ANALYSIS METHOD AND CAPILLAR ELECTROPHORESIS DEVICE

Abstract

It is an object of the invention to provide an analytical method that avoids unnecessary repeat electrophoresis and unnecessary sample preparation. To this end, the invention includes a method for analyzing features contained in a sample by electrophoresis of the sample using a capillary and a sieve matrix, wherein the method comprises: a reading step for reading an analytical condition; a sieve matrix filling step for filling the capillary with the sieve matrix; a sample injection step for injecting the sample into the capillary containing the sieve matrix loaded in the sieve matrix filling step; and an electrophoresis step for electrophorising the sample in the sieve matrix, wherein the sample is injected in the sample injection step.an anomaly detection step of statistically determining whether an analysis result obtained in the electrophoresis step is anomalous, by comparing the analysis result with another analysis result obtained under the same analysis condition; and an analysis condition setting step for setting the analysis condition, wherein, if the anomaly detection step determines that the analysis result is anomalous, the procedure proceeds again to the reading step after the analysis condition setting step has been executed.

Description

Technical area

[0001] The present invention relates to a capillary electrophoresis apparatus and an analysis method using the capillary electrophoresis apparatus. background

[0002] Recently, a capillary electrophoresis device, which has a capillary filled with a sieving matrix, such as a polymer gel or a polymer solution, has been widely used as an electrophoresis device. The capillary electrophoresis device enables automatic filling of the sieving matrix and automatic injection of a sample, enabling continuous analysis. Consequently, it is used in a manner that presets multiple electrophoresis devices to analyze a large number of samples throughout the night.

[0003] A sample electrophoresed within the capillary is irradiated with a laser, and the resulting signal is detected by a detection unit such as a CCD camera. However, in the case of a high concentration of the sample injected into the capillary, the detected signal level is correspondingly high. If the signal level exceeds a counting threshold (e.g., 65,535 counts) of the CCD camera, the signal is saturated and a signal peak is indistinguishable. In this case, a user has previously re-prepared the sample by dilution or the like, or set an assay (information for setting an analysis operation condition and an analysis condition) based on experience.

[0004] A fault may occur in the capillary electrophoresis device, meaning the detection unit fails to detect a signal or can only detect an extremely weak signal. When such a fault occurs, the user must also resolve the issue based on experience, most likely choosing re-sample preparation as a remedy. However, the fault such as no signal detection may be caused by a sample injection error in addition to a faulty sample. If the fault is caused by a sample injection error, a suitable signal can be detected by immediately performing electrophoresis before sample deterioration, thus avoiding re-sample preparation, which is a heavy burden for the user.

[0005] Patent Literature 1 discloses an electrophoresis device that prompts a user to change a parameter for sample concentration, sample injection, or the like when a signal is off-scale (saturated) or when the signal-to-noise ratio is low. Patent Literature 1 also discloses that a low signal-to-noise ratio may indicate poor sample injection or the like, thus prompting the user to adjust the sample injection parameter. List of citationsPatent literature

[0006] Patent Literature 1: US Patent Application Publication No. 2020 / 0003728 Summary of the inventionTechnical problem

[0007] With existing capillary electrophoresis devices, when a fault is encountered, such as signal saturation or, conversely, an undetectable signal, the user has no choice but to determine how to correct the error based on their experience. If electrophoresis is run continuously throughout the night, the user may not realize an error has occurred until early in the morning. Because the sample has deteriorated over time, electrophoresis is less likely to be rerun as the situation arises, even if the error was not caused by a faulty sample. In such a case, the user must re-prepare a sample, resulting in a waste of time and effort, as well as a valuable sample.

[0008] Patent Literature 1 also discloses that the device modifies a sample injection parameter or the like when a signal is off-scale (saturated) or when a signal-to-noise ratio is lower, but does not disclose how the device specifically modifies the parameter or the like. In addition, in Patent Literature 1, if, for example, the signal-to-noise ratio is low for all data, electrophoresis is performed again with the modified injection parameter for all samples because sample injection noise or the like is determined only by the signal-to-noise ratio, that is, only by the signal level. However, if the signal-to-noise ratio is low for all data, it is more likely that an erroneous sample or a missed sample injection occurs instead of the sample injection noise. Consequently, such a low signal-to-noise ratio often cannot be easily resolved by simply rerunning electrophoresis.As a result, unnecessary electrophoresis may be performed, resulting in waste of sieving matrix and time.

[0009] It is an object of the invention to provide an analysis method in which unnecessary re-electrophoresis and unnecessary re-sample preparation are prevented. Solution to the problem

[0010] To achieve the object, the invention includes a method for analyzing features contained in a sample by electrophoresis of the sample using a capillary and a sieve matrix, the method including: a reading step for reading an analysis condition; a sieve matrix filling step for filling the capillary with the sieve matrix; a sample injection step for injecting the sample into the capillary containing the sieve matrix loaded in the sieve matrix filling step; an electrophoresis step for electrophoresing the sample in the sieve matrix, wherein the sample is injected in the sample injection step; an anomaly determination step for statistically determining whether an analysis result obtained in the electrophoresis step is anomalous by comparing the analysis result with another analysis result obtained under the same analysis conditions;and an analysis condition setting step for setting the analysis condition, wherein, if it is determined in the abnormality determination step that the analysis result is abnormal, the process proceeds to the reading step again after the analysis condition setting step is executed.; Advantageous effects of the invention

[0011] According to the invention, it is possible to provide an analysis method in which unnecessary re-electrophoresis and unnecessary re-sample preparation are suppressed. Brief description of the drawings Fig. 1 is a perspective diagram showing an overview of a configuration of a capillary electrophoresis apparatus. Fig. 2 is a plan view of the Fig. Capillary electrophoresis device shown in Figure 1. Fig. Figure 3 is a graphical cross-sectional view along line AA according to Fig. 2. Fig. 4 is a flowchart showing an analysis method of the first embodiment. Fig. 5 is a flowchart showing an analysis method of the second embodiment. Fig. Figure 6 is a diagram showing an example waveform in a saturated state. Fig. Figure 7 is a diagram showing an enlarged view of the section in the dashed line after Fig. 6 shows. Fig. Figure 8 is a diagram showing an image when a signal peak is estimated. Fig. Figure 9 is a diagram showing an example waveform in the case of sample injection failure. Fig. Figure 10 shows an example graph of quality data (EQ) when a sample injection failure occurs. Fig. Figure 11 shows a table of example quality data (EQ) when a sample injection failure occurs. Fig. 12 is a flowchart showing an analysis method of the third embodiment. Description of the embodiments

[0012] An embodiment of the invention will now be described with reference to the drawings.

[0013] First, a configuration of a capillary electrophoresis device with respect to the Fig. 1 to 3.

[0014] Fig. Figure 1 is a perspective diagram showing an overview of a configuration of the capillary electrophoresis apparatus. The capillary electrophoresis apparatus is configured with two units: a detection / thermostat oven unit 150 on the top and an automatic sampler unit 160 on the bottom.

[0015] In the automatic sampler unit 160, a Y-axis moving device 85 is mounted on a sampler base 80 to enable movement along the Y-axis. A Z-axis moving device 90 is mounted on the Y-axis moving device 85 to enable movement along the Z-axis. A sample tray 100 is mounted on the Z-axis moving device 90, with a sieve matrix container 20, an anode-side buffer solution container 30, a cathode-side buffer solution container 40, and a sample container 50 placed on the sample tray 100 by a user. The sample container 50 is placed on an X-axis moving device 95 mounted on the sample tray 100 and can thus move along the X-axis on the sample tray 100. A liquid supply mechanism 60 is also mounted on the Z-axis moving device 90.The liquid supply mechanism 60 is arranged below the sieve matrix container 20.

[0016] The detection / thermostat furnace unit 150 includes a thermostat furnace unit 110 and a thermostat furnace door 120, and can maintain the interior of the thermostat furnace unit 110 at a constant temperature. The detection / thermostat furnace unit 150 further includes a detection unit 130 behind the thermostat furnace unit 110, and can thus apply a laser and detect a signal generated during such laser application. When a capillary assembly 10 is placed into the thermostat furnace unit 110 by a user, a sample is electrophoresed in the thermostat furnace unit 110 while the capillary assembly 10 is maintained at a constant temperature. At this time, the detection unit 130 detects, with a CCD camera or the like, a signal generated when the sample is irradiated with the laser while being electrophoresed within the capillary assembly 10.The thermostatic furnace unit 110 also has an electrode 115 for grounding when a high voltage is applied for electrophoresis.

[0017] As described above, the capillary assembly 10 is attached to the thermostatic furnace unit 110. The sieve matrix container 20, the anode-side buffer solution container 30, the cathode-side buffer solution container 40, and the sample container 50 can be moved along the Y-axis and Z-axis by the automatic sampling unit 160, although only the sample container 50 can be moved even along the X-axis. The automatic sampling unit 160 moves to automatically connect the sieve matrix container 20, the anode-side buffer solution container 30, the cathode-side buffer solution container 40, and the sample container 50 to the attached capillary assembly 10.

[0018] Fig. 2 is a plan view of the Fig. 1. The anode-side buffer solution tank 30, which is mounted on the sample tray 100, includes an anode-side washing tank 31, an anode-side electrophoresis buffer solution tank 32, and a sample introduction buffer solution tank 33. The cathode-side buffer solution tank 40, which is mounted on the sample tray 100, includes a waste liquid tank 41, a cathode-side cleaning tank 42, and a cathode-side electrophoresis buffer solution tank 43.

[0019] The sieve matrix container 20, the anode-side buffer solution container 30, the cathode-side buffer solution container 40 and the sample container 50 are arranged on the sample tray 100 in a Fig. 2. As a result, the anode side / cathode side combinations to be connected to the capillary assembly 10 include the "sieve matrix tank 20 / waste tank 41," the "anode side wash tank 31 / cathode side wash tank 42," the "anode side electrophoresis buffer solution tank 32 / cathode side electrophoresis buffer solution tank 43," and the "sample introduction buffer solution tank 33 / sample tank 50."

[0020] Fig. Figure 3 is a graphical cross-sectional view along line AA according to Fig. 2, which shows a cross-section of the capillary electrophoresis device, wherein the anode side / cathode side combination to be connected to the capillary assembly 10 is the "sieve matrix container 20 / waste container 41." The sieve matrix container 20 is inserted into a guide 101 embedded in the sample tray 100 and secured therein. The liquid feed mechanism 60 is arranged such that a piston 61 built into the liquid feed mechanism 60 is located below the sieve matrix container 20. During electrophoresis, the right side of the capillary assembly 10 is in Fig. 3 is the cathode side, while its left side is the anode side.

[0021] The capillary electrophoresis device is controlled by a control section. The control section includes a control board 122 and a computer 123 connected to the control board 122. Although in the exemplary case according to Fig. 1, the control board 122 is located on the back of the thermostatic furnace unit 110, and the computer 123 is located on the outside (front) of the thermostatic furnace door 120, but such locations are not limited thereto. According to instructions from the computer 123, the control board 122 controls the operations of the high-voltage power supply 121, the thermostatic furnace unit 110, the detection unit 130, and the like. The computer 123 calculates the analysis results based on the signals detected by the detection unit 130 and includes an input section for inputting settings (including an assay described later) of the capillary electrophoresis device, and an output section (display section) for displaying the analysis results and the like. The analysis results can be calculated by the control board 122. First embodiment

[0022] A flow of a sequence of analyses performed by the capillary electrophoresis apparatus according to the present embodiment will be described with respect to Fig. 4 described. Fig. 4 is a flowchart showing the analysis method of the first embodiment.

[0023] First, a user uses the input unit to specify an assay to be used in the sequence of analyses (step S200). The assay refers to information defining an operating condition for analysis and an analysis condition. The operating condition includes the laser power of the detection unit 130, the temperature of the thermostatic furnace unit 110, a type of sieve matrix, the voltage applied in a pre-run step described later and the application time of the voltage, the voltage applied in a sample injection step described later and the application time of the voltage, the voltage applied in an electrophoresis step, the application time of the voltage, the laser irradiation time, and a sampling time during data acquisition, and the like.The analysis condition includes an analysis width specified by the data points or the base size, a signal height threshold, a base call mobility file specified to read a sequence in the case of sequencing analysis, a fragment size used when analyzing in the case of fragment analysis, and the like.

[0024] If it is subsequently determined that the analysis result is abnormal, the user sets a function for executing re-analysis (re-analysis function) as needed. The control section determines whether the re-analysis function is enabled (step S201), and if it is determined that it is not enabled, executes steps S102 to S110. On the other hand, if it is determined in step S201 that the re-analysis function is enabled, the control section executes steps S202 to S212. Because steps S102 to S110 are the same as steps S202 to S210, only steps S202 to S210 will be described below.

[0025] When the analysis process is started in step S202, the sequence of analyses is executed. According to the present embodiment, one analysis is defined as a process from sample injection to the end of electrophoresis by voltage application. In other words, the user can specify not only one analysis but also multiple sequences of analyses. The following describes a case where three analyses, one analysis A, one analysis B, and one analysis C, are specified as an example, but the number of analyses is not limited to this.

[0026] When the first analysis, Analysis A, is started, the control section executes a step of reading the assay specified in step S200 (assay reading step) (step S203).

[0027] Subsequently, the control section controls the thermostatic furnace unit 110 to perform a step of maintaining the capillary assembly 10 at a constant temperature (capillary temperature control step) (step S204).

[0028] Subsequently, the control section starts a laser of the detection unit 130 (step S205).

[0029] Further, the control section controls the automatic sampling unit 160 and the piston 61 to perform a step of filling the capillary assembly 10 with the sieve matrix contained in the sieve matrix container 20 (sieve matrix filling step) (step S206). The sieve matrix filling step will now be described in more detail. First, the automatic sampling unit 160 moves so that the anode side / cathode side combination to be connected to the capillary assembly 10 is the "sieve matrix container 20 / waste container 41." Subsequently, a capillary head 11, which is the anode side end of the capillary assembly 10, is inserted into the sieve matrix container 20, and a loading head 12, which is the cathode side end of the capillary assembly 10, is inserted into the waste liquid tank 41.In this state, the piston 61 incorporated in the liquid supply mechanism 60 moves upward, feeding the sieve matrix contained in the sieve matrix container 20 into the capillary assembly 10 through the capillary head 11. If the capillary assembly 10 has been filled with the sieve matrix before the liquid supply, or if the sieve matrix has been sufficiently fed into the capillary assembly 10 and remains in excess, the sieve matrix is ​​emptied from the loading head 12 into the waste tank 41.

[0030] Subsequently, the control section performs pre-run (step S207). Specifically, first, the automatic sampling unit 160 moves so that the anode-side / cathode-side combination to be connected to the capillary assembly 10 is the "anode-side electrophoresis buffer solution tank 32 / cathode-side electrophoresis buffer solution tank 43." Subsequently, the capillary head 11 is inserted into the anode-side electrophoresis buffer solution tank 32, while the charging head 12 is inserted into the cathode-side electrophoresis buffer solution tank 43. In this state, the high-voltage power supply 121 applies a high voltage to the capillary assembly 10. The pre-run is the same as the electrophoresis after sample injection described later, but is performed before sample injection to stabilize the electrophoresis performance.

[0031] Next, the control section performs a step of injecting the sample placed in the sample container 50 into the capillary assembly 10 (sample injection step) (step S208). Specifically, first, the automatic sampler unit 160 moves so that the anode side / cathode side combination to be connected to the capillary assembly 10 is the "sample introduction buffer solution tank 33 / sample container 50." Subsequently, the capillary head 11 is inserted into the sample introduction buffer solution tank 33, while the charging 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 assembly 10. As a result, the negatively charged sample is injected from the charging head 12 into the capillary assembly 10.

[0032] A cleaning step may be performed to clean the capillary head 11 and the loading head 12, which correspond to both ends of the capillary assembly 10, before or after the sieve matrix filling step or the sample injection step. In this case, the automatic sampling unit 160 first moves so that the anode side / cathode side combination to be connected to the capillary assembly 10 is the "anode side cleaning tank 31 / cathode side cleaning tank 42." Subsequently, the capillary head 11 is inserted into the anode side cleaning tank 31, while the loading head 12 is inserted into the cathode side cleaning tank 42. The capillary assembly 10 is left in this state for a few seconds, making it possible to clean both ends.

[0033] Subsequently, the control section performs a step of applying a voltage to the capillary assembly 10 into which the sample has been injected to electrophorese the sample (electrophoresis step) (step S209). Specifically, first, the automatic sampling unit 160 moves so that the anode-side / cathode-side combination to be connected to the capillary assembly 10 is the "anode-side electrophoresis buffer solution tank 32 / cathode-side electrophoresis buffer solution tank 43." Subsequently, the capillary head 11 is inserted into the anode-side electrophoresis buffer solution tank 32, while the charging head 12 is inserted into the cathode-side electrophoresis buffer solution tank 43. In this state, the high-voltage power supply 121 applies a high voltage to the capillary assembly 10.As a result, a high voltage is applied to the capillary array 10 on the cathode side, causing a current to flow through the electrode 115 via the cathode-side buffer solution tank 40 and the anode-side buffer solution tank 30 to ground, thereby performing electrophoresis. In the electrophoresis step, the sample flowing through the capillary array 10 at the molecular scale due to the molecular sieving effect of the sieve matrix 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 period of time defined in the assay specified in step S200.

[0034] When electrophoresis has been performed for a predetermined period of time in step S209, the first analysis, Analysis A, is completed, and the second analysis, Analysis B, begins. In Analysis B, steps S203 to S209 are also executed as in the previous Analysis A. When Analysis B has been completed, the third analysis, Analysis C, begins. In Analysis C, steps S203 to S209 are also executed as in Analyses A and B.

[0035] When the sequence of analyses including analyses A, B, and C is completed, the control section calculates all analysis results (step S210). If the reanalysis function is not enabled in step S201, the process ends once all analysis results have been calculated (step S213).

[0036] If the reanalysis function is enabled in step S201, once all analysis results have been calculated, the control section executes a step (anomaly determination step) to statistically determine whether an abnormal analysis result is included based on the calculated analysis results of the sequence of analyses and the results of previous analyses performed under the same condition (same assay) (step S211). For example, if the result of analysis B has a significantly smaller value than each of the results of the other analyses A and C, the result of analysis B is determined to be statistically abnormal. A significantly small value among values ​​of multiple analysis results can be determined as an outlier, for example, obtained by the Smirnov-Grubbs test.

[0037] The analysis results used for the determination in step S211 include quality data in addition to the signal height data detected by the detection unit 130. In fragment analysis, the quality data refers to EQ, which indicates the maximum base length within a range that allows base separation, while in sequencing analysis, the quality data refers to QV 20CRL, which indicates the maximum continuous base length with the average QV (base calling reliability) value of more than 99% in a 20 bp window of the decoded sequence, where the window is shifted from the short base side to the long base side.

[0038] If it is determined in step S211 that a statistically abnormal analysis result is included, the control section executes a step of creating a new assay for objective analysis (first assay setting step) (step S212). Creating the new assay means setting the assay that was initially set.

[0039] Subsequently, the control section returns to step S202 and starts re-analysis for the analysis with an abnormal result. For example, if the subject of the abnormality is Analysis B, the control section operates the high-voltage power supply 121, etc., based on the set assay for Analysis B, injects a sample corresponding to Analysis B into the capillary assembly 10, and re-analyzes only for Analysis B. When the re-analysis is completed, the analysis results are recalculated in step S210, and it is determined in step S211 whether the results are statistically abnormal. If it is determined in step S211 that the results are not abnormal, the analysis process ends (step S213).

[0040] As described above, the capillary electrophoresis device according to the present embodiment automatically adjusts the assay and performs a re-analysis if the results of the sequence of analyses contain an abnormality. Therefore, even if a sample injection error occurs, an appropriate signal can be detected by immediately performing electrophoresis before the sample deteriorates. This makes it possible to avoid re-sample preparation, which imposes a heavy burden on the user. Second embodiment

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

[0042] The flow of a sequence of analyses carried out by the capillary electrophoresis apparatus according to the present embodiment will now be described with respect to Fig. 5 described. Fig. 5 is a flowchart showing the analysis method of the second embodiment.

[0043] First, a user uses the input unit to specify an assay to be used in the sequence of analyses (step S200).

[0044] The user then specifies a reanalysis function if necessary. The control section determines whether the reanalysis function is enabled (step S201), and if it is not enabled, it executes steps S103 to S109 in Fig. 4 of the first embodiment for all analyses (step S100) as soon as the analysis has been started (step S102). On the other hand, if the reanalysis function is enabled in step S201, the control section executes steps S202 to S209 in Fig. 4 of the first embodiment for all analyses (step S300) once the analysis has begun (step S202). When all sequences of analyses have been completed, the control section calculates the results of all analyses (step S210). If the reanalysis function is not enabled in step S201, the analysis process ends once all analysis results have been calculated (step S213).

[0045] If the reanalysis function is enabled in step S201, once all analysis results have been calculated, the control section performs a step of determining whether a signal is saturated (signal saturation determination step) (step S301). The signal level data detected by the detection unit 130 in the electrophoresis step is used in the signal saturation determination step.

[0046] Fig. Figure 6 is a diagram showing an example waveform in a saturated state, and Fig. Figure 7 is a diagram showing the section in dashed lines according to Fig. 6 shows an enlarged view. In the Fig. 6 and Fig. 7, the horizontal axis is in units of the number of data points of the CCD camera, while the vertical axis is in units of RFU for the signal height (signal intensity). As in the Fig. 6 and Fig. As shown in Figure 7, a signal peak is less likely to be visually identified once the signal is saturated.

[0047] Therefore, if it is determined in step S301 that the signal is saturated, the control section performs a step of estimating a peak of a signal waveform (signal peak estimation step) (step S302). At this time, the control section approximates the peak by fitting with the Gaussian function, the Lorentz function, or the like, an edge portion of a signal-saturated waveform and a waveform before or after the peak.

[0048] Fig. Figure 8 is a diagram showing an image of the estimation of a signal peak. As shown by the dashed line after Fig. As shown in Figure 8, the signal height is estimated by a peak approximation, while it has been invisible due to saturation.

[0049] Subsequently, based on the peak of the saturated signal estimated in step S302, the control section executes a step of adjusting the assay to prevent signal saturation (second assay adjusting step) (step S303).

[0050] The second assay setup step will now be described using a specific example. It is assumed that signal saturation occurs when an initial sample injection voltage using an unadjusted assay is 1.6 kV. The peak signal intensity estimated by fitting the Gaussian function in step S302 is assumed to be 50,000 RFU. It is further assumed that the user sets an unsaturated signal level of 20,000 RFU as a target value. If the detection unit 130 includes a CCD camera, the unsaturated signal level should be approximately half the count threshold or less (e.g., approximately 30,000 or less if the count threshold is 65,535 counts), and the user can set any signal level as the target value within this range.

[0051] If 20,000 RFU is set as the unsaturated signal level, the control section determines a setting factor of 0.4 from 20,000 RFU divided by 50,000 RFU. The control section multiplies the original sample injection voltage by this setting factor, i.e., 0.4 × 1.6 kV, to obtain a new, set sample injection voltage of 0.64 kV. As a result, the control section generates a new assay with a modified sample injection voltage of 0.64 kV.

[0052] Although the assay was adjusted by changing the sample injection voltage in the specific example, the assay can be adjusted by changing analysis conditions other than the sample injection voltage, such as the sample injection time (voltage application time), the laser irradiation time, and the laser power. In each case, the control section adjusts the assay by multiplying the parameter value of the original analytical condition by the same adjustment factor.

[0053] Once the assay is set, the control section returns to step S202 and performs a re-analysis based on the set assay. Consequently, even if the user cannot visually verify the signal peak due to signal saturation, the peak is automatically estimated and a re-analysis is performed based on the new assay. This makes it possible to acquire data while avoiding signal saturation without unnecessarily repeating electrophoresis. As a result, it is possible to avoid wasting the sieve matrix and time.

[0054] Subsequently, if it is determined in step S301 that the signal is not saturated, the control section performs a step of determining whether the signal level is less than a predetermined threshold (signal level determination step) (step S304). If the signal level is less than the threshold, sample injection failure may occur.

[0055] Fig. Figure 9 is a diagram showing an example waveform in the case of a sample injection disturbance. In Fig. 9, the horizontal axis is in units of the number of data points, while the vertical axis is in units of RFU. As in Fig. 9, only the baselines are output, and no signal caused by the sample is observed. Although in Fig. 9, there may be a case where a signal is partially extremely low. The threshold used in the signal level determination step is set to, for example, 100 RFU, considering the noise. The threshold can be determined based on a signal-to-noise ratio instead of the signal level.

[0056] If it is determined in step S304 that the signal level is smaller than the threshold value, the control section executes a step (anomaly determination step) of statistically determining whether an abnormal analysis result is included based on the results of the sequences of analyses, etc. (step S305).

[0057] Fig. Figure 10 shows an example graph of quality data (EQ) when a sample injection failure occurs, and Fig. Figure 11 is an example table of quality data (EQ) when a sample injection failure occurs. Fig. Figure 11 shows the data obtained by repeating the analysis 24 times with the capillary arrangement 10 with four charging heads 12. In Fig. 11, the four charging heads 12 are shown as CH1, CH2, CH3 and CH4. In Fig. 10 the same data as in Fig. 11, where all data are represented linearly regardless of the four loading heads 12. While most of the data from Analysis 1 to Analysis 24 lie approximately between EQ 450 and EQ 535, some data show EQ 0. Zero is an example of an outlier, where it is statistically determined that the data from CH4 in Analysis 2, CH2 in Analysis 9, and CH3 in Analysis 22 are more likely to indicate sample injection interference because they are significantly smaller than the other data in the sequence. The outlier can be determined using the Smirnov-Grubbs test.

[0058] In this way, by comparing individual data with other data to determine if the data is statistically anomalous, rather than comparing the data with a single threshold, the accuracy of error determination is improved. For example, if all of the data show a low signal level, re-electrophoresis is not an effective measure because there is likely some other error, such as a faulty sample or a missed sample injection, rather than sample injection error. However, if the signal level is low for only some of the data, there is likely a sample injection error, and re-electrophoresis is an effective measure. The control section therefore only reanalyzes the samples for Analysis 2, Analysis 9, and Analysis 22. The assay used for such reanalysis can be the same as the one initially specified.This is because if the sample injection disturbance is caused by air bubbles, the air bubbles can be removed even by repeating electrophoresis under the same conditions, resulting in normal sample injection.

[0059] According to the present embodiment, the results of a particular analysis are compared with the results of other analyses included in a sequence of analyses performed 24 times (Analysis 1 to Analysis 24) to statistically determine whether the results are abnormal. However, instead of comparing the results of the sequence of analyses, the results of a particular analysis may also be compared with the results of previous analyses performed under the same conditions (the same assay) to statistically determine whether the results are abnormal.

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

[0061] According to the second embodiment, if a statistically abnormal analysis result is found in the abnormality determination step, reanalysis is performed using the same assay. If an abnormal analysis result is also found in the abnormality determination step after the reanalysis, the reanalysis is repeated using the same assay. However, according to the third embodiment, if a statistically abnormal analysis result is found in the first abnormality determination step, reanalysis is performed using the same assay. If a statistically abnormal analysis result is also found in the second abnormality determination step, a first assay adjustment step is performed.In addition, according to the third embodiment, re-analysis is performed using the set assay, and if a statistically abnormal analysis result is found even in the third abnormality determination step, a step is performed to output a display to prompt the user to prepare the sample again (re-preparation display step).

[0062] A flow of a sequence of analyses performed by the capillary electrophoresis apparatus according to the present embodiment will now be described with respect to Fig. 12 described. Fig. 12 is a flowchart showing the analysis method of the third embodiment. In Fig. 12, however, steps S200, S102, S100, S110, S302 and S303 are in Fig. 5 (second embodiment) is omitted. Only the differences from the second embodiment are described below.

[0063] If a statistically abnormal analysis result is found in the first abnormality determination step at step S305, the control section does not adjust the assay, but returns to step S202 and performs a new analysis based on the initially set assay. If a statistically abnormal analysis result is also found in the second abnormality determination step at step S305, the control section performs a first assay adjustment step (step S306).

[0064] The first assay setting step will now be described using a specific example. Assume that if an original sample injection voltage is 1.6 kV using an unadjusted assay, a statistically abnormal analytical result is reported. Further assume that, for example, a value of 10 is set as an adjustment factor to increase the signal strength. In this case, the control section 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 section generates a new assay with an updated sample injection voltage of 16 kV.

[0065] Although the assay according to the specific example was adjusted by changing the sample injection voltage, the assay can be adjusted by changing analysis conditions other than the sample injection voltage, such as the sample injection time (voltage application time), the laser irradiation time, and the laser power. In each case, the control section adjusts the assay by multiplying the parameter value of the original analytical condition by the same adjustment factor.

[0066] If the assay is set in the first assay setting step, the control section returns to step S202 and performs a re-analysis based on the set assay. If a statistically abnormal analysis result is found in step S305 even in the third abnormality determination step, it is likely that a faulty sample is occurring instead of a sample injection failure. The control section therefore performs a step of outputting a display to prompt re-sample preparation on the display section (re-preparation display step) (step S307), and the process ends (step S213). Although this is Fig. 12 is not shown, the process also ends (step S213) when no statistically abnormal analysis result is found in step S305.

[0067] According to the present embodiment, it is possible to resolve sample injection failure by automatically performing a reanalysis while avoiding resample preparation as much as possible. Although according to the third embodiment, a reanalysis is performed once using the same assay and once using the set assay, each reanalysis can be performed multiple times as long as the sample does not deteriorate.

[0068] Although the invention has been described herein with the first to third embodiments, the invention is not limited thereto, and various modifications may be made.

[0069] For example, although statistical determination using quality data (EQ) is performed in the anomaly determination step according to the second embodiment, statistical determination may be performed using signal height data. According to an example of determination using signal height data, when most signal height data are each approximately 10,000 RFU, signal height data of approximately 300 RFU are determined to be abnormal. Furthermore, in the anomaly determination step, statistical determination may be performed using both quality data and signal height data.

[0070] The capillary electrophoresis device of the first embodiment determines statistical anomalies to perform a reanalysis to correct a sample injection error, whereas the capillary electrophoresis devices of the second and third embodiments each perform a reanalysis to correct a signal saturation in addition to the sample injection error. However, the capillary electrophoresis device may be one that does not correct a sample injection error but only corrects a signal saturation. List of reference symbols 10 Capillary arrangement 11 Capillary head 12 Charging head 20 sieve matrix containers 30 anode-side buffer solution container 31 anode-side wash tank 32 anode side electrophoresis buffer solution tank 33 Sample introduction buffer solution tank 40 cathode-side buffer solution tank 41 Waste liquid tank 42 cathode-side cleaning tank 43 cathode side electrophoresis buffer solution tank 50 sample containers 60 Fluid supply mechanism 61 pistons 80 sampler base 85 Y-axis movement device 90 Z-axis movement device 95 X-axis movement device 100 sample trays 101 Leadership 110 Thermostatic oven unit 115 Electrode 120 Thermostat oven door 121 High-voltage power supply 122 Control board 123 computers 130 detection unit 150 Detection / Thermostat Oven Unit 160 automatic sampling unit QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Cited patent literature

[0000] US 2020 / 0003728

[0006]

Claims

[1] A method for analyzing characteristics contained in a sample by electrophoresis of the sample using a capillary and a sieve matrix, the method comprising: a read step to read an analysis condition; a sieve matrix filling step for filling the capillary with the sieve matrix; a sample injection step for injecting the sample into the capillary containing the sieve matrix loaded in the sieve matrix filling step; an electrophoresis step for electrophoresing the sample in the sieve matrix, wherein the sample is injected in the sample injection step; an abnormality determination step for statistically determining whether an analysis result obtained in the electrophoresis step is abnormal by comparing the analysis result with another analysis result obtained under the same analysis condition; and an analysis condition setting step for setting the analysis condition, wherein, if it is determined in the abnormality determination step that the analysis result is abnormal, the process proceeds to the reading step again after the analysis condition setting step is executed. [2] The method of claim 1, further comprising: a signal saturation determination step for determining whether the signal detected in the electrophoresis step is saturated; and a signal peak estimation step for estimating a peak of a waveform when it is determined in the signal saturation determination step that the signal is saturated, wherein in the analysis condition setting step, the analysis condition is set to an analysis condition under which no signal is saturated based on the peak estimated in the signal peak estimation step. [3] The method according to claim 1, wherein, even if it is determined in the abnormality determining step that the analysis result is abnormal, the process proceeds to the reading step while the analysis condition setting step is not executed, while if it is again determined in the abnormality determining step that the analysis result is abnormal, the process proceeds to the reading step after the analysis condition setting step has been executed. [4] The method according to claim 1, further comprising a re-preparation display step of outputting a display to cause re-preparation of the sample, wherein, after proceeding to the reading step after the analysis condition setting step has been carried out, if it is determined in the abnormality determining step that the analysis result is abnormal, the method proceeds to the re-preparation display step. [5] Capillary electrophoresis device comprising: a capillary to be filled with a sieve matrix; a power supply that applies a voltage to the capillary to electrophorese a sample; and a detection unit that detects a signal when a laser is applied to the sample being electrophoresed in the capillary; and a control section that controls the operations of the power supply and the detection unit and calculates an analysis result, where the tax section: compares the calculated analysis result with another analysis result obtained under the same analysis condition and consequently statistically determines whether the calculated analysis result is abnormal; and if it is determined that the calculated analysis result is abnormal, sets the analytical condition and operates the power supply and the detection unit under the set analysis condition and performs re-analysis. [6] The capillary electrophoresis apparatus according to claim 5, wherein, when the signal detected by the detection unit is saturated, the control section estimates a peak of a waveform and sets the analysis condition to an analysis condition under which no signal is saturated based on the estimated peak. [7] The capillary electrophoresis apparatus according to claim 6, wherein, when the signal detected by the detection unit is saturated, the control section adjusts an application condition of the voltage to the capillary by the power supply during the injection of the sample or an application condition of the laser by the detection unit and thus reduces the intensity of the signal detected by the detection unit. [8] The capillary electrophoresis apparatus according to claim 5, wherein, when it is determined that the analysis result is abnormal, the control section adjusts an application condition of the voltage to the capillary by the power supply during the injection of the sample or an application condition of the laser by the detection unit and consequently increases the intensity of the signal detected by the detection unit. [9] The capillary electrophoresis apparatus according to claim 5, wherein, even if it is determined that the analysis result is abnormal, the control section reanalyzes the sample under the same analysis condition, and if it is determined that a result of the reanalysis is again abnormal, the control section reanalyzes the sample under the set analysis condition. [10] The capillary electrophoresis apparatus according to claim 5, further comprising a display section that displays an analysis result, wherein the control section outputs an indication to cause the sample to be prepared again to the display section when it is determined that the analysis result is abnormal even if the sample is analyzed again under the set analysis condition.

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