JPH11160279A - Microchip electrophoresis device - Google Patents

Abstract

(57) [Problem] To improve the S / N ratio of a microchip electrophoresis apparatus. A peak of the detected component x at any time t _k is detected element number f that is detected by the integrated end time t _n
(T _n , e _kx ) is f (t _n , e _kx ) = (e _kx −e _ka ) · ( _en b −e _na ) / (e _kb −
e _ka) + e _a can be represented, as measured at time t _k data y (t _k,
e) is Thus, the element axis can be converted to the integration end time t _n . Each element peak of the component x shown in FIG. 4A is element-axis transformed to the position of the detection element number _enx as shown in FIG. By calculating integrated data at the detection element number e _nx using the following equation, as shown in FIG.
/ N ratio can be improved.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an electrophoresis apparatus for analyzing a very small amount of sample at ultra-high speed and high separation, and more particularly, to bonding two transparent plate members to form a separation channel inside. The present invention relates to a microchip electrophoresis apparatus using a microchip that has been made to fall.

[0002]

2. Description of the Related Art In the case of analyzing a very small amount of protein, nucleic acid, or the like, an electrophoresis method has been conventionally used, and a capillary electrophoresis apparatus is an example of a technique for realizing the apparatus. A capillary electrophoresis device is a device in which a glass capillary having an inner diameter of 100 μm or less is filled with an electrophoresis buffer, a sample is introduced at one end side, and a high voltage is applied between both ends to develop an analyte in the capillary. is there. Since the inside of the capillary has a large surface area with respect to the volume, that is, high cooling efficiency, a high voltage can be applied, and D
A very small amount of sample such as NA can be analyzed at high speed and with high resolution.

[0003] In recent years, the use of fused silica
DJ Harrison et al./Ana is a form in which faster analysis and smaller equipment can be expected in place of capillaries.
As shown in l. Chim. Acta 283 (1993) 361-366, a capillary electrophoresis chip (referred to as a microchip) formed by joining two substrates has been proposed. FIG. 1 shows an example of the microchip. A pair of transparent substrates (generally, glass, quartz, resin, etc.) 51, 52 are formed, and electrophoresis capillary grooves 54, 55 that cross each other are formed on the surface of one substrate 52, and the grooves 54 are formed on the other substrate 51. , 55 at the position corresponding to the end of the reservoir 53
Are provided as through holes.

When this microchip is used, both substrates 51 and 52 are overlapped as shown in FIG. 1C, and the electrophoresis running solution is injected into the grooves 54 and 55 from one of the reservoirs 53.
Thereafter, the sample is injected into the reservoir 53 at one end of the shorter groove 54, and a high voltage is applied between the reservoirs 53 at both ends of the groove 54 for a predetermined time. As a result, the sample is dispersed in the groove 54. Next, an electrophoresis voltage is applied to the reservoirs 53 at both ends of the longer groove 55. This allows
The sample existing at the intersection 56 between the two grooves 54 and 55 is
Electrophoresis inside. By arranging a detector at an appropriate position in the groove 55, separated samples carried in order by electrophoresis are sequentially detected.

[0005]

In the case of microchip electrophoresis, the optical path length is only about 10 times smaller than that of general-purpose capillary electrophoresis, ie, about 10 to 20 μm. Has become. In addition, since the sample is migrating, the time during which the sample is present in front of the detection region is short, and the sensitivity cannot be improved by performing a long-time integration process.

Accordingly, an object of the present invention is to improve the S / N ratio of a microchip electrophoresis apparatus and increase the detection sensitivity.

[0007]

A microchip electrophoresis apparatus according to the present invention includes a pair of transparent plate members, wherein at least one of the plate members has a surface on which a groove for flowing a liquid is formed, and the other plate member. Are provided with through holes at positions corresponding to the grooves, these plate-like members are bonded together with the grooves inside, and a microchip formed by the grooves to form a separation channel, and both ends of the separation channel An electrophoresis power supply unit that applies a flowing voltage therebetween, a light irradiation unit that collects light from a light source over a predetermined range of the separation channel, and absorbs or emits light by a target component separated in the separation channel. For detection, an array light detection unit in which a plurality of light detection elements are arranged along the separation channel, and a data processing unit that performs data processing by taking in the output of each detection element of the array light detection unit, Data processing unit detects each light A storage unit for storing the output of the child,
An integrated average operation unit for calculating an integrated average value of the output of the same sample portion with respect to the output of the light detection element stored in the storage unit.

[0008] Light is condensed on the entire separation channel by the light irradiation unit, and light transmitted through the separation channel is detected by an array light detection unit arranged along the separation channel. Since each separated sample moves in the liquid injected into the separation channel with time, the detection peak of each sample detected at each time shifts little by little. Therefore, while the peak of each moving sample is tracked by computer processing, the peak of the same sample at each time is integrated and averaged by the integrated average calculation unit.

[0009]

FIG. 2 is a schematic structural view showing one embodiment.
For example D ₂ lamp or a light source 1 such as a tungsten lamp is provided with, in the optical path of the light from the light source 1 mirror 3,
A lens 5, a slit 7, and a mirror 9 are provided.
The lens 5 converts the light from the light source 1 reflected by the mirror 3 into parallel light. On the optical path of the light reflected by the mirror 9 in the optical path, the light source 1 made parallel by the lens 5
A grating 11 that splits the light emitted from the grating 11 is provided, and a mirror 13 that guides the light split by the grating 11 to the slit 15 is provided. In order to irradiate light only to the separation channel of the microchip 17, the slit 15 has a rectangular opening.
Are provided on the light incident side. On the opposite side of the microchip 17, a PDA (photodiode array) 1 in which a plurality of photodetectors for detecting light transmitted through the separation channel are arranged.
9 are provided.

A data processing unit 21 is provided which takes in the detection output of each detection element from the PDA 19, performs signal processing such as absorbance conversion, and performs integration processing. Data processing unit 2
1 includes a signal conversion unit 23 that converts the captured detection signal into, for example, absorbance, a storage unit 25 that temporarily stores data from the signal conversion unit 23, and an integration average calculation unit 2 that performs integration processing.
7 are provided. The high-voltage power supply 29 is connected to the data processing unit 2
1 to apply an electrophoresis voltage to the separation channel of the microchip 17.

Next, the operation will be described. Light emitted from the light source 1 is incident on the grating 11 via the mirror 3, the lens 5, the slit 7, and the slit 9. The light split by the grating 11 is reflected by the mirror 13 and the slit 1.
The light is condensed on the entire separation flow path of the microchip 17 through 5. The light is incident on the PDA 19 through the separation channel. The time range in which the integrated averaging is performed is determined, and the electrophoresis is performed by applying a migration voltage to the separation flow channel of the microchip 17 by the high-voltage power supply 29 in that range. At this time, light detection is performed by the PDA 19 at equal time intervals. The obtained data is subjected to, for example, absorbance conversion by the signal conversion unit 23 to perform signal processing, and then is stored in the storage unit 25. After the electrophoresis is completed, the data stored in the storage unit 25 is integrated by the integrated average calculation unit 27 for each sample.

For example, the following two methods can be considered for the method of detecting and integrating the movement of the peak of a certain sample in the integrated average calculating section 27. (1) A method that focuses on the peak of each sample, tracks the peak that moves with time, and integrates data from the photodetector corresponding to the peak. (2) A method of focusing on peaks of at least two or more samples and detecting a moving speed of a peak of the entire detected image based on a moving speed of the peaks. In (1), it is possible to perform a reliable process on a sample having a relatively high concentration or a sample which can be easily detected, in which a peak can be detected without performing integration averaging.
Unless integrated averaging is performed, it is difficult to handle a trace sample for which peak detection is difficult. Therefore, in this embodiment, the method (2) will be described.

FIG. 3 shows an example of data stored in the storage unit. The horizontal axis is the element number, and the vertical axis is the detection intensity (absorbance).
It is arranged in the order of detected time. In the data stored in the storage unit 25, in the data of the integration start time t _{0 and} the data of the integration end time t _n , at least two peaks of the sample commonly included in the element number section for performing both integration averaging are selected,
Track how each sample peak moves over time. Here, for example, the components a and b are selected, the detection element numbers e _0a and e _0b at which the components a and b are detected at the integration start time t _0, and the components a and b are detected at the integration end time t _n. the detection element number was an e _na, e _nb.

Here, from FIG. 3, the electrophoresis start time t _start
Can be represented by the following equation (1). peak _{- {(e nb -e 0b)} (e na -e 0a)} ... (1) t start t start = {t n (e 0b -e 0a) -t 0 (e na -e nb)} / _Is assumed to be e _start .

[0015] In the data of FIG. 3, constant velocity to migrate with peak positions selected time, since migration distance and the elapsed time is proportional relationship, the detection for detecting a peak of component a at an arbitrary time t _k The element e _ka and the detection element e _kb for detecting the peak of the component b can be expressed by the following equations (2) and (3), respectively. e _ka = (e _0a −e _start ) · (t _k −t _start ) / (t _na −t _start ) + e _start (2) e _kb = (e _0b −e _start ) · (t _k −t _start ) / (T _nb −t _start ) + e _start (3)

At an arbitrary time t _k , for example, the peak of the component x detected by the detection element e _kx is detected at the detection element number f (t _n , e _n) detected when electrophoresis is continued until the integration end time t _n.
kx ) can be expressed by the following equation (4). f (t _n , e _kx ) = (e _kx −e _ka ) · ( _en b −e _na ) / (e _kb −e _ka ) + e _na (4)

Data y measured at an arbitrary time t _k
When (t _k , e) (e is a detection element number) is converted into an integration end time t _n by using equation (4), Becomes

When equation (5) is integrated and averaged, That is, the light detection data y for any time t _k, the data of a detection element, the function f (t _n of whether the accumulation end time t _n has been detected by the ordinal number of the detecting element (4),
e _kx ), a plurality of peaks whose element axes are corrected for the detection element are integrated and averaged, and finally, the detection element is normalized by the score. For more accurate calculation, it is preferable to select a plurality of peaks and perform a multidimensional regression calculation.

FIG. 4 is a diagram showing an example in which the peak of a certain sample x is corrected for the element axis to the detection element number at which the peak is located at the integration end time. (A) is data before correction, (B) is data after correction, and (C) is data after integration. The horizontal axis represents the detection element number, and the vertical axis represents the detection intensity. The respective peaks from the integration start time t ₀ to the integration end time t _n shown in FIG.
Using the equation, the detection element is moved to the position of the detection element number _enx as shown in FIG. Detection element number e _nx is integrated end time t _n
Is the detection element where the peak is located. Detection element number e _nx
By integrating the peaks corrected for the element axis,
As shown in (C), the S / N ratio can be improved to make a clear peak.

[0020]

According to the present invention, a data processing unit is provided, and the data processing unit is provided with a storage unit and an integrated average operation unit. In the integrated averaging operation unit, by computer processing, while tracking the peak of each moving sample, the deviation due to migration is corrected on the element axis, and the peak of the same sample at each time is integrated and averaged by the integrated averaging operation unit. it can. As a result, the noise level decreases in inverse proportion to the square root of the integration time.
/ N ratio can be improved.

[Brief description of the drawings]

FIG. 1 is a diagram illustrating an example of a microchip.

FIG. 2 is a schematic configuration diagram illustrating an embodiment.

FIG. 3 is an example of data stored in a storage unit of the embodiment.

FIG. 4 is a diagram illustrating an example in which a peak of a certain sample is subjected to element axis correction to a detection element number at which the peak is located at an integration end time. (A) is data before correction, (B) is data after correction, and (C) is data after integration.

Claims (1)

[Claims] 1. A pair of transparent plate-shaped members, wherein a groove through which a liquid flows is formed on a surface of at least one of the plate-shaped members, and a hole which penetrates the other plate-shaped member at a position corresponding to the groove. A microchip in which the plate members are bonded together with the groove inside, and a separation channel is formed by the groove; a migration power supply unit for applying a flow voltage between both ends of the separation channel; A light irradiation unit that collects light from the light source over a predetermined range of the path, and a plurality of light irradiation units along the separation channel for detecting absorption or emission of the light by the target component separated in the separation channel. An array light detection unit in which the light detection elements are arranged, and a data processing unit that captures the output of each detection element of the array light detection unit and performs data processing, wherein the data processing unit outputs the output of each light detection element The storage unit to store and the storage unit Microchip electrophoresis apparatus characterized by and an integration average calculator for calculating an accumulated average value of the output of the same sample portion for the output of 憶 light detecting element.