be introduced into the electrophoresis path by pressure or dispensing.

  At the time of electrophoretic analysis, the buffer container 144 is disposed immediately below the sample introduction unit 104, and the sample introduction end 105 is immersed in the buffer solution held in the buffer container 144. Thus, between the positive electrode and the negative electrode of the high-voltage power supply 111, the hollow electrode 106, the buffer solution in the buffer container 144, the migration path in the capillary 102, the polymer flow path 155 of the polymer filling block 151, the tube 153, and the anode buffer container 112 A current path composed of the buffer solution and the anode electrode 113 is formed. A high voltage of about 15 kV is applied to this energization path with the hollow electrode 106 as a negative potential and the anode electrode 113 as a positive potential. As a result, an electric field is generated in the direction from the irradiation unit 103 to the sample introduction unit 104, and the negatively charged sample component introduced into the migration path is electrophoresed in the direction of the irradiation unit 103.

  The temperature control unit 161 controls the temperature of the migration path that affects the migration speed of the sample components. The temperature control unit of this example accommodates the capillary 102 in a thermostat (not shown). Then, air maintained at a constant temperature by a temperature control mechanism such as Peltier is circulated in the thermostatic chamber by a blowing mechanism such as a fan, and the capillary 102 is held at a predetermined temperature.

  In this example, a capillary position measuring light source 127 is further provided between the glass substrate 107 on which the capillaries 102 are arranged and the detection optical unit 131. The capillary position measuring light source 127 is a light source that irradiates a single wavelength, and may be, for example, a laser diode. Each time the capillary is replaced, the capillary 102 is irradiated by the capillary position measuring light source 127. The reflected light from the capillary 102 is detected by the detection optical unit 131. A reflected image of the capillary 102 is formed on the two-dimensional detector 136, and the position of the capillary is accurately measured. Thereby, it is possible to measure the displacement or inclination of the capillary 102 when the capillary is replaced.

  If the capillary 102 is misaligned or inclined, the fluorescence spectrum on the screen of the two-dimensional detector 136 is displayed deviated. However, in this example, since the displacement or inclination of the capillary 102 is measured, the displacement of the fluorescence spectrum can be corrected. Thus, it is not necessary to acquire new wavelength calibration data when the capillary is replaced.

  The irradiation unit 103 will be described in detail with reference to FIG. As shown in FIG. 2A, a glass substrate 107 is provided at a position close to the capillary head 108. As shown in FIG. 2B, the plurality of capillaries 102 are arranged on a glass substrate 107. The capillaries 102 are arranged on the glass substrate 107 so as to be parallel to each other to some extent and substantially parallel to the glass substrate. The term “a certain degree” means that there may be an inclination of several degrees, and the term “substantially” means that it is within the accuracy error.

  Excitation light from the irradiation optical unit 121 is irradiated along the axial direction of the capillary 102 or along a direction inclined at a predetermined angle with respect to the axial direction of the capillary 102. In the irradiation unit 103, the polyimide film of the capillary 102 is removed. Therefore, the excitation light is totally reflected from the outer surfaces of the plurality of capillaries 102 and propagates through the capillaries 102 to excite the samples in the capillaries 102 simultaneously. By the excitation light propagating in the capillary 102, the sample in the capillary 102 generates fluorescence in the range of several mm to several tens mm. Thus, in this example, a linear light emitting region is formed.

  Fluorescence generated from the sample in the capillary 102 is detected by a detection optical unit 131 arranged along a direction perpendicular to the axial direction of the capillary 102 as shown in FIG.

  The detection optical unit 131 will be described with reference to FIG. As shown in FIG. 3A, the detection optical unit 131 includes a first camera lens 132, a detection filter 133, a diffraction grating 134, a second camera lens 135, and a two-dimensional detector 136. In this example, a diffraction grating 134 is used as a wavelength dispersion method. The fluorescence emitted from the light emitting portion of the capillary 102 becomes a parallel light beam by the first camera lens 132. This parallel light beam is guided to the detection filter 133. The detection filter 133 transmits only fluorescence in the wavelength region used for analysis. The fluorescence transmitted through the detection filter 133 is wavelength-dispersed by the diffraction grating 134 and imaged on the two-dimensional detector 136 by the second camera lens 135. The two-dimensional detector 136 is a CCD camera, for example. The image signal from the two-dimensional detector 136 is processed by a computer and the sample is analyzed.

  FIG. 3B shows an image obtained by the two-dimensional detector 136. In this example, two images are obtained corresponding to two capillaries. The horizontal axis of this image is the chromatic dispersion direction, and the vertical axis is the axial direction of the capillary.

  In place of the diffraction grating 134, wavelength dispersion means in which prisms are appropriately combined may be used. The two-dimensional detector 136 may be configured by appropriately combining a one-dimensional detector, a photomultiplier, a photodiode, and the like and an optical mechanism instead of the CCD camera.

  Hereinafter, a method for measuring the position of the capillary using the capillary electrophoresis apparatus according to the present invention and correcting the deviation of the fluorescence spectrum based on the position of the capillary will be described. In the first method, a data acquisition area is set. In the second method, the reference fluorescence spectrum acquired as the wavelength calibration data is corrected. Here, a case where a single capillary is used and four color fluorescent dyes are used will be described.

  FIG. 4 shows the characteristics of the red laser diode. In this example, a red laser diode is used as the capillary position measuring light source 127. The red laser diode is a monochromatic light source having a narrow emission band with a peak wavelength of 655 nm and a half width of about 2 nm.

  FIG. 5 shows a reference fluorescence spectrum acquired as wavelength calibration data. Here, four color dyes, dR110, dR6G, dTAMRA, and dROX are used. The fluorescence from the four color fluorescent dyes is wavelength-dispersed by the diffraction grating 134 and becomes a spectrum. In the actual analysis, these reference fluorescence spectra are used. In this way, the fluorescence spectra of four colors can be separated from the detection light of the DNA sample, and the four types of DNA can be accurately identified.

  With reference to FIG. 6, the wavelength shift caused by the displacement of the capillary will be described. The position of the fluorescence spectrum image formed on the two-dimensional detector 136 is determined by the relative positions of the capillary 102 and the two-dimensional detector 136. It is assumed that the two-dimensional detector 136 is fixed. When the position of the capillary 102 is changed by exchanging the capillary, the shape of the fluorescence spectrum image on the image of the two-dimensional detector 136 does not change, but the position is translated in the wavelength dispersion direction or the capillary axis direction.

  A thick curve 61 in FIG. 6 shows a fluorescence spectrum image formed on the two-dimensional detector 136 when the capillary 102 is at the reference position. The reference position is the position of the capillary 102 when wavelength calibration data is acquired.

  The vertical axis represents signal intensity, and the horizontal axis represents wavelength. However, the wavelength scale on the horizontal axis is set when wavelength calibration data is acquired. Therefore, the horizontal axis represents the position in the wavelength dispersion direction in the image of the two-dimensional detector 136. A thin line curve 62 in FIG. 6 shows a fluorescence spectrum image formed on the two-dimensional detector 136 when the position of the capillary 102 moves in the wavelength dispersion direction. When the position of the capillary 102 changes, the fluorescence spectrum image shifts in the wavelength direction. This phenomenon occurs when the capillary array 101 is attached and replaced.

  When the data is acquired and analyzed with the position of the fluorescence spectrum image at the time of analysis shifted from the position of the reference fluorescence spectrum image acquired as wavelength calibration data, the fluorescence spectrum from the DNA sample is accurately separated. I can't. For example, when detecting the fluorescence of the fluorescent dye dR110, the fluorescence signal of dR6G is generated as a pseudo signal, and the reliability of the analysis is reduced. The greater the deviation in the fluorescence spectrum, the larger the pseudo signal and the worse the analysis accuracy.

  Therefore, in the present invention, the position of the capillary 102 at the time of analysis is accurately measured, and a predetermined operation is performed so that the fluorescence spectrum image does not deviate from the reference fluorescence spectrum image acquired as wavelength calibration data. This will be described later.

  A method for measuring the position of the capillary will be described with reference to FIG. FIG. 7A shows the capillary image on the image of the two-dimensional detector 136 when the capillary 102 is irradiated with the capillary position measuring light source 127, the vertical axis is the position in the capillary axis direction, and the horizontal axis is the position in the wavelength dispersion direction. It is. FIG. 7B is a spectrum of the capillary image, where the vertical axis represents the light intensity and the horizontal axis represents the wavelength. In this example, a red laser diode that is a single wavelength light source is used as the capillary position measuring light source 127. Therefore, even if the reflected light from the capillary 102 is split by the diffraction grating 134, a single wavelength image and spectrum can be obtained. Two peaks in the spectrum of FIG. 7B represent the reflected light at the outer surface of the capillary 102. The distance between the two peaks is the same as the outer diameter of the capillary 102.

  In this example, a capillary having an outer diameter of 326 μm was used. The distance between the peaks is about 330 μm. A wavelength indicating the center position of the capillary on the image of the two-dimensional detector 136 is obtained from the image or spectrum of the capillary in FIG. In this example, an image having a certain length with respect to the capillary axis direction can be acquired, so that not only can the position of the capillary be accurately obtained, but also the inclination angle of the capillary can be accurately obtained.

  With reference to FIG. 8, a method for setting a data acquisition region on an image obtained by the two-dimensional detector 136 will be described. FIG. 8 is the same as FIG. 7A, and shows the capillary image 81 on the image of the two-dimensional detector 136 when the capillary 102 is irradiated with light from the capillary position measurement light source 127, and the vertical axis indicates the capillary axis direction. The horizontal axis represents the position of the two-dimensional detector 136 in the wavelength direction on the image.

  First, a method for setting the horizontal range of the data acquisition area will be described. As described above, the capillary center position on the image of the two-dimensional detector 136 is obtained by using a red laser diode as the capillary position measuring light source 127. The capillary center position represents the wavelength 655 nm of the capillary position measuring light source 127. That is, the position of the wavelength 655 nm in the two-dimensional image obtained by the two-dimensional detector 136 can be specified. A data acquisition region on the two-dimensional detector 136 can be set based on the position of the wavelength 655 nm.

  In the analysis experiment, the analysis wavelength region is predetermined. For example, it is assumed that the analysis wavelength region is 525 nm to 715 nm. Based on the position of wavelength 655 nm, the position of wavelength 525 nm and the position of wavelength 715 nm are specified. For example, a CCD having a resolution corresponding to 2 nm per pixel is used as the two-dimensional detector 136. The difference between the wavelength 655 nm and the wavelength 525 nm is 130 nm, which corresponds to 65 pixels. Therefore, the position shifted to the left of 65 pixels from the position of the wavelength 655 nm represents the wavelength 525 nm. Similarly, the difference between the wavelength 655 nm and the wavelength 715 nm is 60 nm, which corresponds to 30 pixels. Therefore, the position shifted to the right of 30 pixels from the position of the wavelength 655 nm represents the wavelength 715 nm. The analysis wavelength region of 525 nm to 715 nm corresponds to 95 pixels. That is, the data acquisition region in the two-dimensional detector 136 is set from a position shifted 65 pixels to the left from the position of wavelength 655 nm to a position shifted 30 pixels to the right. Here, the case where the analysis wavelength region is 525 nm to 715 nm has been described, but a data acquisition region is similarly obtained even in other analysis wavelength regions.

  According to the present invention, since the data acquisition region on the two-dimensional detector 136 is set with reference to the wavelength 655 nm of the capillary position measurement light source 127, a spectrum image in a preset analysis wavelength region can be obtained.

  Once the horizontal range of the data acquisition area is set, next, the vertical range of the data acquisition area is set. The image area of the two-dimensional detector 136 is equally divided into a plurality of areas by lines parallel to the horizontal axis. In this example, five data acquisition areas 82 are set.

  With reference to FIG. 9, the 1st example of the analysis method using the capillary electrophoresis apparatus by this invention is demonstrated. FIG. 9A shows the case where the capillary 102 is at the reference position. The reference position is the position of the capillary at the time of wavelength calibration data acquisition. At the reference position, the capillary 102 is not displaced or inclined. From left to right, an arrangement state of the capillaries 102, an image 201 showing an image 91 of the capillary, and an image 202 showing a reference fluorescence spectrum 92 acquired as wavelength calibration data. In the image 202, five data acquisition areas are set.

  FIG. 9B shows a case where the position of the capillary 102 is shifted from the reference position in the wavelength dispersion direction due to the replacement of the capillary. From left to right, an arrangement state of the capillaries 102, an image 203 showing an image 93 of the capillary, and an image 204 showing a fluorescence spectrum 94 of the analysis sample. In the image 204, five data acquisition areas are set. Since the position of the capillary image 91 in the image 201 of FIG. 9A is different from the position of the capillary image 93 in the image 203 of FIG. 9B, the position of the data acquisition region in the image 204 of FIG. It is shifted laterally from the position of the data acquisition area.

  FIG. 9C shows a case where the capillary 102 is tilted with respect to the capillary axis from the position at the time of wavelength calibration data acquisition by exchanging the capillary. From left to right, an arrangement state of the capillaries 102, an image 205 showing an image 95 of the capillary, and an image 206 showing a fluorescence spectrum 96 of the analysis sample.

  When the capillary 102 is inclined with respect to the capillary axis, it can be assumed that the capillary 102 is composed of a plurality of short portions, and that each portion is sequentially shifted in the wavelength dispersion direction. In this example, the image is divided into five data acquisition areas in the vertical direction. Therefore, the capillary 102 may be divided into five parts so as to correspond to the five data acquisition regions, and each part may be assumed to be shifted in the wavelength dispersion direction. However, the shift amount of each portion is 0 at the center position in the vertical direction of the image, and increases as the distance from the center position increases.

  FIG. 9D is a graph 207 of the fluorescence spectrum 71 extracted from each data acquisition region. Five reference fluorescence spectra are obtained as wavelength calibration data from the five data acquisition regions included in the image 202 of FIG. 9A. Similarly, five fluorescence spectra are obtained from the five data acquisition regions included in the image 204 in FIG. 9B. Five fluorescence spectra are obtained from the five data acquisition regions included in the image 206 of FIG. 9C. The five spectra in FIG. 9A and the five spectra in FIG. 9B are the same for each data acquisition region divided in the capillary axis direction. Although the five spectra in FIG. 9A and the five spectra in FIG. 9C are slightly different depending on the number of vertical divisions, the pseudo signal is small. The more finely divided, the smaller the pseudo signal.

  As described above, according to the present invention, the capillary position is measured and the data acquisition area is set based on the capillary position even when the capillary is displaced due to the replacement of the capillary or when the capillary is inclined. Therefore, a fluorescence spectrum is obtained at the same position as the reference fluorescence spectrum acquired as wavelength calibration data. Therefore, it is not necessary to acquire wavelength calibration data every time the capillary is replaced. Moreover, a pseudo signal can be prevented by using the fluorescence spectrum thus obtained.

  Note that the light emitting part of the capillary may thermally expand due to the influence of the temperature control unit 161. Therefore, the measurement of the capillary position is preferably performed immediately before acquisition of analysis data after the temperature of the temperature control unit 161 is stabilized and the capillary position is fixed.

  With reference to FIG. 10, the detailed procedure of the operation of the first example of the analysis method using the capillary electrophoresis apparatus according to the present invention will be described. In step 200, wavelength calibration data is acquired. Wavelength calibration data is usually acquired at the manufacturing plant before shipment. For example, a wavelength-calibrated DNA sample labeled with four color dyes is electrophoresed to obtain a reference fluorescence spectrum. When the reference fluorescence spectrum is obtained as the wavelength calibration data in this way, electrophoretic analysis is performed as follows.

  The basic procedure of electrophoretic analysis is: preparation before analysis in step 201, start of analysis in step 202, loading of electrophoresis medium in step 203, preliminary migration in step 204, sample introduction in step 205, capillary position measurement in step 206A and data The acquisition area setting, electrophoresis in step 207, and analysis in step 208 are performed on the user side.

  The operator makes preparations before analysis in step 201. When the capillaries 102 are deteriorated or when the length of the capillaries 102 needs to be changed, the capillary array 101 is replaced. Here, it is assumed that the capillary has been replaced. First, the buffer container 144 and the anode buffer container 112 are filled with a buffer solution. The buffer solution is, for example, an electrolyte solution that is commercially available for electrophoresis. Next, a sample to be analyzed is dispensed into the well of the sample container 143. The sample is, for example, a DNA PCR product. In addition, the cleaning liquid is dispensed into the cleaning container 145. The cleaning liquid is pure water, for example. Further, the electrophoresis medium is injected into the syringe 152. The electrophoresis medium is, for example, a polyacrylamide separation gel that is commercially available for electrophoresis.

  The operator starts analysis in step 202. In step 203, the electrophoresis medium is filled, and a new electrophoresis medium is filled in the capillary 102 to form an electrophoresis path. First, the waste liquid container 146 is transported directly under the sample introduction unit 104 by the auto sampler unit. Next, the syringe 152 is driven to fill the capillary 102 with a new electrophoresis medium, and the used electrophoresis medium is discarded in the waste liquid container 146. Finally, the auto sampler unit transports the cleaning container 145 directly below the sample introduction unit 104, immerses the sample introduction end 105 in the cleaning liquid, and cleans the sample introduction end 105 contaminated by the electrophoresis medium.

In the preliminary electrophoresis in step 204, a predetermined voltage is applied to the electrophoresis medium so that the electrophoresis medium is in a state suitable for electrophoresis. First, the buffer container 144 is transported directly below the sample introduction unit 104 by the autosampler unit, and the sample introduction end 105 is immersed in the buffer solution to form a current path.
Next, a voltage of several to several tens of kilovolts is applied to the electrophoresis medium for several to several tens of minutes by the power supply unit. Thereby, the electrophoresis medium is in a state suitable for electrophoresis. Finally, the auto sampler unit transports the cleaning container 145 directly below the sample introduction unit 104, immerses the sample introduction end 105 in the cleaning solution, and cleans the sample introduction end 105 contaminated with the buffer solution with the cleaning solution.

  In step 205, sample components are introduced into the migration path. First, the sample container 143 is transported directly below the sample introduction unit 104 by the autosampler unit, and the sample introduction end 105 is immersed in the sample held in the well of the sample container 143. As a result, an energization path is formed, and a sample component can be introduced into the migration path. A pulse voltage is applied to the energization path by the power supply unit, and the sample component is introduced into the migration path. Finally, the auto sampler unit transports the cleaning container 145 directly below the sample introduction unit 104, immerses the sample introduction end 105 in the cleaning liquid, and cleans the sample introduction end 105 contaminated with the sample with the cleaning liquid.

  In step 206A, the capillary position is measured and the data acquisition area is set. In the capillary position measurement, a capillary position measurement light source 127 is used to measure the capillary position. In setting the data acquisition area, the data acquisition area of the two-dimensional detector 136 is set based on the capillary position. Details of step 206A have been described with reference to FIG.

  In the electrophoresis of step 207, each sample component contained in the sample is separated and analyzed by electrophoresis. First, the buffer container 144 is transported directly under the sample introduction unit 104 by the autosampler unit, and the sample introduction end 105 is immersed in the buffer solution to form a current path. Next, a high voltage of about 15 kV is applied to the energization path by the power supply unit to generate an electric field in the migration path.

  Due to the generated electric field, the sample component in the migration path moves to the irradiation unit 103 at a speed depending on the property of each sample component. That is, the sample components are separated by the difference in movement speed. And it detects in order from the sample component which reached the irradiation part 103. For example, when the sample includes a large number of DNAs having different base lengths, the moving speed varies depending on the base length, and reaches the irradiation unit 103 in order from the DNA having the shorter base length. The irradiation unit 103 irradiates excitation light. The terminal base sequence of each fluorescently labeled DNA generates fluorescence in the order in which it reaches the irradiation unit 103.

  The two-dimensional detector 136 detects the fluorescence spectrum. The detection of the fluorescence spectrum is performed based on the data acquisition area set in step 206A.

  In the analysis in step 208, the wavelength calibration data obtained in step 200 is used to normalize the data obtained by electrophoresis to obtain target chromatic dispersion data. If the wavelength calibration data is inaccurate, a pseudo signal is generated, and the reliability of the analysis result is lowered.

  When the planned data is collected, the process is terminated. The voltage application is stopped and the electrophoretic analysis is terminated. The above is a series of analysis procedures. If further analysis is desired, the analysis procedure is repeated from filling of the migration medium in step 203. When performing another analysis, the analysis procedure is repeated from the preparation before the analysis in step 201. In any case, the acquisition of wavelength calibration data in step 200 is not repeated.

  With reference to FIG. 11, the 2nd example of the analysis method using the capillary electrophoresis apparatus by this invention is demonstrated. In this example, when the position of the capillary changes due to the replacement of the capillary, the position of the capillary is measured, and the wavelength calibration data is corrected based on the measured position. That is, instead of setting the data acquisition area, the reference fluorescence spectrum at the time of wavelength calibration data acquisition is corrected. The corrected reference fluorescence spectrum is substantially the same as the wavelength calibration data that would have been obtained if it was newly acquired when the capillary was replaced.

  FIG. 11A shows the case where the capillary 102 is at the reference position. That is, the capillary 102 at the time of wavelength calibration data acquisition is shown. In order from the left, there are an arrangement state of the capillaries 102, an image 91 of the capillary and an image 301 showing the reference fluorescence spectrum 92 acquired as wavelength calibration data, and a graph 302 of the reference fluorescence spectrum 71 obtained from the image 301.

  In the image 301, five data acquisition areas are set. In this example, the data acquisition area is fixed and is not set based on the position of the capillary 102. These five data acquisition areas are set in advance on the image of the two-dimensional detector 136, for example.

  Although a graph of the reference fluorescence spectrum is obtained from each data acquisition region, they are almost the same although there are differences due to aberrations of the optical system. Therefore, the graph 302 shows one of the graphs of the reference fluorescence spectrum 71 acquired as the wavelength calibration data.

  FIG. 11B shows a case where the position of the capillary 102 is shifted from the reference position in the wavelength dispersion direction by replacing the capillary. From left to right, the arrangement state of the capillaries 102, the image 303 showing the capillary image 93 and the fluorescence spectrum 94 of the analysis sample, the graph 304 of the fluorescence spectrum 72 of the analysis sample obtained from the image 303, and the fluorescence spectrum 73 of the analysis sample after correction The graph 305 of FIG. The data acquisition area of the image 303 is the same as the data acquisition area of the image 301.

  Although a graph of the fluorescence spectrum of the analysis sample is obtained from each data acquisition region, they are almost the same although there are differences due to aberrations of the optical system. Therefore, the graph 304 shows one of the graphs of the fluorescence spectrum 72 of the analysis sample.

  The position of the fluorescence spectrum 72 indicated by the graph 304 is shifted from the position of the reference fluorescence spectrum 71 indicated by the graph 302. This shift amount corresponds to the shift amount between the position of the capillary image 91 in the image 301 and the position of the capillary image 93 in the image 303. The corrected reference fluorescence spectrum 73 shown in the graph 305 is obtained by moving the reference fluorescence spectrum 71 of the graph 302 in the horizontal direction by this shift amount.

  FIG. 11C shows a case where the capillary 102 is inclined with respect to the capillary axis from the reference position by replacing the capillary. From left to right, the arrangement state of the capillary 102, the image 306 showing the capillary image 95 and the fluorescence spectrum 96 of the analysis sample, the graphs 307 and 309 of the fluorescence spectra 74 and 76 of the analysis sample obtained from the image 306, and the corrected analysis sample The graphs 308 and 310 of the fluorescence spectra 75 and 77 of FIG. The data acquisition area of the image 306 is the same as the data acquisition area of the image 301.

  The fluorescence spectra 96 of the analysis samples in the five data acquisition regions are shifted corresponding to the inclination of the capillary 102. Five fluorescence spectra are obtained from the five data acquisition regions, but they are laterally offset from each other. A graph 307 is a fluorescence spectrum obtained from the uppermost data acquisition region, and a graph 309 is a fluorescence spectrum obtained from the lowermost data acquisition region.

  The position of the graph of the fluorescence spectrum 74 indicated by the graph 307 is shifted in the horizontal direction from the position of the fluorescence spectrum 71 indicated by the graph 302. This shift amount corresponds to the shift amount between the position of the capillary image 91 in the image 301 and the position of the capillary image 95 in the uppermost data acquisition region of the image 306. By moving the graph of the reference fluorescence spectrum 71 of the graph 302 to the left in the horizontal direction by this shift amount, the corrected graph 308 of the reference fluorescence spectrum 75 is obtained.

  Similarly, the position of the graph of the fluorescence spectrum 76 indicated by the graph 309 is shifted in the horizontal direction from the position of the reference fluorescence spectrum 71 indicated by the graph 302. This shift amount corresponds to a shift amount between the position of the capillary image 91 in the image 301 and the position of the capillary image 95 in the lowermost data acquisition region of the image 306. By moving the graph of the reference fluorescence spectrum 71 indicated by the graph 302 to the right in the horizontal direction by this amount of deviation, the corrected graph 310 of the reference fluorescence spectrum 77 is obtained.

  However, since the capillary image 95 in each data acquisition region is inclined, the position of the capillary image 95 is obtained by obtaining the position of the center of the capillary image 95 in each data acquisition region.

  In this way, in this example, the reference fluorescence spectrum acquired as the wavelength calibration data is corrected based on the displacement amount or inclination of the capillary. Since the analysis is performed using the corrected reference fluorescence spectrum, it is not necessary to acquire wavelength calibration data every time the capillary is replaced.

  By using the corrected reference fluorescence spectrum for the analysis of the fluorescence spectrum obtained by electrophoresis, generation of a pseudo signal can be prevented.

  With reference to FIG. 12, the detailed procedure of the operation of the second example of the analysis method using the capillary electrophoresis apparatus according to the present invention will be described. In step 200, wavelength calibration data is acquired. Wavelength calibration data is usually acquired at the manufacturing plant before shipment. For example, a wavelength-calibrated DNA sample labeled with four color dyes is electrophoresed to obtain a reference fluorescence spectrum. When the reference fluorescence spectrum is obtained as the wavelength calibration data in this way, electrophoretic analysis is performed as follows.

  The basic procedure of electrophoretic analysis is as follows: preparation before analysis in step 201, start of analysis in step 202, loading of electrophoresis medium in step 203, preliminary migration in step 204, sample introduction in step 205, capillary position measurement in step 206B and wavelength This includes correction of calibration data, electrophoresis in step 207, and analysis in step 208, and is performed on the user side.

  The preparation before analysis in step 201, the start of analysis in step 202, filling of the migration medium in step 203, preliminary migration in step 204, sample introduction in step 205, and electrophoresis in step 207 have been described with reference to FIG.

  In step 206B, the capillary position is measured and the wavelength calibration data is corrected. In the capillary position measurement, a capillary position measurement light source 127 is used to measure the capillary position. In the correction of the wavelength calibration data, the wavelength calibration data is corrected based on the position of the capillary.

  In the analysis of step 208, the wavelength calibration data obtained in step 206B is used to normalize the data obtained by electrophoresis to obtain target chromatic dispersion data.

  With reference to FIG. 13, another example of the capillary position measuring light source will be described. In this example, the capillary position measuring light source 171 has a certain emission wavelength band. Examples of such a light source include a light emitting diode (LED) and a Ne lamp. As shown in FIG. 13A, a capillary position measuring filter 172 is provided on the output side of the capillary position measuring light source 171. The capillary position measurement filter 172 blocks a predetermined wavelength region in the emission wavelength band of the capillary position measurement light source 171. The light transmitted through the capillary position measurement filter 172 thus reflects the surface of the capillary 102 and reaches the first camera lens 132.

  13B shows the emission wavelength band of the capillary position measuring light source 171, and FIG. 13C shows the transmission characteristics of the capillary position measuring filter 172. Since this filter characteristic does not depend on temperature, it is not affected by the wavelength shift of the light source due to temperature change.

  The capillary position measurement filter 172 may be a band-pass filter that passes a predetermined narrow wavelength region, but may be a long-pass filter that passes a wavelength region larger than a predetermined wavelength. When the band-pass filter is used, a spectrum in a narrow wavelength region as shown in FIG. 7 is obtained. Therefore, the position of the capillary 102 can be specified from the position of this spectrum. When a long pass filter is used, a spectrum in a wavelength region larger than a predetermined wavelength position is obtained, so that the position of the capillary 102 can be specified from the start position of this spectrum.

  With reference to FIG. 14, another example of the capillary electrophoresis apparatus of the present invention will be described. In the capillary electrophoresis apparatus of this example, the fluorescence from the light emitting portion of the capillary 102 is emitted from the end of the capillary. The detection optical unit 131 is arranged along the axial direction of the capillary so that fluorescence from the end of the capillary can be detected.

  The excitation light 122 from the light source 123 irradiates the capillary 102, and the detection light is emitted from the end of the capillary. This detection light reaches the detection optical unit 131 via the detection window 181 of the electrophoresis medium filling unit 150.

  The detection optical unit 131 of this example includes a first camera lens 132, a detection filter 133, a prism 182, a second camera lens 135, and a two-dimensional detector 136. In this example, a prism 182 is used as the wavelength dispersion element instead of the diffraction grating. The fluorescence emitted from the end of the capillary 102 becomes a parallel light beam by the first camera lens 132. This parallel light beam is guided to the detection filter 133. The detection filter 133 transmits only fluorescence in the wavelength region used for analysis. The fluorescence transmitted through the detection filter 133 is wavelength-dispersed by the prism 182 and imaged on the two-dimensional detector 136 by the second camera lens 135. The two-dimensional detector 136 is a CCD camera, for example. The image signal from the two-dimensional detector 136 is processed by a computer and the sample is analyzed.

  Also in the capillary electrophoresis apparatus of this example, the position of the capillary may be displaced by the replacement of the capillary. However, by setting the data acquisition area of the two-dimensional detector or correcting the reference fluorescence spectrum acquired as the wavelength calibration data by the above-described method, generation of a pseudo signal due to the displacement of the capillary is prevented. be able to.

  As mentioned above, although the example of this invention was demonstrated, this invention is not limited to this, It is understood by those skilled in the art that various changes are possible in the range of the invention described in the claim. The It is also within the scope of the present invention to appropriately combine the embodiments.

It is a perspective view which shows the outline of a capillary electrophoresis apparatus. It is a perspective view which shows the outline of the irradiation part of the capillary of a capillary electrophoresis apparatus. It is a perspective view which shows the outline of the detection optical unit of a capillary electrophoresis apparatus. It is a figure which shows the wavelength characteristic of the light source for capillary position measurement of a capillary electrophoresis apparatus. It is a figure which shows the reference | standard fluorescence spectrum acquired as wavelength calibration data by a capillary electrophoresis apparatus. In a capillary electrophoresis apparatus, it is explanatory drawing for demonstrating the shift of the fluorescence spectrum resulting from the position shift of a capillary. An example of an image for detecting the position of a capillary in a capillary electrophoresis apparatus is shown. It is a figure which shows the method to set a data acquisition area | region with the image by a two-dimensional detector in a capillary electrophoresis apparatus. It is explanatory drawing for demonstrating the 1st example of the analysis method using a capillary electrophoresis apparatus. It is a figure which shows the detailed procedure of operation of the 1st example of the analysis method using a capillary electrophoresis apparatus. It is explanatory drawing for demonstrating the 2nd example of the analysis method using a capillary electrophoresis apparatus. It is explanatory drawing for showing the detailed procedure of operation of the 2nd example of the analysis method using a capillary electrophoresis apparatus. It is a figure which shows the other example of the light source for capillary position measurement of a capillary electrophoresis apparatus. It is a perspective view which shows the outline of the other example of a capillary electrophoresis apparatus.

Explanation of symbols

DESCRIPTION OF SYMBOLS 101 Capillary array 102 Capillary 103 Irradiation part 104 Sample introduction part 105 Sample introduction end 106 Hollow electrode 107 Glass substrate 108 Capillary head 110 Power supply unit 111 High voltage power supply 112 Anode buffer container 113 Anode electrode 114 Ammeter 121 Irradiation optical unit 122 Excitation light 123 Light source 124 Condensing lens 125 Illumination filter 126 Condensing lens 127 Capillary position measuring light source 131 Detection optical unit 132 First camera lens 133 Detection filter 134 Diffraction grating 135 Second camera lens 136 Two-dimensional detector 141 Autosampler unit 143 Sample container 144 Buffer container 145 Cleaning container 146 Waste liquid container 150 Electrophoresis medium filling unit 151 Polymer filling block 152 Syringe 153 Tube 154 Solenoid valve 15 Polymer flow path 161 temperature control unit 171 capillary position measuring light source 172 capillary position measuring filter 181 detection window 182 prisms

Claims (18)

With replaceable capillaries;
An irradiation optical system for irradiating the capillary with excitation light;
A detection optical system comprising: a wavelength dispersion element that separates fluorescence from the capillary; and a two-dimensional detector that detects a fluorescence spectrum image obtained from the wavelength dispersion element;
In a capillary electrophoresis apparatus having
Furthermore, when the capillary is replaced by providing a light source for capillary position measurement, irradiating the capillary with light from the light source for capillary position measurement, and detecting the image of the capillary with the two-dimensional detector, the capillary Detecting the deviation of the capillary position relative to the axial reference position of
A capillary electrophoresis apparatus , wherein a data acquisition region in the two-dimensional detector is set based on the deviation, and a fluorescence spectrum image of a sample is detected in the data acquisition region . 2. The capillary electrophoresis apparatus according to claim 1, wherein the data acquisition region includes a plurality of data acquisition regions arranged along the capillary axis direction, and each data acquisition region is determined by a range in the wavelength dispersion direction and a range in the capillary axis direction. Capillary electrophoresis apparatus characterized by being made. 2. The capillary electrophoresis apparatus according to claim 1, wherein when the capillary is displaced in the wavelength dispersion direction with respect to the reference position, the data acquisition region is moved by a distance corresponding to the deviation in the wavelength dispersion direction. A capillary electrophoresis apparatus characterized by comprising: 2. The capillary electrophoresis apparatus according to claim 1, wherein when the capillary is inclined with respect to the reference position, the data acquisition region is inclined at an angle corresponding to the inclination. Capillary electrophoresis device. 2. The capillary electrophoresis apparatus according to claim 1, wherein a scale of a wavelength is set in the image of the two-dimensional detector so that the wavelength of the light source for capillary position measurement corresponds to the position of the image of the capillary by the two-dimensional detector. And a data acquisition region corresponding to the analysis wavelength range is set by reading a long wavelength end and a short wavelength end of the analysis wavelength range given in advance by the wavelength scale. 2. The capillary electrophoresis apparatus according to claim 1, wherein the capillary position measuring light source includes a single wavelength light source. 2. The capillary electrophoresis apparatus according to claim 1, wherein the capillary position measuring light source includes a light source having a predetermined emission wavelength band and a filter for blocking a predetermined wavelength region of light from the light source. Capillary electrophoresis device. 2. The capillary electrophoresis apparatus according to claim 1, wherein the irradiation optical system irradiates the capillary with excitation light from a direction along an axis of the capillary or from a direction inclined at a predetermined angle with respect to the axis of the capillary. A capillary electrophoresis device characterized. 2. The capillary electrophoresis apparatus according to claim 1, wherein the irradiation optical system includes a light emitting diode as an excitation light source. 2. The capillary electrophoresis apparatus according to claim 1, wherein the reference position is a position of the capillary when wavelength calibration data is acquired at the time of shipment. With replaceable capillaries;
An irradiation optical system for irradiating the capillary with excitation light;
A detection optical system comprising: a wavelength dispersion element that separates fluorescence from the capillary; and a two-dimensional detector that detects a fluorescence spectrum image obtained from the wavelength dispersion element;
In a capillary electrophoresis apparatus having
Furthermore, when the capillary is replaced by providing a light source for capillary position measurement, irradiating the capillary with light from the light source for capillary position measurement, and detecting the image of the capillary with the two-dimensional detector, the capillary Detecting the deviation of the capillary position relative to the axial reference position of
A capillary electrophoresis apparatus, wherein the position of the fluorescence spectrum at the reference position is corrected based on the deviation. 12. The capillary electrophoresis apparatus according to claim 11, wherein the capillary position measuring light source includes a single wavelength light source. 12. The capillary electrophoresis apparatus according to claim 11, wherein the capillary position measuring light source includes a light source having a predetermined emission wavelength band and a filter for blocking a predetermined wavelength region of light from the light source. Capillary electrophoresis device. 12. The capillary electrophoresis apparatus according to claim 11, wherein the irradiation optical system irradiates the capillary with excitation light from a direction along an axis of the capillary or from a direction inclined at a predetermined angle with respect to the axis of the capillary. A capillary electrophoresis device characterized. 12. The capillary electrophoresis apparatus according to claim 11, wherein the irradiation optical system includes a light emitting diode as an excitation light source. 2. The capillary electrophoresis apparatus according to claim 1, wherein the reference position is a position of the capillary when wavelength calibration data is acquired at the time of shipment. The sample is electrophoresed in the capillary, the capillary is irradiated with excitation light, the fluorescence from the capillary is dispersed to generate a fluorescence spectrum, the fluorescence spectrum is detected by a two-dimensional detector, and based on the detection result In the capillary electrophoresis method for analyzing a sample, when the capillary is replaced, detecting the position of the capillary;
Measuring the deviation of the capillary position relative to the capillary reference position;
Setting a data acquisition region in the two-dimensional detector based on the deviation, and detecting a fluorescence spectrum image of the analysis sample in the data acquisition region . The sample is electrophoresed in the capillary, the capillary is irradiated with excitation light, the fluorescence from the capillary is dispersed to generate a fluorescence spectrum, the fluorescence spectrum is detected by a two-dimensional detector, and based on the detection result In a capillary electrophoresis method for analyzing a sample, Detecting the position of the capillary when the capillary is replaced;
Measuring the deviation of the capillary position relative to the capillary reference position;
Correcting the position of the fluorescence spectrum image at the reference position based on the deviation, and a capillary electrophoresis method.

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