JPH05223778A - Electrophoretic device - Google Patents

Abstract

PURPOSE:To obtain an electrophoretic device which can detect a sample which is subjected to molecular weight separation with high sensitivity. CONSTITUTION:A sample condensing section 2 constituted of a molecular sieve film is installed to part of an electrophoretic path constituted of capillaries 1 and 3. A sample is gathered into and condensed in the section 2 by applying a voltage across electrode tanks 5 and 4 after the sample is injected into the capillary 1. Then the sample is electrophoretically moved by applying a voltage across electrode tanks 6 and 5 and a molecular weight separating pattern is measured. Since the sample condensing section is installed to the electrophoretic path, an electrophoretic device, in the electrophoretic path of which the concentration of the sample is substantially increased and which can detect DNA, protein, sugar, etc., with high sensitivity can be obtained.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an electrophoretic device for separating and analyzing nucleic acids, proteins, sugars and the like.

[0002]

2. Description of the Related Art Electrophoresis is effective as a means for separating and analyzing bio-related substances such as DNA, proteins and sugars. As a medium for electrophoresis, agarose, polyacrylamide gel and the like are mainly used. These are used in the form of a flat gel formed between two glass plates, a capillary gel formed inside a capillary, or the like.
In addition, a method of using only a buffer solution without using a gel as an electrophoretic medium has been investigated. Currently, the inner diameter is 100 μm.
Capillary electrophoresis using the following capillaries is also drawing attention.

In electrophoresis using capillaries such as capillary gel electrophoresis and capillary zone electrophoresis, since the diameter of the capillaries is small, the surface area per volume of the electrophoretic medium is large and the Joule heat can be easily dissipated. Therefore, even if a high voltage is applied, the temperature rise is suppressed, and as a result, a higher voltage can be applied as compared with normal electrophoresis, and high resolution can be obtained in a short time. Also, due to the small diameter of the capillaries, the absolute amount of sample is several tens nL.
It is small (nanoliter) and suitable for trace analysis. UV is used for detection of samples separated by electrophoresis.
Absorbance by a detector, fluorescence intensity by a laser-excited fluorescence measuring device, or the like is used. For these capillary electrophoresis, see, for example, Bunseki, No. 8, 59.
Pages 9-606 (1991) (Bunseki, No)
8, 599-606 (1991)).

Further, capillary gel electrophoresis is applied to DNA.
There are also attempts to apply it to the separation and analysis of fragments. For example, Nucleic Acid Research, Vol. 18, pp. 1415-1419 (1990) (Nucleic Aci)
d Research, 18, 1415-1419 (1
990)), a capillary with an inner diameter of 75 μm is used, and a high voltage of 9 kV is applied to obtain a D
High-speed and high-resolution detection of NA fragments and the like is aimed at. The detection part of this method tilts the axis of the capillary and the irradiation axis of the laser light by about 25 degrees from the vertical direction, and irradiates the central part of the capillary with the laser light focused to a diameter of about 20 μm and irradiates the generated fluorescence. A bandpass interference filter or the like is used for spectral detection.

[0005]

In the electrophoretic device, it is desired to increase the detection sensitivity with the miniaturization of the sample. Generally, in fluorescence measurement in an electrophoretic state, in addition to fluorescence from the target phosphor itself, scattered light of excitation light by the gel (Rayleigh scattering, etc.) as the background light, the gel support such as the inner wall of the capillary, the outer wall Scattered light at, and fluorescence of the gel itself occur. In particular, the capillary has a circular cross section, and has a shape in which excitation light is easily scattered. In addition, since the capillary diameter is small, the inner and outer walls of the capillary where scattered light is generated are very close to the fluorescence emission position from the sample,
It is also difficult to spatially separate the two. for that reason,
There is a problem that the background becomes high and the detection sensitivity is likely to be lowered.

As described above, the capillary electrophoresis is characterized in that the absolute amount of the sample to be analyzed is small, and an extremely small amount of sample can be detected. However, since the capillary itself is generally used as an optical cell for optical detection, the optical path length is less than or equal to the inner diameter of the capillary, and the concentration sensitivity is not always good. The density sensitivity is proportional to the optical path length, and the optical path length of a normal optical cell is 2 mm to 10 mm, whereas in the case of a capillary, it is about 25 to 100 μm or less, so the sensitivity is lowered. Therefore, a highly sensitive detection method suitable for capillary electrophoresis has been an important issue.

Generally, the sensitivity in fluorescence measurement is higher than that by absorbance, and Nucleic Acid Research, Vol. 18, pp. 1415-1419 (1990).
(Nucleic Acid Research, 1
8, 1415-1419 (1990)), the sample is detected by fluorescence measurement. However, even in the fluorescence measurement, the total fluorescence intensity is proportional to the optical path length, so that the sensitivity is lower than that of a normal optical cell. In capillary gel electrophoresis, the sample is concentrated when it is introduced into the gel, and the sample concentration in the gel is effectively increased, which can be expected to improve the sensitivity, but conversely, the scattered light and fluorescence from the gel are expected. Also increases. Therefore, a highly sensitive detection method has been an important issue. An object of the present invention is to solve the above problems and provide an electrophoretic device for highly sensitively detecting and analyzing a sample separated by electrophoresis.

[0008]

SUMMARY OF THE INVENTION The above object is to provide an electrophoretic device for electrophoresing a sample for detection and analysis, and a first electrophoretic path into which the sample is injected and a sample to which the sample is concentrated. The concentrating section and the second migration path through which the concentrated sample is electrophoretically separated do not branch, and the first migration path, the sample concentrating section, and the second migration path are connected in order.
That is, this can be achieved by an electrophoretic device having a structure in which one end of the first migration path and one end of the sample concentration section are connected, and the other end of the sample concentration section is connected to one end of the second migration path. At least a part of the sample concentrating portion is a tubular carrier having a molecular sieving effect, and can be achieved by having a structure in which the sample concentrating portion is connected substantially coaxially with the first and second migration paths. The sample concentrating section can be achieved by a molecular sieving membrane arranged near the intersection of the axis of the first migration path and the axis of the second migration path.

Further, the first step of applying a voltage between the first migration path end and the sample concentrating section to inject the sample, and the step of applying a voltage between the sample concentrating section and the second migration path end Can be achieved by an electrophoretic device having a second step of electrophoretically separating. In the first step, no voltage is applied to the end of the second migration path at least so that the sample migrates to the end of the second migration path. No voltage is applied to the ends. Further, the purpose is further achieved by making the length of the first migration path shorter than the length of the second migration path.

[0010]

In the electrophoretic device for detecting and analyzing a sample by electrophoresis, the migration path is composed of the first migration path, the second migration path and the sample concentrating section, and the first migration path and the sample concentrating section. By connecting the second migration path and the second migration path in order, it is possible to effectively increase the amount of the sample that can be injected into the migration path and further increase the sample concentration in the migration path by concentrating the sample in the sample concentrating unit. Therefore, it is possible to enhance the concentration sensitivity of the detectable sample. In an electrophoresis device using a conventional capillary, etc.,
The amount of sample liquid that can be injected into the capillary is on the order of nL (nanoliter). This limits the small diameter of the capillary and the length of the sample injection region (the length of the region containing the sample injected into the migration path in the migration direction) so as not to impair the resolution. This is because it is necessary. That is, as the length of the sample injection region is increased, the migration band width is increased and the resolution is deteriorated. Normally, the length of the sample injection region is about 1 mm, and if the inner diameter of the capillary is 50 μm, the injection amount of the sample solution will be about 2 nL. However, the total amount of the sample prepared during the injection operation needs to be at least several μL (microliter). This is determined by factors such as the performance and operability of the pipette for dispensing into the injection container. That is, the amount of the sample solution injected into the capillary is 1/1 compared to the sample solution to be prepared.
It is very small, about 000.

In the present invention, the sample is injected from one end of the first migration path, the sample is concentrated by the sample concentrating section, and the sample is electrophoretically separated at the second migration path, detected and analyzed. Therefore, if the length of the sample injection region in the second migration path, which is the portion to be electrophoretically separated, is the same as the conventional one, the amount of the sample liquid that can be injected is as much as the amount concentrated in the sample concentrating portion as compared with the conventional one. Will increase. Moreover, as mentioned above,
Since the sample amount to be prepared is originally in a large excess, it is not necessary to increase the sample amount to be prepared by the operation of the present invention.
On the contrary, it is also possible to inject the entire amount of the prepared sample.
With this, the concentration of the sample in the second migration path is increased, and the detection sensitivity of the sample can be effectively increased. Further, when the amount of the sample to be injected is the same as the conventional one, the length of the sample injection region in the second migration path can be reduced by further concentrating, and the resolution can be improved. ..

The sample concentrating section is composed of a tubular carrier having a molecular sieving effect in whole or in part, and is efficiently concentrating the sample by connecting the first and second migration paths substantially coaxially. Therefore, the sample concentrating section can be easily configured. As the tubular carrier having a molecular sieving effect, for example, a sample to be analyzed, for example, a tubular sieving of a molecular sieving membrane having a molecular weight cut-off smaller than the molecular weight of protein, DNA, or the like is used. This molecular sieving membrane allows a substance having a molecular weight smaller than the cut-off molecular weight, for example, a component of the electrolytic solution such as a buffer solution (salt, etc.) to pass through,
Do not pass the sample you want to analyze. Therefore, by inserting one end of the first migration path into the sample solution and applying a voltage between the sample concentration section and the sample concentration section, the sample is injected into the first migration path in the same manner as in normal electrophoresis, and the sample concentration is performed. It is possible to migrate in the direction of the part. By continuing the migration, the sample remains at the molecular sieving membrane, and as a result, the sample is concentrated near the molecular sieving membrane in the sample concentrating section. The shape of the sample concentrating portion may be substantially the same as that of the first or second migration path. For example, when the first or second migration path is a capillary having a diameter of 50 μm, a tubular molecular sieve membrane having an inner diameter of about 50 μm can be used. The length of the sample concentrating portion in the migration direction, more specifically, the length of the molecular sieving membrane portion facing the migration portion of the sample in the migration direction (the migration end of the first migration path and the second migration path) The distance from the migration start end) is preferably short, and is preferably about 1 to 3 mm or less.

The sample concentrating section is a flat molecular sieving film, and the molecular sieving film is disposed near the intersection of the axis of the first migration path and the axis of the second migration path to facilitate sample concentration. Parts can be configured and the sample can be efficiently concentrated. For example, the ends of the first migration path and the second migration path on the side of the sample concentrating portion are brought into close contact with each other so that their axes intersect at 90 degrees, and the end faces of the first and second migration paths are provided near the intersection of the axes. Place and fix the planar molecular sieving membrane so that a small space is created with and. By doing this, the sample stays at the molecular sieving membrane by performing electrophoresis in the same manner as above,
As a result, the sample is concentrated in the minute space formed by the end faces of the first and second migration paths and the molecular sieving membrane surface. The molecular sieving membrane is a membrane having a property of sieving molecules, and various carriers such as ultrafiltration membranes, dialysis membranes such as cellulose ester membranes, and microporous membranes can be used depending on the molecular weight cutoff. In the electrophoretic procedure of the electrophoretic device of the present invention, first, as a first step, the first migration path end is inserted into the sample solution, and a voltage is applied between the sample concentrating portion and the sample is injected. By this operation, the sample can be migrated to the sample concentrating section and concentrated. In particular, by increasing the voltage application time, the sample can be concentrated more and the sample concentration in the sample concentrating section can be increased. The first
In the step of (2), the sample leaks from the sample concentrating unit in the second migration path direction by not applying a voltage to the second migration path edge so that the sample migrates to the second migration path edge at least. Can be prevented, and can be concentrated efficiently. In addition, since the voltage application time is not limited in principle, the sample can be sufficiently injected and concentrated, and the effective concentration of the sample in the sample concentrating portion can be increased.

Next, as a second step, a voltage is applied to the sample concentrating portion and the second migration path end without applying a voltage to the first migration path end, thereby collecting the concentrated sample. It is possible to efficiently perform electrophoretic separation in a concentrated state, that is, in a state where the concentration is increased. Therefore, it is possible to easily and accurately detect the sample to be electrophoresed by the ordinary detection means provided in a part of the second migration path, to concentrate the sample prepared at the beginning and to detect it with high sensitivity. Can be analyzed. As the sample detecting means, various devices such as an optical detecting means for fluorescence intensity and absorbance of the sample, an electrochemical detecting means, or the like can be used. In addition, the length of the first migration path can be made shorter than the length of the second migration path to allow high-speed migration between the end of the first migration path and the sample concentrating section,
The time required for injection / concentration can be shortened and the sample can be concentrated efficiently. The first and second migration paths are preferably gel-filled capillaries or electrolyte-filled capillaries, and may be flat gels formed between glass plates.

[0015]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment (first embodiment) of the present invention will be described below. In this example, a method of separating the molecular weight of a fluorescently labeled DNA fragment by capillary gel electrophoresis and detecting fluorescence will be described. Fluorescein isothiocyanate (FITC) is used as a fluorescent substance for labeling. First, an outline of an electrophoresis device will be described. Figure 1
A configuration diagram of the electrophoretic device of this example is shown in FIG. The device was arranged in the capillary 1 as the first migration path, the capillary 3 as the second migration path, the sample concentrating section 2, the electrode tanks 4 and 5 and 6, the high voltage power supply 7, and the electrode tanks 4 and 5 and 6. The electrodes 8 and 9 and 10, the excitation light irradiation unit 11, the fluorescence detection unit 12, the data processing unit 13, the display unit 14, and the recording unit 15. FIG. 2 is an enlarged cross-sectional view near the sample concentrating unit 2.
The sample concentrating portion 2 is formed by a cylindrical carrier 25 having a molecular sieving effect, and both ends thereof are connected to the capillaries 1 and 3 so that their central axes are substantially aligned with each other as shown in the figure to form a continuous migration path. Further, the capillaries 1 and 3 are adjusted so as to hold a constant gap 30. For the cylindrical carrier 25, a carrier that allows only low molecules such as an electrolyte to pass therethrough and does not allow a sample to pass therethrough is selected, and the cylindrical carrier 25 is dipped in a buffer solution in the electrode tank 5 to perform electrophoresis with the sample concentrating section 2 as the migration end. be able to. The other ends of the capillaries 1 and 3 are immersed in the electrode baths 4 and 6, respectively.

The sample is injected from the end of the capillary 1 by a usual method. That is, the electrode tank 4 is replaced with a container containing the sample solution, the end of the capillary 1 and the electrode 8 are inserted into the sample solution, and a voltage is applied between the sample solution and the electrode tank 5. When the migration path is a gel, a voltage is applied with the sample solution on the cathode side and the electrode tank 5 on the anode side. The polarity of the power source needs to be changed depending on the type of electrophoretic medium. The sample is injected into the capillary 1 by applying a voltage. After that, the end of the capillary 1 and the electrode 8 are returned to the original electrode tank 4, and a voltage is applied between the electrode tanks 4 and 5 with the same polarity to cause the sample to migrate in two directions of the sample concentrating section. By performing electrophoresis for a sufficiently long time, the injected sample is collected in the gap 30, which is the distance between the migration end of the first migration path and the migration start end of the second migration path. In principle, the length of the region containing the sample collected in the sample concentrating unit 2 is the width of the gap 30 at the maximum, and therefore the width of the gap 30 is sufficient for the length of the sample injection region in which the sample is injected into the capillary 1. By shortening it to 1, the sample solution will be concentrated. By increasing the time of voltage application, that is, the time of injecting the sample into the capillary 1, the amount of injected sample can be increased, and this can be collected in the sample concentrating unit 2. In this way, by utilizing the molecular sieving effect, the sample concentrating section can be easily constructed.

Next, the concentrated sample is electrophoresed using the capillary 3 as a separation unit. At the time of this operation, a voltage is applied between the electrode tank 5 and the electrode tank 6 to cause the concentrated sample to migrate toward the electrode tank 6. The sample is electrophoresed while the molecular weights are separated, and the fluorescence is detected and analyzed at the fluorescence detection position 24. A sample passing through the fluorescence detection position 24 is excited by the excitation light irradiation unit 11, and the generated fluorescence is detected by the fluorescence detection unit 12.
Detect with. The excitation light irradiation unit 11 is composed of an excitation laser light source 16 and a condenser lens 17, and irradiates the capillary with the laser light focused. The fluorescence detection unit 12 includes a lens 18, a spectral filter 19, a lens 20, a photomultiplier tube 21, and an amplifier 22, which are arranged in a direction perpendicular to the irradiation axis of the excitation light. The measured fluorescence intensity is processed by the data processing unit 13 such as a computer, and the results are output to the display unit 14 and the recording unit 15. An example of measuring a fluorescence-labeled DNA fragment with the device having the above configuration will be described. Fluorescent labeled DNA
Fragments are prepared by the well known Sanger et al. Dideoxy method using fluorescently labeled primers and a DNA polymerase reaction. A 21-mer primer (labeled primer) conjugated with fluorescein isothiocyanate (FITC) is used. A labeled primer is added to the single-stranded DNA serving as a template and annealed to bind the labeled primer to the single-stranded DNA. Next, four bases (adenine, cytosine, guanine,
Thymine) corresponding to four deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP), and
In order to obtain a DNA fragment whose end corresponds to adenine, dideoxyadenosine triphosphate (ddATP) was added, and DN was added.
A polymerase reaction is performed. By the above operation, fluorescently labeled DNA fragments having adenine at various ends and having various lengths are obtained.

The capillaries 1 and 3 have an inner diameter of 100 μm.
and a quartz tube having an outer diameter of 200 μm and lengths of 3 cm and 40 cm, respectively. In the cylindrical carrier 25,
A cellulosic molecular sieving membrane having a molecular weight cut off of 2000 and having a tubular shape with an inner diameter of 200 μm is used. The width of the gap 30 is 0.1 mm. The molecular weight of the fluorescence-labeled DNA fragment of the sample is larger than the molecular weight cut-off of the tubular carrier 25, and the sample can be concentrated by electrophoresis. Inside the capillary 1 serving as the migration path, the sample concentrating portion 2 and the capillary 3, a polyacrylamide gel 23 containing urea as a denaturant and having a concentration of 4% is formed to serve as a migration medium for the sample or a medium for molecular weight separation. .. Since the capillary is covered with polyimide and interferes with the fluorescence measurement, it is necessary to remove the polyimide coating corresponding to the part of the fluorescence detection position 24 prior to the gel filling, which is separated from the sample concentrating portion 2 by 30 cm. The position is set as the fluorescence detection position 24, and the coating of 1 mm before and after the position is peeled off and used. Buffers (Tris, boric acid, E
A buffer solution containing DTA) is injected to immerse the capillary end, the sample concentrating portion and the electrode. The operations of injecting a fluorescently labeled DNA fragment, which is a sample, into the capillary, concentrating, and separating the molecular weight will be described.

First, about 5 μL of the fluorescence labeled DNA fragment solution obtained by the above operation is prepared as a sample solution in a sampling tube (sample container). The end of the capillary 1 and the electrode 8 dipped in the electrode tank 4 are transferred to a sampling tube, the sample solution is set to the cathode side, the electrode tank 5 is set to the anode side, and a voltage of 150 V is applied for 2 minutes to inject the sample into the capillary. Next, the end of the capillary 1 and the electrode 8 are returned to the electrode tank 4, the electrode tank 4 is on the cathode side, and the electrode tank 5 is on the anode side.
A voltage of 600 V is applied for 15 minutes for electrophoresis, and the injected sample is collected in the sample concentrating unit 2 and concentrated. It is desirable to adjust the migration time and the migration voltage so that almost all of the injected sample reaches the sample concentrating portion.

Next, the electrode tank 5 is set to the cathode side and the electrode tank 6 is set to the anode side, and a voltage of 6 kV is applied to migrate the sample to separate the molecules. Next, the fluorescence intensity of the fluorescence-labeled DNA fragment that has passed through the fluorescence detection position 24 while being separated in molecular weight is measured. As the laser light source 16, an argon laser light source with an oscillation wavelength of 488 nm is used for FITC excitation. As the spectral filter 19, by using a bandpass interference filter that passes a wavelength range of 500 nm to 540 nm, the FIT
The fluorescence intensity of C can be detected, and the molecular weight separation pattern of DNA fragments can be measured.

Compared with the conventional apparatus in which the sample injection time is several seconds, in this embodiment, the amount of the sample injected is large and the sample is concentrated, so that the sample concentration in the gel is substantially increased. Therefore, the fluorescence intensity increases, and the fluorescence can be detected with high accuracy. As a result, the detection sensitivity of the sample is improved. In the case of this example, a fluorescence intensity increase of about 10 times or more that of the conventional apparatus can be obtained. In addition, since the area where the samples gather is narrowed by the concentration, the molecular weight separation pattern can be measured without impairing the resolution. Furthermore, by making the sample injection time sufficiently long, it is possible to inject the entire prepared sample into the capillary, and the sample can be easily detected. Also,
Even when the injection amount of the sample is the same as in the conventional case, the concentration of the sample in the gel is substantially increased by concentrating, the sample is easily detected, and the detection sensitivity is improved. Furthermore, by adjusting the width of the gap 30 to be narrow, the migration band width in the gel of the second migration path can be further narrowed, and the resolution can be improved. The laser device and the phosphor used are the argon laser and the FIT in the embodiment.
Not limited to C, any phosphor and a suitable laser device can be used. It is also possible to detect fluorescence from not only one type of fluorescent substance but also two or more types of fluorescent substances at the same time. For that purpose, it is only necessary to provide photodetection units corresponding to the number of phosphors in FIG. 1 and detect fluorescence in different wavelength regions.

By applying this device, the base sequence of DNA can also be determined. In other words, primers labeled with different fluorophores are used for each type of terminal base, and
After carrying out the A polymerase reaction, the reaction solutions are mixed and electrophoresed. The base species can be identified and the base sequence can be determined by identifying the fluorescence peak of the DNA fragment that passes through the fluorescence detection position by electrophoresis. In addition, in order to identify the fluorescence peak, it is possible to provide a means such as providing four photodetection sections for detecting four types of fluorescence wavelength ranges in FIG. In this example, the measurement of the DNA fragment was described as an example.
As a matter of course, it can also be used for analysis of sugar and the like. In this embodiment, an example in which the sample is detected by the fluorescence intensity is shown.
The same effect can be obtained when the absorbance is detected using a UV monitor or the like. The same applies to other detection means. As the molecular sieving membrane, various materials such as ultrafiltration membrane can be used in addition to the above-mentioned cellulose. Moreover, the shape of the capillary is not limited. Further, in the present embodiment, the sample was injected into the capillary by applying a voltage, but various methods other than this are possible. For example, the sample can be injected into the capillary by the usual pressurizing method or drop method. The sample after injection can be concentrated in the same manner as in this example.

Furthermore, in this embodiment, polyacrylamide gel was used as the carrier for electrophoresis, but the same operation can be performed with other gels such as agarose. It is also possible to use a buffer solution or the like as a carrier for migration without using a gel. In this case, the migration direction of the sample is mainly opposite to that of the gel, so
It is necessary to reverse the polarity of voltage application. It is also possible to use a buffer solution in a part of the migration path instead of a gel for the entire migration path and to detect a sample that migrates in this part. Also,
By making the length of the capillary 1 sufficiently shorter than the length of the capillary 3 as in the present embodiment, the time required for the injection and concentration of the sample can be shortened. Also,
Since no voltage is applied between the electrode tank 6 and the electrode tank 5 or 4 during the sample injection and concentration operation, the sample does not pass through the sample concentration section 2 and migrate into the capillary 3 which is the molecular weight separation section. , Can be effectively concentrated. Furthermore, during the molecular weight separation operation, the electrode tank 4 and the electrode tank 5 or 6
Since no voltage is applied during the period, only the concentrated sample in the sample concentrating unit 2 migrates into the capillary 3 which is the molecular weight separating unit, and the capillary 1 does not migrate to the concentrating position.
The sample that may be present inside does not migrate in the direction of the capillary 3 and enables measurement with high separation accuracy.

If a voltage in the opposite direction is applied between the electrode tank 6 and the electrode tank 5 at the time of injecting and concentrating the sample, it is possible to forcibly prevent the migration of the sample into the capillary 3. It becomes possible to improve the accuracy. According to the present embodiment, by providing the sample concentrating section, the sample can be detected with higher sensitivity. Further, it is possible to narrow the area where the sample is concentrated in the sample concentrating section and the sample is collected, and the sample can be analyzed with high accuracy without impairing the separation performance in electrophoresis. Another embodiment (second embodiment) of the present invention will be described. As in the first embodiment, the end is adenine,
The case of detecting fluorescently labeled DNA fragments having various lengths as a sample will be described. The outline of the electrophoretic device is basically the same as that of FIG. In the present embodiment, the structure of the sample concentrating section and the voltage application method are the first.
Is different from the embodiment described above.

FIG. 3A shows the sample concentrating unit 2 in this embodiment.
The expanded sectional view of the vicinity of a is shown. An ultrafiltration tube having a molecular weight cutoff of 3,000 and an inner diameter of 500 μm is used as the molecular sieving tube 26. A capillary 1a having an inner diameter of about 100 μm, an outer diameter of about 500 μm and a length of 3 cm is used for the first migration path for injecting the sample. The second migration path for electrophoresis separation has an inner diameter of about 100 μm and an outer diameter of about 500 μm.
A capillary 3a having a length of m and a length of 40 cm is used. For the capillaries 1a and 3a, polyacrylamide gels 27 and 28 containing urea as a denaturant and having a concentration of 4% were used.
Is filled. The tube for molecular sieving 26 is a pair of holes 5b provided in the walls of the electrode tank 5a which face each other at an interval of 1 mm.
And 5c. Tube 2 for molecular sieve in this state
The gel-filled capillaries 1a and 3a are pushed into 6 from both sides, and the sample concentrating portion 2a
Make up. The gap 29 between the ends of the capillaries 1a and the ends of the capillaries 3a facing each other and the gap 29 has a length of 1 mm, and the inside thereof is filled with a buffer solution in advance. Although the buffer solution in the gap 29 and the buffer solution in the external electrode tank 5a are separated via the molecular sieving tube 26, they are electrically connected to each other because electrolytes and the like pass therethrough. Normal electrophoresis can be performed using the migration end.

Each operation of injecting and concentrating the sample fluorescence-labeled DNA fragment into the capillary, and separating the molecular weight will be described. Approximately 5 μL of the fluorescent labeled DNA fragment solution is used as a sample solution,
Prepare in a sampling tube. The end of the capillary 1a immersed in the electrode tank 4 and the electrode 8 were transferred to a sampling tube containing a sample solution, and a potential voltage of -150V was applied to the sample solution part, 0V was applied to the electrode tank 5a, and -2000V was applied to the electrode tank 6. Continue applying for 1 minute. By this operation, the sample is injected from the sampling tube into the capillary 1a and migrates toward the sample concentrating section 2a. Since the direction of the electric field in the gel portion inside the capillary 3a is opposite to that in the capillary 1a,
It is possible to prevent the migrated sample from leaking into the capillary 3a. Next, the end of the capillary 1a and the electrode 8 were returned to the electrode tank 4, and a voltage of −450 V to the electrode tank 4, 0 V to the electrode tank 5a, and −6000 V to the electrode tank 6 was applied for about 20 minutes, and the injected sample Are collected in the gap 29 of the sample concentrating section 2a and concentrated. It is desirable to adjust the migration time and the migration voltage so that almost all of the injected sample reaches the sample concentrating section 2a. Also in this case, since the direction of the electric field in the gel portion in the capillary 3a is opposite to that in the capillary 1a, it is possible to prevent the migrated sample from leaking into the capillary 3a. That is, the diffusion of the sample in the sample concentrating section 2a becomes small,
The sample can be efficiently concentrated at the position of the gap 29 of the sample concentrating unit 2a.

Next, a voltage is applied so that the electrode tank 5 has a voltage of -6 kV and the electrode tank 6 has a voltage of 0 V.
The sample concentrated to a can be guided to the capillary 3a, and the molecular weight can be separated. The inside of the gap 29 of the sample concentrating portion 2a is a buffer solution, while the capillary 3
Since the inside of a is the polyacrylamide gel 28, when the concentrated sample enters the capillary 3a, it will be concentrated again, so that the concentration of the sample in the gel is further increased,
It becomes possible to detect with higher accuracy. According to this example, in the operation of injecting and concentrating the sample into the capillary, by applying electric fields in opposite directions with respect to the sample concentrating portion as a boundary, diffusion of the sample in the concentrating portion is suppressed, resulting in higher accuracy. In addition, the fluorescence intensity from the sample can be detected. Moreover, in addition to the concentration of the sample in the sample concentrating section, the first migration path for injecting the sample is a gel, the sample concentrating section is a buffer solution, and the second migration path for separating the molecular weight of the sample is a gel. Since the sample is concentrated during the molecular weight separation, the practical concentration of the sample in the gel is increased, and the sample can be detected more easily with high sensitivity. In this embodiment, the sample concentrating section 2a is integrated with the electrode tank 5a, but it may be arranged in the same manner as in the first embodiment.

Further, it becomes possible to arrange a plurality of concentrating sections in the same electrode tank and simultaneously condense and measure a plurality of samples. FIG. 3B shows a perspective view of multiple sample concentrating units for simultaneously concentrating a plurality of samples. A plurality of (four in the figure) sample concentrating portions are formed in the lower portion of the electrode tank 105, and each sample concentrating portion has a cross-sectional structure similar to that of FIG. 3A. That is, the tube for molecular sieving 126
The capillary 101a and the capillary 103a are connected to a to form one sample concentrating portion. Similarly,
Molecular sieving tubes 126b-d, capillary 10
Three sample concentrators are formed by 1b-d and capillaries 103b-d. The plurality of sample concentrating portions are arranged in a row in one electrode tank, and all are electrically connected via the buffer solution in the electrode tank. When injecting a sample, if the capillaries 101a to 101d are on the sample injecting side,
Each of the capillaries 101a to 101d is dipped in a different sample solution, an electric field is applied between the capillaries 101a to 101d, and the sample is injected. At this time, the number of electrodes required for injecting the sample is the same as the number of samples when they are simultaneously used. Electrode tank 1
One electrode for 05 is sufficient. The concentration procedure is the same as described above. Regarding the electrophoretic separation, the ends of the capillaries 103a to 103d, which are electrophoretic separations, are inserted into one electrode tank (electrode tank 6 shown in FIG. 1), a voltage is applied between the electrode tanks 105, and the electrophoresis is performed at a fixed distance. Detect at the migrated position. If multiple sample concentrators are used as in this example,
For a plurality of samples, at least one concentrating section, one electrode tank on the electrophoretic separation side, and one electrode are sufficient. Further, since the capillaries and the tubes for molecular sieving are thin, it is not necessary to increase the size of the entire electrode tank even if a plurality of tubes are combined as in this example, and the size can be reduced.

Another embodiment (third embodiment) of the present invention will be described. The measurement sample, the sample injection method, the fluorescence detection method and the like are performed in the same manner as in the first embodiment. The overall configuration of the electrophoresis device is
This is basically the same as the case of the first embodiment, and in this embodiment, the structure of the sample concentrating portion will be mainly described. The sample concentrator is composed of a membrane holder, an electrode tank, and a molecular sieve membrane.
Each part will be described below. FIG. 4 shows a part view of the membrane holder 39. 4A, 4B,
(C) and (d) are a top view, a front view (showing a sectional view), a bottom view, and a side view, respectively, which are illustrated based on trigonometry.
The membrane holder is formed by processing the bottom of a cubic tetrafluoroethylene resin having a size of about 8 mm × 8 mm × 8 mm so that the front cross section becomes a pentagon. The membrane holder has an inner diameter of 150 μm for inserting and fixing the capillary.
m capillary insertion holes 31 and 32 are provided. Further, a screw hole 33 (screw size M5, depth 4 mm) for holding and pressing the molecular sieve film is provided. A molecular sieving film is laid on the bottom of the screw hole 33 and fixed by a push screw.
The bottom of the screw hole 33 and the capillary insertion holes 31 and 32 are connected to each other, and the bottom of the screw hole 33 has a center of about 15 mm.
It has a substantially circular connection port 34 of 0 μm to 200 μm.

FIG. 5 shows a component diagram of the electrode tank 40. Figure 5
(A), (b), (c) is the top view, the front view (illustrating sectional drawing), and the bottom view, respectively. The electrode tank is a cylindrical tetrafluoroethylene resin having an outer diameter of about 10 mm, and has a push screw portion 35 (screw size M5) corresponding to the screw hole 33 of the membrane holder and a buffer solution reservoir 36 (inner diameter 8 mm, 20m depth
m) and a through hole 37 (inner diameter 1 mm). Further, the bottom surface of the electrode tank is used as the membrane pressing surface 38. Figure 6
2 is an assembly diagram (cross-sectional view) of a sample concentrating unit constructed by the membrane holder 39, the electrode tank 40, and the molecular sieving membrane 41. FIG.
The molecular sieving film 41 is provided on the bottom of the screw hole 33 of the film holder 39.
(Outer diameter 4 mm) is placed, and the molecular sieving film 41 is pressed and held by the film pressing surface 38 of the pressing screw portion 35 of the electrode tank 40. At the time of actual migration, inner diameter 100 μm,
Capillaries 42 and 43 having an outer diameter of 200 μm are inserted and fixed in the capillary insertion holes 31 and 32. The sample is concentrated in the space surrounded by the capillaries 42 and 43 and the molecular sieving film 41. Further, the buffer solution 36 in the electrode tank 40 is filled with the buffer solution and the electrode is inserted. The capillaries 42 and 43 and the capillary insertion holes 31 and 32
The inside surrounded by the connection port 34 is filled with a carrier for electrophoresis such as gel or buffer solution for use. In this state, since the capillaries 42, 43 and the buffer solution in the electrode tank 40 electrically contact with each other by the molecular sieving film 41, it is possible to perform electrophoresis using the sample concentrating portion as the migration end.

The electrophoretic medium of the capillaries 42 and 43 and the molecular sieving film 41 are in contact with each other at the connecting port 34. By the same operation as in the first embodiment, this connection port 34
The sample is electrophoresed and concentrated on the sample, but since the size of this portion is as small as the size of the cross section of the capillary cut obliquely, the sample can be concentrated efficiently. Using the sample concentrating unit of this example, various operations such as the carrier inside the migration path including the capillary, the type of the molecular sieving membrane, the sample injection method, the sample concentrating method, and the electrophoretic separation method can be performed using the first or second method. By performing the same as in the embodiment, the first
Alternatively, the sample can be detected with high sensitivity as in the second embodiment. According to this embodiment, since a sheet-shaped film can be used as the molecular sieving film, various molecular sieving films can be used. Further, according to the sample concentrating unit having the structure of this embodiment, the sample concentrating unit in which the electrode tank and the molecular sieving membrane are integrated can be easily configured. further,
It is easy to connect the capillaries and fix the molecular sieving membrane, and it is detachable, which improves the operability and reduces the cost by repeated use. Further, similarly to FIG. 3 (b) of the second embodiment, also in this embodiment, it is possible to construct a multiple concentrating section in which a plurality of sample concentrating sections are arranged, and downsizing can be achieved.

[0032]

According to the present invention, by providing a sample concentrating portion in the migration path, the concentration of the sample in the migration path is substantially increased, so that the sample DNA, protein, sugar, etc. can be separated in molecular weight. It is possible to realize an electrophoretic device capable of highly sensitive detection without damaging the electrophoretic device.

[Brief description of drawings]

FIG. 1 is a configuration diagram of an electrophoretic device according to a first embodiment of the present invention.

FIG. 2 is an enlarged cross-sectional view of the vicinity of the sample concentrating portion according to the first embodiment of the present invention.

FIG. 3A is an enlarged cross-sectional view of the vicinity of a sample concentrating unit according to the second embodiment of the present invention, and FIG. 3B is a perspective view of multiple sample concentrating units.

FIG. 4 is a component diagram of a membrane holder according to a third embodiment of the present invention.

FIG. 5 is a component diagram of an electrode tank according to a third embodiment of the present invention.

FIG. 6 is an assembly diagram of a sample concentrating unit according to a third embodiment of the present invention.

[Explanation of symbols]

1, 1a, 3, 3a ... Capillary 2, 2a ... Sample concentrating section 4, 5, 5a, 6 ... Electrode tank, 5b, 5c ... Hole, 7
High voltage power source, 8, 9, 10 ... Electrode, 11 ... Excitation light irradiation unit, 12 ... Fluorescence detection unit, 13 ... Data processing unit, 14 ... Display unit, 15 ... Recording unit, 16 ... Laser light source, 17, 18 ,
20 ... Lens, 19 ... Spectral filter, 21 ... Photomultiplier tube, 22 ... Amplifier, 23 ... Polyacrylamide gel, 2
4 ... Fluorescence detection position, 25 ... Cylindrical carrier, 26 ... Molecular sieving tubes, 27, 28 ... Polyacrylamide gel, 2
9, 30 ... Gap, 31, 32 ... Capillary insertion hole, 33 ... Screw hole, 34 ... Connection port, 35 ... Push screw part,
36 ... Buffer reservoir, 37 ... Through hole, 38 ... Membrane pressing surface, 39
... Membrane holder, 40 ... Electrode tank, 41 ... Molecular sieving membrane, 4
2, 43, 101a to 101d, 103a to 103d ...
Capillary, 105 ... Electrode tank, 126a-126d ...
Tube for molecular sieve.

Claims (6)

[Claims] 1. An electrophoretic device for detecting and analyzing a sample by electrophoresing the sample, wherein the migration path is a first migration path into which the sample is injected, a sample concentrating section in which the sample is concentrated, and the concentrated sample. The sample is configured without branching with a second migration path for electrophoretic separation, and the first migration path, the sample concentrating section, and the second migration path are sequentially connected to each other. Electrophoretic device. 2. The sample concentrating portion is a tubular carrier having at least a part thereof having a molecular sieving effect, and is connected to the first and second migration paths in a substantially coaxial manner. The electrophoretic device according to claim 1. 3. A sample concentrating portion is provided by arranging the axis of the first migration path and the axis of the second migration path so as to intersect with each other, and disposing a molecular sieving membrane at the intersecting position. The electrophoretic device according to claim 1, which is characterized in that: 4. A first step of introducing a sample by applying a voltage between the end of the first migration path and the sample concentrating section, and the ends of the sample concentrating section and the second migration path. The electrophoretic device according to any one of claims 1 to 3, further comprising a second step of applying a voltage to the portion to electrophoretically separate the sample. 5. In the first step, between the sample concentrating section and the end of the second migration path, a voltage having a polarity opposite to that of the voltage between the first migration path end and the sample concentrating section is provided. The electrophoretic device according to claim 5, wherein a voltage is applied. 6. The electrophoretic device according to claim 1, wherein the length of the first migration path is shorter than the length of the second migration path.