Analysis method and capillary electrophoresis apparatus

Abstract

The purpose of the present invention is to provide an analysis method that suppresses unnecessary repetition of electrophoresis or sample preparation. An analysis method according to the present invention involves analyzing the characteristics of a sample by electrophoresis of the sample using a capillary and a migration medium and includes a read-in step for reading-in analysis conditions, a migration medium filling step for filling the capillary with the migration medium, a sample injection step for injecting the sample into the capillary filled with the migration medium at the migration medium filling step, an electrophoresis step for electrophoresing the sample injected at the sample injection step through the migration medium, an abnormality determination step for comparing analysis results obtained at the electrophoresis step with other analysis results for the same analysis conditions to statistically determine whether there is an abnormality, and an analysis conditions adjustment step for adjusting the analysis conditions. When it has been determined that there is an abnormality at the abnormality determination step, the method returns to the read-in step via the analysis conditions adjustment step.

Description

[Technical Field]

[0001] The present invention relates to a capillary electrophoresis apparatus and an analytical method using the same. [Background technology]

[0002] In recent years, capillary electrophoresis devices, in which capillaries are filled with a migration medium such as a polymer gel or a polymer solution, have become widely used as electrophoresis devices. These capillary electrophoresis devices are capable of automatic filling of the migration medium and automatic injection of samples, making continuous analysis possible. Multiple electrophoresis runs can be set up in advance, allowing for the analysis of a large number of samples overnight.

[0003] A laser is irradiated onto the sample undergoing electrophoresis inside the capillary, and the resulting signal is detected by a detection unit such as a CCD camera. However, if the concentration of the sample injected into the capillary is high, the detected signal will be high, and if it exceeds the CCD camera's count threshold (e.g., 65,535 counts), the signal will saturate and the peak of the signal will become indistinguishable. In this case, the user would traditionally reconstitute the sample by diluting it or adjust the assay (information that sets the analytical operating and analysis conditions) based on their experience.

[0004] Furthermore, capillary electrophoresis devices can also experience errors in which the detection unit detects no signal or only a very low signal. When such errors occur, users have traditionally dealt with them based on their experience, with the most common solution being re-preparation of the sample. However, errors such as no signal being detected can also be caused by improper sample injection, in addition to sample defects. If the error is due to improper sample injection, an appropriate signal can be detected by immediately performing electrophoresis before the sample deteriorates, thereby avoiding the need for sample re-preparation, which places a significant burden on the user.

[0005] Patent Document 1 discloses an electrophoresis device that, when the signal is off-scale (saturated) or the signal-to-noise ratio is low, prompts the user to change the sample concentration, sample injection parameters, etc. Patent Document 1 also discloses that, when the signal-to-noise ratio is low, there is a possibility of a sample injection failure, etc., and prompts the user to adjust the sample injection parameters. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] US Patent Application Publication No. 2020 / 0003728 Summary of the Invention [Problem to be solved by the invention]

[0007] In conventional capillary electrophoresis devices, when an error such as signal saturation or no signal can be detected occurs, the user has no choice but to rely on their own experience to resolve the issue. Furthermore, if electrophoresis is performed continuously overnight, the user may not notice the error until early morning. Because the sample has deteriorated over time, it is difficult to perform electrophoresis again, even if the error is not due to a defective sample. In this case, the user must reprepare the sample, wasting not only time and effort but also valuable sample.

[0008] Patent Document 1 also discloses that the device changes sample injection parameters, etc., when the signal is off-scale (saturated) or the signal-to-noise ratio is low, but does not specifically disclose how the device changes these parameters. Furthermore, Patent Document 1 determines sample injection failures, etc., based solely on the signal-to-noise ratio, i.e., signal height. Therefore, if the signal-to-noise ratio is low for all data, for example, electrophoresis is performed again for all samples using the changed injection parameters. However, if the signal-to-noise ratio is low for all data, the possibility of sample failure or forgetting to insert a sample is higher than the possibility of sample injection failure, and simply performing electrophoresis again often does not solve the problem. As a result, unnecessary electrophoresis may be performed, wasting migration medium and time.

[0009] An object of the present invention is to provide an analytical method that suppresses unnecessary re-electrophoresis and sample re-preparation. [Means for solving the problem]

[0010] In order to solve the above-mentioned problems, the present invention provides an analytical method for analyzing characteristics of a sample by electrophoresing the sample using a capillary and a migration medium, the method comprising: a reading step for reading analytical conditions; a migration medium filling step for filling the capillary with the migration medium; a sample injection step for injecting the sample into the capillary filled with the migration medium in the migration medium filling step; an electrophoresis step for electrophoresing the sample injected in the sample injection step in the migration medium; an abnormality determination step for statistically determining whether or not the analysis result obtained in the electrophoresis step is abnormal by comparing it with other analysis results under the same analytical conditions; and an analytical condition adjustment step for adjusting the analytical conditions; if an abnormality is determined in the abnormality determination step, the method proceeds to the reading step again via the analytical condition adjustment step. [Effects of the Invention]

[0011] According to the present invention, an analytical method can be provided that suppresses unnecessary re-electrophoresis and sample re-preparation. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a perspective view showing an outline of the configuration of a capillary electrophoresis apparatus. [Figure 2] FIG. 2 is a top view of the capillary electrophoresis apparatus shown in FIG. 1. [Figure 3] Cross section AA in Figure 2. [Figure 4] 1 is a flowchart showing the analysis method of Example 1. [Figure 5] 1 is a flowchart showing an analysis method of Example 2. [Figure 6] 10 is a graph showing an example of a signal waveform in a saturated state. [Figure 7] A graph enlarging the dotted line area in Figure 6. [Figure 8] 10 is a graph showing an image when estimating the peak of a signal. [Figure 9] 10 is a graph showing an example of a signal waveform in the case of a sample injection failure. [Figure 10] FIG. 10 is a diagram showing an example of a plot of quality data (EQ) when a sample injection failure occurs. [Figure 11] FIG. 10 is a diagram showing an example of a quality data (EQ) table when a sample injection failure occurs. [Figure 12] 1 is a flowchart showing the analysis method of Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0013] Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

[0014] First, the configuration of a capillary electrophoresis apparatus will be described with reference to Figures 1 to 3. Figure 1 is a perspective view showing an outline of the configuration of a capillary electrophoresis apparatus. The capillary electrophoresis apparatus is divided into two units: a detection / constant temperature bath unit 150 at the top and an autosampler unit 160 at the bottom.

[0015] The autosampler unit 160 has a Y-axis driver 85 mounted on the sampler base 80, allowing for drive in the Y axis. A Z-axis driver 90 is mounted on the Y-axis driver 85, allowing for drive in the Z axis. A sample tray 100 is mounted on the Z-axis driver 90, and the user places the loading medium container 20, the anode-side buffer container 30, the cathode-side buffer container 40, and the sample container 50 on the sample tray 100. The sample container 50 is placed on an X-axis driver 95 mounted on the sample tray 100, allowing it to move in the X axis on the sample tray 100. A liquid delivery mechanism 60 is also mounted on the Z-axis driver 90. This liquid delivery mechanism 60 is located below the loading medium container 20.

[0016] The detection / thermostat unit 150 includes a thermostat unit 110 and a thermostat door 120, and can maintain the inside of the thermostat unit 110 at a constant temperature. The detection / thermostat unit 150 also includes a detection unit 130 behind the thermostat unit 110, which can irradiate a laser and detect the resulting signal. When a user places the capillary array 10 in the thermostat unit 110, the sample undergoes electrophoresis while the capillary array 10 is maintained at a constant temperature by the thermostat unit 110. The detection unit 130 detects a signal generated when a laser is irradiated onto the sample undergoing electrophoresis in the capillary array 10, using a CCD camera or the like. The thermostat unit 110 also includes an electrode 115 for grounding when a high voltage is applied for electrophoresis.

[0017] As described above, the capillary array 10 is fixed to the thermostatic bath unit 110. The running medium container 20, the anode side buffer container 30, the cathode side buffer container 40, and the sample container 50 can be driven in the Y-axis and Z-axis by the autosampler unit 160, and only the sample container 50 can be driven in the X-axis. When the autosampler unit 160 is driven, the running 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] Fig. 2 is a top view of the capillary electrophoresis apparatus shown in Fig. 1. The anode-side buffer container 30 set on the sample tray 100 contains an anode-side washing tank 31, an anode-side electrophoresis buffer container 32, and a sample introduction buffer container 33. The cathode-side buffer container 40 set on the sample tray 100 contains a waste tank 41, a cathode-side washing tank 42, and a cathode-side electrophoresis buffer container 43.

[0019] The running medium container 20, the anode buffer container 30, the cathode buffer container 40, and the sample container 50 are arranged on the sample tray 100 in the positional relationship shown in Fig. 2. As a result, the anode-cathode combinations connected to the capillary array 10 are "running medium container 20-waste tank 41," "anode washing tank 31-cathode washing tank 42," "anode electrophoresis buffer container 32-cathode electrophoresis buffer container 43," and "sample introduction buffer container 33-sample container 50."

[0020] 3 is a cross-sectional view taken along the line AA in FIG. 2, showing a cross section of a capillary electrophoresis device in which the anode-cathode combination connected to the capillary array 10 is "loading medium container 20-waste tank 41." The loading medium container 20 is inserted into a guide 101 embedded in the sample tray 100. The liquid delivery mechanism 60 is positioned so that the plunger 61 built into the liquid delivery mechanism 60 is below the loading medium container 20. During electrophoresis, the right side of the capillary array 10 in FIG. 3 is the cathode side, and the left side is the anode side.

[0021] The capillary electrophoresis apparatus is controlled by a control unit. The control unit is composed of a control board 122 and a computer 123 connected to the control board 122. FIG. 1 shows an example in which the control board 122 is installed on the rear side of the thermostatic chamber unit 110 and the computer 123 is installed outside (on the front side of) the thermostatic chamber door 120, but the installation locations are not limited to this. The control board 122 controls the operations of the high-voltage power supply 121, the thermostatic chamber unit 110, the detection unit 130, etc. in accordance with instructions from the computer 123. The computer 123 calculates analysis results based on signals detected by the detection unit 130, etc., and also has an input unit for inputting settings for the capillary electrophoresis apparatus (including assays, which will be described later), etc., and an output unit (display unit) for displaying analysis results, etc. Note that the calculation of analysis results may be performed by the control board 122. [Example]

[0022] A series of analytical steps performed by the capillary electrophoresis apparatus according to the present embodiment will be described with reference to Fig. 4. Fig. 4 is a flowchart showing the analytical method of the first embodiment.

[0023] First, the user sets the assay to be used in the series of analyses using the input unit (step S200). An assay is information that defines the operating conditions and analysis conditions for the analysis. Operating conditions include the laser power of the detection unit 130, the temperature of the thermostatic chamber unit 110, the type of electrophoresis medium, the voltage and time applied in the pre-run (described below), the voltage and time applied in the sample injection step (described below), the voltage and time applied in the electrophoresis step, the laser irradiation time and sampling period during data acquisition, etc. Analysis conditions include the analysis width specified by the size of data points or bases, the signal height threshold, a base call mobility file set for reading the sequence in the case of sequencing analysis, and the fragment size used in the case of fragment analysis.

[0024] Next, the user sets a function (reanalysis function) that performs reanalysis if the analysis results are determined to be abnormal, as needed. The control unit determines whether the reanalysis function is enabled (step S201), and if it is not enabled, executes steps S102 to S110. On the other hand, if it is determined in step S201 that the reanalysis function is enabled, the control unit executes steps S202 to S212. Steps S102 to S110 are the same as steps S202 to S210, so only steps S202 to S210 will be described below.

[0025] When the analysis is started in step S202, a series of analyses is performed. In this embodiment, one analysis is defined as the period from sample injection to one electrophoresis run performed by voltage application. In other words, the user can preset not only one analysis but also a series of multiple analyses. The following description will be given using an example in which three analyses, Analysis A, Analysis B, and Analysis C, are preset, but the number of analyses is not limited to this.

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

[0027] Next, the control unit controls the thermostatic bath unit 110 to execute a step of keeping the capillary array 10 at a constant temperature (capillary temperature control step) (step S204).

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

[0029] Furthermore, the control unit controls the autosampler unit 160 and the plunger 61 to execute a step (a loading medium filling step) of filling the capillary array 10 with the loading medium contained in the loading medium container 20 (step S206). The loading medium filling step will now be described in more detail. First, the autosampler unit 160 moves, and the anode-cathode combination connected to the capillary array 10 becomes the loading medium container 20-waste tank 41. Next, the capillary head 11, which is the end of the anode side of the capillary array 10, is inserted into the loading medium container 20, and the load header 12, which is the end of the cathode side of the capillary array 10, is inserted into the waste tank 41. In this state, the plunger 61 built into the liquid delivery mechanism 60 moves upward, and the loading medium contained in the loading medium container 20 is delivered into the capillary array 10 through the capillary head 11. If the capillary array 10 is already filled with migration medium before the liquid is transferred, or if the migration medium is sufficiently transferred into the capillary array 10 and becomes surplus, the migration medium is discharged from the load header 12 to the waste liquid tank 41.

[0030] Next, the control unit executes a pre-run (step S207). Specifically, first, the autosampler unit 160 moves, and the anode-cathode combination 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 load header 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 an operation similar to that of electrophoresis after sample injection, which will be described later, but is executed before sample injection to stabilize the performance of electrophoresis.

[0031] Thereafter, the control unit executes a step (sample injection step) 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 anode-cathode combination connected to the capillary array 10 becomes "sample introduction buffer solution tank 33-sample container 50." Next, the capillary head 11 is inserted into the sample introduction buffer solution tank 33, and the load header 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 from the load header 12 into the capillary array 10.

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

[0033] Next, the control unit executes a step (electrophoresis step) of applying a voltage to the capillary array 10 into which the sample has been injected to electrophorese the sample (step S209). Specifically, first, the autosampler unit 160 moves, and the anode-cathode combination 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 load header 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. As a result, a high voltage is applied to the cathode-side capillary array 10, and the high voltage is passed through the cathode-side buffer tank 40 and the anode-side buffer tank 30 to GND at the electrode 115, thereby performing electrophoresis. In the electrophoresis process, the sample flows through the capillary array 10 in order of size due to the molecular sieving effect of the migration medium, and is detected by the detection unit 130. The voltage application by the high-voltage power supply 121 and the signal detection by the detection unit 130 continue for a predetermined time defined in the assay set in step S200.

[0034] When electrophoresis is performed for a predetermined time in step S209, analysis A, which is the first analysis, ends and analysis B, which is the second analysis, begins. In analysis B, steps S203 to S209 are performed, just like in analysis A, which was the previous analysis. When analysis B ends, analysis C, which is the third analysis, begins. In analysis C, steps S203 to S209 are performed, just like in analyses A and 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 reanalysis function is not enabled in step S201, the analysis ends when all analysis results are calculated (step S213).

[0036] If the reanalysis function is enabled in step S201, once all analysis results have been calculated, the control unit executes a step (abnormality determination step) of statistically determining whether any abnormal analysis results are included based on the calculated analysis results of the series of analyses and the analysis results of past analyses performed under the same conditions (assays) (step S211). For example, if the analysis result of analysis B is significantly smaller than the analysis results of other analyses, such as analysis A and analysis C, the analysis result of analysis B is determined to be statistically abnormal. Note that significantly smaller values ​​among multiple analysis results may be determined using outliers obtained, for example, by the Smirnoff-Grubbs test.

[0037] The analysis results used for the determination in step S211 include quality data as well as signal height data detected by the detection unit 130. In fragment analysis, the quality data is, for example, EQ, which indicates the maximum base length within the range in which single base separation is possible, and in sequencing analysis, QV20CRL, which indicates the maximum consecutive base length for which the average QV value (base call reliability) in a window of 20 bp of the decoded sequence is slid from the short base side to the long base side and exceeds 99%.

[0038] If it is determined in step S211 that a statistically abnormal analysis result is included, the control unit executes a step of creating a new assay for the target analysis (first assay adjustment step) (step S212). Note that creating a new assay also means adjusting the initially set assay.

[0039] Next, the control unit returns to step S202 and starts a reanalysis of the analysis for which the analysis result was abnormal. For example, if the abnormality was in analysis B, the control unit operates the high-voltage power supply 121 and the like based on the adjusted assay for analysis B, injects a sample corresponding to analysis B into the capillary array 10, and performs a reanalysis of only analysis B. Once the reanalysis is complete, the analysis results are calculated again in step S210, and whether or not they are statistically abnormal is determined in step S211. If an abnormality is not determined in step S211, the analysis ends (step S213).

[0040] As described above, in this embodiment, if the analysis results of a series of analyses contain an abnormality, the capillary electrophoresis device adjusts the assay and performs a reanalysis. Therefore, even if a sample injection error occurs, an appropriate signal can be detected by performing electrophoresis immediately before the sample deteriorates. This avoids the need for sample repreparation, which places a significant burden on the user. [Example]

[0041] In Example 1, if a statistically abnormal analysis result is found in the anomaly determination step, the first assay adjustment step is executed. However, in Example 2, even if a statistically abnormal analysis result is found in the anomaly determination step, the first assay adjustment step is not executed, and reanalysis is performed using the same assay. Furthermore, in Example 2, unlike Example 1, signal saturation is determined based on signal height data detected by the detection unit 130, and if the signal is saturated, the second assay adjustment step is executed and reanalysis is performed using a new assay. Furthermore, in Example 1, a sample injection failure is determined only by the anomaly determination step, but in Example 2, a sample injection failure is determined not only by the anomaly determination step but also by a step of comparing signal height data with a threshold value (signal height determination step).

[0042] A series of analytical steps performed by the capillary electrophoresis apparatus according to the second embodiment will be described with reference to Fig. 5. Fig. 5 is a flowchart showing the analytical method of the second embodiment.

[0043] First, the user sets the assays to be used in a series of analyses using the input unit (step S200).

[0044] Next, the user sets the reanalysis function as necessary. The control unit determines whether the reanalysis function is enabled (step S201). If it is not enabled, when the analysis is started (step S102), steps S103 to S109 in FIG. 4 of the first embodiment are executed for all analyses (step S100). On the other hand, if the reanalysis function is enabled in step S201, when the analysis is started (step S202), the control unit executes steps S202 to S209 in FIG. 4 of the first embodiment for all analyses (step S300). When the entire series of analyses is completed, the control unit calculates all analysis results (step S210). Note that if the reanalysis function is not enabled in step S201, when all analysis results are calculated, the analysis ends (step S213).

[0045] If the reanalysis function is enabled in step S201, once all analysis results have been calculated, the control unit executes a step of determining whether or not the signal is saturated (signal saturation determination step) (step S301). In the signal saturation determination step, 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 when saturated, and Figure 7 is a graph enlarging the dotted line portion of Figure 6. In Figures 6 and 7, the horizontal axis represents the number of data points from the CCD camera, and the vertical axis represents RFU, which is related to signal height (signal intensity). As shown in Figures 6 and 7, when the signal is saturated, it is difficult to visually confirm the peak.

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

[0048] Fig. 8 is a graph showing an image of estimating the peak of a signal. As shown by the dotted line in Fig. 8, by approximating the peak, the signal height that was not visible due to saturation can be estimated.

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

[0050] The second assay adjustment step will be described using a specific example. Assume that signal saturation occurs when the original sample injection voltage in the assay before adjustment is 1.6 kV. Assume also that the peak signal intensity estimated by fitting with a Gaussian function in step S302 is 50,000 RFU. Assume also that the user sets a target value of 20,000 RFU as the signal height that will not saturate. Note that, if the detection unit 130 is a CCD camera, the signal height that will not saturate may be approximately half the count threshold or less (for example, approximately 30,000 or less when the count threshold is 65,535 counts), and the user can arbitrarily set the target value within this range.

[0051] If 20,000 RFU is set as the non-saturating signal height, the controller calculates an adjustment factor of 0.4 by dividing 20,000 RFU by 50,000 RFU. The controller then multiplies the original sample injection voltage by this adjustment factor, i.e., 0.4 x 1.6 kV, to calculate a new adjusted sample injection voltage of 0.64 kV. As a result, the controller creates a new assay with the sample injection voltage changed to 0.64 kV.

[0052] In the specific example, the assay was adjusted by changing the sample injection voltage, but the assay may also be adjusted by changing analysis conditions other than the sample injection voltage, such as the sample injection time (voltage application time), laser irradiation time, laser power, etc. In either case, the control unit adjusts the assay by multiplying the parameter values ​​of the original analysis conditions by the same adjustment coefficient.

[0053] Once the assay is adjusted, the process returns to step S202, and the control unit performs reanalysis based on the adjusted assay. In this way, even if the signal saturates and the signal peak cannot be visually confirmed by the user, the peak is automatically estimated and reanalysis is performed based on a new assay. This allows data to be obtained without signal saturation and without needlessly repeating electrophoresis. As a result, it is possible to prevent the waste of electrophoresis medium and time.

[0054] Next, if 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, there is a possibility that a sample injection failure has occurred.

[0055] FIG. 9 is a graph showing an example of a signal waveform in the case of a sample injection failure. In FIG. 9, the horizontal axis is in units of data points, and the vertical axis is in units of RFU. As shown in FIG. 9, only the baseline is output, and no signal due to the sample is observed. Although no signal is observed at all in FIG. 9, there are also cases where the signal is extremely low in some areas. The threshold used in the signal height determination step is set to, for example, 100 RFU, taking noise into consideration. Note that instead of threshold determination based on signal height, threshold determination based on the signal-to-noise ratio may also be performed.

[0056] If it is determined in step S304 that the signal height is less than the threshold, the control unit executes a process (abnormality determination process) of statistically determining whether or not any abnormal analysis results are included based on a series of analysis results, etc. (step S305).

[0057] Figure 10 shows an example plot of quality data (EQ) when a sample injection failure occurs, and Figure 11 shows an example table of quality data (EQ) when a sample injection failure occurs. Figure 11 shows data from 24 analyses using a capillary array 10 with four load headers 12. In Figure 11, each of the four load headers 12 is designated CH1, CH2, CH3, and CH4. Figure 10 uses the same data as Figure 11, but plots all data together, regardless of the four load headers 12. While most of the data from Analysis-1 to Analysis-24 ranges from EQ 450 to 535, there are also occasional data with an EQ of 0. An EQ of 0 is an example of an outlier. The data for CH4 in Analysis-2, CH2 in Analysis-9, and CH3 in Analysis-22 are significantly smaller than the rest of the data, and are therefore statistically determined to be highly likely to be due to a sample injection failure. The Smirnoff-Grubbs test may be used to identify outliers.

[0058] In this way, by comparing individual data with other data and statistically determining whether or not they are abnormal, rather than comparing individual data with a single threshold, the accuracy of error detection is improved. For example, if the signal height of all data is low, this is likely not due to a sample injection error but rather to other errors such as a defective sample or a missing sample, and therefore re-running electrophoresis is not an effective solution. However, if the signal height of only some data is low, this is likely due to a sample injection error, and re-running electrophoresis is an effective solution. Therefore, the control unit re-analyzes only the samples in Analysis-2, Analysis-9, and Analysis-22. The assay used in this case can be the same as the assay initially set. If the cause of the sample injection error is air bubbles, re-running electrophoresis under the same conditions may remove the bubbles and successfully inject the sample.

[0059] In this example, the results of a certain analysis were compared with the results of other analyses included in a series of analyses performed 24 times (Analysis-1 to Analysis-24) to statistically determine whether or not they are abnormal. However, in addition to the series of analyses, the results of past analyses performed under the same conditions (assay) may also be compared with the results of previous analyses to statistically determine whether or not they are abnormal.

[0060] If, as a result of the re-electrophoresis, it is determined in step S304 that the signal height is equal to or greater than the threshold value, the analysis ends (step S213). [Example]

[0061] In Example 2, if a statistically abnormal analysis result is found in the anomaly determination step, reanalysis is performed using the same assay, and if a similar abnormal analysis result is found in the anomaly determination step after reanalysis, reanalysis using the same assay is repeated. However, in Example 3, if a statistically abnormal analysis result is found in the first anomaly determination step, reanalysis is performed using the same assay, and if a statistically abnormal analysis result is found in the second anomaly determination step, a first assay adjustment step is executed. Furthermore, in Example 3, reanalysis is performed using the adjusted assay, and if a statistically abnormal analysis result is found in the third anomaly determination step, a step of outputting a display prompting repreparation of the sample (repreparation display step) is executed.

[0062] A series of analytical procedures performed by a capillary electrophoresis apparatus according to this embodiment will be described with reference to Fig. 12. Fig. 12 is a flowchart showing an analytical method according to Example 3. However, in Fig. 12, steps S200, S102, S100, S110, S302, and S303 shown in Fig. 5 (Example 2) are omitted. Only the differences from Example 2 will be described below.

[0063] In step S305, if a statistically abnormal analysis result is found in the first abnormality determination process, the control unit does not adjust the assay, returns to step S202, and the control unit performs reanalysis based on the initially set assay. In step S305, if a statistically abnormal analysis result is found in the second abnormality determination process, the control unit executes the first assay adjustment process (step S306).

[0064] The first assay adjustment step will be described using a specific example. Suppose that a statistically abnormal analysis result was obtained when the original sample injection voltage of the assay before adjustment was 1.6 kV. Furthermore, suppose an adjustment factor for increasing signal strength was set to, for example, 10. In this case, the control unit multiplies the original sample injection voltage by the adjustment factor, i.e., 10 × 1.6 kV, to obtain a new adjusted sample injection voltage of 16 kV. As a result, the control unit creates a new assay in which the sample injection voltage is updated to 16 kV.

[0065] In the specific example, the assay was adjusted by changing the sample injection voltage, but the assay may also be adjusted by changing analysis conditions other than the sample injection voltage, such as the sample injection time (voltage application time), laser irradiation time, laser power, etc. In either case, the control unit adjusts the assay by multiplying the parameter values ​​of the original analysis conditions by the same adjustment coefficient.

[0066] Once the assay is adjusted in the first assay adjustment step, the process returns to step S202, and the control unit performs reanalysis based on the adjusted assay. If a statistically abnormal analysis result is found in the third abnormality determination step in step S305, it is assumed that a sample defect has occurred, rather than a sample injection defect. Therefore, the control unit executes a step of outputting a message to the display unit (repreparation display step) prompting the user to repreparate the sample (step S307), and the analysis ends (step S213). Although not shown in FIG. 12, the analysis also ends (step S213) if no statistically abnormal analysis result is found in step S305.

[0067] According to this embodiment, it is possible to deal with sample injection failures by automatically performing reanalysis while avoiding sample repreparation as much as possible. Note that in this embodiment, reanalysis is performed once using the same assay and once using the adjusted assay, but each may be performed multiple times as long as the sample does not deteriorate.

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

[0069] For example, in the abnormality determination step in Example 2, a statistical determination was made using quality data (EQ), but a statistical determination may also be made using signal height data. As an example of a determination using signal height data, signal height data of approximately 300 RFU is determined to be abnormal when other signal height data are approximately 10,000 RFU. Furthermore, in the abnormality determination step, a statistical determination may also be made using both quality data and signal height data.

[0070] Furthermore, Example 1 is a capillary electrophoresis apparatus that determines statistical abnormalities and performs reanalysis to address sample injection defects, while Examples 2 and 3 are capillary electrophoresis apparatus that perform reanalysis to address signal saturation in addition to sample injection defects. However, a capillary electrophoresis apparatus that only addresses signal saturation without addressing sample injection defects may also be used. [Explanation of symbols]

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

Claims

1. An analytical method for analyzing characteristics of a sample by electrophoresing the sample using a capillary and a migration medium, comprising: a reading step of reading analysis conditions; a loading medium filling step of filling the capillary with the loading medium; a sample injection step of injecting the sample into the capillary filled with the migration medium in the migration medium filling step; an electrophoresis step of electrophoresing the sample injected in the sample injection step in the electrophoresis medium; a signal height determination step of determining whether the signal height detected by the detection unit when the laser is irradiated in the electrophoresis step is less than a predetermined threshold value; an abnormality determination step of statistically determining whether or not the analysis result obtained in the electrophoresis step is abnormal by comparing the analysis result with other analysis results obtained under the same analysis conditions; an analysis condition adjusting step of adjusting analysis conditions, an analysis method characterized in that, when the signal height is determined to be less than a predetermined threshold value in the signal height determination process and an abnormality is determined in the abnormality determination process, the analysis condition adjustment process adjusts the conditions for applying voltage to the capillary by a power supply when injecting the sample or the conditions for irradiating the laser by the detection unit to conditions that increase the intensity of the signal detected by the detection unit, and then proceeds to the reading process again.

2. In claim 1, a signal saturation determination step of determining whether or not the signal detected in the electrophoresis step is saturated; a signal peak estimation step of estimating a peak of a signal waveform when it is determined that the signal is saturated in the signal saturation determination step, The analysis method is characterized in that the analysis condition adjustment step adjusts the analysis conditions to avoid saturating the signal based on the peak estimated in the signal peak estimation step.

3. In claim 1, An analysis method characterized in that, even if an abnormality is determined in the abnormality determination step, the method first proceeds to the reading step without going through the analysis condition adjustment step, and if an abnormality is again determined in the abnormality determination step, the method proceeds to the reading step via the analysis condition adjustment step.

4. In claim 1, a reconstitution display step of outputting a display prompting the user to reconstitute the sample; An analytical method characterized in that, after passing through the analysis condition adjustment step and proceeding to the reading step, if an abnormality is again determined in the abnormality determination step, the method proceeds to the re-preparation display step.

5. a capillary filled with a migration medium; a power supply that applies a voltage to the capillary to cause electrophoresis of the sample; a detection unit that detects a signal when a laser is irradiated onto the sample undergoing electrophoresis in the capillary; a control unit that controls the operation of the power supply and the detection unit and calculates an analysis result; In a capillary electrophoresis apparatus comprising: The control unit determining whether the signal height detected by the detection unit is less than a predetermined threshold value, and statistically determining whether the calculated analysis result is abnormal by comparing it with other analysis results under the same analysis conditions; When the signal height is determined to be less than a predetermined threshold and is determined to be abnormal, the capillary electrophoresis apparatus adjusts the conditions for applying voltage to the capillary by the power supply when injecting the sample or the conditions for irradiating the laser by the detection unit to conditions that increase the intensity of the signal detected by the detection unit, and operates the power supply and the detection unit under the adjusted analysis conditions to perform the analysis again.

6. In claim 5, a control unit that, when the signal detected by the detection unit is saturated, estimates the peak of the signal waveform and, based on the estimated peak, adjusts the analysis conditions to prevent signal saturation.

7. In claim 6, a control unit for controlling a capillary electrophoresis apparatus, the control unit adjusting, when the signal detected by the detection unit is saturated, the conditions for applying a voltage to the capillary by the power supply when the sample is injected, or the conditions for irradiating the laser by the detection unit, thereby reducing the intensity of the signal detected by the detection unit.

8. (delete)

9. In claim 5, The capillary electrophoresis apparatus is characterized in that, even if the control unit determines that the analysis results are abnormal, it first performs the analysis again under the same analysis conditions, and if it determines that the analysis results are abnormal again, it performs the analysis again under the adjusted analysis conditions.

10. In claim 5, Further provided is a display unit for displaying the analysis results, The capillary electrophoresis apparatus is characterized in that, when the analysis result is determined to be abnormal even after re-analysis under the adjusted analysis conditions, the control unit outputs a message to the display unit urging the user to re-prepare the sample.

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