Analysis method and capillary electrophoresis device

Abstract

It is an object of the present invention to provide an analytical method that suppresses unnecessary re-electrophoresis and re-preparation of a sample. To this end, the analysis method according to the present invention analyzes characteristics relating to a sample by subjecting the sample to electrophoresis using a capillary tube and an electrophoresis medium, and comprises: a reading step for reading analysis conditions; an electrophoresis medium filling step in which the capillary tube is filled with the electrophoresis medium; a sample injection step for injecting the sample into the capillary tube filled with the migration medium in the migration medium filling step; an electrophoresis step in which the sample injected in the sample injection step is subjected to electrophoresis in the electrophoresis medium; an abnormality determination step for statistically determining whether or not there is an abnormality by comparing an analysis result obtained in the electrophoresis step with another analysis result having the same analysis condition; and an analysis condition adjustment step for adjusting an analysis condition, and when an abnormality is determined in the abnormality determination step, the process is transferred to the reading step again via the analysis condition adjustment step.

Description

Technical Field

[0001] The invention relates to a capillary electrophoresis device and an analysis method using the device. Background Art

[0002] In recent years, capillary electrophoresis devices in which a capillary is filled with a moving medium such as a polymer gel or a polymer solution have been widely used as electrophoresis devices. This capillary electrophoresis device can automatically fill the moving medium and automatically inject the sample, so continuous analysis can be performed, and multiple electrophoresis can be set in advance to analyze a large number of samples overnight.

[0003] The sample being electrophoresed in the capillary is irradiated with laser light, and the signal at this time is detected by a detection unit such as a CCD camera. However, when the concentration of the sample injected into the capillary is high, the detected signal becomes higher. If it exceeds the counting threshold of the CCD camera (for example, 65535 counts), the signal is saturated and the peak of the signal cannot be determined. In this case, in the past, users have re-prepared samples such as dilution and adjusted measurement conditions (information for setting the action conditions and analysis conditions for analysis) based on experience.

[0004] In addition, in capillary electrophoresis devices, errors may occur in which no signal is detected by the detection unit or only a very low signal is detected. Even in the case of such an error, in the past, users have handled it based on experience, and the easiest treatment method to choose is to re-prepare the sample. However, in addition to poor samples, errors such as no signal detection may also be caused by poor sample injection. When the cause of the error is poor sample injection, if electrophoresis is performed immediately before the sample deteriorates, an appropriate signal can be detected, thereby avoiding the need to re-prepare the sample, which places a heavy burden on the user.

[0005] Patent document 1 discloses an electrophoresis device that prompts a user to change sample concentration, sample injection parameters, etc. when the signal exceeds the range (saturation) or when the signal-to-noise ratio is low. In addition, Patent document 1 also discloses that when the signal-to-noise ratio is low, there is a possibility of poor sample injection, etc., prompting the user to adjust the sample injection parameters. Prior art literature Patent Literature

[0006] Patent Document 1: U.S. Patent Application Publication No. 2020 / 0003728 Summary of the invention Technical problem to be solved by the invention

[0007] In previous capillary electrophoresis devices, when errors such as signal saturation or, conversely, failure to detect signals occur, users can only judge the treatment method based on their own experience. In addition, when electrophoresis is performed continuously overnight, the user will not notice the error until the early morning, and the sample will deteriorate over time, so it is difficult to perform electrophoresis again directly even if it is not caused by a bad sample. In this case, the user must prepare the sample again, which not only wastes time and energy, but also wastes precious samples.

[0008] In addition, Patent Document 1 also discloses that when the signal exceeds the range (saturation) or the signal-to-noise ratio is low, the device changes the parameters of sample injection, etc., but does not specifically disclose how the device changes. In addition, in Patent Document 1, poor sample injection, etc. is determined only by the signal-to-noise ratio, that is, only by the signal height, so, for example, when the signal-to-noise ratio of all data is low, all samples are electrophoresed again using the changed injection parameters. However, when the signal-to-noise ratio of all data is low, the possibility of a bad sample or forgetting to put in the sample, etc. is higher than the possibility of a poor sample injection, so it is often not possible to solve it only by re-electrophoresis. As a result, useless electrophoresis is performed, which may result in a waste of electrophoresis medium and time. An object of the present invention is to provide an analytical method which suppresses unnecessary re-electrophoresis and re-preparation of samples. Technical means for solving technical problems

[0010] In order to solve the above-mentioned problems, the analysis method of the present invention performs analysis on characteristics related to the sample by subjecting the sample to electrophoresis using a capillary and a phoretic medium, and the analysis method includes: a reading step of reading analysis conditions; a phoretic medium filling step of filling the capillary with the phoretic medium; a sample injection step of injecting the sample into the capillary filled with the phoretic medium in the phoretic medium filling step; an electrophoresis step of subjecting the sample injected in the sample injection step to electrophoresis in the phoretic medium; an abnormality determination step of statistically determining whether an analysis result obtained in the electrophoresis step is abnormal by comparing it with other analysis results under the same analysis conditions; and an analysis condition adjustment step of adjusting the analysis conditions, and when an abnormality is determined in the abnormality determination step, the analysis is transferred to the reading step again via the analysis condition adjustment step. Effects of the Invention

[0011] According to the present invention, it is possible to provide an analysis method which suppresses unnecessary re-electrophoresis and re-preparation of samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is a perspective view showing the schematic structure of a capillary electrophoresis device. Figure 2 yes Figure 1 A top view of the capillary electrophoresis apparatus is shown. Figure 3 yes Figure 2 AA section view in. Figure 4 : is a flowchart showing the analysis method of Example 1. Figure 5 This is a flowchart showing the analysis method of Example 2. Figure 6 : is a graph showing an example of a signal waveform at the time of saturation. Figure 7 It is magnified Figure 6 The dashed part of the chart. Figure 8 It is a graph showing an image when the peak of the estimated signal is reached. Fig. 9 This is a graph showing an example of a signal waveform when sample injection is poor. Fig.10 1 is a diagram showing an example of plotting quality data (EQ) when poor sample injection occurs. Fig.11 1 is a diagram showing an example of plotting quality data (EQ) when poor sample injection occurs. Fig.12 : is a flowchart showing the analysis method of Example 3. DETAILED DESCRIPTION

[0013] Hereinafter, embodiments of the present invention are described using the drawings.

[0014] First, use Figure 1 to Figure 3 The structure of a capillary electrophoresis device will be described. Figure 1 1 is a perspective view schematically showing the structure of a capillary electrophoresis apparatus. The capillary electrophoresis apparatus is divided into two units: a detection / constant temperature chamber unit 150 located at an upper portion and an autosampler unit 160 located at a lower portion.

[0015] In the automatic sampler unit 160, the Y-axis driver 85 is mounted on the sampler base 80 and can be driven along the Y-axis. The Z-axis driver 90 is mounted on the Y-axis driver 85 and can be driven along the Z-axis. The sample tray 100 is mounted on the Z-axis driver 90, and the phoretic medium container 20, the anode side buffer container 30, the cathode side buffer container 40 and the sample container 50 are set on the sample tray 100 by the user. The sample container 50 is set on the X-axis driver 95, and the X-axis driver 95 is mounted on the sample tray 100, and the sample container 50 can move along the X-axis on the sample tray 100. The liquid delivery mechanism 60 is also mounted on the Z-axis driver 90. The liquid delivery mechanism 60 is set below the phoretic medium container 20.

[0016] The detection / constant-temperature bath unit 150 includes a constant-temperature bath unit 110 and a constant-temperature bath door 120, and can maintain the interior of the constant-temperature bath unit 110 at a constant temperature. In addition, the detection / constant-temperature bath unit 150 also includes a detection unit 130 located behind the constant-temperature bath unit 110, which can irradiate laser and detect the signal at this time. When the capillary array 10 is set in the constant-temperature bath unit 110 by the user, the sample is electrophoresed in the state where the capillary array 10 is kept at a constant temperature in the constant-temperature bath unit 110. At this time, the detection unit 130 detects the signal when the laser is irradiated on the sample for electrophoresis in the capillary array 10 through a CCD camera or the like. In addition, an electrode 115 is also installed in the constant-temperature bath unit 110, and the electrode 115 is used to be connected to GND (ground) when a high voltage for electrophoresis is applied.

[0017] As described above, the capillary array 10 is fixed to the thermostatic bath unit 110. The phoretic medium container 20, the anode side buffer container 30, the cathode side buffer container 40, and the sample container 50 can be driven along the Y axis and the Z axis by the automatic sampler unit 160, and only the sample container 50 can be further driven along the X axis. By driving the automatic sampler unit 160, the phoretic medium container 20, the anode side buffer container 30, the cathode side buffer container 40, and the sample container 50 are automatically connected to the fixed capillary array 10.

[0018] Figure 2 yes Figure 1 The anode side buffer container 30 provided on the sample tray 100 includes an anode side cleaning tank 31, an anode side electrophoresis buffer tank 32, and a sample introduction buffer tank 33. In addition, the cathode side buffer container 40 provided on the sample tray 100 includes a waste liquid tank 41, a cathode side cleaning tank 42, and a cathode side electrophoresis buffer tank 43.

[0019] The phoretic medium container 20, the anode side buffer container 30, the cathode side buffer container 40 and the sample container 50 are arranged as follows: Figure 2 The positional relationship shown is arranged on the sample tray 100. Therefore, the combination of the anode side-cathode side connected to the capillary array 10 is "electrophoresis medium container 20-waste liquid tank 41", "anode side cleaning tank 31-cathode side cleaning tank 42", "anode side electrophoresis buffer tank 32-cathode side electrophoresis buffer tank 43", "sample introduction buffer tank 33-sample container 50".

[0020] Figure 3 yes Figure 2The cross-sectional view at AA in FIG. 1 shows a cross section of the capillary electrophoresis device when the combination of the anode side-cathode side connected to the capillary array 10 is "the eluting medium container 20-the waste liquid tank 41". The eluting medium container 20 is inserted and set in the guide 101 embedded in the sample tray 100. In addition, the liquid delivery mechanism 60 is configured so that the plunger 61 built in the liquid delivery mechanism 60 is located below the eluting medium container 20. When performing electrophoresis, the capillary array 10 is Figure 3 The right side is the cathode side and the left side is the anode side.

[0021] The capillary electrophoresis device is controlled by a control unit including a control substrate 122 and a computer 123 connected to the control substrate 122 . Figure 1 An example is shown in which the control substrate 122 is arranged on the back side of the thermostatic chamber unit 110 and the computer 123 is arranged on the outside (front side) of the thermostatic chamber door 120, but the setting position is not limited to this. The control substrate 122 controls the operation of the high-voltage power supply 121, the thermostatic chamber unit 110, the detection unit 130, etc. according to the instructions from the computer 123. The computer 123 calculates the analysis result based on the signal detected by the detection unit 130, etc., and also includes an input unit for inputting the settings of the capillary electrophoresis device (including the measurement conditions described later) and an output unit (display unit) for displaying the analysis result. In addition, the calculation of the analysis result can be performed by the control substrate 122. [Example 1]

[0022] based on Figure 4 The flow of a series of analyses performed by the capillary electrophoresis apparatus of this embodiment will be described. Figure 4 This is a flow chart showing the analysis method of Example 1.

[0023] First, the user uses the input unit to set the measurement conditions used in a series of analyses (step S200). The measurement conditions are information that defines the action conditions and analysis conditions of the analysis. The action conditions are the laser power of the detection unit 130, the temperature of the thermostatic bath unit 110, the type of electrophoretic medium, the voltage and time applied in the pre-run described later, the voltage and time applied in the sample injection process described later, the voltage and time applied in the electrophoresis process, the laser irradiation time when acquiring data, the sampling period, etc. The analysis conditions are the analysis width specified by the size of the data point or base, the threshold of the signal height, the base call mobility file set for reading the sequence if it is sequencing analysis, and the fragment size used for analysis if it is fragment analysis.

[0024] Then, the user sets a function (re-analysis function) for re-analyzing when the analysis result is determined to be abnormal as needed. The control unit determines whether the re-analysis function is valid (step S201), and if it is not valid, executes steps S102 to S110. On the other hand, if the re-analysis function is valid in step S201, the control unit executes steps S202 to S212. Since steps S102 to S110 are the same as steps S202 to S210, only steps S202 to S210 are described below.

[0025] If the analysis is started in step S202, a series of analyses are performed. In this embodiment, one analysis is performed before one electrophoresis is performed by applying a voltage after the sample is injected. That is, the user can not only pre-set one analysis, but also pre-set a plurality of series of analyses. The following example illustrates the case where three analyses, namely, analysis A, analysis B, and analysis C, are pre-set, but the number of analyses is not limited thereto.

[0026] When analysis A, which is the first analysis, starts, the control unit executes a step of reading the measurement conditions set in step S200 (measurement condition reading step) (step S203).

[0027] Next, the control unit controls the constant temperature chamber unit 110 to execute a process of maintaining the capillary array 10 at a constant temperature (capillary temperature adjustment process) (step S204 ).

[0028] Then, the control unit activates the laser of the detection unit 130 (step S205).

[0029] Furthermore, the control unit controls the automatic sampler unit 160 and the plunger 61 to perform a process (a moving medium filling process) of filling the moving medium contained in the moving medium container 20 into the capillary array 10 (a moving medium filling process) (step S206). Here, the moving medium filling process will be described in more detail. First, the automatic sampler unit 160 moves, and the combination of the anode side and the cathode side connected to the capillary array 10 becomes "moving medium container 20-waste liquid tank 41". Next, the capillary head 11 as the end of the anode side of the capillary array 10 is inserted into the moving medium container 20, and the loading head 12 as the end of the cathode side of the capillary array 10 is inserted into the waste liquid tank 41. In this state, the plunger 61 built into the liquid feeding mechanism 60 moves upward, and the moving medium contained in the moving medium container 20 is transported to the capillary array 10 through the capillary head 11. When the phoretic medium has been filled into the capillary array 10 before the liquid is delivered, or when the phoretic medium is sufficiently delivered into the capillary array 10 and there is excess, the phoretic medium is discharged from the loading head 12 into the waste tank 41 .

[0030] Next, the control unit performs a pre-run (step S207). Specifically, first, the autosampler unit 160 moves, and the combination of the anode side and the cathode side connected to the capillary array 10 becomes "anode side electrophoresis buffer tank 32-cathode side electrophoresis buffer tank 43". Next, the capillary head 11 is inserted into the anode side electrophoresis buffer tank 32, and the loading head 12 is inserted into the cathode side electrophoresis buffer tank 43. In this state, the high voltage power supply 121 applies a high voltage to the capillary array 10. The pre-run is the same action as the electrophoresis after the sample injection described later, but because it is performed before the sample injection, it plays a role in stabilizing the electrophoresis performance.

[0031] Thereafter, the control unit executes a process (sample injection process) of injecting the sample set in the sample container 50 into the capillary array 10 (step S208). Specifically, first, the autosampler unit 160 moves, and the combination of the anode side and the cathode side connected to the capillary array 10 becomes "sample introduction buffer tank 33-sample container 50". Next, the capillary head 11 is inserted into the sample introduction buffer tank 33, and the loading head 12 is inserted into the sample container 50. In this state, the high voltage power supply 121 applies a high voltage to the capillary array 10. As a result, the negatively charged sample is injected into the capillary array 10 from the loading head 12.

[0032] In addition, a cleaning process may be performed before and after the electrophoretic medium filling process and the sample injection process, in which the capillary head 11 and the loading head 12, which are both ends of the capillary array 10, are cleaned. In this case, first, the autosampler unit 160 moves, and the anode side-cathode side combination connected to the capillary array 10 becomes the "anode side cleaning tank 31-cathode side cleaning tank 42". Next, the capillary head 11 is inserted into the anode side cleaning tank 31, and the loading head 12 is inserted into the cathode side cleaning tank 42. By leaving it in this state for a few seconds, both ends of the capillary array 10 can be cleaned.

[0033] Next, the control unit executes a process (electrophoresis process) of applying voltage to the capillary array 10 injected with the sample to perform electrophoresis on the sample (step S209). Specifically, first, the autosampler unit 160 moves, and the combination of the anode side and the cathode side connected to the capillary array 10 becomes "anode side electrophoresis buffer tank 32-cathode side electrophoresis buffer tank 43". Next, the capillary head 11 is inserted into the anode side electrophoresis buffer tank 32, and the loading head 12 is inserted into the cathode side electrophoresis buffer tank 43. In this state, the high voltage power supply 121 applies a high voltage to the capillary array 10. Thus, the high voltage is applied to the capillary array 10 on the cathode side, and flows to GND through the cathode side buffer tank 40 and the anode side buffer tank 30 through the electrode 115, thereby performing electrophoresis. In the electrophoresis process, the sample flowing in the capillary array 10 in order of size due to the molecular sieving effect of the electrophoretic medium is detected by the detection unit 130. The voltage application by the high-voltage power source 121 and the signal detection by the detection unit 130 are continued until a predetermined time defined in the measurement conditions set in step S200 .

[0034] When electrophoresis is performed for a predetermined time in step S209, analysis A as the first analysis ends, and analysis B as the second analysis begins. In this analysis B, steps S203 to S209 are performed in the same manner as the previous analysis A. When analysis B ends, analysis C as the third analysis begins. In this analysis C, steps S203 to S209 are performed in the same manner as the analysis A and the analysis B.

[0035] When a series of analyses consisting of analysis A, analysis B, and analysis C are all completed, the control unit calculates all analysis results (step S210). If the re-analysis function is not enabled in step S201, the analysis ends when all analysis results are calculated (step S213).

[0036] When the re-analysis function is enabled in step S201, if all the analysis results are calculated, the control unit performs a process (abnormality determination process) of statistically determining whether an abnormal analysis result is included based on the calculated analysis results of a series of analyses, the analysis results of past analyses performed under the same conditions (measurement conditions), etc. (step S211). For example, when the analysis result of analysis B is a significantly smaller value than the analysis results of other analyses A or C, it is determined that the analysis result of analysis B is statistically abnormal. In addition, the significantly smaller value among the plurality of analysis results can be determined, for example, by a deviation value obtained by a Smirnov-Grubbs test or the like.

[0037] The analysis result used for determination in step S211 includes not only the signal height data detected by the detection unit 130, but also the quality data. In fragment analysis, the quality data is EQ, etc., which indicates the maximum base length within the range in which single base separation can be performed, and in sequencing analysis, it is QV20CRL, etc., which indicates the maximum continuous base length in which the average QV value (reliability of base calling) in the window exceeds 99% when the sequence being interpreted is slid from the short base side to the long base side in a window of 20 bp.

[0038] If it is statistically determined in step S211 that an abnormal analysis result is included, the control unit executes a process of creating new measurement conditions for the target analysis (first measurement condition adjustment process) (step S212). In addition, creating new measurement conditions may also be adjusting the measurement conditions initially set.

[0039] Next, the control unit returns to step S202 and starts reanalyzing the analysis with abnormal analysis results. For example, when the abnormal object is analysis B, the control unit operates the high voltage power supply 121 and the like based on the adjusted measurement conditions of analysis B, injects the sample corresponding to analysis B into the capillary array 10, and reanalyzes only analysis B. When the reanalysis is completed, the analysis result is calculated again in step S210, and it is statistically determined whether there is an abnormality in step S211. If it is not determined to be abnormal in step S211, the analysis ends (step S213).

[0040] As described above, in this embodiment, when the analysis results of a series of analyses include abnormalities, the capillary electrophoresis device automatically adjusts the measurement conditions and re-analyzes. Therefore, even in the case of poor sample injection, an appropriate signal can be detected by performing electrophoresis immediately before the sample deteriorates. Therefore, it is possible to avoid re-preparing the sample, which imposes a large burden on the user. [Example 2]

[0041] In Example 1, when there are statistically abnormal analysis results in the abnormality determination process, the first measurement condition adjustment process is executed, while in Example 2, even when there are statistically abnormal analysis results in the abnormality determination process, the first measurement condition adjustment process is not executed, but re-analysis is performed using the same measurement conditions. In addition, in Example 2, unlike Example 1, signal saturation is determined based on signal height data detected by the detection unit 130, and when the signal is saturated, the second measurement condition adjustment process is executed, and re-analysis is performed using new measurement conditions. In addition, in Example 1, poor sample injection is determined only in the abnormality determination process, but in Example 2, poor sample injection is determined not only in the abnormality determination process, but also in a process (signal height determination process) that compares the signal height data with a threshold value.

[0042] based on Figure 5 The flow of a series of analyses performed by the capillary electrophoresis apparatus of this embodiment will be described. Figure 5 This is a flow chart showing the analysis method of Example 2.

[0043] First, the user uses the input unit to set measurement conditions used in a series of analyses (step S200 ).

[0044] Next, the user sets the re-analysis function as needed. The control unit determines whether the re-analysis function is valid (step S201). If it is not valid, if the analysis is started (step S102), the method of Example 1 is executed for all analyses. Figure 4 On the other hand, when the re-analysis function is enabled in step S201, the control unit starts the analysis (step S202), and then executes the steps of Example 1 for all the analyses. Figure 4 Steps S202 to S209 in (step S300). When a series of analyses are all completed, the control unit calculates all analysis results (step S210). In addition, if the re-analysis function is not effective in step S201, if all analysis results are calculated, the analysis ends (step S213).

[0045] When the reanalysis function is enabled in step S201, once all analysis results are calculated, the control unit executes a step of determining whether the signal is saturated (signal saturation determination step) (step S301). In the signal saturation determination step, the signal height data detected by the detection unit 130 in the electrophoresis step is used.

[0046] Figure 6 is a graph showing an example of a signal waveform in the case of saturation, Figure 7 Is magnification Figure 6 The dashed line portion of the diagram. Figure 6 and Figure 7 In the figure, the unit of the horizontal axis is the number of data points of the CCD camera, and the unit of the vertical axis is RFU which is related to the signal height (signal strength). Figure 6 and Figure 7 As shown, when the signal is saturated, it is difficult to visually confirm the apex.

[0047] Therefore, when it is determined that the signal is saturated in step S301, the control unit executes a process of estimating the vertex of the signal waveform (signal vertex estimation process) (step S302). At this time, the control unit approximates the vertex from the end of the waveform of the signal saturation or the waveform before and after the peak by fitting a Gaussian function or a Lorentzian function.

[0048] Figure 8is a graph showing an image at the peak of the estimated signal. Figure 8 The middle dashed line indicates that the signal height that is invisible due to saturation is estimated by approximating the apex.

[0049] Next, the control unit executes a step (second measurement adjustment step) of adjusting the measurement conditions so that the signal is not saturated based on the peak of the saturated signal estimated in step S302 (step S303 ).

[0050] The second measurement condition adjustment process is explained by giving specific examples. When the original sample injection voltage based on the measurement condition before adjustment is 1.6 kV, the signal is set to saturation. In addition, the signal intensity of the vertex inferred by fitting the Gaussian function in step S302 is set to 50000 RFU. Then, as the unsaturated signal height, it is assumed that the user sets 20000 RFU as the target value. In addition, in the case where the detection unit 130 is a CCD camera, the unsaturated signal height can be about half of the counting threshold (for example, when the counting threshold is 65535 counts, it is about 30000 or less). If it is within this range, the user can arbitrarily set it as the target value.

[0051] When 20000RFU is set as the non-saturated signal height, the control unit obtains an adjustment coefficient of 0.4 by 20000RFU÷50000RFU. In addition, the control unit multiplies the adjustment coefficient by the original sample injection voltage, that is, 0.4×1.6kV, to obtain 0.64kV as the adjusted new sample injection voltage. As a result, the control unit creates a new measurement condition in which the sample injection voltage is changed to 0.64kV.

[0052] In addition, although the measurement conditions are adjusted by changing the sample injection voltage in the specific example, the measurement conditions can also be adjusted by changing the sample injection time (voltage application time), the laser irradiation time, the laser power, and other analysis conditions in addition to the sample injection voltage. In any case, the control unit adjusts the measurement conditions by multiplying the parameter value of the original analysis condition by the same adjustment coefficient. When the measurement conditions are adjusted, the process returns to step S202, and the control unit re-analyzes based on the adjusted measurement conditions. Thus, even when the signal is saturated and the user cannot visually confirm the apex of the signal, since the apex is automatically estimated and re-analyzed based on the new measurement conditions, data that avoids signal saturation can be obtained without uselessly repeating electrophoresis. As a result, waste of electrophoresis medium and time can be prevented.

[0054] Next, when it is determined in step S301 that the signal is not saturated, the control unit executes a step of determining whether the signal height is less than a predetermined threshold (signal height determination step) (step S304). If the signal height is less than the threshold, sample injection failure may have occurred.

[0055] Fig. 9 This is a graph showing an example of a signal waveform when sample injection is poor. Fig. 9 In the example, the horizontal axis is the number of data points, and the vertical axis is RFU. Fig. 9 As shown, only the baseline is output and the signal caused by the sample cannot be seen. Fig. 9 In the example, although no signal is seen at all, there are also cases where the signal is extremely low. Considering the noise, the threshold used in the signal height determination process is set to, for example, 100RFU. In addition, a threshold determination based on the signal-to-noise ratio can be performed instead of a threshold determination based on the signal height.

[0056] When it is determined in step S304 that the signal height is smaller than the threshold value, the control unit executes a step of statistically determining whether an abnormal analysis result is included based on a series of analysis results or the like (abnormality determination step) (step S305 ).

[0057] Fig.10 is a diagram showing an example of plotting quality data (EQ) in the case where poor sample injection occurs, Fig.11 FIG. 1 is a diagram showing an example of a table of quality data (EQ) when poor sample injection occurs. Fig.11 24 times of analysis are repeated using the capillary array 10 with four loading heads 12. Fig.11 , each of the four loading heads 12 is denoted as CH1, CH2, CH3, CH4. Fig.10 In, use Fig.11 The same data, all data are plotted together, regardless of the four loading heads 12. Although the data from Analysis-1 to Analysis-24 are almost all around EQ450 to 535, data with EQ 0 occasionally appear. 0 is an example of a deviation value, and the values ​​of the data of CH4 of Analysis-2, CH2 of Analysis-9, and CH3 of Analysis-22 are significantly smaller than the values ​​of a series of other data, so it is statistically determined that the possibility of poor sample injection is high. In addition, the Smirnov-Grubbs test can be used to determine the deviation value.

[0058] Thus, instead of comparing a single data with a single threshold, it is compared with other data, and statistically determines whether the data is abnormal, thereby improving the accuracy of error determination. For example, when the signal height of all data is low, it can be considered that it is not a poor sample injection, but other errors such as a poor sample or forgetting to put in the sample, so performing electrophoresis again is not an effective treatment method. However, when there is only a part of the data and only the signal height is low, it is considered that the possibility of poor sample injection is high, so performing electrophoresis again is an effective treatment method. Therefore, the control unit only re-analyzes the samples of Analysis-2, Analysis-9 and Analysis-22. The measurement conditions used at this time can be the same as the measurement conditions initially set. This is because in the case where the cause of poor sample injection is bubbles, even if electrophoresis is performed again under the same conditions, the bubbles may be removed and the sample may be injected normally.

[0059] In addition, in the present embodiment, the result of a certain analysis is compared with the results of other analyses included in a series of analyses (Analysis-1 to Analysis-24) performed 24 times to statistically determine whether an abnormality exists, but in addition to a series of analyses, it is also possible to compare with the results of past analyses performed under the same conditions (measurement conditions) and statistically determine whether an abnormality exists.

[0060] As a result of the electrophoresis being performed again, if it is determined in step S304 that the signal height is equal to or higher than the threshold value, the analysis is terminated (step S213). [Example 3]

[0061] In Example 2, when there are statistically abnormal analysis results in the abnormality determination process, re-analysis is performed using the same measurement conditions, and when there are also abnormal analysis results in the abnormality determination process after the re-analysis, the same measurement conditions are repeatedly used for re-analysis. However, in Example 3, when there are statistically abnormal analysis results in the first abnormality determination process, re-analysis is performed using the same measurement conditions, and when there are also statistically abnormal analysis results in the second abnormality determination process, a first measurement condition adjustment process is performed. In addition, in Example 3, re-analysis is performed using the adjusted measurement conditions, and when there are also statistically abnormal analysis results in the third abnormality determination process, a process of outputting a display prompting re-preparation of samples (re-preparation display process) is performed.

[0062] based on Fig.12 The flow of a series of analyses performed by the capillary electrophoresis apparatus of this embodiment will be described. Fig.12 is a flow chart showing the analysis method of Example 3. Fig.12 In, omitted Figure 5Illustration of step S200, step S102, step S100, step S110, step S302, and step S303 in (Example 2). Only the parts different from Example 2 will be described below.

[0063] In step S305, when there are statistically abnormal analysis results in the first abnormality determination process, the control unit does not adjust the measurement conditions, but returns to step S202, and the control unit re-analyzes based on the measurement conditions initially set. In step S305, when there are also statistically abnormal analysis results in the second abnormality determination process, the control unit executes the first measurement condition adjustment process (step S306).

[0064] The first measurement condition adjustment process is described by giving a specific example. When the original sample injection voltage based on the measurement condition before adjustment is 1.6 kV, a statistically abnormal analysis result is obtained. In addition, as an adjustment coefficient for increasing the signal strength, a value such as 10 is set. In this case, the control unit multiplies the adjustment coefficient by the original sample injection voltage, that is, 10×1.6 kV, to obtain 16 kV as the adjusted new sample injection voltage. As a result, the control unit creates a new measurement condition in which the sample injection voltage is updated to 16 kV.

[0065] In addition, although the measurement conditions are adjusted by changing the sample injection voltage in the specific example, the measurement conditions can also be adjusted by changing the sample injection time (voltage application time), the laser irradiation time, the laser power, and other analysis conditions in addition to the sample injection voltage. In any case, the control unit adjusts the measurement conditions by multiplying the parameter value of the original analysis condition by the same adjustment coefficient.

[0066] If the measurement conditions are adjusted in the first measurement condition adjustment process, the process returns to step S202, and the control unit re-analyzes based on the adjusted measurement conditions. In step S305, when there are statistically abnormal analysis results in the third abnormality determination process, it is considered that a sample defect has occurred rather than a sample injection defect. Therefore, the control unit executes a process of outputting a display prompting the display unit to re-prepare the sample (re-preparation display process) (step S307), and the analysis ends (step S213). In addition, although in Fig.12 Although not shown in the figure, in step S305, even if there is no statistically abnormal analysis result, the analysis ends (step S213).

[0067] According to this embodiment, poor sample injection can be dealt with by avoiding sample re-preparation as much as possible and automatically performing re-analysis. In addition, in this embodiment, re-analysis based on the same measurement conditions is performed once, and re-analysis based on the adjusted measurement conditions is performed once, but each can be performed multiple times as long as it is before the sample deteriorates.

[0068] Although Embodiments 1 to 3 have been described above, the present invention is not limited to these embodiments, and various modifications are possible.

[0069] For example, in the abnormality determination process of Example 2, statistical determination using quality data (EQ) is performed, but statistical determination using signal height data may also be performed. As an example of determination using signal height data, when other signal height data are about 10000RFU, the case where the signal height data is about 300RFU is determined to be abnormal. In addition, in the abnormality determination process, both quality data and signal height data may be used for statistical determination.

[0070] In addition, Example 1 is a capillary electrophoresis apparatus that determines statistical abnormality and performs re-analysis to cope with poor sample injection, and Examples 2 and 3 are capillary electrophoresis apparatuses that perform re-analysis to cope with signal saturation in addition to poor sample injection. However, a capillary electrophoresis apparatus that only copes with signal saturation but not poor sample injection may also be used. Description of symbols

[0071] 10 capillary array, 11 capillary head, 12 loading head, 20 electrophoresis medium container, 30 anode side buffer container, 31 anode side cleaning tank, 32 anode side electrophoresis buffer tank, 33 sample introduction buffer tank, 40 cathode side buffer container, 41 waste liquid tank, 42 ​​cathode side cleaning tank, 43 cathode side electrophoresis buffer tank, 50 sample container, 60 liquid delivery mechanism, 61 plunger, 80 sampler base, 85 Y-axis drive body, 90 Z-axis drive body, 95 X-axis drive body, 100 sample tray, 101 guide rail, 110 constant temperature bath unit, 115 electrode, 120 constant temperature bath door, 121 high voltage power supply, 122 control substrate, 123 computer, 130 detection unit, 150 detection / constant temperature bath unit, 160 automatic sampler unit.

Claims

1. An analysis method for analyzing characteristics related to a sample by subjecting the sample to electrophoresis using a capillary and a phoretic medium, the analysis method comprising: a reading step of reading analysis conditions; A step of filling the capillary with the electrophoretic medium; a sample injecting step of injecting the sample into the capillary filled with the electrophoretic medium in the electrophoretic medium filling step; an electrophoresis step of subjecting the sample injected in the sample injection step to electrophoresis in the electrophoretic medium; an abnormality determination step of statistically determining whether or not an analysis result obtained in the electrophoresis step is abnormal by comparing the analysis result obtained in the electrophoresis step with other analysis results obtained under the same analysis conditions; as well as An analysis condition adjustment step for adjusting the analysis conditions, When it is determined that an abnormality is present in the abnormality determination step, the process proceeds to the reading step again via the analysis condition adjustment step.

2. The analysis method according to claim 1, characterized in that Also includes: a signal saturation determination step of determining whether the signal detected in the electrophoresis step is saturated; as well as If saturation is determined in the signal saturation determination step, a signal peak estimating step of estimating the peak of the signal waveform is performed, The analysis condition adjustment step adjusts the analysis conditions to such an extent that the signal is not saturated based on the peak estimated in the signal peak estimation step.

3. The analysis method according to claim 1, characterized in that Even when abnormality is determined in the abnormality determination step, the process first proceeds to the reading step without passing through the analysis condition adjustment step. When abnormality is determined again in the abnormality determination step, the process proceeds to the reading step via the analysis condition adjustment step.

4. The analysis method according to claim 1, characterized in that The method further includes the step of outputting a display prompting re-preparation of the sample, After the analysis condition adjustment step is passed and the process moves to the reading step, if the process is judged to be abnormal again in the abnormality judgment step, the process moves to the re-preparation display step.

5. A capillary electrophoresis device, comprising: a capillary tube filled with a phoretic medium; A power source for applying voltage to the capillary and performing electrophoresis on the sample; a detection unit that detects a signal when laser light is irradiated to the sample electrophoresed in the capillary; and a control unit that controls the operation of the power supply and the detection unit and calculates the analysis result, The capillary electrophoresis device is characterized in that: In the control unit, By comparing the calculated analysis results with other analysis results under the same analysis conditions, it is statistically determined whether there is an abnormality. When it is determined that an abnormality exists, the analysis conditions are adjusted, the power supply and the detection unit are operated under the adjusted analysis conditions, and the analysis is performed again.

6. The capillary electrophoresis device according to claim 5, characterized in that: When the signal detected by the detection unit is saturated, the control unit estimates a peak of the signal waveform and adjusts the analysis conditions to a condition that does not saturate the signal based on the estimated peak.

7. The capillary electrophoresis device according to claim 6, characterized in that: When the signal detected by the detection unit is saturated, the control unit adjusts the application conditions of the voltage applied by the power supply to the capillary when injecting the sample, or the irradiation conditions of the laser by the detection unit to reduce the intensity of the signal detected by the detection unit.

8. The capillary electrophoresis device according to claim 5, characterized in that: When the analysis result is determined to be abnormal, the control unit adjusts the application conditions of the voltage applied by the power supply to the capillary when injecting the sample or the irradiation conditions of the laser by the detection unit to increase the intensity of the signal detected by the detection unit.

9. The capillary electrophoresis device according to claim 5, characterized in that: Even when the analysis result is determined to be abnormal, the control unit first performs analysis again under the same analysis conditions, and when the analysis result is determined to be abnormal again, performs analysis again under the adjusted analysis conditions.

10. The capillary electrophoresis device according to claim 5, characterized in that: It also includes a display unit, which is used to display the analysis results. When the analysis result is determined to be abnormal even if the analysis is performed again under the adjusted analysis conditions, the control unit outputs a display to the display unit to prompt re-preparation of the sample.

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