Electrophoresis apparatus and analysis method

Abstract

The electrophoresis apparatus of the present disclosure is characterized by comprising: an electrophoresis path for a sample; a spectroscopic element that spectrally disperses light from the sample within the electrophoresis path; a light detector that detects the light spectrally dispersed by the spectroscopic element; and an arithmetic unit that obtains a spectrum of the light based on a signal from the light detector, wherein the arithmetic unit corrects the spectrum using a correction coefficient determined for each electrophoresis condition or fluorescent dye.

Description

Technical Field

[0001] The present disclosure relates to an electrophoresis apparatus and an analysis method. Background Art

[0002] As a method for analyzing the base sequence or base length of DNA, electrophoresis is well known. As one of the analysis methods using electrophoresis, there is capillary electrophoresis. Capillary electrophoresis is a technique in which a separation medium such as acrylamide is filled in a thin tube called a capillary to perform electrophoresis. More specifically, when a sample containing DNA is placed at one end of the capillary and a high voltage is applied to both ends of the capillary in this state, the negatively charged charged particles, i.e., DNA, move toward the anode side in the capillary depending on their own size, i.e., the base length. Then, by measuring the time required for the sample to migrate a fixed distance (usually from the sample injection end of the capillary to the signal detection unit), the base length of the DNA can be analyzed. Each DNA is labeled with a fluorescent dye and emits fluorescence upon irradiation with excitation light. The fluorescence is detected by a light detector.

[0003] In the analysis of DNA based on capillary electrophoresis, for the purpose of speeding up the analysis, sometimes multiple fluorescent dyes are used. The multiple fluorescent dyes are irradiated with excitation light and emit different fluorescences respectively. The spectrum obtained by spectroscopically analyzing the fluorescence on the light detector is called a fluorescence spectrum. Each fluorescent dye has a different fluorescence spectrum, but they are not sharp and there is an overlap between each fluorescent dye. Therefore, when DNA fragments labeled with different fluorescent dyes have the same fragment length in the light detector, the spectrum of the fluorescence obtained by the light detector is the linear sum, i.e., the weighted sum, of the fluorescence spectra of the multiple fluorescent dyes. In order to obtain the signal intensity (fluorescence intensity) of each fluorescent dye from this state, it is only necessary to obtain the linear coefficient, i.e., the weight value, of the spectrum of each fluorescent dye constituting the spectrum from the spectrum obtained by the light detector.

[0004] In order to obtain this weight value, each fluorescence spectrum must be known in advance. Each fluorescence spectrum is originally determined by the fluorescent dye and the separation medium in a unified manner regardless of the device. However, in an actual device, for various reasons, the fluorescence spectrum changes. One of the well-known reasons is the positional relationship between the capillary and the light detector. Therefore, when replacing the capillary, before performing electrophoresis on a sample to be analyzed (hereinafter referred to as "actual sample"), an operation of obtaining the fluorescence spectrum in this device and this capillary in advance is required. This operation is called "spectrum calibration". In addition, when using a capillary array in which multiple capillaries are arranged to perform electrophoresis on multiple samples simultaneously, it is necessary to obtain the fluorescence spectrum for each capillary.

[0005] Here, an example of spectrum calibration of the prior art will be described.

[0006] Figure 1It is a diagram showing a diffraction grating image (lower part) imaged on a photodetector of a multi-capillary electrophoresis device and the signal intensity distribution of capillaries corresponding to the A-A' direction of the diffraction grating image (upper part). The multi-capillary electrophoresis device separates the fluorescence emitted from each fluorescent pigment in the wavelength direction by irradiating a laser of a specific wavelength onto the capillaries and using a diffraction grating, detects the separated light using a photodetector such as a CCD, and obtains a diffraction grating image. Then, the signal intensity distribution (spectrum) is obtained from the diffraction grating image.

[0007] Figure 1 The lower part shows the diffraction grating image when a laser is irradiated onto a capillary array with 4 capillaries arranged. The vertical axis represents the arrangement direction of the capillaries, and the horizontal axis represents the wavelength direction. In Figure 1 In the upper part, the vertical axis represents the signal intensity (brightness value (RFU)), and the horizontal axis represents the wavelength. Additionally, Figure 1 An example of continuously (actually discretely for each pixel) measuring the spectrum using a diffraction grating is shown, but it can also be data obtained by sampling the above spectrum at relatively wide wavelength intervals. For example, as shown in the diffraction grating image of Figure 1 , for each capillary, it is also possible to obtain only the signal intensities at 20 wavelengths λ(0) to λ(19). Additionally, it is also possible to take the arithmetic mean of the signal intensities near each of the wavelengths λ(0) to λ(19).

[0008] Figure 2 It is a flowchart showing an existing spectral calibration method.

[0009] In step S101, the operator performs electrophoresis of a matrix standard. The matrix standard is a reagent used to obtain a fluorescence spectrum and obtain a matrix described later. The matrix standard contains 4 DNA fragments of different lengths labeled with different fluorescent pigments respectively. Information on the length or the order of the lengths of the DNA fragments corresponding to each fluorescent pigment is known.

[0010] Figure 3A It is a diagram showing the waveform of the signal intensity obtained by performing electrophoresis of the matrix standard. The vertical axis represents the signal intensity, and the horizontal axis represents time. In step S101, it is assumed that fluorescence spectra of 4 fluorescent pigments (ROX, TMR, R110, R6G) are obtained, Figure 3A It shows a state where the signal intensity waveforms of each fluorescent pigment are overlapped on one graph. As shown in Figure 3A , sharp peaks appear at times corresponding to the lengths of the DNA fragments labeled with each fluorescent pigment. Since the DNA fragments of different lengths are labeled with different fluorescent pigments respectively, each fluorescent pigment emits light alone at each peak time (t0, t1, t2, t3). Therefore, by obtaining the time when only a specific fluorescent pigment emits light (at Figure 3AThe spectra at t0, t1, t2, t3, and t4) are obtained to get the fluorescence spectra of the respective fluorescent dyes.

[0011] Return to Figure 2 , in step S102, the arithmetic control circuit of the multi-capillary electrophoresis device calculates the fluorescence intensity based on the spectra at each moment of the signal intensity obtained in step S101. The processing of this step can be performed for each scanning moment or after accumulating spectral data at a fixed time interval.

[0012] In step S103, the arithmetic control circuit detects Figure 3A the peak moments of the signal intensity waveform. As described above, since the order of appearance of the peaks corresponding to the lengths of the DNA fragments labeled with the respective fluorescent dyes is known, the type of fluorescent dye can be identified based on the appearance moments of the peaks. In Figure 3A , it shows the case where ROX emits light alone at time t0, TMR emits light alone at time t1, R110 emits light alone at time t2, and R6G emits light alone at time t3. The spectra at each moment correspond to the respective fluorescence spectra. That is, by obtaining the spectra at each peak moment, the respective fluorescence spectra can be known.

[0013] Figure 3B is the fluorescence spectrum obtained from the Figure 3A signal intensity waveform. The vertical axis represents the fluorescence intensity, and the horizontal axis represents the wavelength. As Figure 3B shown, the arithmetic control circuit obtains the fluorescence spectra of the respective fluorescent dyes based on the signal intensity waveform.

[0014] Return to Figure 2 , in step S104, the arithmetic control circuit uses the respective fluorescence spectra to obtain matrix M. The following Equation 1 shows an example of matrix M when the signal intensities at 20 wavelengths λ(0) to λ(19) are obtained. The elements of matrix M correspond to the intensity ratios of the signal intensities of the respective fluorescent dyes at each wavelength at each peak moment. This ratio is, for example, the ratio to the maximum value between wavelengths for each fluorescent dye. For example, the element WX1 in Equation 1 is the ratio of the fluorescence intensity of the fluorescent dye ROX at wavelength λ(1) at time t0. The higher this value, the higher the contribution of that wavelength to the fluorescence intensity. Matrix M is used to obtain the respective fluorescence intensities based on the spectral waveform obtained by the photodetector.

[0015] [Equation 1]

[0016]

[0017]

[0018]

[0019]

[0020]

[0021] As described above, the operations in steps S101 to S104 are spectral calibration. In the case of multiple capillaries, it is necessary to obtain the matrix M for each capillary. In addition, spectral calibration needs to be performed each time a capillary is set or a component is replaced, etc.

[0022] The matrix M obtained through spectral calibration is also referred to as the reference spectrum, and ideally it is the same as the fluorescence spectrum of the actual sample. However, in reality, sometimes a deviation occurs between the reference spectrum and the fluorescence spectrum of the actual sample. If a deviation occurs, the weight value cannot be correctly calculated, and an incorrect fluorescence intensity is recorded. In severe cases, a spurious peak appears at the same peak time as the main peak.

[0023] Figure 4 is the fluorescence spectrum in the case where a spurious peak appears. The spurious peak is generated due to the overlap of the fluorescence spectra of each color, and when a deviation occurs between the reference spectrum and the fluorescence spectrum of the actual sample, the influence brought by this overlap is observed to be relatively large. In addition, in the case of multiple main peaks, this spurious peak is observed in all of these main peaks.

[0024] The deviation between the reference spectrum and the fluorescence spectrum of the actual sample generally results from the differences in the fluorescent dyes and electrophoresis conditions during spectral calibration and when the actual sample migrates. That is, each time the operator changes the fluorescent dye and electrophoresis conditions used in the actual sample, it is necessary to re-perform spectral calibration, which thus increases the trouble and cost.

[0025] Patent Document 1 discloses "a gene analysis device, characterized in that it uses known DNA fragment information, namely a molecular weight standard and an allele ladder, used during the electrophoresis of an actual sample to obtain a reference fluorescence spectrum, and for a capillary that does not use an allele ladder, detects the displacement amount of the fluorescence spectrum of the molecular weight standard, and uses this displacement amount to displace the reference fluorescence spectrum to calculate the fluorescence spectrum, thereby performing spectral calibration" (refer to the abstract of this document). Thus, it is not necessary to perform electrophoresis using a special matrix standard, and therefore spectral calibration can be achieved in a short time and at low cost.

[0026] The molecular weight standard refers to a mixture of known DNA fragments labeled with a specific fluorescent dye. The allele ladder refers to a mixture of known DNA fragments labeled with the same fluorescent dye as the actual sample. In the application described in Patent Document 1, the molecular weight standard is mixed with all the specimens during electrophoresis. Then, the allele ladder is analyzed through a capillary different from the actual sample.

[0027] Prior Art Documents

[0028] Patent document

[0029] Patent Document 1: Japanese Unexamined Patent Application Publication No. 2014-117222 Summary of the invention

[0030] Problems to be solved by the invention

[0031] However, in Patent Document 1, the shift amount of the fluorescence spectrum between capillaries is calculated using a specific fluorescent dye, and the case where the shift amount varies depending on the fluorescent dye is not assumed. Therefore, depending on the fluorescent dye, an appropriate reference spectrum may not be obtained, resulting in a deviation between the reference spectrum and the fluorescence spectrum of the actual sample, and the generation of false peaks. In addition, in Example 3 of Patent Document 1, an example of spectral calibration for each capillary is given. However, for this purpose, a peak composed of a monochromatic fluorescent dye is required. Therefore, in a sample in which multiple peaks overlap, a reference spectrum may not be obtained. As a result, a deviation may occur between the reference spectrum and the fluorescence spectrum of the actual sample. Based on the above, the method of Patent Document 1 is difficult to apply to any fluorescent dye and any sample, and therefore spectral calibration needs to be performed again whenever the electrophoresis conditions or fluorescent dye are changed. As a result, the trouble and cost for the operator increase.

[0032] Therefore, the present disclosure provides an electrophoresis apparatus and an analysis method that reduce the trouble and cost for the operator.

[0033] Means for solving the problems

[0034] To solve the above problems, the electrophoresis apparatus of the present disclosure is characterized by including: an electrophoresis path for a sample; a spectroscopic element that spectroscopically analyzes light from the sample in the electrophoresis path; a light detector that detects the light spectroscopically analyzed by the spectroscopic element; and an arithmetic unit that obtains the spectrum of the light based on a signal from the light detector, and the arithmetic unit corrects the spectrum using a correction coefficient determined for each electrophoresis condition or fluorescent dye.

[0035] In addition, another electrophoresis apparatus of the present disclosure is characterized by including: an electrophoresis path for a sample; a spectroscopic element that spectroscopically analyzes light from the sample in the electrophoresis path; a light detector that detects the light spectroscopically analyzed by the spectroscopic element; and an arithmetic unit that calculates the signal intensity of the light based on the signal of the light detector, and the light detector obtains the signal with a signal acquisition width, and the signal acquisition width is set so that the correlation coefficient between the spectra of multiple fluorescent dyes is equal to or greater than a predetermined value.

[0036] Further features related to the present disclosure will become clear from the description and the drawings of this specification. In addition, the aspects of the present disclosure are achieved by elements and combinations of various elements, as well as the following detailed description and the appended claims.

[0037] The description in this specification is merely a typical illustration and does not limit the scope of the claims or the application examples of the present disclosure in any sense.

[0038] Advantages of the Invention

[0039] According to the present disclosure, it is not necessary to perform spectral calibration again every time the migration conditions or the fluorescent dye are changed. As a result, the trouble and cost of the operator are reduced. Other problems, configurations, and effects will become clear from the following description of the embodiments. Brief Description of the Drawings

[0040] Figure 1 It is a graph showing the signal intensity (upper part) and wavelength (lower part) of fluorescence detected by a multi-capillary electrophoresis device.

[0041] Figure 2 It is a flowchart showing an existing spectral calibration method.

[0042] Figure 3A It is a diagram for explaining the outline of spectral calibration of the prior art.

[0043] Figure 3B It is a diagram for explaining the outline of spectral calibration of the prior art.

[0044] Figure 4 It is a diagram for explaining a pseudo peak.

[0045] Figure 5 It is a schematic diagram showing a multi-capillary electrophoresis device according to the first embodiment.

[0046] Figure 6 It is a schematic diagram showing the structure of an optical system in a thermostat.

[0047] Figure 7 It is a flowchart showing a method for calculating a correction coefficient according to the first embodiment.

[0048] Figure 8A It is a diagram for explaining the outline of the calculation of matrix M' in the first embodiment.

[0049] Figure 8B It is a diagram for explaining the outline of the calculation of matrix M' in the first embodiment.

[0050] Figure 9 It is a flowchart showing a method for applying a correction coefficient in electrophoresis of an actual sample.

[0051] Figure 10 It is a flowchart of the electrophoresis method for actual samples.

[0052] Figure 11 It is a graph for explaining Gaussian fitting.

[0053] Figure 12 It is a flowchart showing the analysis method of the sample of the second embodiment.

[0054] Figure 13 It is a graph showing the results of Experimental Example 1.

[0055] Figure 14 It is a flowchart showing the analysis method of the sample of the third embodiment.

[0056] Figure 15A It is a graph showing the fluorescent dyes used in Experimental Example 2.

[0057] Figure 15B It is a graph showing the results of Experimental Example 2.

[0058] Figure 16 It is a flowchart showing the analysis method of the sample of the fifth embodiment.

[0059] Figure 17A It is the fluorescence spectrum obtained in Experimental Example 3.

[0060] Figure 17B It is the fluorescence spectrum obtained in the control experiment of Experimental Example 3.

[0061] Figure 18 It is a flowchart showing the analysis method of the sample of the sixth embodiment. Detailed implementation manners

[0062] Hereinafter, the embodiments will be described with reference to the accompanying drawings. In the accompanying drawings, sometimes elements having the same function are also denoted by the same reference numerals. In addition, the accompanying drawings show embodiments and installation examples that follow the technical principles of the present disclosure, but these are for understanding the present disclosure and are never used to limitatively interpret the technology of the present disclosure. The description of this specification is merely a typical illustration and does not limit the scope of the claimed invention or application examples of the present disclosure in any sense.

[0063] In the present embodiment, those skilled in the art have described it in sufficient detail for implementing the present disclosure. However, it should be understood that other installations and forms are also possible, and changes in structure and configuration and replacement of various elements can be made without departing from the scope and spirit of the technical idea of the present disclosure. Therefore, the following description should not be construed as being limited thereto.

[0064] [First Embodiment]

[0065] As described in the background art, when there is a deviation between the reference spectrum and the fluorescence spectrum of the actual sample, the correct weight value cannot be calculated, and the incorrect fluorescence intensity is recorded. This deviation is mainly caused by the change in the spectrum due to the denaturation of the fluorescent dye. The denaturation of the fluorescent dye is caused by inappropriate pH, storage at inappropriate temperature, and overexcitation of the dye. In addition, the denaturation of the fluorescent dye may also occur during spectral calibration and when the electrophoresis voltage is different during the migration of the actual sample. In a capillary electrophoresis apparatus having a plurality of capillaries, the intensity of the excitation light is different for each capillary, and thus a deviation may occur. It should be noted that in each of the above-listed examples, the degree of denaturation varies depending on the fluorescent dye. In addition, when the actual sample is labeled with a fluorescent dye different from the matrix standard, a deviation will of course occur.

[0066] Therefore, in the first embodiment, the operation (correction of the fluorescence spectrum) of the operator who has purchased a multi-capillary electrophoresis apparatus when the electrophoresis voltage is different during spectral calibration and the migration of the actual sample will be described. In addition, in this specification, the spectral calibration performed by the manufacturer of the multi-capillary electrophoresis apparatus before the shipment of the apparatus is sometimes referred to as "first spectral calibration", and the spectral calibration performed by the operator who has purchased the multi-capillary electrophoresis apparatus is referred to as "second spectral calibration".

[0067] <Example of the Structure of a Multi-Capillary Electrophoresis Apparatus>

[0068] Figure 5 is a schematic diagram showing the structure of the multi-capillary electrophoresis apparatus 500 of the first embodiment. As Figure 5 shown, the multi-capillary electrophoresis apparatus 500 includes a device main body 501 and a control computer 502.

[0069] The device main body 501 includes an arithmetic control circuit 503, a photodetector 504, a thermostat 505, a capillary array 506, a light source 507, a light irradiation unit 508, a loading head 509, a cathode buffer container 511, a sample container 512, a polymer cassette 513, an anode buffer container 514, an anode 515, a high voltage power supply 516, an array head 517, a transporter 518, an injection mechanism 520, a heating and cooling mechanism 523, and a diffraction grating 524.

[0070] The device main body 501 is communicably connected to the control computer 502. The operator can operate the control computer 502 to control each unit included in the device main body 501. The control computer 502 receives data (such as the detection signal of the photodetector 504) acquired by the device main body 501. The control computer 502 includes a display for displaying the received data. In addition, the control computer 502 may be built in the device main body 501.

[0071] The arithmetic control circuit 503 performs arithmetic processing of the measured value (fluorescence intensity) based on the detection signal of the optical detector 504, and corrects the measured value (fluorescence intensity). In addition, the arithmetic control circuit 503 controls the apparatus main body 501 in accordance with the input and command from the control computer 502.

[0072] The optical detector 504 is an optical sensor that detects fluorescence generated by the laser, which is the excitation light irradiated from the light source 507 to the capillary array 506. As the light source 507, a liquid laser, a gas laser, a semiconductor laser, etc. can be appropriately used, or an LED can be used instead. The light source 507 can irradiate the excitation light from both sides of the arrangement of the capillary array 506, and can also be configured to irradiate the excitation light time-divisionally.

[0073] The thermostat 505 is a temperature control mechanism for controlling the temperature of the capillary array 506. The thermostat 505 is covered with a heat insulating material to keep the temperature fixed in the tank, and the temperature is controlled by the heating and cooling mechanism 523. Thereby, the temperature of most of the capillary array 506 can be maintained at a fixed temperature of about 60°C, for example.

[0074] The capillary array 506 is constituted by arranging a plurality of (4 in the Figure 5 example) capillaries 519 (electrophoresis paths). The capillary array 506 can be configured to be appropriately replaced with a new one in the case of confirming breakage or deterioration of quality. In addition, the capillary array 506 can be replaced with another capillary array having different numbers and lengths of capillaries according to the measurement.

[0075] Each of the plurality of capillaries 519 constituting the capillary array 506 can be constituted by a glass tube having an inner diameter of several tens to several hundreds of μm and an outer diameter of several hundreds of μm. In addition, in order to improve the strength, the surface of the glass tube can also be covered with a polyimide film. However, the polyimide film on the surface of the capillary 519 is removed at the part where the laser is irradiated and in its vicinity. The inside of the capillary 519 is filled with a separation medium for separating DNA molecules in a biological sample (sample). Here, a commercially available polyacrylamide-based separation gel for electrophoresis (hereinafter referred to as "polymer") is used.

[0076] The light irradiation unit 508 is disposed on a part of the capillary array 506. As described later, the light irradiation unit 508 is configured to allow the laser light (excitation light) from the light source 507 to be incident on a plurality of capillaries 519 in common, and guide the fluorescence emitted from the plurality of capillaries 519 to the light detector 504. Specifically, in order to irradiate the light irradiation part provided on the capillary array 506 with the laser light as the measurement light, the light irradiation unit 508 includes a light projection optical system such as an optical fiber and a lens. The diffraction grating 524 (spectral element) spectroscopically analyzes the light from the capillary 519 and makes it incident on the light detector 504.

[0077] In the present disclosure, an example in which the fluorescence from the fluorescent dye based on the irradiation of the excitation light is detected by the light detector 504 is described, but the detected light is not limited to fluorescence, and may be light absorption, luminescence, etc.

[0078] The loading head 509 is provided at one end of the capillary array 506. The loading head 509 functions as a cathode to which a negative voltage for introducing a biological sample (sample) into the capillary 519 is applied. An array head 517 is provided at the other end of the capillary array 506, and the array head 517 bundles a plurality of capillaries 519 into one bundle. In addition, the array head 517 has a tip 521 for inserting the polymer cassette 513 on its lower surface.

[0079] The transporter 518 is configured to place the cathode buffer solution container 511, the sample container 512, the polymer cassette 513, and the anode buffer solution container 514 on its upper surface and transport them. As an example, the transporter 518 includes three electric motors and a linear actuator, and can move in three axial directions of up and down, left and right, and front and back.

[0080] The cathode buffer solution container 511 and the anode buffer solution container 514 are containers for holding the buffer solution for electrophoresis, and the sample container 512 is a container for holding the sample to be measured.

[0081] The polymer cassette 513 is a container for holding the polymer for electrophoresis. The upper part 522 of the polymer cassette 513 is sealed with a highly plastic material such as rubber or silicone, and is connected to the injection mechanism 520 for filling the polymer and the transporter 518.

[0082] The order of filling the polymer into the capillary 519 from the polymer cassette 513 is as shown in the following (1) to (3).

[0083] (1) The transporter 518 operates, and the array head 517 moves to the upper side of the polymer cassette 513.

[0084] (2) The tip 521 of the array head 517 penetrates the upper portion 522 of the polymer cartridge 513. At this time, the upper portion 522 of the polymer cartridge 513 with high plasticity wraps around the tip 521 of the array head 517, whereby the two are in close contact, and the polymer cartridge 513 and the capillary 519 are connected in a sealed state.

[0085] (3) The injection mechanism 520 pushes up the polymer inside the polymer box 513 and injects the polymer into the capillary 519.

[0086] An anode 515 for applying a positive voltage for electrophoresis is arranged in the anode buffer container 514 so as to be in contact with the buffer. A high voltage power supply 516 is connected between the anode 515 and the loading head 509 serving as a cathode.

[0087] The transporter 518 transports the cathode buffer container 511 and the sample container 512 to the cathode end 510 of the capillary 519. At this time, the anode buffer container 514 moves to the tip 521 corresponding to the anode end of the capillary 519 in conjunction therewith.

[0088] The sample container 512 contains the same number of sample tubes as the capillary tubes 519. The operator dispenses DNA into the sample tubes.

[0089] The calculation control circuit 503 (calculation unit) includes a measurement value calculation unit 5032 , a correction coefficient calculation unit 5033 , a correction coefficient database 5034 , and a correction unit 5035 .

[0090] The measurement value calculation unit 5032 calculates the measurement value (fluorescence intensity) based on the detection signal of the light detector 504. The correction coefficient calculation unit 5033 calculates the correction coefficient for correcting the measurement value calculated by the measurement value calculation unit 5032. The correction coefficient database 5034 stores the correction coefficient calculated by the correction coefficient calculation unit 5033. In addition, the correction unit 5035 applies the correction coefficient stored in the correction coefficient database 5034 to the measurement value of the measurement value calculation unit 5032, and calculates the corrected measurement value. The calculation processing of each unit of the above-mentioned calculation control circuit 503 can be realized by, for example, a processor such as a CPU or an MPU executing a program.

[0091] Figure 6 505 is a schematic diagram showing the structure of the optical system in the constant temperature chamber 505. Figure 6 As shown, as an example, the light irradiation unit 508 has a plurality of (in Figure 6There are two mirrors 602 and a condenser lens 603 in the middle. The mirror 602 changes the traveling direction of the laser 601 from the light source 507. In addition, the condenser lens 603 condenses the laser on the light irradiation part of the capillary array 506. Thus, the laser 601 is incident on the plurality of capillaries 519 in sequence. The fluorescent dye in each capillary 519 is excited by the laser 601 and emits information light (fluorescence having a wavelength depending on the sample). The information light is spectroscopically separated in the wavelength direction by the diffraction grating 524. The spectroscopically separated information light is detected by the light detector 504. At this time, the light detector 504 can also continuously (actually discretely for each pixel) measure the spectrum, but in this embodiment, as an example, only the signal intensities at 20 wavelengths λ(0) to λ(19) are obtained.

[0092] In this way, by observing the fluorescence intensity of the fluorescence emitted by the incidence of the laser 601 with the light detector 504, the analysis of DNA in electrophoresis can be performed. Electrophoresis means that a mobility is imparted to the sample in the capillary 119 by the electric field generated between the cathode and anode buffers, and the sample is separated by the difference in mobility depending on the nature of the sample. Here, the case where the sample is DNA will be described as an example.

[0093] DNA has a negative charge in the polymer through the phosphodiester bond equivalent to the double helix backbone. Therefore, DNA moves toward the anode side in the electric field. At this time, since the polymer has a mesh-like structure, the mobility of DNA depends on the ease of penetration into the mesh, in other words, depends on the size of DNA. DNA with a short base length easily passes through the mesh-like structure and has a higher mobility, while DNA with a long base length is the opposite. Since a fluorescent substance (fluorophore) is pre-labeled on DNA, optical detection is performed with the light detector 504 in order from the DNA with the shortest base length. Usually, the measurement time and the voltage application time are set according to the sample with the longest migration time.

[0094] <Calculation method of correction coefficient>

[0095] As described above, this embodiment proposes a method for correcting the fluorescence spectrum when the migration voltage is different during spectral calibration and actual sample migration. The manufacturer of the multi-capillary electrophoresis device 500 obtains a correction coefficient for correcting the fluorescence spectrum obtained during actual sample migration before the device leaves the factory and registers it in the correction coefficient database 5034 of the arithmetic control circuit 503.

[0096] Figure 7It is a flowchart showing a calculation method of a correction coefficient. An overview of the calculation method of the correction coefficient is as follows. First, in step S1, the manufacturer performs spectral calibration using a matrix standard, and obtains a reference matrix M through the arithmetic control circuit 503. Next, in step S2, the arithmetic control circuit 503 obtains a matrix M' for correction. Finally, in step S3, the arithmetic control circuit 503 obtains a correction coefficient matrix K.

[0097] (Step S1)

[0098] In step S1, the manufacturer performs spectral calibration (first spectral calibration) using a matrix standard containing DNA fragments labeled with arbitrary fluorescent dyes. In the present embodiment, as an example, ROX, TMR, R110, and R6G are used as fluorescent dyes. The migration voltage should be the same as the migration voltage in the spectral calibration (second spectral calibration) before the actual sample migrates, which will be described later. In the present embodiment, as an example, it is set to 15 kV, but the migration voltage is not limited thereto.

[0099] The manufacturer registers the types of fluorescent dyes and the migration voltage in the arithmetic control circuit 503 by controlling the operation of the input device of the computer 502. The measurement value calculation unit 5032 calculates the matrix M under this condition.

[0100] Here, one of the problems to be solved by the present disclosure is that if the migration voltage is different during the spectral calibration performed by the operator and during the migration of the actual sample, a deviation will occur between the reference spectrum and the fluorescence spectrum of the actual sample. The migration voltage affects the time required for electrophoresis and the separation ability, which is one of the important quality indicators during analysis. Therefore, when using a multi-capillary electrophoresis device, the operator frequently changes the migration voltage of the actual sample as needed. Moreover, whenever the migration voltage of the actual sample is changed, the operator needs to perform spectral calibration again with the same migration voltage as the actual sample.

[0101] To solve this problem, in the present embodiment, it is proposed to perform the first spectral calibration at various migration voltages before the multi-capillary electrophoresis device leaves the factory, quantify the deviation between the spectra found here, and thus pre-register the correction coefficient that minimizes the deviation in the arithmetic control circuit 503. The correction coefficient is registered together with information such as the fluorescent dyes used and the migration voltage.

[0102] The operator who has purchased the device can select an arbitrary migration voltage from the signals registered in the arithmetic control circuit 503, and after performing the second spectral calibration, make the actual sample migrate at the same arbitrary migration voltage registered in the arithmetic control circuit 503. That is, within the range registered in the arithmetic control circuit 503, even if the migration voltage of the actual sample is changed several times, the operator does not need to perform spectral calibration again each time.

[0103] If the above application is envisioned, in step S1, the manufacturer should not only transfer the matrix standard at 15 kV, but also transfer the matrix standard at multiple voltages. Then, all the obtained matrices M should be registered in the arithmetic control circuit 503 together with the information on the migration voltage and the fluorescent dye.

[0104] The calculation method for matrix M is as described above.

[0105] (Step S2)

[0106] In step S2, the manufacturer migrates the matrix standard with the same fluorescent dye and the same migration conditions as the actual sample. Here, it is assumed that the actual sample is labeled with the same fluorescent dye as the matrix standard used in step S1 and migrated at 7.5 kV. At this time, the manufacturer registers the type of the fluorescent dye and the migration voltage in the arithmetic control circuit 503 by controlling the operation of the input device of the computer 502.

[0107] As described above, the matrix standard contains DNA fragments of different lengths labeled with different fluorescent dyes respectively. Therefore, at each peak time (t0’, t1’, t2’, t3’), each fluorescent dye emits light independently. In addition, since the order of appearance of the peak times corresponding to the respective fluorescent dyes is known, the type of the fluorescent dye corresponding to each peak time can be identified.

[0108] Figure 8A is a graph showing the waveform of the signal intensity obtained by performing electrophoresis of the matrix standard. The vertical axis represents the signal intensity, and the horizontal axis represents the time. As Figure 8A shown, ROX emits light independently at time t'0, TMR emits light independently at time t'1, R110 emits light independently at time t'2, and R6G emits light independently at time t'3. The spectrum at each time corresponds to the fluorescence spectrum of each fluorescent dye. Therefore, the arithmetic control circuit 503 obtains the fluorescence spectra of the respective fluorescent dyes by obtaining the spectra at each peak time.

[0109] Figure 8B is Figure 8A the fluorescence spectrum obtained from the signal intensity waveform, with the vertical axis representing the fluorescence intensity and the horizontal axis representing the wavelength.

[0110] The measurement value calculation unit 5032 calculates the matrix M’ using the respective fluorescence spectra. The following mathematical formula 2 shows an example of the matrix M’ when the signal intensities at 20 wavelengths λ(0) to λ(19) are obtained. The elements of the matrix M’ correspond to the intensity ratios of the respective fluorescent dyes at each wavelength at each peak time (t’0, t’1, t’2, t’3). For example, the element W’X1 in the mathematical formula 2 is the ratio of the fluorescence intensity of the fluorescent dye ROX at time t’0 and wavelength λ(1).

[0111] [Mathematical Formula 2]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117] In addition, in step S2, due to the reasons described in step S1, in actual operation, the matrix standard is electrophoresed through multiple voltages including 7.5 kV, and all the obtained matrices M' are registered in the arithmetic control circuit 503 together with information such as the electrophoresis voltage and the fluorescent dye.

[0118] (Step S3)

[0119] Return to Figure 7 , in step S3, the measured value calculation unit 5032 sends the calculated matrices M and M' to the correction coefficient calculation unit 5033. The correction coefficient calculation unit 5033 obtains the correction coefficient matrix K based on matrices M and M'. The element of the correction coefficient matrix K is defined as the element k(ij) = w'(ij) / w(ij) in the fluorescent dye i and wavelength j. As already described, the fluorescent dyes and electrophoresis voltages used in steps S1 and S2 are registered in the arithmetic control circuit 503. Therefore, k(ij) can be stored in the correction coefficient database 5034 together with the information on the electrophoresis conditions and fluorescent dyes used in the calculation. At this time, as described in steps S1 and S2, when matrices M and M' are obtained through multiple electrophoresis voltages, the correction coefficient calculation unit 5033 calculates the correction coefficient matrix K through all their combinations and registers it in the correction coefficient database 5034 together with the information on the electrophoresis voltage and fluorescent dye.

[0120] <Analysis Method of Electrophoresis of Actual Sample>

[0121] Figure 9 It is a flowchart showing the application method of the correction coefficient in the electrophoresis of the actual sample performed by the operator.

[0122] (Step S11)

[0123] The above steps S1 to S3 have ended at the time point when the operator purchases the multi-capillary electrophoresis device 500. The operator only needs to perform the operations after step S11. In addition, at the time of purchase (after step S3), in order to transport the device, the capillary is loaded and unloaded, and the positional relationship between the photodetector 504 and the capillary 519 changes. That is, the device is in a state where spectral calibration needs to be performed again.

[0124] In step S11, the operator performs spectral calibration using the matrix standard as in step S1. For convenience, the spectral calibration performed by the operator is called "second spectral calibration". The migration voltage in the second spectral calibration can be arbitrarily selected as long as it is the migration voltage registered in the correction coefficient database 5034. In the present embodiment, as an example, it is assumed that the migration is performed at 15 kV. In addition, regarding the fluorescent dyes, the matrix standard is labeled with ROX, TMR, R110, and R6G. Let the matrix M obtained by the measurement value calculation unit 5032 through the second spectral calibration in step S11 be the matrix M(r).

[0125] (Step S12)

[0126] In step S12, the operator performs the migration of the actual sample. It is assumed that the actual sample is an unknown sample, but the types of fluorescent dyes and the migration voltage are known. The migration conditions of the actual sample are 7.5 kV in step S2. Regarding the fluorescent dyes, like the matrix standard, the actual sample is also labeled with ROX, TMR, R110, and R6G.

[0127] Figure 10 is a flowchart of the electrophoresis method of the actual sample in step S12. As Figure 10 shown, the basic sequence of electrophoresis includes sample preparation (step S121), start of analysis (step S122), filling of the separation medium (step S123), preliminary migration (step S124), sample introduction (step S125), and migration analysis (step S126).

[0128] (Step S121)

[0129] In step S121, as sample preparation before the start of analysis, the operator sets the sample and reagents in the multi-capillary electrophoresis device 500. More specifically, first, the operator Figure 5The cathode buffer container 511 and the anode buffer container 514 shown are filled with a buffer that forms a part of the current path. The buffer can use, for example, a commercially available electrolyte solution for electrophoresis. In addition, the operator dispenses an actual sample to be analyzed into the hole of the sample container 512. The actual sample is, for example, a PCR product of DNA. In addition, the operator injects a separation medium for electrophoresis of the sample into the injection mechanism 520. The separation medium uses the above-mentioned polymer. Moreover, in the case of expected deterioration of the capillary 519 or the case of changing the length of the capillary 519, the operator replaces the capillary array 506.

[0130] (Step S122)

[0131] In step S122, the operator registers the type of fluorescent dye used in the actual sample and the electrophoretic voltage in the calculation control circuit 503 by operating the input device of the control computer 502. Then, the operator inputs an instruction to start analysis to the control computer 502. When the instruction to start analysis is input, the control computer 502 transmits the instruction to the device body 501. Thus, the device body 501 starts the analysis.

[0132] (Step S123)

[0133] In step S123, the device main body 501 starts filling the capillary 519 with a polymer. The polymer filling is a step of filling the capillary 519 with a new polymer to form a migration path.

[0134] In the polymer filling of this embodiment, first, Figure 5 The conveyor 518 shown carries the cathode buffer container 511 to the right below the loading head 509 to receive the used polymer discharged from the cathode end 510 of the capillary 519. Then, the injection mechanism 520 is driven to fill the capillary 519 with new polymer and discard the used polymer. Finally, in order to prevent the separation medium from drying, the cathode end 510 is immersed in the buffer in the cathode buffer container 511.

[0135] (Step S124)

[0136] In step S124, the device main body 501 performs preliminary electrophoresis. The preliminary electrophoresis is a step of applying a predetermined voltage to the polymer to make the polymer suitable for electrophoresis.

[0137] In the preliminary electrophoresis of this embodiment, first, the cathode end 510 is immersed in the buffer solution in the cathode buffer solution container 511 by the transporter 518 to form an electric conduction path. Then, a voltage of several to several tens of kV is applied to the polymer by the high-voltage power supply 516 for several to several tens of minutes to make the polymer in a state suitable for electrophoresis. Finally, in order to prevent the drying of the polymer, the cathode end 510 is immersed in the buffer solution in the cathode buffer solution container 511.

[0138] (Step S125)

[0139] In step S125, the device main body 501 introduces the sample components into the electrophoresis path. This step can be performed automatically or by sequentially sending control signals from the control computer 502.

[0140] In the sample introduction of this embodiment, first, the cathode end 510 is immersed in the sample held in the hole of the sample container 512 by the transporter 518. Thereby, an electric conduction path is formed and it becomes a state where the sample components can be introduced into the electrophoresis path. Then, a pulse voltage is applied to the electric conduction path by the high-voltage power supply 516 to introduce the sample components into the electrophoresis path. Finally, in order to prevent the drying of the polymer, the cathode end 510 is immersed in the buffer solution in the cathode buffer solution container 511.

[0141] (Step S126)

[0142] In step S126, the device main body 501 performs electrophoresis analysis. In the electrophoresis analysis, various sample components contained in the sample are separated and analyzed by electrophoresis.

[0143] In the electrophoresis analysis of this embodiment, first, the cathode end 510 is immersed in the buffer solution in the cathode buffer solution container 511 by the transporter 518 to form an electric conduction path. Then, a high voltage of 7.5 kV is applied to the electric conduction path by the high-voltage power supply 516 to generate an electric field in the electrophoresis path. Through the generated electric field, various sample components in the electrophoresis path move toward the light irradiation unit 508 at a speed depending on the properties of the respective sample components. That is, the sample components are separated by the difference in their moving speeds. Then, the light detector 504 sequentially detects the sample components reaching the light irradiation unit 508.

[0144] For example, when the sample contains multiple DNAs with different base lengths, a difference in moving speed is generated according to the base length, and the DNAs reach the light irradiation unit 508 in sequence starting from the DNA with a short base length. Each DNA is bound to a fluorescent dye corresponding to the analysis object. When excitation light is irradiated from the light source 507 to the light irradiation unit 508, information light (fluorescence having a wavelength depending on the sample) is generated from the sample and released to the outside. This information light is spectroscopically separated in the wavelength direction by the diffraction grating 524 and detected by the light detector 504.Figure 1 This is an example of the image detected by the photodetector 504. In the electrophoresis analysis, in the photodetector 504, the information light is detected at fixed time intervals, and the image data is sent to the arithmetic control circuit 503. Alternatively, in order to reduce the amount of information sent, the photodetector 504 may not send the image data, but only send the brightness (signal intensity) of a partial area in the image data. For example, the signal intensity at the wavelength positions at fixed intervals may be sent for each capillary only.

[0145] In the present embodiment, as described in Figure 1 , in the above-mentioned image data, only the signal intensity data of the wavelengths λ(0) to λ(19) of 20 for each capillary are sent to the arithmetic control circuit 503. This signal intensity data represents the spectra of the respective DNA samples in each capillary, and these spectra are stored in the measured value calculation unit 5032. The spectra of all the capillaries 519 at all the detection times in the above-mentioned electrophoresis analysis are stored in the measured value calculation unit 5032. In addition, although the spectra at all the detection times can be stored in the measured value calculation unit 5032, when only specific peak times are important for the operator, the spectra around only the specific times may be stored.

[0146] (Step S127)

[0147] In step S127, after the device main body 501 finishes acquiring the predetermined image data, the voltage application is stopped, and the electrophoresis analysis is ended.

[0148] The above is Figure 9 an example of the processing of the electrophoresis process (step S12). In addition, steps S123 to S127 may be automatically performed by the device main body 501, or may be performed by sequentially sending control signals from the control computer 502.

[0149] (Step S13)

[0150] Return to Figure 9 , in step S13, the correction unit 5035 retrieves the correction coefficient matrix K having the acquisition time of the matrix M(r) and the combination of the electrophoresis voltage and the fluorescent dye that are the same as those of the actual sample in step S12 from the correction coefficient database 5034, and multiplies each element of the matrix M(r) by each element k(ij) of the matrix K to calculate the matrix M(r)k.

[0151] (Step S14)

[0152] In step S14, the calibration unit 5035 calculates the fluorescence intensity. Specifically, the calibration unit 5035 calculates the intensity of each fluorescent pigment based on the image data obtained in the above electrophoresis process (step S12). In this step S14, the spectrum of each capillary 519 at each moment is multiplied by the intensity ratio of each fluorescent pigment in wavelengths λ(0) to λ(19) and then added together. If expressed in matrix form, it becomes the following Equation 3.

[0153] [Equation 3]

[0154] c = M(r)kf

[0155] c = c X c T c R c G

[0156] f = [f0 f1 f2 ······ f 18 f 19

[0157]

[0158]

[0159]

[0160]

[0161]

[0162] Here, the vector C represents the fluorescence intensity of each fluorescent pigment used. Therefore, the elements CX, CT, CR, and CG of the vector C represent the fluorescence intensities of ROX, TMR, R110, and R6G, respectively. The vector f represents the signal intensity observed by the photodetector 504. The elements f0 to f19 of the vector f represent the signal intensities at wavelengths λ(0) to λ(19), respectively. The elements f0 to f19 can be, for example, the arithmetic mean of the signal intensities near wavelengths λ(0) to λ(19).

[0163] In addition, among the measurement signals at each wavelength λ(0) to λ(19) detected by the photodetector 504, in addition to the signals based on the fluorescent pigments, Raman scattered light from the polymer filled in the capillary 519 is included as a baseline signal. Therefore, when calculating the vector f, it is necessary to remove this baseline signal in advance.

[0164] ​​As an example of a method for removing the baseline signal, the spectrum of Raman scattered light is obtained in advance before the device is shipped, and is stored as the baseline signal in the arithmetic control circuit 503. Then, by subtracting this baseline signal from the measurement signals at respective times, the signal based on the fluorescent dye is obtained, and this is taken as the vector f. Alternatively, the minimum value in the vicinity of each time may be taken as the baseline signal value at that time.

[0165] When transforming the measurement spectrum f into a fluorescence intensity vector, the matrix M(r)k is used.

[0166] The correction unit 5035 calculates the fluorescence intensity of each fluorescent dye from the measurement spectrum according to the above formula 3. By performing this process on the spectra of the respective capillaries 519 at each time, time series data of the fluorescence intensity of each capillary 519 can be obtained. Hereinafter, this time series data of the fluorescence intensity is referred to as a fluorescence intensity waveform.

[0167] (Step S15)

[0168] In step S15, the correction unit 5035 performs peak detection on the above fluorescence intensity waveform. In peak detection, the center position (peak time), the height of the peak, and the width of the peak are mainly important. The center position of the peak corresponds to the DNA fragment length. The height of the peak is used for quality evaluation such as the magnitude of the DNA concentration in the sample. The width of the peak is also important in evaluating the quality of the sample and the electrophoresis result. As one method for estimating the peak parameters of such actual data, Gaussian fitting, which is a known technique, can be used.

[0169] Figure 11 is a diagram showing the concept of Gaussian fitting. As Figure 11 shown, Gaussian fitting is a process of calculating parameters (average value μ, standard deviation σ, and maximum amplitude value A) such that the Gaussian function g best approximates the actual data for the actual data in a fixed interval. As an index indicating the approximation degree of the actual data, the least squares error between the actual data and the Gaussian function value is often used. As a numerical calculation method for minimizing this least squares error, methods such as the Gauss-Newton method can be used to optimize the parameters. In addition, methods for improving the accuracy, such as the case where two or more peak waveforms are mixed and the case where the data around the peak is asymmetric, can also be applied. And if the variance σ of the Gaussian function g is determined, its full width at half maximum (FWHM: Full Width at Half Maximum) is obtained from the formula shown in Figure 11 . This value can be set as the peak width.

[0170] In this way, the correction unit 5035 obtains the peak parameters for the fluorescence intensity waveforms of all the fluorescent dyes. At this time, when the peak width and the height of the peak do not satisfy the predetermined threshold conditions, they may be excluded from the peaks.

[0171] Through the above operations, using the matrix M obtained with a migration voltage of 15 kV, the signal intensity of the actual sample obtained with a migration of 7.5 kV is correctly calculated. In this embodiment, a specific combination of migration voltages is exemplified. In practice, however, the operator can arbitrarily select the migration voltages for the second spectral calibration (step S11) and the actual sample migration (step S12) within the range registered in the correction coefficient database 5034.

[0172] <Technical effects>

[0173] As described above, in the first embodiment, before the multi-capillary electrophoresis device 500 is shipped from the factory, first spectral calibration is performed at multiple migration voltages and migration is performed under the same conditions as the actual sample. For each combination of migration voltages, a correction coefficient matrix K for correcting spectral deviation is obtained and registered in the correction coefficient database 5034 together with the information of the fluorescent dye. The operator who has purchased the device can perform the second spectral calibration and the migration of the actual sample with any combination of migration voltages registered in the correction coefficient database 5034. In addition, even if the operator changes the voltage during the migration of the actual sample, the reference spectrum and the fluorescence spectrum of the actual sample do not deviate. Therefore, correct fluorescence intensity can be obtained without re-performing the second spectral calibration.

[0174] [Second Embodiment]

[0175] In the first embodiment, a matrix standard is used to obtain the matrix M'. However, in the second embodiment, a method of obtaining the matrix M' using a known DNA sample is proposed. The known DNA sample refers to a PCR product of DNA, a commercially available standard sample, etc. In this embodiment, as an example, the matrix standard, the known DNA sample, and the actual sample are all labeled with ROX, TMR, R110, and R6G. In addition, during the migration of the known DNA sample, the times (t0', t1', t2', t3') when each fluorescent dye emits light alone are known.

[0176] Figure 12 It is a flowchart showing the analysis method of the sample of the second embodiment.

[0177] In step S21, the manufacturer performs spectral calibration using the matrix standard in the same manner as in step S1, and the measurement value calculation unit 5032 obtains the matrix M. The migration voltage is 15 kV.

[0178] In step S22, the manufacturer migrates the known DNA sample.

[0179] In step S23, the measured value calculation unit 5032 obtains the spectra at the times (t0’, t1’, t2’, t3’) when each fluorescent dye emits light alone, and creates a matrix M’ based on the intensity ratios of the respective fluorescent dyes. The migration voltage is 7.5 kV.

[0180] In step S24, similarly to step S3, the correction coefficient calculation unit 5033 calculates a correction coefficient matrix K based on matrices M and M’. The correction coefficient matrix K is registered in the correction coefficient database 5034 together with the information on the migration voltage and the fluorescent dyes. As described in step S1 of the first embodiment, in actual operation, steps S21 and S22 are performed at various migration voltages, and a plurality of matrices M and M’ are obtained. When migration is performed at a plurality of voltages, all the correction coefficient matrices K are registered.

[0181] Similarly to the first embodiment, steps S21 to S24 are performed on the manufacturer side before the multi-capillary electrophoresis device 500 is shipped, and the correction coefficient matrix K has already been registered in the correction coefficient database 5034. The operations actually performed by the operator who has purchased the device are the subsequent steps S25 and later. Here, after step S24, the capillary 519 is loaded and unloaded during transportation, and the positional relationship between the photodetector 504 and the capillary 519 changes. If the capillary 519 is not loaded and unloaded after step S24, a matrix with the same migration voltage as the actual sample migration (step S26) can be selected from the matrix M obtained in step S21 and set as the matrix M(r)k described later.

[0182] In step S25, similarly to step S11, the operator performs second spectral calibration, and the measured value calculation unit 5032 obtains the matrix M(r). As an example, the migration voltage in step S25 is set to 15 kV, but in actual operation, any migration voltage registered in the correction coefficient database 5034 can be selected.

[0183] In step S26, similarly to step S12, the operator performs actual sample migration. As an example, the migration voltage here is set to 7.5 kV, but in actual application, any migration voltage registered in the correction coefficient database 5034 can be arbitrarily selected.

[0184] Regarding steps S27 to S29, they are the same as steps S13 to S15 ([[]] Figure 9 ) described in the first embodiment, so the description is omitted.

[0185] Through the above operations, even if the migration voltages are different during the first spectral calibration (step S21) and the actual sample migration (step S25), the reference spectrum and the fluorescence spectrum of the actual sample will not deviate, and the fluorescence intensity of the actual sample can be correctly calculated. Although a specific combination of migration voltages is illustrated here, in fact, the operator can arbitrarily select the migration voltages for the second spectral calibration (step S25) and the actual sample migration (step S26) within the range registered in the calibration coefficient database 5034.

[0186] <Technical Effect>

[0187] As described above, in the second embodiment, similar to the first embodiment, the operator who has purchased the device can perform the second spectral calibration and the actual sample migration through any combination of migration voltages registered in the calibration coefficient database 5034. Additionally, even if the operator changes the voltage during the actual sample migration, the reference spectrum and the fluorescence spectrum of the actual sample will not deviate. Therefore, even without re-performing the second spectral calibration, the correct fluorescence intensity can be obtained.

[0188] <Experimental Example 1>

[0189] The effects of the second embodiment were confirmed according to the following steps.

[0190] (Sample)

[0191] As the matrix standard during the first spectral calibration (step S21), BigDye (registered trademark) Terminator v3.1 Matrix Standards (Dye Set Z) (manufactured by Applied Biosystems) was used. For both the known DNA sample (step S22) and the actual sample (step S26), 3500 / 3500xL Sequencing Standards and BigDye (registered trademark) Terminator v3.1 (manufactured by Applied Biosystems) were used. ROX, TMR, R110, and R6G were used as fluorescent dyes for the above samples.

[0192] (Analysis Steps)

[0193] In Experimental Example 1, as a verification of the second embodiment, steps S21 to S26 were carried out according to the procedures described in steps S1, S12, S2, S3, S11, and S12, respectively. The capillary length during migration was 36 cm, the applied voltage during sample injection was 1.6 kV, the applied voltage during migration was 15 kV during the first spectral calibration (step S21), and the voltages during the known sample migration and the actual sample migration were 7.5 kV.

[0194] Next, Steps S27 to S29 are executed in the same manner as described in Steps S13 to S15.

[0195] As a comparison for the second embodiment, the light intensity of the actual sample is calculated and the peak is detected using matrix M without applying the calibration coefficient matrix K. The signal intensities of the pseudo-peaks were compared between the second embodiment and its comparison.

[0196] (Experimental results)

[0197] Figure 13 It is a graph showing the results of Experimental Example 1. Figure 13 It shows the matrix M, matrix M', and calibration coefficient matrix K obtained in Steps S21, S23, and S24.

[0198] In Figure 13 In the graph in, the horizontal axis represents the peak time and the vertical axis represents the fluorescence intensity. Pseudo-peaks were confirmed in the comparison, but were clearly reduced in the method of the second embodiment.

[0199] [Third Embodiment]

[0200] In the first and second embodiments, the case where the migration voltages are different during the second spectral calibration and during the migration of the actual sample was described, but in the third embodiment, the case where the fluorescent dyes are different is described. In this embodiment, as an example, the matrix standards used in the first spectral calibration are labeled with FAM, JOE, TMR, and CXR. In addition, the actual sample is labeled with R6G, R110, TMR, and ROX.

[0201] Figure 14 It is a flowchart showing the analysis method of the sample of the third embodiment.

[0202] In Step S31, in the same manner as in Step S1, the manufacturer performs spectral calibration using the matrix standard, and the measurement value calculation unit 5032 obtains the matrix M. However, the sample uses the matrix standard labeled with FAM, JOE, TMR, and CXR.

[0203] In Step S32, in the same manner as in Step S1, the manufacturer obtains the matrix M'. However, the sample uses the matrix standard labeled with R6G, R110, TMR, and ROX.

[0204] In step S33, similarly to step S3, the correction coefficient calculation unit 5033 calculates a correction coefficient matrix K based on matrices M and M'. The correction coefficient matrix K is registered in the correction coefficient database 5034 together with information on the migration voltage and the fluorescent dye. As described in step S1 of the first embodiment, in actual operation, in steps S31 and S32, multiple matrices M and M' are obtained through combinations of various fluorescent dyes. When migration is performed with combinations of multiple fluorescent dyes, all the correction coefficient matrices K are registered.

[0205] Similarly to the first embodiment, steps S31 to S33 are implemented by the manufacturer before the multi-capillary electrophoresis device 500 is shipped, and the correction coefficient matrix K has already been registered in the correction coefficient database 5034. The operations actually performed by the operator who has purchased the device are the subsequent steps S34 and later. Here, after step S33, during transportation, the capillary 519 is loaded and unloaded, and the positional relationship between the optical detector 504 and the capillary 519 changes. If the capillary 519 is not loaded and unloaded after step S33, a matrix of the same fluorescent dye as the actual sample migration (step S35) can be selected from the matrix M obtained in step S31 and used as the matrix M(r)k described later.

[0206] In step S34, similarly to step S11, the operator performs a second spectral calibration, and the measurement value calculation unit 5032 obtains the matrix M(r). The fluorescent dyes in step S34 are FAM, JOE, TMR, and CXR in this embodiment, but in actual application, any fluorescent dye can be selected from the fluorescent dyes registered in the correction coefficient database 5034.

[0207] In step S35, similarly to step S12, the operator performs the migration of the actual sample. As an example, the actual sample is labeled with R6G, R110, TMR, and ROX. However, in actual application, any fluorescent dye can be selected from the fluorescent dyes registered in the correction coefficient database 5034.

[0208] Regarding steps S36 to S38, since they are the same as steps S13 to S15 ( Figure 9 ) described in the first embodiment, the description is omitted.

[0209] Through the above operations, even if the fluorescent dyes are different during spectral calibration (step S31) and actual sample migration (step S35), the reference spectrum and the fluorescent spectrum of the actual sample will not deviate, and the fluorescent intensity of the actual sample can be calculated correctly. Here, specific combinations of fluorescent dyes are illustrated, but in reality, the operator can arbitrarily change the fluorescent dyes for the second spectral calibration (step S34) and actual sample migration (step S35) within the range registered in the correction coefficient database 5034.

[0210] <Technical effects>

[0211] As described above, in the third embodiment, before the multi-capillary electrophoresis device 500 leaves the factory, samples labeled with a set of different fluorescent dyes are used to perform the first spectral calibration and electrophoresis under the same conditions as the actual samples. For each combination of fluorescent dyes, a correction coefficient matrix K for correcting spectral deviation is obtained and registered in the correction coefficient database 5034. An operator who has purchased the device can perform the second spectral calibration and electrophoresis of the actual samples using any combination of fluorescent dyes registered in the correction coefficient database 5034. In addition, even if the operator changes the fluorescent dye during the electrophoresis of the actual samples, the reference spectrum and the fluorescent spectrum of the actual samples will not deviate. Therefore, correct fluorescence intensities can be obtained without re-performing the second spectral calibration.

[0212] <Experimental Example 2>

[0213] The effects of the third embodiment were confirmed according to the following steps.

[0214] (Specimen)

[0215] As the matrix standard for the first spectral calibration (step S31), PowerPlex (registered trademark) 4C Matrix Standards (manufactured by Promega Corporation) was used. To obtain the matrix M' (step S32), BigDye (registered trademark) Terminator v3.1 Matrix Standards (Dye Set Z) (manufactured by Applied Biosystems) was used. For the actual samples (step S35), 3500 / 3500xL Sequencing Standards, BigDye (registered trademark) Terminator v3.1 (manufactured by Applied Biosystems) was used.

[0216] Figure 15A is a diagram showing the fluorescent dyes used in Experimental Example 2. As Figure 15A shown, in the matrix standard (step S31), FAM, JOE, TMR, and CXR were used as fluorescent dyes. In addition, in the samples of steps S32 and S35, ROX, TMR, R110, and R6G were used as fluorescent dyes.

[0217] (Analysis steps)

[0218] In Experimental Example 2, as verification of the third embodiment, steps S31, S32, S33, and S34 were carried out in the same manner as described in steps S1, S1, S3, and S11, respectively. The capillary length during electrophoresis was 36 cm, the applied voltage during sample injection was 1.6 kV, and the applied voltage during electrophoresis was 15 kV in all steps during the first spectral calibration (step S31).

[0219] Next, steps S35 to S38 were carried out in the same manner as described in steps S12 to S15.

[0220] As a control for the third embodiment, instead of applying the calibration coefficient matrix K, the matrix M was used for calculating the light intensity and peak detection of the actual sample. The signal intensities of the false peaks were compared between the third embodiment and its control.

[0221] (Experimental results)

[0222] Figure 15B It is a graph showing the results of Experimental Example 2. Figure 15B It shows the matrix M, matrix M', and calibration coefficient matrix K obtained in steps S31 to S33.

[0223] In Figure 15B In the graph of, the horizontal axis represents the peak time and the vertical axis represents the fluorescence intensity. False peaks were confirmed in the control, but were clearly reduced in the method of the third embodiment.

[0224] [Fourth Embodiment]

[0225] In the first embodiment, the calibration coefficient matrix K obtained by a specific device was applied to the data of the actual sample obtained by the same device. In the fourth embodiment, a method of applying the calibration coefficient matrix K obtained by a certain specific device to the data of the actual sample obtained by another device is proposed.

[0226] In the present embodiment, as an example, the case where the fluorescent dyes are different from that in the third embodiment ( Figure 14 ) is taken as an example for explanation. As an example, the matrix standards used in the first spectral calibration (step S31) were labeled with FAM, JOE, TMR, and CXR. Moreover, the actual samples were labeled with R6G, R110, TMR, and ROX.

[0227] The first spectral calibration (step S31), acquisition of the matrix M' (step S32), and calculation of the correction coefficient matrix K (step S33) are performed on the manufacturer side in a specific multi-capillary electrophoresis device A in the same manner as in the third embodiment. The device A, for example, sends the correction coefficient matrix K to different devices (multiple devices) via a network and registers it in each correction coefficient database 5034. For example, the correction coefficient matrix K can also be registered in all multi-capillary electrophoresis devices before shipment.

[0228] After step S34, it can be implemented in any device in which the same correction coefficient matrix K as that of device A is registered.

[0229] <Technical effects>

[0230] As described above, in the fourth embodiment, the correction coefficient matrix K obtained using a specific multi-capillary electrophoresis device is also registered in other devices. Thus, it is not necessary to measure the correction coefficient matrix K in each device, so the costs and time on the manufacturer side can be reduced.

[0231] [Fifth embodiment]

[0232] In the first embodiment, by multiplying the matrix M(r) obtained from the second spectral calibration by the correction coefficient matrix K, the deviation between the matrix M(r) and the fluorescence spectrum of the actual sample is prevented. In the fifth embodiment, a method of preventing deviation by changing the wavelength width (signal acquisition width) of the signal detected by the photodetector is proposed. For the same processing as in the first embodiment, the description is omitted.

[0233] Figure 16 It is a flowchart showing the analysis method of the sample in the fifth embodiment.

[0234] In this embodiment, when the photodetector 504 of the multi-capillary electrophoresis device 500 samples data, it measures the signal intensities at 20 wavelengths λ(0) to λ(19). Here, 20 wavelengths are cited as an example, but actually, the arithmetic mean of the signal intensities near each of the wavelengths λ(0) to λ(19) can also be taken. In addition, the matrix standard is labeled with CXR, and the actual sample is labeled with ROX. The fluorescence spectra of these fluorescent dyes are known and are inconsistent.

[0235] Since the photodetector 504 in this embodiment only detects 20 wavelengths, the fluorescence spectrum of CXR is represented by a vector Vm composed of 20 elements, and the fluorescence spectrum of ROX is represented by a vector Vs composed of 20 elements.

[0236] In step S51, the measurement value calculation unit 5032 defines 20 wavelengths (signal acquisition widths) in such a way that the correlation coefficient between the vector Vm and the vector Vs is maximized. At this time, it is also possible to assign weights to the maximum value of the spectrum or its vicinity. Additionally, if there are no problems in practical applications, it is sufficient to sufficiently increase the correlation coefficient, and it is not necessarily required to set it to the maximum value. That is, the signal acquisition width is defined in such a way that the correlation coefficient becomes a predetermined value or more.

[0237] In step S52, similarly to step S1, the operator performs spectral calibration. At this time, the measurement value calculation unit 5032 calculates a vector Vc composed of 20 elements.

[0238] In step S53, similarly to step S12, the operator performs electrophoresis of the actual sample. Here, the vector f obtained by the measurement value calculation unit 5032 represents the signal intensity observed by the photodetector 504. Its elements f0 to f19 respectively represent the signal intensities at wavelengths λ(0) to λ(19).

[0239] In step S54, the correction unit 5035 calculates the fluorescence intensity. Specifically, it is sufficient to multiply the spectrum of each capillary 519 at each moment by the intensity ratio of each fluorescent dye at each wavelength of wavelengths λ(0) to λ(19) and add them. If this is expressed in matrix form, it becomes the following mathematical formula 4.

[0240] [Mathematical formula 4]

[0241] c = Vmf

[0242] f = [f0 f1 f2 ······ f 18 f 19

[0243] Vm = [w0 w1 w2 ······ w 18 w 19

[0244] The vector c is a fluorescence intensity vector. The vector f represents the signal intensity detected by the photodetector 504. Its elements f0 to f19 respectively represent the signal intensities at wavelengths λ(0) to λ(19).

[0245] In addition, as described in step S14 of the first embodiment, in the measurement signals at each wavelength λ(0) to λ(19) detected by the photodetector 504, in addition to the signals based on the fluorescent dye, Raman scattered light from the polymer filled in the capillary is included as a baseline signal. Therefore, when calculating the vector f, it is necessary to remove this baseline signal in advance. The baseline removal can also be performed by the method described in step S14.

[0246] ​​In step S55, the calibration unit 5035 performs peak detection in the same manner as in step S15.

[0247] Through the above operations, even if the fluorescent dyes are different during spectral calibration (step S1) and actual sample migration (step S12), the reference spectrum and the fluorescence spectrum of the actual sample will not deviate, and the fluorescence intensity of the actual sample can be correctly calculated.

[0248] <Technical effect>

[0249] As described above, in the fifth embodiment, the photodetector 504 detects the light from the capillary 519 with a wavelength width at which the correlation coefficient of the fluorescence spectra of multiple fluorescent dyes increases. Therefore, the matrix M(r)k is not required during migration, so the analysis time can be shortened, and the burden on the arithmetic control circuit 503 can be reduced.

[0250] <Experimental example 3>

[0251] The effects of the fifth embodiment were confirmed according to the following steps.

[0252] (Apparatus)

[0253] The multi-capillary electrophoresis apparatus 500 described in the first embodiment can be used ( Figure 5 ). However, as an example in this embodiment, the photodetector 504 detects the signal intensities at 20 wavelengths between 520 nm and 690 nm. In this experimental example 3, the signal acquisition width for sufficiently increasing the cross-correlation coefficient of the two spectra is known. Let the values of these 20 wavelengths represented by a vector be λtest. In addition, as a control, it is assumed that signals are acquired at equal intervals of 8.9 nm in the same interval, and these 20 wavelengths are represented by a vector as λctrl. The following Equation 5 represents the elements of λtest and λctrl.

[0254] [Equation 5]

[0255] λtest =(520 529 538 547 556 565 574 583 602 627 634 640 646 653 659 665 671 678 684 690)

[0256] λctrl =(520 529 538 547 556 565 574 583 592 601 605 618 627 636 645 654 663 672 680 690)

[0257] (Specimen)

[0258] For spectral calibration (step S52), one out of the four peaks contained in PowerPlex (registered trademark) 4C Matrix Standards (manufactured by Promega Corporation) and labeled with CXR is used. For the electrophoresis of the actual sample (step S53), one out of the four peaks contained in BigDye (registered trademark) Terminator v3.1 Matrix Standards (Dye Set Z) (manufactured by Applied Biosystems) and labeled with ROX is used.

[0259] (Analysis step)

[0260] In Experimental Example 3, as verification of the fifth embodiment, steps S52 and S53 are carried out according to the procedures described in steps S11 and S12 respectively. The capillary length during electrophoresis is 36 cm, the applied voltage during sample injection is 1.6 kV, and the applied voltage during electrophoresis is 15 kV both during spectral calibration (step S52) and during the electrophoresis of the actual sample (step S53).

[0261] (Experimental results)

[0262] Figure 17A is the fluorescence spectrum obtained in Experimental Example 3. Figure 17A Shows the fluorescence spectrum obtained at λtest. The following Equation 6 represents the signal intensities of vector Vm and vector Vs at λtest. As shown in Equation 6, the correlation coefficient (corr.) between vector Vm and vector Vs when the fifth embodiment of λtest is applied is 0.998.

[0263] [Equation 6]

[0264] Vm Vm=(0.01 0.02 0.02 0.03 0.03 0.04 0.06 0.06 0.16 1.00 0.86 0.71 0.58 0.43 0.35 0.29 0.24 0.21 0.19 0.17)

[0265] Vs=(0.01 0.01 0.02 0.02 0.02 0.02 0.04 0.04 0.09 1.00 0.89 0.75 0.62 0.46 0.36 0.29 0.24 0.21 0.19 0.17)

[0266] corr.=0.998

[0267] Figure 17B is the fluorescence spectrum obtained in the control experiment of Experimental Example 3. Figure 17BThe fluorescence spectrum obtained through λctrl is shown. The following Equation 7 represents the signal intensities of vector Vm and vector Vs in λctrl. As shown in Equation 7, the correlation coefficient (corr.) between vector Vm and vector Vs in the case of λctrl is 0.986.

[0268] [Equation 7]

[0269] Vm = (0.01 0.02 0.02 0.03 0.03 0.04 0.06 0.06 0.16 0.38 0.70 1.00 0.99 0.79 0.59 0.39 0.30 0.23 0.20 0.17)

[0270] Vs = (0.01 0.01 0.02 0.02 0.02 0.02 0.04 0.04 0.09 0.24 0.54 0.87 1.00 0.83 0.62 0.42 0.30 0.23 0.20 0.17)

[0271] corr. = 0.986

[0272] As can be seen from Equation 6 and Equation 7, in the case where λtest, i.e., the fifth embodiment, is applied, the cross-correlation coefficient becomes higher compared to the control (λctrl).

[0273] [Sixth Embodiment]

[0274] In the first to third embodiments, the case where the migration voltage or the fluorescent dye is different during the second spectral calibration and when the actual sample migrates is described. In the sixth embodiment, the case where both the migration voltage and the fluorescent dye are different is described. In this embodiment, as an example, the matrix standard used in the first spectral calibration is labeled with FAM, JOE, TMR, and CXR. In addition, the actual sample is labeled with R6G, R110, TMR, and ROX. The migration voltage during spectral calibration is 15 kV, and the migration voltage when the actual sample migrates is 7.5 kV.

[0275] Figure 18 It is a flowchart showing the analysis method of the sample of the sixth embodiment.

[0276] In step S61, similarly to step S1, the manufacturer performs spectral calibration using the matrix standard, and the measurement value calculation unit 5032 acquires matrix M. The migration voltage is 15 kV.

[0277] In step S62, similarly to step S2, the manufacturer acquires matrix M'. However, the sample uses the matrix standard labeled with R6G, R110, TMR, and ROX. The migration voltage at this time is 7.5 kV.

[0278] In step S63, similarly to step S3, the correction coefficient calculation unit 5033 calculates a correction coefficient matrix K based on matrices M and M'. The correction coefficient matrix K is registered in the correction coefficient database 5034 together with information on the migration voltage and the fluorescent dye. As described in step S1 of the first embodiment, in actual operation, steps S61 and S62 are performed with various combinations of migration voltages and fluorescent dyes to obtain a plurality of matrices M and M'. When performing electrophoresis using a plurality of migration voltages and a plurality of fluorescent dyes, all the correction coefficient matrices K are registered.

[0279] Similarly to the first embodiment, steps S61 to S63 are performed on the manufacturer side before the multi-capillary electrophoresis device 500 is shipped, and the correction coefficient matrix K has already been registered in the correction coefficient database 5034. The operations actually performed by the operator who has purchased the device are the subsequent steps S64 and later. Here, after step S63, the capillary 519 is loaded and unloaded during transportation, and the positional relationship between the optical detector 504 and the capillary 519 changes. If the capillary 519 is not loaded and unloaded after step S63, a matrix of the same fluorescent dye as that in the actual sample electrophoresis (step S65) can be selected from the matrix M obtained in step S61 and used as the matrix M(r)k described later.

[0280] In step S64, similarly to step S11, the operator performs second spectral calibration, and the measurement value calculation unit 5032 obtains the matrix M(r). As an example, the migration voltage and the fluorescent dye in step S64 can be set to be the same as those in the first spectral calibration (step S61), but in practice, any migration voltage and fluorescent dye can be selected from the migration voltages and fluorescent dyes registered in the correction coefficient database 5034.

[0281] In step S65, the operator performs electrophoresis of the actual sample. The migration voltage and the fluorescent dye used here are the same as those at the time of obtaining the matrix M' (step S62) as an example, but in practice, any migration voltage and fluorescent dye can be selected from the migration voltages and fluorescent dyes registered in the correction coefficient database 5034.

[0282] Regarding steps S66 to S68, they are also the same as steps S13 to S15 described in the first embodiment ( Figure 9 ), so the description is omitted.

[0283] By the above operations, even if both the fluorescent dye and the migration voltage used during spectral calibration (step S61) and actual sample migration (step S65) are different, the reference spectrum and the fluorescence spectrum of the actual sample do not deviate, and the fluorescence intensity of the actual sample can be correctly calculated. Here, a specific combination of fluorescent dye and migration voltage is illustrated, but in reality, the operator can arbitrarily change the migration voltage and fluorescent dye for the second spectral calibration (step S64) and actual sample migration (step S65) within the range registered in the calibration coefficient database 5034.

[0284] <Technical effects>

[0285] As described above, in the sixth embodiment, before the multi-capillary electrophoresis device 500 leaves the factory, samples labeled with different sets of fluorescent dyes are used to perform the first spectral calibration and actual sample migration at different migration voltages. For each combination of fluorescent dye and migration voltage, a calibration coefficient matrix K for correcting spectral deviation is obtained and registered in the calibration coefficient database 5034. An operator who has purchased the device can perform the second spectral calibration and actual sample migration through any combination of fluorescent dye and migration voltage registered in the calibration coefficient database 5034. Thus, compared with the first to third embodiments, the degree of freedom of the fluorescent dye and migration voltage used by the operator in this embodiment is increased.

[0286] [Seventh Embodiment]

[0287] In the first to third embodiments, the case where the migration voltage or fluorescent dye is different during the second spectral calibration and actual sample migration is described, but in the seventh embodiment, the case where the chemical properties or composition of the polymer are different is described. As described above, the polymer is merely an example of the separation medium, so of course the same application can be applied to separation media other than polymers.

[0288] The analysis method of the seventh embodiment can be implemented, for example, in the same process as the first embodiment, so only the differences will be described below.

[0289] In the seventh embodiment, as an example, the polymer used during spectral calibration (steps S1 and S11) contains 4% polyacrylamide. Moreover, the polymer used during actual sample migration (steps S2 and S12) contains 7% polyacrylamide. Both the matrix standard and the actual sample are labeled with R6G, R110, TMR, and ROX, and the migration voltage is set to 15 kV.

[0290] As described in the first embodiment, in actual applications, steps S1 and S2 are performed with various combinations of polymers to obtain a plurality of matrices M and M'. The various polymers mentioned here refer to polymers containing various concentrations of polyacrylamide as an example.

[0291] In step S3, the correction coefficient calculation unit 5033 calculates a correction coefficient matrix K based on matrices M and M'. The correction coefficient matrix K is registered in the correction coefficient database 5034 together with information such as the type of polymer. When electrophoresis is performed using multiple polymers, all the correction coefficient matrices K are registered.

[0292] According to the method of the present embodiment, even if the composition of the polymer is different during spectral calibration (step S1) and actual sample electrophoresis (step S12), the reference spectrum and the fluorescence spectrum of the actual sample do not deviate, and the fluorescence intensity of the actual sample can be correctly calculated. Here, a combination of specific compositions is illustrated, but in practice, the operator can arbitrarily change the chemical characteristics of the polymer for the second spectral calibration (step S11) and actual sample electrophoresis (step S12) within the range registered in the correction coefficient database 5034. In addition, the method of the present embodiment can also be applied to cases where the compositions of the polymers are different.

[0293] [Embodiment 8]

[0294] In the first to third embodiments, the case where the electrophoresis voltage or the fluorescent dye is different during spectral calibration and actual sample electrophoresis is described, but in the eighth embodiment, the case where the length of the capillary 519 is different is described.

[0295] The analysis method of the eighth embodiment can be implemented, for example, in the same process as the first embodiment, so the differences will be described below.

[0296] In the eighth embodiment, as an example, the length of the capillary during spectral calibration (steps S1 and S11) is set to 50 cm, and the length of the capillary during actual sample electrophoresis (steps S2 and S12) is set to 36 cm.

[0297] As described in the first embodiment, in actual operation, steps S1 and S2 are performed with various combinations of capillary lengths to obtain multiple matrices M and M'.

[0298] In step S3, the correction coefficient calculation unit 5033 calculates a correction coefficient matrix K based on matrices M and M'. The correction coefficient matrix K is registered in the correction coefficient database 5034 together with information on the capillary length. When electrophoresis is performed with multiple capillary lengths, all the correction coefficient matrices K are registered.

[0299] According to the method of the present embodiment, even if the capillary lengths are different during spectral calibration (step S1) and actual sample migration (step S12), the reference spectrum and the fluorescence spectrum of the actual sample do not deviate, and the fluorescence intensity of the actual sample can be correctly calculated. Here, a specific combination of lengths is illustrated, but in fact, the operator can arbitrarily change the capillary lengths of the second spectral calibration (step S11) and actual sample migration (step S12) within the range registered in the calibration coefficient database 5034.

[0300] [Embodiment 9]

[0301] In the first to third embodiments, the cases where the migration voltage or the fluorescent dye is different during spectral calibration and actual sample migration are described, but in the ninth embodiment, the cases where the composition or chemical properties of the anode buffer are different are described.

[0302] The analysis method of the ninth embodiment can be implemented, for example, in the same process as the first embodiment, so the differences will be described below.

[0303] In the ninth embodiment, as an example, the pH of the anode buffer used in spectral calibration (steps S1 and S11) is set to 7.5. The pH of the anode buffer during actual sample migration (steps S2 and S12) is set to 8.0.

[0304] As described in the first embodiment, in actual operation, steps S1 and S2 are performed with combinations of anode buffers at various pHs to obtain a plurality of matrices M and M'.

[0305] In step S3, the calibration coefficient calculation unit 5033 calculates the calibration coefficient matrix K based on the matrices M and M'. The calibration coefficient matrix K is registered in the calibration coefficient database 5034 together with the information on the pH of the anode buffer. In the case of performing migration with anode buffers at multiple pHs, all the calibration coefficient matrices K are registered.

[0306] According to the method of the present embodiment, even if the pH of the anode buffer is different during spectral calibration (step S1) and actual sample migration (step S12), the reference spectrum and the fluorescence spectrum of the actual sample do not deviate, and the fluorescence intensity of the actual sample can be correctly calculated. Here, a specific combination of pHs is illustrated, but in fact, the operator can arbitrarily change the pH of the anode buffer for the second spectral calibration (step S11) and actual sample migration (step S12) within the range registered in the calibration coefficient database 5034. In addition, the method of the present embodiment can also be applied to the case where the composition of the anode buffer is different.

[0307] [Embodiment 10]

[0308] In the ninth embodiment, the case where the chemical properties of the anode buffer are different was described. However, in the tenth embodiment, the case where the composition or chemical properties of the cathode buffer are different will be described.

[0309] In the tenth embodiment, as an example, the pH of the cathode buffer used in spectral calibration (steps S1 and S11) is 7.5. The pH of the cathode buffer during actual sample electrophoresis (steps S2 and S12) is set to 8.0. Other aspects are the same as those in the ninth embodiment, and thus the description thereof is omitted.

[0310] According to the method of the present embodiment, even if the pH of the cathode buffer is different during spectral calibration (step S1) and actual sample electrophoresis (step S12), the reference spectrum and the fluorescence spectrum of the actual sample do not deviate, and the fluorescence intensity of the actual sample can be correctly calculated. Here, a specific combination of pH values is exemplified. However, in practice, the operator can arbitrarily change the pH of the anode buffer for the second spectral calibration (step S11) and actual sample electrophoresis (step S12) within the range registered in the calibration coefficient database 5034. In addition, the method of the present embodiment can also be applied to the case where the composition of the cathode buffer is different.

[0311] [Eleventh Embodiment]

[0312] In the ninth embodiment, the case where the chemical properties of the anode buffer are different was described, and in the tenth embodiment, the case where the chemical properties of the cathode buffer are different was described. However, in the eleventh embodiment, the case where the chemical properties or composition of the sample solution are different will be described.

[0313] The analysis method of the eleventh embodiment can be implemented, for example, in the same procedure as that of the first embodiment. Therefore, the differences will be described below.

[0314] In the eleventh embodiment, as an example, the pH of the solution of the matrix standard used in spectral calibration (steps S1 and S11) is set to 7.5. The pH of the solution of the actual sample used in steps S2 and S12 is set to 8.0.

[0315] As described in the first embodiment, in actual operation, steps S1 and S2 are performed with combinations of samples at various pH values to obtain a plurality of matrices M and M'.

[0316] In step S3, the calibration coefficient calculation unit 5033 calculates a calibration coefficient matrix K based on matrices M and M'. The calibration coefficient matrix K is registered in the calibration coefficient database 5034 together with the information on the pH of the sample solution. When electrophoresis is performed using samples at multiple pH values, all the calibration coefficient matrices K are registered.

[0317] According to the method of the present embodiment, even if the pH of the sample solution is different during spectral calibration (step S1) and actual sample migration (step S12), the reference spectrum and the fluorescence spectrum of the actual sample do not deviate, and the fluorescence intensity of the actual sample can be correctly calculated. Here, a combination of sample solutions with specific pH values is exemplified, but in practice, the operator can arbitrarily change the pH of the sample solution for the second spectral calibration (step S11) and actual sample migration (step S12) within the range registered in the calibration coefficient database 5034. In addition, the method of the present embodiment can also be applied to cases where the composition of the sample solution is different.

[0318] [Embodiment 12]

[0319] In Embodiment 12, the case where the temperature of the thermostat 505 is different is described.

[0320] The analysis method of Embodiment 12 can be implemented, for example, in the same process as Embodiment 1, so the differences will be described below.

[0321] In Embodiment 12, as an example, the temperature of the thermostat 505 during spectral calibration (steps S1 and S11) is set to 42°C. Moreover, the temperature of the thermostat 505 during actual sample migration (steps S2 and S12) is set to 60°C.

[0322] As described in Embodiment 1, in actual operation, steps S1 and S2 are performed with various temperature combinations to obtain a plurality of matrices M and M'.

[0323] In step S3, the calibration coefficient calculation unit 5033 calculates the calibration coefficient matrix K based on the matrices M and M'. The calibration coefficient matrix K is registered in the calibration coefficient database 5034 together with the information on the temperature of the thermostat 505. When migration is performed with the thermostat 505 set to a plurality of temperatures, all the calibration coefficient matrices K are registered.

[0324] According to the method of the present embodiment, even if the temperature of the thermostat 505 is different during spectral calibration (step S1) and actual sample migration (step S12), the reference spectrum and the fluorescence spectrum of the actual sample do not deviate, and the fluorescence intensity of the actual sample can be correctly calculated. Here, a specific combination of the temperature of the thermostat 505 is exemplified, but in practice, the operator can arbitrarily change the temperature of the thermostat 505 for the second spectral calibration (step S11) and actual sample migration (step S12) within the range registered in the calibration coefficient database 5034.

[0325] [Regarding Manifestation]

[0326] As an example of the method for confirming infringement of the present disclosure, the following verification can be cited. Based on Figure 9An explanation will be given.

[0327] In the target device, step S11 (spectral calibration) is performed. At this time, although the migration voltage is 15 kV, the analysis is actually performed at a migration speed equivalent to 7.5 kV. The migration speed can be adjusted by adding an appropriate amount of salt to the sample. Alternatively, it can be registered as 15 kV on the device while actually migrating at 7.5 kV. Then, according to the method of the first embodiment, the actual sample is analyzed (steps S13 to S15). At this time, if the pseudo-peak increases compared to the first embodiment, it is highly likely that the target device applies the correction coefficient determined for each migration voltage to the fluorescence spectrum of the matrix standard.

[0328] In addition, the following verification can also be performed. Based on Figure 14 An explanation will be given. In this case, a fluorescent dye different from the actual one is registered in step S31. If the pull-up increases compared to the third embodiment after the actual sample analysis (steps S34 to S36), it is highly likely that the correction coefficient determined for each fluorescent dye is applied to the fluorescence spectrum of the matrix standard.

[0329] [Modification Example]

[0330] The present disclosure is not limited to the above-described embodiments and includes various modification examples. For example, the above-described embodiments are embodiments described in detail for easy understanding of the present disclosure and do not necessarily need to have all the structures described. In addition, a part of one embodiment can be replaced with the structure of another embodiment. In addition, the structure of another embodiment can be added to the structure of one embodiment. In addition, for a part of the structure of each embodiment, a part of the structure of another embodiment can also be added, deleted, or replaced.

[0331] Symbol Explanation

[0332] 101... device main body, 102... control computer, 103... arithmetic control circuit, 104... photodetector, 105... thermostat, 106... capillary array, 107... light source, 108... light irradiation unit, 109... loading head, 110... cathode end, 111... cathode buffer solution container, 112... sample container, 113... polymer cassette, 114... anode buffer solution container, 115... anode, 116... DC power supply, 117... array head, 118... transporter, 119... capillary, 120... injection mechanism, 121... tip, 122... upper part of polymer cassette, 123... heating and cooling mechanism, 201... laser, 202... mirror, 203... condenser lens.

Claims

1. An electrophoresis apparatus, characterized in that, the electrophoresis apparatus includes: an electrophoresis path for a sample; a spectroscopic element that spectroscopically analyzes light from the sample within the electrophoresis path; a light detector that detects the light spectroscopically analyzed by the spectroscopic element; and an arithmetic unit that obtains a spectrum of the light based on a signal from the light detector, the arithmetic unit corrects the spectrum using a correction coefficient determined for each electrophoresis condition or fluorescent dye, the arithmetic unit calculates a value representing a relative relationship between a first spectrum of a first fluorescent dye and a second spectrum of a second fluorescent dye as the correction coefficient, the arithmetic unit applies the correction coefficient to a third spectrum of a third fluorescent dye identical to the first fluorescent dye, thereby correcting the third spectrum according to the relative relationship.

2. An electrophoresis apparatus, characterized in that, the electrophoresis apparatus includes: an electrophoresis path for a sample; a spectroscopic element that spectroscopically analyzes light from the sample within the electrophoresis path; a light detector that detects the light spectroscopically analyzed by the spectroscopic element; and an arithmetic unit that obtains a spectrum of the light based on a signal from the light detector, the arithmetic unit corrects the spectrum using a correction coefficient determined for each electrophoresis condition or fluorescent dye, the arithmetic unit calculates a value representing a relative relationship between a first spectrum obtained under a first electrophoresis condition and a second spectrum obtained under a second electrophoresis condition as the correction coefficient, the arithmetic unit applies the correction coefficient to a third spectrum obtained under a third electrophoresis condition identical to the first electrophoresis condition, thereby correcting the third spectrum according to the relative relationship.

3. The electrophoresis apparatus according to claim 1 or 2, characterized in that, the correction coefficient is determined for each voltage during electrophoresis of the sample.

4. The electrophoresis apparatus according to claim 1 or 2, characterized in that, the correction coefficient is determined according to the pH of the buffer solution during electrophoresis of the sample or the pH of the solution of the sample.

5. The electrophoresis apparatus according to claim 1 or 2, characterized in that, the correction coefficient is determined according to each length of the electrophoresis path.

6. The electrophoresis apparatus according to claim 1 or 2, characterized in that, the electrophoresis apparatus further includes a thermostat for accommodating the electrophoresis path, the correction coefficient is determined according to each set temperature of the thermostat.

7. The electrophoresis apparatus according to claim 1 or 2, characterized in that, the correction coefficient is determined according to each composition or chemical property of the separation medium within the electrophoresis path.

8. The electrophoresis apparatus according to claim 1 or 2, characterized in that, the electrophoresis apparatus further includes a plurality of the electrophoresis paths, the arithmetic unit sets the correction coefficient for each of the plurality of electrophoresis paths.

9. An electrophoresis apparatus, characterized in that, the electrophoresis apparatus includes: an electrophoresis path for a sample; a spectroscopic element that spectroscopically analyzes light from the sample within the electrophoresis path; a light detector that detects the light spectroscopically analyzed by the spectroscopic element; and An arithmetic unit that calculates the signal intensity of the light based on the signal from the photodetector. The photodetector acquires the signal with a signal acquisition width that is set such that the correlation coefficient between the spectra of a plurality of fluorescent dyes is equal to or greater than a predetermined value.

10. An analysis method, characterized in that The analysis method includes the following steps: Electrophoresing a sample in an electrophoresis path provided in an electrophoresis apparatus; Spectrally splitting the light from the sample in the electrophoresis path by a spectral splitting element; Detecting the light spectrally split by the spectral splitting element by a photodetector; And By an arithmetic unit, based on the signal from the photodetector, obtaining the spectrum of the light, The step of obtaining the spectrum of the light includes: by the arithmetic unit, correcting the spectrum using a correction coefficient determined for each electrophoresis condition or fluorescent dye, The arithmetic unit calculates a value representing the relative relationship between a first spectrum of a first fluorescent dye and a second spectrum of a second fluorescent dye as the correction coefficient, The arithmetic unit applies the correction coefficient to a third spectrum of a third fluorescent dye identical to the first fluorescent dye, thereby correcting the third spectrum according to the relative relationship.

11. An analysis method, characterized in that The analysis method includes the following steps: Electrophoresing a sample in an electrophoresis path provided in an electrophoresis apparatus; Spectrally splitting the light from the sample in the electrophoresis path by a spectral splitting element; Detecting the light spectrally split by the spectral splitting element by a photodetector; And By an arithmetic unit, based on the signal from the photodetector, obtaining the spectrum of the light, The step of obtaining the spectrum of the light includes: by the arithmetic unit, correcting the spectrum using a correction coefficient determined for each electrophoresis condition or fluorescent dye, The arithmetic unit calculates a value representing the relative relationship between a first spectrum obtained under a first electrophoresis condition and a second spectrum obtained under a second electrophoresis condition as the correction coefficient, The arithmetic unit applies the correction coefficient to a third spectrum obtained under a third electrophoresis condition identical to the first electrophoresis condition, thereby correcting the third spectrum according to the relative relationship.

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