JP3887963B2 - Isoelectric focusing device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an electrophoresis apparatus for separating and analyzing amphoteric electrolytes such as peptides and proteins at their isoelectric points, and more particularly to an electrophoresis apparatus capable of detecting sample components separated over a predetermined range of a separation channel. is there.
[0002]
[Prior art]
Conventionally, there is a technique called capillary isoelectric focusing as a technique for separating and analyzing peptides and proteins. FIG. 1 is a schematic diagram showing a conventional capillary isoelectric focusing apparatus.
[0003]
An aqueous solution in which a large number of amphoteric electrolyte mixtures (polyaminopolycarboxylic acid mixture, polyaminopolysulfonic acid mixture, etc .: polybuffer, etc.) having various ionization degrees are dissolved in the capillary 1 which has been subjected to inner surface treatment so as not to generate electroosmotic flow. 3, one end of the capillary 1 is immersed in an acidic solution (such as phosphoric acid aqueous solution) 5 that gives a lower pH than the most acidic electrolyte in the aqueous solution 3, and the other end is contained in the aqueous solution 3. Soaked in an alkaline solution (such as an aqueous sodium hydroxide solution) 7 that gives a higher pH than the most basic electrolyte. Electrodes are immersed in the solution 5 and the solution 7, and the solution 5 is an anolyte and the solution 7 is a catholyte. Although not shown, a detector by ultraviolet absorption detection, electrical conductivity detection or the like is provided at the detection point position of the capillary 1.
[0004]
When a voltage is applied to the solution 5 and the solution 7, each amphoteric electrolyte stops after moving to the position of the isoelectric point, and a pH gradient is formed in the capillary. At this time, if an amphoteric electrolyte sample such as protein is added to the solution 3, the component is concentrated in a narrow zone at the isoelectric point on the pH gradient.
After reaching the steady state, the liquid in the capillary is moved by various methods in order to move the zone to the detection point, and the zone of each component is detected. As a method of moving the zone, there are a method of continuing the voltage application by changing the electrode liquid and a method of pushing out by the pressure difference.
[0005]
In the prior art shown in FIG. 1, there are two processes: a process of concentrating the ampholyte component to the isoelectric point while applying a voltage, and a process of moving the concentrated component zone to the detection point. In order to achieve this, it is necessary to continue the voltage application by changing the electrode solution or to extrude with a pressure difference. Both of these methods of moving the zone are time consuming, and it is unavoidable to disturb the zone by moving the correctly concentrated zone on the pH gradient to the detection point.
[0006]
In order to solve such a problem, a method for detecting without moving the zone has been proposed. In the method, after the separation reaches a steady state, light is irradiated over a predetermined range of the separation flow path, and light absorption or emission at each position is detected over the predetermined range. As the light detection means, a detector having an array of light receiving elements arranged along a separation channel is used. In this method, since the zone is not moved after sample separation, measurement can be performed at high speed without disturbing the separation state.
[0007]
Since the positional accuracy and reproducibility of the pH gradient formed in the separation channel vary depending on the composition of the ampholyte, the injection state, etc., it is impossible to accurately determine the pH only from the separation position. In order to solve this, a marker sample having a known pH is injected into the separation channel together with the sample, and the pH of the separated sample component is determined using the marker sample as a reference point of the pH axis.
[0008]
[Problems to be solved by the invention]
Normally, since the pH of the separated sample component is determined manually from the separation position relationship of the marker sample, it is difficult to automatically identify the sample component on the pH axis.
In addition, when a large number of marker samples are injected to grasp the pH gradient in detail, there is a problem that it is difficult to distinguish between the sample and the marker sample.
Therefore, an object of the present invention is to accurately and automatically identify the pH of sample components in an isoelectric focusing apparatus that identifies a sample without moving the zone after sample separation.
[0009]
[Means for Solving the Problems]
FIG. 2 is a block diagram showing the present invention.
The light detection means 2 that detects light from the separation channel over a predetermined range is connected to a detection signal storage unit 4 that stores a detection signal corresponding to the detection position of the separation channel. The detection signal storage unit 4 is a pH gradient function calculation unit 6 that calculates a pH gradient function f (x), and a pH gradient function calibration unit 8 that calibrates the pH gradient function f (x) to obtain a pH gradient function g (x). , And the sample component identification unit 10 that identifies the separated sample component based on the pH gradient function g (x). The pH gradient function f (x) calculated by the pH gradient function calculation unit 6 is stored in the pH gradient function storage unit 12 and then sent to the pH gradient function calibration unit 8 when the pH gradient function g (x) is calculated.
Here, the pH gradient function represents a pH gradient formed when a large number of amphoteric electrolyte mixtures are filled in a separation channel and a voltage is applied, and a pH function with respect to a detection position (separation position) x. It is.
[0010]
An isoelectric focusing device according to the present invention includes an electrophoresis member having a separation channel formed inside a transparent member, an electrophoresis power supply device that applies an electrophoresis voltage between both ends of the separation channel, and a predetermined separation channel. Irradiating means for irradiating light over a range, a light detecting means for detecting light absorption or emission by each component separated in the separation flow path over a predetermined range, and a detection signal of the light detection means for the separation flow path The detection signal storage unit that stores the signal corresponding to each position and a first analysis for introducing a plurality of types of marker samples with known isoelectric points into a separation channel filled with a polybuffer for separation. A pH gradient function calculation unit that calculates a pH gradient function f (x) from the detected peak position and isoelectric point, a pH gradient function storage unit that stores the pH gradient function f (x), and a sample together with a plurality of types of marker samples Above poly The second analysis is performed by introducing the sample into the separation channel filled with the same polybuffer as the buffer, and the pH gradient function f (x) is calibrated based on the detected peak position and isoelectric point of the detected marker sample. A pH gradient function calibration unit that obtains a pH gradient function g (x) and a sample component identification unit that determines the pH of the separated sample component based on the pH gradient function g (x) are provided.
[0011]
A plurality of types of marker samples are injected into the separation channel and separated to perform the first analysis, the separation state is detected by the light detection means 2 and stored in the detection signal storage unit 4, and the detection position of each marker sample and Based on the known isoelectric point, the pH gradient function f (x) in the separation channel is calculated by the pH gradient function calculation unit 6. The number of types of marker samples to be injected is preferably large within a range that can be reliably separated, and a more accurate pH gradient function f (x) can be calculated.
The pH gradient function f (x) is stored in the pH gradient function storage unit 12.
[0012]
Next, a plurality of types of marker samples are injected together with the sample into the separation channel and separated to perform a second analysis, and the separation state is detected by the light detection means 2 and stored in the detection signal storage unit 4. The pH gradient function f (x) read from the pH gradient function storage unit 12 by the pH gradient function calibration unit 8 is paralleled as necessary based on the detection position of the marker sample and the known isoelectric point in the second analysis. Calibration such as movement and expansion / contraction is performed to obtain a pH gradient function g (x). A small number of marker samples to be injected, such as a few. If the number of marker samples is small, the sample peak and the marker sample peak can be easily distinguished.
[0013]
Here, in the present invention, the same polybuffer is used for the first analysis and the second analysis. Since the influence of the marker in the second analysis on the isoelectric point of the marker is weak, the basic shape of the pH gradient functions f (x) and g (x) in the first analysis and the second analysis is not so much. As it is, it can be assumed that the pH gradient function g (x) is subjected only to translation and expansion / contraction deformation as compared with the pH gradient function f (x).
Based on the calibrated pH gradient function g (x), the sample component identification unit 10 determines the pH of each sample component separated from the second analysis data stored in the detection signal storage unit 4.
[0014]
【Example】
FIG. 3 is a schematic configuration diagram showing an embodiment. In this embodiment, the present invention is applied to a microchip electrophoresis apparatus using a microchip in which a separation channel is formed inside a transparent plate member. For the measurement, an array-type optical sensor is used, and light irradiation is performed by irradiating light in a line shape along the flow path or by optically scanning along the flow path. As a detection method, any method can be used as long as it can obtain concentration information, such as a method for obtaining absorbance by absorption of ultraviolet rays or a laser fluorescence method. In this embodiment, an absorbance method is used, and an array type photodetector in which photodiodes are arranged along a separation channel is used as a detector.
[0015]
A microchip 18 in which a separation channel 14 and a sample introduction channel 16 intersect with each other is provided inside the glass substrate. On one surface of the microchip 18, holes reaching the separation channel 14 or the sample introduction channel 16 are formed at positions corresponding to both ends of the separation channel 14 and both ends of the sample introduction channel 16. An electrophoresis power supply device 20 that applies an electrophoresis voltage to both ends of the separation channel 14 and to both ends of the sample introduction channel 16 is provided.
[0016]
A light irradiating means 26 configured to irradiate light over a predetermined range of the separation channel 14 is provided, which includes a light source 22 and a spectroscope 24. On the opposite side of the microchip 18 from the irradiation means 26, there is provided an array type photodetector 28 in which light receiving elements for detecting light from the separation channel 14 are arranged along the separation channel 14 at respective positions. ing. The array-type photodetector 28 is connected to the CPU 34 via an amplification circuit 30 that amplifies a detection signal and an A / D converter 32 that converts an analog signal into a digital signal. The detection signal storage unit 4, the pH gradient function calculation unit 6, the pH gradient function calibration unit 8, the sample component identification unit 10 and the pH gradient function storage unit 12 shown in FIG.
[0017]
Containers 36 each containing a marker sample, a sample, and a polybuffer are installed. A needle 38 that moves between the microchip 18 and the container 36 and injects a marker sample, sample, or polybuffer into one end of the separation channel 14 or the sample introduction channel 16 is provided. The operation of the needle 38 is driven by a motor 40. The needle 38 is connected to a syringe 42 that sucks / discharges a set amount of marker sample, specimen or polybuffer. The operation of the syringe 42 is driven by a motor 44. The motors 40 and 44 are connected to the CPU 34 via the motor control circuit 44 and are controlled by the CPU 34.
[0018]
When the material of the microchip 18 is quartz, borosilicate glass, or the like, the inner wall surfaces of the separation channel 14 and the sample introduction channel 16 are previously treated with linear acrylamide, polyvinyl alcohol, or the like so as not to generate an electroosmotic flow. It is preferable to chemically modify the silanol group.
[0019]
Next, the operation of this embodiment will be described.
(Determination of pH gradient function f (x) (first analysis))
The CPU 34 drives the motor 40 via the motor control circuit 44 to move the needle 38 to the container 36 containing the polybuffer. The motor 44 is driven and a predetermined amount of polybuffer is sucked by the syringe 42. The needle 38 is moved to the hole at one end of the separation channel 14 of the microchip 18 and the syringe 42 is operated to fill the separation channel 14 and the sample introduction channel 16 with the polybuffer.
After the needle 38 is withdrawn from the microchip 18, the light irradiating means 26 irradiates a predetermined range of the separation channel 14 with light. The light from the separation channel 14 is detected by the array-type photodetector 28 and stored as a background in the detection signal storage unit 4 of the CPU 34 via the amplifier circuit 30 and the A / D converter 32.
[0020]
Next, for example, the needle 38 is moved to a container 36 containing a solution (marker sample solution) containing four types of marker samples having different isoelectric points, and a predetermined amount of the marker sample solution is sucked by the syringe 42. The needle 38 is moved to the hole at one end of the sample introduction channel 16 of the microchip 18 and the syringe 42 is operated to inject the marker sample solution into the sample introduction channel 16.
After the needle 38 has been withdrawn from the microchip 18, a predetermined voltage is applied for a predetermined time from the separation channel 14 and the sample introduction channel 16 by the migration power supply device 20, and the marker sample is separated from the separation channel 14 and the sample introduction flow. Move to the intersection of the roads 16. Further, a predetermined voltage is applied for a predetermined time, and the four types of marker samples are separated according to the pH gradient of the separation channel 14. The marker sample is concentrated at each isoelectric point position.
[0021]
After completion of the separation, the light irradiation means 26 irradiates a predetermined range of the separation channel 14 with light. Light from the separation channel 14 is detected by the array type photodetector 28, and the detection signal is sent to the detection signal storage unit 4 of the CPU 34 via the amplifier circuit 30 and the A / D converter 32.
The detection signal storage unit 4 of the CPU 30 stores the amplified and A / D converted detection signal by subtracting the background corresponding to the detection position.
[0022]
FIG. 4A is data representing separation of marker samples in the first analysis. The vertical axis represents the detection intensity, and the horizontal axis represents the detection position (x). The detection position (x) is the order of detection elements arranged along the separation flow path of the array-type photodetector 28 from the intersection of the separation flow path 14 and the sample introduction flow path 16.
Marker samples with isoelectric points pH1, pH2, pH3, and pH4 are detected at detection positions x1, x2, x3, and x4, respectively. Each marker sample can be identified from the separation order. The magnitude relationship between the conduction points is pH1 <pH2 <pH3 <pH4.
[0023]
FIG. 4B is a graph showing the pH gradient function f (x) of the first analysis. The vertical axis represents pH, and the horizontal axis represents the detection position (x).
The pH gradient function calculation unit 6 plots the pH with respect to the detection position x in each marker sample from the first analysis data stored in the detection signal storage unit 4, and uses polynomial approximation or spline interpolation by the least square method. Estimate the pH gradient function f (x).
The pH gradient function f (x) is stored in the pH gradient function storage unit 12.
[0024]
(Measurement of actual sample (second analysis))
In the same manner as in the first analysis, the same polybuffer as in the first analysis is injected into the separation channel 14 and the sample introduction channel 16, and light is irradiated to a predetermined range of the separation channel 14. Is detected by the array-type photodetector 28 and stored as a background in the detection signal storage unit 4 of the CPU 34 via the amplifier circuit 30 and the A / D converter 32.
The needle 38 is moved to a container 36 containing a solution (sample solution) containing a sample and two types of marker samples having different isoelectric points that can be distinguished from the sample, and a predetermined amount of the sample solution is sucked by the syringe 42. . The needle 38 is moved to the hole at one end of the sample introduction channel 16 of the microchip 18 and the syringe 42 is operated to inject the sample solution into the sample introduction channel 16.
[0025]
After the needle 38 is withdrawn from the microchip 18, a predetermined voltage is applied from the separation power supply device 20 from the separation channel 14 and the sample introduction channel 16 for a predetermined time, and the marker sample and the sample are separated from the separation channel 14 and the sample. It moves to the intersection of the introduction flow path 16. Further, a predetermined voltage is applied for a predetermined time, and the marker sample and the sample are separated according to the pH gradient of the separation channel 14. The marker sample and the sample component are concentrated at each isoelectric point position.
After the separation is completed, the separation state is detected in the same manner as in the first analysis, and the detection signal is stored in the detection signal storage unit 4 with the background subtracted.
[0026]
FIG. 5A is data representing separation of marker samples in the second analysis. The vertical axis represents the detection intensity, and the horizontal axis represents the detection position (x).
Marker samples having isoelectric points of pH 0 and pH 00 are detected at detection positions x0 and x00, respectively, and sample components separated at the detection positions in between are detected. The magnitude relationship between the conduction points is pH0 <pH00.
[0027]
FIG. 5B is a graph in which the pH gradient function f (x) of the first analysis is represented by a solid line, and the pH gradient function g (x) of the second analysis is represented by a one-dot chain line. The vertical axis represents pH, and the horizontal axis represents the detection position (x).
The pH gradient function calibration unit 8 extracts only the marker sample data from the second analysis data stored in the detection signal storage unit 4, and plots the pH with respect to the detection position x. The pH gradient function f (x) is read from the pH gradient function storage unit 12, and the pH gradient function f (x) is calibrated by translation or linear expansion / contraction as necessary based on the pH plot of the second analysis, and pH A gradient function g (x) is determined.
The sample component identification unit 10 determines the pH of each sample component using the pH gradient function g (x) and the second analysis data stored in the detection signal storage unit 4.
[0028]
In this embodiment, four types of marker samples having different isoelectric points were used in the first analysis. However, the present invention is not limited to this. If three or more types of marker samples are used, the pH gradient function f (x) is used. Is obtained. When there are three or more types of marker samples at the time of the second analysis, the pH axis is not simply proportional expansion and contraction, and the pH accuracy can be further improved by performing higher-order function fitting using a spline curve or the like.
[0029]
【The invention's effect】
The present invention relates to an isoelectric focusing apparatus that identifies a sample without moving the zone after sample separation, and introduces a plurality of types of marker samples into a separation channel filled with a polybuffer, and detects the detection peak position of the marker sample. And a first analysis for calculating the pH gradient function f (x) from the isoelectric point, and a marker sample detected by introducing a sample together with a plurality of types of marker samples into a separation channel filled with the same polybuffer as the polybuffer. Based on the detected peak position and isoelectric point, the pH gradient function f (x) is translated and stretched and calibrated to obtain the pH gradient function g (x). Based on the pH gradient function g (x), Since the second analysis to determine the pH of the separated sample components was performed and the pH of the sample components was automatically determined, the efficiency of the analysis work can be improved and the reliability can be improved. Data can be obtained.
[Brief description of the drawings]
FIG. 1 is a schematic diagram showing a conventional capillary isoelectric focusing apparatus.
FIG. 2 is a block diagram illustrating the present invention.
FIG. 3 is a schematic configuration diagram illustrating an embodiment.
FIG. 4A is data representing the separation of the marker sample of the first analysis, and FIG. 4B is a graph representing the pH gradient function f (x) of the first analysis.
FIG. 5A is data representing the separation of the marker sample of the second analysis, and FIG. 5B is a solid line representing the pH gradient function f (x) of the first analysis. Is a graph showing the pH gradient function g (x) of
[Explanation of symbols]
2 Photodetection means 4 Detection signal storage unit 6 pH gradient function calculation unit 8 pH gradient function calibration unit 10 Sample component identification unit 12 pH gradient function storage unit

Claims (1)

An electrophoretic member having a separation channel formed inside the transparent member;
An electrophoretic power supply device for applying an electrophoretic voltage between both ends of the separation channel;
Irradiating means for irradiating light over a predetermined range of the separation channel;
A light detection means for detecting absorption or light emission of the light by each component separated in the separation channel over a predetermined range;
A detection signal storage unit for storing the detection signal of the light detection means in correspondence with each position of the separation channel;
A plurality of types of marker samples with known isoelectric points are introduced into the separation channel filled with the amphoteric electrolyte mixture to perform a first analysis, and the detection peak position of the marker sample and the pH and detection position from the isoelectric point. a pH gradient function calculation unit for calculating a pH gradient function f (x) indicating a relationship with x;
A pH gradient function storage unit for storing the pH gradient function f (x);
A plurality of types of marker samples are introduced together with the sample into the separation channel filled with the same amphoteric electrolyte mixture as the amphoteric electrolyte mixture, and then subjected to a second analysis to detect the detected peak position and isoelectric point of the marker sample. A pH gradient function calibration unit that calibrates the pH gradient function f (x) based on the above to obtain a pH gradient function g (x) indicating the relationship between the pH and the detection position x;
A microchip electrophoresis apparatus comprising: a sample component identification unit that determines a pH of a separated sample component based on the calibrated pH gradient function g (x).

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