MXPA97003680A

MXPA97003680A - Multicapi electroforetic device

Info

Publication number: MXPA97003680A
Application number: MXPA/A/1997/003680A
Authority: MX
Inventors: Funato Masayoshi; Nagashima Taku
Original assignee: Otsuka Pharmaceut Co Ltd
Priority date: 1995-09-18
Filing date: 1997-05-16
Publication date: 1997-08-01

Abstract

Using a plurality of light emitting portions, lights driven by different electrical signal components are applied to the respective light transmitting portions of the plurality of capillaries, the lights passing through the light transmitting parts are focused at a certain point When the focused light is detected, the respective electrical signal components included in the detected signal are separated, consequently, a plurality of samples can be analyzed by arranging a plurality of capillaries in parallel

Description

DEVICE ELECTROFORETTCO MULTICPPILPR TECHNICAL FIELD The present invention relates to a ulticapillary electrophoretic device having the ability to analyze a plurality of samples at the same time by arranging a plurality of capillaries, which are thin glass tubes, in parallel.

TECHNICAL BACKGROUND A capillary electrophoretic device is a device for separating a sample component by filling an electrophoretic solvent in a capillary and charging a solution containing the sample component dissolved therein from one end of the capillary and starting a "potential gradient along the capillary. . An object of the measurement of the capillary electrophoretic device varies in various fields such as co-ions, biopolymers, biornonomers, chemical compounds and the like. The capillary electrophoretic device comprises a light detector for detecting an intensity distribution of a fluorescent image or an image of absorbed light of a sample component by applying light to a portion of the sample component during electrophoresis. Consequently, a state of distribution will last + e the movement of the sample component in the capillary is detected at a high resolution so that the presence of the motive + e + component and the concentration thereof can be determined with base in the state of distribution. Afterwards, it has recently been required to carry out an increase in processing speed and an increase in the amount of processing of the electrophoretic device. Therefore, the prior art proposes a construction wherein a plurality of capillaries is disposed in a row and the light is applied to the respective capillaries from one end in a direction of the arrangement, and then the lights applied from the respective capillaries are detected with a light receiving device provided in 1 to 1 correspondence on each capillary (Japanese Utility Model Publication No. 7-20591 ()). Consequently, a plurality of analysis processing can be carried out, simultaneously, with a relatively simple construction, thus shortening the processing time. In the technique described in the aforementioned publication, it is necessary to provide the light receiving device corresponding to each capillary. In reality, a sensitivity varies with each light receiving device and a gain of an electrical processing circuit also varies so an adjustment work is required to correct the sensitivity and gain in the circuit. In addition, there is also a problem in that a large number of light receiving elements must be arranged and the size of the detection part can not be reduced.

DESCRIPTION OF THE INVENTION An object of the invention is to provide a ulticapillary electrophoretic device, which has no space to produce dispersion in sensitivity of the light receiving device and a gain distribution of the electrical processing circuit and which can reduce the size of the detection part. The rnulti.capil electrophoretic device of the present invention, to achieve the aforementioned object, comprises a plurality of capillaries having light transmitting parts held in parallel, a plurality of light emitting portions for applying lights to the light transmitting parts. respective of the aforementioned plurality of the capillaries, a light emitting driving part for driving the aforesaid plurality of light emitting portions by different electric signal components, a light focusing part for focusing lights passing through the light transmitting parts mentioned above at a certain point, a light detecting part for sensing the focused light, a signal processing part for separating the respective electrical signal components included in an electrical signal detected by the light detecting part aforementioned, and a part of application of voltage to apply a voltage to the capillaries mentioned above (claim i). According to said construction, the light emitted and driven by different electrical signal components can be applied to the respective light transmission portions of the capillaries. On the other hand, when the component demonstrates it is subjected to electrophoresis by filling an electrophoretic solvent in a capillary and filling a solution containing a sample component dissolved in it from one end of the capillary and starting a potential gradient along the capillary-, the attenuation of light intensity corresponding to a fluorescent image and an image of absorbed light of the sample component is generated in the respective light transmitting parts. Then, the lights that pass through the aforementioned light transition parts are focused at a certain point. As a means for focusing the lights that pass through the light transmitting parts to a certain point, a packet of optical fibers can be used. In addition, the focused light is detected and the respective electrical signal components included in the detected signals are separated. Therefore, it is fungible to individually determine the amount of attenuation of the light intensity whose intensity is focused by passing through the light transmitting portions of the respective capillaries. Accordingly, it is possible to determine the amount of attenuation of the light intensity of the respective capillaries only by preparing a light detecting portion even when the plurality of capillaries is not provided with the light detecting part. Accordingly, as in a conventional case where a light receiving device corresponding to the respective capillaries is provided and an electrical circuit is provided, there is no space to produce a scattering and sensitivity of the light receiving element and a dispersion in gain of the light. light receiving processing circuit. Therefore, an adjustment work can be facilitated. Furthermore, it is not necessary to arrange a large number of light receiving parts and the size of the detection part can be reduced. Incidentally, it is preferred that the electrical signal generated in the aforementioned light emitting driving part is a continuity signal of a system of mutually octagonal functions whereby the electrical signal can be separated by the signal processing part mentioned above (claim 2). This "system" of mutually orthogonal functions "refers to any known system of orthogonal functions, and examples thereof include a system of sinusoidal functions having different frequencies, and a system of pulse functions having different forms. The pulse function system includes a system of pulse functions in which repeated frequencies are in a relationship of equal number of times with respect to each other (see figure 6) .In addition, a system of orthogonal pulse functions. From each column (each row) of the matrix of H am rd is also known (see figure 7.) In addition, a time division system of pulse functions can also be used (see figure 8). signal continuity of a system of functions in which the electrical signals generated in the driving part of light emission are mutually orthogonal, an influence of the signal of the other pillar can reduce * to zero, in principle. Therefore, there is no fear of crosstalk. Therefore, the reliability of the measurement can be increased. The aforementioned signal processing part may include a synchronous rectification circuit for separating the respective electrical signals (claim 3). In addition, the electrical signals generated in the aforementioned light emitting driving part are sinusoidal signals having different frequencies, and the signal processing part includes a frequency filter circuit to separate the respective frequency components (claim 4). An optical modulation part for modulating lights to torque + ir of the plurality of torque + is light emitters with different electrical signal components can be provided in place of the aforementioned light emitter + torque + (claim 5). For example, the lights of the respective light emitting portions can be modulated using an optical shutter comprising an optical lens device, a liquid crystal device, etc. As shown in Figure 9, mechanical shredding can be carried out using a disc provided with a plurality of openings in the row corresponding to the number of capillaries to represent pulse functions of different systems. The aforementioned object as well as other objects of the present invention will be apparent with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram showing a system for measuring compliance with a multicapillary electrophoretic method. Figure 2 is a diagram showing a construction of the present invention, wherein a fluorescent image or an image of light absorbed from a measuring zone of multi-pillars illuminated by a plurality of LED light sources is collected. detector through a package of op + cas fibers. Figure 3 is a cross-sectional view showing a construction of a light collection system in which the light source lights of LFD are collected on an area of measurement of polymers and a fluorescent light image or an absorbed light emitted from the measurement zone is conducted to an optical fiber. Rig 4 is a circuit block diagram showing a light emitting driving part for supplying a light emitting driving signal to LED and a signal processing part for processing a light detecting signal of a light detector. according to one embodiment of the present invention. Fig. 5 is a circuit block diagram showing a light emitting driving part for supplying a light emitting driving signal to LED and a signal processing pair + e for processing a detection signal of a light detector in accordance with another modality. Fig. 6 is a waveform diagram showing a system of pulse functions in which the repeated frequencies are in a ratio of equal number of times, one with respect to another, as a modality of the system of octagonal functions. Figure 7 is a waveform diagram showing a system of pulse functions as another modality of the octagonal function system. Figure 9 is a waveform diagram showing a time division system of pulse functions as another modality of the octagonal function system. Fig. 9 is a diagram showing an embodiment for making a system of functions carrying out mechanical crushing using a disc provided with rows of openings in multiple stages. Figure 10 is a graph showing the measurement results in the case where an intensity of a detection signal is determined using a multicapillary electrophoretic method of measurement of the present invention. Figure 10 (a) is a graph showing the measurement results in channel 1 and figure 10 (d) is a graph showing the measurement results in channel 2.

BETTER RISE FOR CARRYING OUT THE FUCK OF THE INVENTION The mode for carrying out the present invention will be explained in detail with reference to the accompanying drawings. Figure 1 is a diagram showing a system for measuring compliance with a multiplecpillary electrophoretic method, in which a sample solution is loaded into multicapillars C made of fused quartz and a high voltage is applied to both ends of the multicapillars. In the vicinity of the terminal of the pliers C, a zone of measurement Z to which light is applied is present. The intensity distribution of the fluorescent light image and the absorbed light image of the sample component generated in the Z zone is detected with the light detector, and the distribution is made in the signaling portion. Incidentally, an ammeter A is provided to monitor the intermission of a current produced by the generation of foams in the polycyclar pillars C. Figure 2 is an enlarged diagram showing a plurality of light emitting parts 1 for applying lights to the multicapiars C and a light detector 10. The plurality of light emitting parts 1 comprise a blue LED 2, a multilayer and multilayer film web path filter 3 for extracting only light having a predetermined wavelength, and a focus 4 to focus the light to the measurement zone Z. Incidentally, the light-emitting device is not limited to the blue LED, and any light-emitting device such as LED of another color, laser diode, etc. can be used. Figure 3 is a cross section showing a construction in the views of a light focusing part of the capillaries C. The construction comprises a ball lens 6 for focusing lights emitted from the light emitting parts 1, a slot 5 for isolating excess light, and two-ball lenses 7 and 8 to introduce light exiting through the center of the capillaries C towards a light focusing fiber 9. The light introduced in the aforementioned focus light fiber 9 is packaged as shown in Figure 2, and the incidence on the light sensing part 10 as shown in Figure 2. The light sensing part 10 is provided with a sharp cut filter 11 to extract only light that it has a predetermined wavelength, and a light detector 12. As the light detector 12, a photomultiplier and a PIN photodiode can be used. Fig. 4 shows a light emitting driving part 19 for supplying a light emitting driving signal to the LED 2, and a signal processing part 20 for processing a detection signal of the light detector 12. The driving part of The light emission 19 is composed of a waveform generator circuit 21 and an LED driver 22. The waveform generator circuit 21 generates sinusoidal signals having different frequencies, and the LED driver 22 emits and drives the LED 2 on the basis of this sinusoidal signal. The light detection signal which has passed through the respective ilapillary membranes (known as "channel") and which has entered the light detector 12 is converted into an electrical signal. In this electrical signal, a number of sinusoidal waves are overlapped. The electrical signal is input to a synchronous rectification circuit 24 after passing through the direct current cut filter 13. On the other hand, a rectangular wave signal having the same frequency as the sine wave signal generated by the circuit -Wire waveform 21 is formed in a synchronous signal circuit 23 and then is input to the synchronous rectification circuit 24. The synchronous rectification circuit 24 is specifically a multiplier, and a product of the aforementioned electrical signal and signal Rectangular wave generated in the synchronous signal circuit 23 is a picked up. As a consequence, only the signal component generated in the channel waveform generation circuit 21 can be extracted. This output signal is stabilized in a stabilizer circuit 25, and then output as measurement data. Using the aforementioned function, only the signal component of each channel can be separated and removed from the stabilizer circuit 25. Incidentally, the signal processing part 20 for processing the electrical signal of the light detector 12 is not limited to the circuit before mentioned shown in Figure 4. In the above-mentioned circuit shown in Figure 4, the synchronous signal rectification circuit 24 of the multiplier is used, but a band path filter circuit 26 corresponding to sinusoidal signals having different frequencies generated in each wavelength generator circuit 21, as shown in Figure 5. Consequently, only the corresponding frequency signal can be separated and then extracted. In the aforementioned mode, sinusoidal signals having different frequencies are generated in each circuit <waveform generator 21, but the waveform of the signal is not limited thereto. Instead of the sinusoidal signal, a rectangular wave signal can be used. It is preferred that the signals generated in each waveform generator circuit 21 be mutually orthogonal. That is, when each signal is denoted by a symbol »(i = 1, 2, 3), the signals are multiplied with each other to carry out an integration for a certain period, the following equations are p efferly met to alleviate a alteration of another channel signal. fai 2 dt = 1 faiajdt = 0 (ij) As the system of mutually orthogonal functions, in addition to the aforementioned sinusoidal signals having different frequencies, as shown in Figure 6, a system of pulse functions can be used where the Repeated frequencies are in a ratio of equal number of times to each other (v. gr., 1kHz, 2kHz, 4kHz, 8kHz ....). A system of pulse functions of binary codes 1 and -l can be used, as shown in Fig. 7. In addition, a time division system of pulse functions can be used, as shown in Fig. 8. In addition, in the method mentioned above, the signal was generated in the passage of the part that drives the emission Je light 19 to drive the LED 2. However, the lights emitted from the LED 2 at a certain intensity of light can be modulated with different electrical components of signal. For example, the lights can be modulated from the respective light-emitting portions, using an optical-shutter comprising an electro-optical device, a liquid-crystal device, etc. As shown in Figure 9, mechanical trituration can be carried out using a disk provided with n plurality of openings in the row, which correspond to the number of capillaries so as to represent pulse functions of different systems. Then, using a measurement system (Figure 1 to Figure 4) in accordance with the multiply-capillary electrophoretic method referred to above and using as sample an aqueous solution of fluoroscein, the intensity of the detection signal was measured. However, to see the wavelength of the signal, the measurement was carried out after removing the stabilizer circuit 25 shown in Figure 4. In the case of the initiation of the measurement, the container was filled with water while the other The container was closed and suctioned using a pump, and then the cultures were filled with water. The fluoroscein solution (5 x 10-7 moles) was loaded from one end of the C ultilapillars to electrophoresis the sample component. The number of C capillaries was set at two, and LEDs were emitted and driven using a 4 kHz sine wave on one channel (referred to as "channel 1"), and a 2 kHz sine wave on the other channel (preferred as " channel 2"), respectively. Since a change with the time of the sonusoidal wave mentioned above is sufficiently shorter than a change with the intensity time of the fluorescent Juz image and image of absorbed light (usually in the order of seconds), one can ignore influence of the change over time of the intensity of the fluorescent light image and image of absorbed light exerted on the signal processing part 20. An output of a photomultiplier PfT was measured in channel 1 and channel 2 with the step of time. Figure 10 (a) is a graph showing the results obtained by measuring in channel 1, and Figure 10 (b) is a graph showing the results measuring in channel 2. The numbering unit is millivolts (pp value) ). In channel 1, when the last C cells are filled with water, the measurement signal does not appear and only noises (1.6 V) are generated in the light emitting driving part 19 and the signal processing part 20. It is considered that the difference between this "2" and "1.6" is caused p >; or a difference in the light emission intensity of LED 2 and a difference in the degree of amplification of the electrical circuit. When the sample component is subjected to electrophoresis in channel 1, a large signal (100 rnV) appears in channel i. At this time, the noise component in channel 2 increases to 4 rnV. That is, a diaphony (interference) having an amplitude of 4 is generated with respect to the amplitude of 100. This crosstalk value is -28dB and it is a sufficiently small value »C? A or the component of the sample in channel 1 has been exhausted J the component of the sample is subjected to eleot roforeeis in channel 2, a large signal appears (77 rnV) in channel 2. At this time, the amount of diaphysis is -26 dB and this is a sufficiently small value. Incidentally, in the circuit used in the measurement, the stabilizing circuit 25 is removed. It is assumed that the amount of crosstalk can be further reduced by providing a stabilizing circuit 25 having a truly optimized constant time. As described above, when the multi-capillary electrophoretic device of the present invention is used, the respective signal components can be separated after the lights passing through the respective channels are focused to a certain point and are detected in a single part. of light detection. Therefore, the signal component that appears in the corresponding channel can be measured without being affected by other channels.

Claims

NOVELTY OF THE INVENTION CLAIMS

1. A multi-capped electrophoretic device Jar comprising a plurality of capillaries having light transmitting parties maintained in parallel, a plurality of light emitting portions for applying lights to the respective light transmitting portions of the plurality of capillaries, an emitting driving part of light to drive the plurality of light emitting parts by means of different electric signal components, a light focusing part to focus the lights passing through the light transmitting parts towards a certain point, a part of light detection to detect the focused light, a signal processing part to separate the respective components of the electrical signal included in an electrical signal detected by the light detecting part, and a voltage application part to apply a voltage to the capillaries.

2. The ulticapillary electrophoretic device of conformity with claim 1, further characterized in that the electrical signal generated in the light emitting driving part is a signal constituted by a system of mutually orthogonal functions of odo that the electrical signal can be separated by means of the signal processing part.

3. The multicopillary electrophoretic device according to claim 1, further characterized in that the signal processing part includes a synchronous rectification circuit for separating the respective electrical signals.

4. The multi-capillary electrophoretic device according to claim 1, further characterized in that the electrical signals generated in the light-emitting driving part are sinusoidal signals having different frequencies, and - the signal processing part includes a light-emitting filter circuit. frequencies to separate the respective frequency components. The multi-capillary electrophoretic device according to claim 1, further characterized in that it comprises, in place of the light-emitting driving part, an optical modulating part for modulating lights from the plurality of light-emitting parts with different electrical signal components. .