JP2005201825A - Electrophoretic system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an electrophoretic system manufacturable easily, and allowing high through-put analysis. <P>SOLUTION: This electrophoretic system is a plane type electrophoretic system using active matrix substrates as two opposed substrates 1a, 1b, and having an electrophoretic medium 2 of a plane between the substrates, and a voltage is impressed to the electrophoretic medium 2 by picture element electrodes 4 provided in the active matrix substrates. The optional active matrix substrate is selected, and a migration direction and a migration distance are set to conduct electrophoresis. That is, a channel is set in an optional shape to conduct the electrophoresis. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an electrophoresis apparatus, and more particularly to an electrophoresis apparatus suitable for high-throughput analysis of a sample containing a biological substance (gene (nucleic acid), protein, etc .; biopolymer).

  In the field of life sciences, the decoding of genomes (total gene information) including humans is progressing at a rapid pace, and the decoding results are expected not only to improve new varieties of animals and plants, but also to individual humans. By analyzing the genetic information, it is becoming possible to realize so-called tailor-made medicine that provides the most appropriate treatment and medication based on each person's genetic information, beyond the medical technology that has not gone beyond the symptomatic treatment. is there. If tailor-made medical care is established, it is expected that highly effective disease prevention, medication and treatment can be realized while eliminating waste of medication and treatment. In addition, the use of genetic information analysis technology is progressing extremely rapidly and in a wide range of fields such as infectious disease determination, livestock product identification, and agricultural product production location identification.

  In this genome analysis, a capillary electrophoresis apparatus is a gene testing apparatus that is extremely practical and widely used. Capillary electrophoresis is smaller in size than the conventional gel electrophoresis, that is, by using a capillary tube with an inner diameter of about 25 to 100 microns to reduce the amount of reagents and specimens and simultaneously perform high-speed analysis. It was realized.

  In capillary electrophoresis, a capillary buffer (electrophoresis medium) consisting of a gel or polymer solution is filled into a capillary, a high voltage (100 to 300 V / cm) is applied to both ends, and DNA is a buffer network. The size and concentration of an unknown sample are correctly determined by size separation using molecular sieves and simultaneously passing calibration markers of known size and concentration.

  Fluorescence detection is mainly used for the detection of specimens and markers. In the case of double-stranded DNA, the DNA and fluorescent reagent are intercalated during electrophoresis simply by adding a small amount of fluorescent reagent to the electrophoresis buffer. Fluorescence detection becomes possible. In fact, the excellent high-speed analysis capability of this capillary electrophoresis played an extremely important role in the human genome analysis of about 3 billion base pairs.

  In recent years, a microcapillary array chip has been developed that can be easily filled with a buffer solution for electrophoresis, can be multi-channeled, and can be further increased in speed. This microcapillary array chip is verified not only for gene analysis but also for analysis of proteins, peptides, amino acids, carbohydrates (sugar chains), etc., and is said to be post-genome analysis as well as genome analysis It is expected as a biological sample analysis system that will be applied to proteome analysis.

  In this microcapillary array chip, a groove of several centimeters in length, 100 μm in width, 10 inches in depth and about 10 to 50 μm is formed on a plastic substrate such as quartz, PMMA (polymethylmethacrylate), PC (polycarbonate) or the like. This groove is used as a capillary for electrophoresis (channel for electrophoresis).

  Recently, with the aim of dramatically improving throughput, instead of using plastics and other substrates that have limitations in microfabrication, a single-crystal silicon substrate is used in a semiconductor process, and the electrophoresis channel is changed to a nanometer. There are also chips that have a complicated electrophoretic channel structure having a complicated shape such as a miniaturized size, that is, a range of 1 nm to 1 μm, and a channel shape meandering or zigzag.

In such so-called microchip electrophoresis, unlike conventional capillary electrophoresis, tubular capillaries are not used, so that a plurality of capillaries can be arranged and formed on the substrate in a free positional relationship. For this reason, in spite of being downsized to a substrate shape of about several centimeters, the throughput of the biological sample analysis is dramatically improved.
Bio Venture, 2003 5-6 Vol3 No.3 32-38 Yodosha May 1, 2003

  However, miniaturization of channels (micro- and nano-channel) accompanying multi-channeling intended for high throughput makes it unavoidable to increase costs due to restrictions on processing materials and processing methods. It cannot be said that it has always been adapted to the recent demands for which such information analysis is required. Further, there is a possibility that the problem of relative sensitivity reduction (reduction in signal / noise ratio) of an optical signal becomes obvious due to further miniaturization of reagents and specimens.

  In order to avoid such problems, it may be possible to use a photomultiplier tube or a lock-in amplifier that directly amplifies the optical signal itself, but the signal processing system itself becomes complicated. There are drawbacks.

  Further, as a buffer solution for microchip electrophoresis, a polymer solution or the like is widely used. However, a cellulose derivative or the like is used for an existing polymer solution, and the cellulose is derived from synthetic cellulose or vegetable cellulose. However, a special liquid feeding device is required to fill these channels in the miniaturized channel.

  As described above, the existing method for channel miniaturization and integration has a problem that the high throughput is realized, but the cost of the chip itself is increased and the peripheral device is complicated.

  Therefore, it is necessary to realize high-speed analysis while avoiding the advancement of processing accuracy, the complexity of the manufacturing process, and the increase in the load on the peripheral system associated with the micro- and multi-channel.

  The present invention has been made in view of the above-described conventional problems, and an object of the present invention is to provide an electrophoresis apparatus that can be easily manufactured and can perform high-throughput analysis.

  In order to solve the above-described problems, an electrophoresis apparatus (the present electrophoresis apparatus) of the present invention has a plurality of substrates for applying a voltage to two opposing substrates and an electrophoresis medium sandwiched between the substrates. The electrophoresis medium is in a thin film shape, and a plurality of samples are migrated between the electrodes. In this configuration, the electrophoresis medium (gel or the like) is a thin film (a flat plate of the order of μm or less). Since a thin-film electrophoresis medium is used, a high voltage can be applied. Therefore, high-throughput analysis of a large number of samples is possible. In this electrophoresis apparatus, a plurality of electrophoresis is performed between the electrodes through the substrates.

  In addition, the present electrophoresis apparatus is characterized in that a sample is migrated between electrodes arbitrarily selected from the plurality of electrodes. In this configuration, the electrophoresis direction and the migration distance are arbitrarily set by selecting an arbitrary electrode from among a large number of electrodes arranged throughout the substrate (or the electrophoresis medium), and electrophoresis is performed. It can be carried out. That is, electrophoresis can be performed by setting a channel for electrophoresis in an arbitrary shape.

  Further, since the shape of the migration channel is set by selecting an arbitrary electrode, it is not necessary to design the channel by microfabrication like a microcapillary. Therefore, it can manufacture simply and can reduce manufacturing cost.

  In addition, the electrophoresis apparatus is characterized in that the substrate is a matrix type substrate in which the plurality of electrodes are arranged in a matrix on the opposing surface of the substrate. In this configuration, a plurality of electrodes are arranged in a matrix on the opposing surface of the substrate. For example, the electrode for electrophoresis in this electrophoresis apparatus can also be used as a pixel of a liquid crystal display device as will be described later. This makes it possible to provide a large number of electrophoresis electrodes for applying a voltage to the electrophoresis medium on the opposite surface of the substrate. Therefore, the range of selection of the migration direction and migration distance is further expanded. Further, it is possible to set a channel for electrophoresis having a complicated shape.

  The matrix substrate may be a substrate in which a plurality of electrodes are arranged in a matrix, or a substrate in which a plurality of electrodes are arranged in a matrix by opposing the two substrates. Good.

  The present electrophoresis apparatus is characterized in that the matrix substrate is a simple matrix substrate. In this configuration, since the matrix substrate is a simple matrix substrate in which the electrodes provided on the two substrates are simply orthogonal, the substrate has a simple structure. Therefore, since the electrophoresis apparatus can be easily manufactured, the manufacturing cost can be reduced.

  In addition, the present electrophoresis apparatus is characterized in that the matrix substrate is an active matrix substrate. In this configuration, since the matrix substrate is an active matrix substrate, each pixel can be selected as an electrode for migration even if a large number of pixel electrodes are provided. Therefore, the setting range of the migration direction and migration distance is further expanded. Further, it is possible to set a channel for electrophoresis having a complicated shape.

  In the present electrophoresis apparatus, it is preferable that the two opposing substrates are both active matrix substrates. In this configuration, the two substrates are two active matrix substrates. This makes it possible to detect the sample that has migrated between the opposing electrodes provided on the two substrates. Accordingly, it is not necessary to modify the sample (fluorescence, chemiluminescence, bioluminescence, ultraviolet absorption, isotope labeling, etc.) as in normal electrophoresis. For this reason, it is not necessary to use a carcinogenic substance such as a fluorescent light-emitting reagent. Therefore, it is not necessary to collect the luminescent reagent after the completion of electrophoresis, so that a highly practical (disposable) electrophoresis apparatus can be provided.

  If the two active matrix substrates are the same substrate, the present electrophoresis apparatus can be manufactured with one type of substrate. For this reason, it leads to the reduction of manufacturing cost. Note that the two active matrix substrates may be different substrates.

  Further, the present electrophoresis apparatus is characterized in that the opposing electrodes of the active matrix substrate detect the impedance of the sample. Thereby, the sample can be detected by the change in impedance when the sample is migrated between the counter electrodes provided on the two active matrix substrates. Examples of the impedance detected by the counter electrode include capacitance (dielectric constant), inductance, conductance, and the like.

  In addition, this electrophoresis apparatus is characterized in that a spacer is provided between the substrates. As a result, the distance between the two substrates can be kept constant, and an electrophoretic medium having a uniform thickness can be formed. Further, during migration, the spacer becomes an obstacle that hinders sample migration. Therefore, the migration speed can be controlled by the spacer. For this reason, the separation accuracy can be improved by providing the spacer 5.

  In addition, the present electrophoresis apparatus is characterized in that the spacer is provided regularly. In this configuration, since the spacer interval is uniform, electrophoresis using a sieve can be performed based on the interval. Accordingly, it is possible to separate samples having different molecular sizes without changing the concentration of the electrophoresis medium depending on the molecular size of the sample. Furthermore, it is possible to provide a sample separation / extraction function by changing the size, shape, etc. of the spacer. As a result, even when a small amount of sample is migrated, high-precision separation is possible.

  The electrophoresis apparatus is characterized in that the sample contains a biological substance. Here, the biological substance is a substance derived from a living body (such as a biopolymer) such as a gene (nucleic acid), amino acid, peptide, protein, sugar chain, and the like. Of these, a gene or protein is preferable. These biological substances are often present only in trace amounts, but serve as causative substances for various diseases or diagnostic markers. Therefore, the electrophoresis apparatus can be applied to the medical field such as clinical examination and diagnosis by performing separation / analysis of such biological substances by electrophoresis. For this reason, this electrophoresis apparatus is useful for early detection and early treatment of diseases, and is extremely useful for implementing preventive medicine. It can also be applied to the identification of agricultural products by genetic analysis. In addition, when a sample is a biological substance, this electrophoresis apparatus can be paraphrased as a biological substance detection instrument (a gene or protein analyzer, a clinical test kit, a diagnostic kit, etc.).

  In addition, this electrophoresis apparatus was arbitrarily selected from the plurality of electrodes in the electrophoresis apparatus having a plurality of electrodes for applying a voltage to the electrophoresis medium sandwiched between two opposing substrates. The sample may be migrated between the electrodes. In this configuration, the electrophoresis direction and the migration distance are arbitrarily set by selecting an arbitrary electrode from among a large number of electrodes arranged throughout the substrate (or the electrophoresis medium), and electrophoresis is performed. It can be carried out. That is, electrophoresis can be performed by setting a channel for electrophoresis in an arbitrary shape.

Moreover, since the shape of the electrophoresis channel is set by selecting an arbitrary electrode, it is not necessary to design the channel by microfabrication like a microcapillary. Therefore, it can manufacture simply and can reduce manufacturing cost.

The “gene (nucleic acid)” includes DNA and RNA. DNA includes, for example, cDNA and genomic DNA obtained by cloning, chemical synthesis techniques, or a combination thereof. The DNA may be double-stranded or single-stranded, and the single-stranded DNA may be a coding DNA serving as a sense strand or an anti-coding DNA serving as an antisense strand.

  As described above, the electrophoresis apparatus (the present electrophoresis apparatus) of the present invention is characterized in that a sample is migrated between electrodes arbitrarily selected from the plurality of electrodes. In this configuration, the electrophoresis direction and the migration distance are arbitrarily set by selecting an arbitrary electrode from among a large number of electrodes arranged throughout the substrate (or the electrophoresis medium), and electrophoresis is performed. It can be carried out. That is, there is an effect that electrophoresis can be performed by setting a channel for electrophoresis in an arbitrary shape. In addition, when the substrate or electrode of the present electrophoresis apparatus is used as the substrate or electrode of a liquid crystal display apparatus, an apparatus in which electrophoresis and display are integrated can be provided.

  An embodiment of the present invention will be described with reference to FIGS. In the following, as an example of the present electrophoresis apparatus, a substrate (glass substrate or the like) on which a transparent electrode or TFT (Thin-Film Transistor) used in a liquid crystal display device is formed, and an opposing surface on which a color filter or the like is formed. An electrophoresis apparatus in which an electrophoresis medium (electrophoretic buffer solution; hereinafter referred to as “electrophoresis medium”) is filled between a substrate (such as a plastic substrate) will be described.

  FIG. 7 is a plan view showing a schematic configuration of the present electrophoresis apparatus. This electrophoresis apparatus has a configuration in which an electrophoresis medium (hereinafter referred to as “electrophoresis medium”) 2 is sandwiched between opposing substrates 1a1b. An electrode 7 is provided between the substrates 1a1b. Such a configuration can be realized, for example, by forming a gap in the liquid crystal panel structure as a layer of the migration medium 2, and can be a thin-film migration medium 2 (μm order). FIG. 7 shows an example in which a plurality of samples 6, 6... Are subjected to one-dimensional electrophoresis by applying a voltage to the electrodes 4 provided at both ends between the substrates 1a1b.

  Moreover, this electrophoresis apparatus can also be comprised as follows. FIG. 1 is a plan view showing a schematic configuration of the present electrophoresis apparatus. This electrophoresis apparatus is a flat-plate electrophoresis apparatus, and has a configuration in which an electrophoresis medium (hereinafter referred to as “electrophoresis medium”) 2 is sandwiched between two opposing substrates 1a1b. A plurality of electrodes (corresponding to the pixel electrodes 4 and transparent electrodes of the liquid crystal display device) arranged in a matrix are provided on the inner surface of the counter substrate 1a1b (the surface on the side of the migration medium 2). A voltage is applied to the electrophoresis medium 2. In the present electrophoresis apparatus, it is possible to arbitrarily select an electrode (electrophoresis electrode) for applying a voltage to the electrophoresis medium 2 from a plurality of electrodes. Thereby, the electrophoresis of the sample 6 becomes possible by freely setting the migration direction and the migration distance. Note that a sealing agent (not shown) is provided around the electrophoresis medium 2 except at least the sample insertion opening, and the electrophoresis medium 2 is configured not to leak from between the substrates 1a1b. 1 shows an example in which the sample 6 is two-dimensionally electrophoresed (arrows in the figure), but a plurality of samples 6, 6...

  Here, based on FIGS. 2 to 4, the configuration of the substrate 1a1b of the present electrophoresis apparatus and the sample liquid migration method (separation method) will be described. 2 to 4, the electrode on the substrate 1 a (referred to as the upper substrate) side is indicated by a solid line, and the electrode on the substrate 1 b (referred to as the lower substrate) side is indicated by a one-dot chain line. For convenience of explanation, only the voltage / switching of electrodes related to migration will be described.

  FIG. 2 is a diagram showing a configuration in which the substrate 1a1b is a simple matrix type substrate, FIGS. 2 (a) to 2 (d) are plan views, and FIG. 2 (e) is an A view of FIG. 2 (a). FIG. 3 is a cross-sectional view taken along a line A (showing a charged state in FIG. 2C). In this configuration, the substrate 1a is provided with X electrodes (scanning electrodes) X1 to X4, and the substrate 1b is provided with Y electrodes (signal electrodes) Y1 to Y4 in a stripe shape. Each board | substrate 1a1b is opposingly arranged so that Y1-Y4 may orthogonally cross.

  For example, as shown in FIG. 2 (b), the electrophoresis method of this electrophoresis apparatus is to first select the Y electrodes Y1 and Y3, deselect other electrodes, minus the Y electrode Y1, and plus the Y electrode Y3. Give the charge. As a result, the negatively charged sample moves in the positive direction (from the electrodes Y1 to Y3). Next, as shown in FIG. 2C, a negative charge is applied to the X electrode X1, and a positive charge is applied to the X electrode X3. As a result, similarly, the negatively charged sample moves in the positive direction (from the electrodes X1 to X3). Similarly to FIG. 2B, as shown in FIG. 2D, when a negative charge is applied to the Y electrode Y3 and a positive charge is applied to the Y electrode Y4, the negatively charged sample is moved in the positive direction (Y electrode). Move from Y3 to Y4).

  FIG. 3 is a diagram showing a configuration in which one substrate 1b is an active matrix substrate and the other substrate 1a is a counter substrate, and FIGS. 3 (a) to 3 (d) are plan views. 3 (e) is a cross-sectional view taken along the line AA in FIG. 3 (a) (showing a charged state in FIG. 3 (c)). In this configuration, a common electrode 3 is provided on the substrate 1a, and source electrodes S1 to S4, gate electrodes G1 to G4, and a pixel electrode 4 are provided on the substrate 1b. Although the common electrode 3 can be used for liquid crystal display, the common electrode 3 is not necessary when display is unnecessary.

  In the electrophoresis method of this electrophoresis apparatus, for example, as shown in FIG. 3B, first, the gate electrode G1 is turned on to apply a negative charge to the source electrode S1 and a positive charge to the source electrode S3. As a result, the pixel electrode 4a is negative and the pixel electrode 4b is positive potential, and the negatively charged sample moves in the positive direction (from the pixel electrode 4a to 4b). Next, as shown in FIG. 3C, the gate electrode G1 is turned on, a negative charge is applied to the source electrode S3, and the pixel electrode 4b is set to a negative potential. The negative potential of the pixel electrode 4b is maintained by the pixel capacitance. Subsequently, the gate electrode G1 is turned off, the gate electrode G3 is turned on, a positive charge is applied to the source electrode S3, and the pixel electrode 4c is set to a positive potential. Similarly, the positive potential of the pixel electrode 4c is maintained by the pixel capacitance. When migration is performed in the direction parallel to the source electrode S, such an operation is periodically repeated. As a result, the pixel electrode 4b maintains a negative potential and the pixel electrode 4c maintains a positive potential, so that the negatively charged sample moves in the positive direction (from the pixel electrode 4b to 4c). Similarly to FIG. 3B, when the gate electrode G3 is turned on and a negative charge is applied to the source electrode S3 and a positive charge is applied to the source electrode S4 as shown in FIG. 3D, the pixel electrode 4c is negative. The pixel electrode 4d becomes a positive potential, and the negatively charged sample moves in the positive direction (from the pixel electrode 4c toward 4d).

  FIG. 4 is a diagram showing a configuration in which the two substrates 1a and 1b are both active matrix substrates. FIGS. 4A to 4D are plan views, and FIG. ) Is a cross-sectional view taken along the line AA in FIG. 4A (showing a charged state in FIG. 4C). In this configuration, both substrates 1a1b include source electrodes S1 to S4 (substrate 1a side); S′1 to S′4 (substrate 1b side) and gate electrodes G1 to G4 (substrate 1a side); G′1 to G '4 (substrate 1b side) and the pixel electrode 4 are provided. In FIG. 4, the pixel electrodes of both substrates 1a1b are arranged to face each other, and the overlapping portion is indicated by hatching.

  For example, as shown in FIG. 4B, first, the gate electrode G1 and G′1 are turned on, the source electrode S1 ′ is negative (the source electrode S1 is off), and the source electrode S3 is used as the migration method of this migration apparatus. A positive charge is given to (source electrode S′3 is off). As a result, the pixel electrode 4f on the substrate 1b side is negative (no voltage is applied to the pixel electrode 4e on the substrate 1a side), and the pixel electrode 4g on the substrate 1a side is positive (no voltage is applied to the pixel electrode 4h on the substrate 1b side). Thus, the negatively charged sample moves in the positive direction (from the pixel electrode 4f to 4g). Next, as shown in FIG. 4C, the gate electrodes G′1 and G3 are turned on, a negative charge is applied to the source electrode S′3, a positive charge is applied to the source electrode S3, and the pixel electrode 4h on the substrate 1b side is turned on. The negative potential (no voltage is applied to the pixel electrode 4g on the substrate 1a side) and the pixel electrode 4i on the substrate 1a side are set to the positive potential (no voltage is applied to the pixel electrode 4j on the substrate 1b side). Thereby, the negatively charged sample moves in the positive direction (from the pixel electrode 4h to 4i). 4B, as shown in FIG. 4D, the gate electrodes G3 and G′3 are turned on, the source electrode S3 ′ is negative (the source electrode S3 is off), and the source electrode S4 is turned on. When a positive charge (source electrode S4 ′ is off) is applied, the pixel electrode 4j on the substrate 1b side is negative (no voltage is applied to the pixel electrode 4i on the substrate 1a side), and the pixel electrode 4k on the substrate 1a side is positive (substrate) The pixel electrode 4l on the 1b side is not applied with a voltage), and the negatively charged sample moves in the positive direction (from the pixel electrode 4j to 4k). According to the above configuration, the electrophoresis apparatus of FIG. 4 unifies the source electrodes S1 to S4 of one substrate as a positive potential supply source, and the source electrodes S1 ′ to S4 ′ of the other substrate as a negative potential supply source. Therefore, the apparatus can be simplified. If the potentials of different polarities are supplied for each electrode as shown in FIG. 3 without unifying the potentials of the source electrodes in one substrate in this way, the structure of FIG. For example, electrophoresis can be performed between electrodes 1b only). In addition, the electrode area can be doubled by supplying the same potential to the opposing electrodes of the other substrate.

  As described above, in the present electrophoresis apparatus, a large number of electrodes are arranged throughout the substrate 1a1b (electrophoresis medium 2). Thereby, by selecting an arbitrary electrode from among them, the electrophoresis direction and the migration distance can be arbitrarily set, and electrophoresis can be performed. That is, electrophoresis can be performed by setting a channel for electrophoresis in an arbitrary shape. Signals such as selection of electrodes and setting of charges are controlled by a driver (not shown) as in the liquid crystal display device.

  Further, the present electrophoresis apparatus can be manufactured by filling the electrophoresis medium 2 between the two substrates 1a1b in the same manner as the manufacturing process of the liquid crystal display device. For this reason, this electrophoresis apparatus does not need to design a channel by microfabrication like a microcapillary. Further, when the migration medium 2 does not include liquid crystal, it is not necessary to fill the liquid crystal between the substrates in a vacuum. Therefore, it can manufacture simply and can reduce manufacturing cost.

Furthermore, since the present electrophoresis apparatus is a plate type electrophoresis apparatus, the entire electrophoresis medium 2 can be used as a channel. In the present electrophoresis apparatus, a matrix substrate (simple matrix type substrate, active matrix type substrate) can be applied as the substrate 1a1b. Thereby, since the pixel pitch in the liquid crystal display device can be reduced to form the migration medium 2, it is possible to set much more channels than the channels of the multi-channel capillary electrophoresis chip. . Therefore, it becomes possible to analyze the high throughput dramatically.

  As described above, the present electrophoresis apparatus can simultaneously satisfy the high throughput of analysis and the reduction of manufacturing cost.

  Moreover, in this electrophoresis apparatus, it is preferable to provide the spacer 5 between the board | substrates 1a1b as shown to Fig.5 (a), FIG.5 (b), and FIG. Thereby, the space | interval of board | substrate 1a1b can be kept constant. That is, the flat migration medium 2 having a uniform thickness can be formed. The size and pitch of the spacers 5 can be set to several hundred nm as in the case of the liquid crystal display device, for example.

  Furthermore, as shown in FIG. 6, during the migration, the spacer 5 becomes an obstacle that hinders the migration of the sample. That is, the sample migrates so as to pass through between the spacers 5 (as indicated by arrows in the figure). Therefore, the migration speed can be controlled by adjusting the distance between the spacers (adjusting the distribution density). For example, a sample with a small molecular weight migrates quickly because it can easily pass through this interval, and conversely, a sample with a large molecular weight migrates slowly because it is difficult to pass through this interval. For this reason, the separation accuracy can be improved by providing the spacer 5.

  Thus, the spacer 5 not only simply keeps the distance between the substrates 1a1b constant, but also has a function as the electrophoretic medium 2 that adjusts the migration speed and separation accuracy.

  Such spacers 5 are preferably arranged uniformly (distributed uniformly) between the substrates 1a and 1b. That is, it is preferable that the intervals of the spacers 5 are arranged uniformly. Thereby, the electrophoresis using the sieve by the space | interval of the spacer 5 is possible. In microcapillary electrophoresis, samples having different molecular sizes are separated according to the concentration of the electrophoresis medium packed in the capillary. In other words, it was necessary to change the electrophoresis medium according to the sample. On the other hand, in the present electrophoresis apparatus, the density (size (diameter), pitch) of the spacers 5 that are uniformly arranged is changed according to the molecular size of the sample without changing the concentration of the electrophoresis medium 2. Samples with different molecular sizes can be separated. In addition, if a plurality of regions in which the density of the spacer 5 is changed are arranged in one electrophoresis apparatus, appropriate electrophoresis according to a plurality of molecular sizes can be performed in one apparatus.

  Further, it is possible to provide a sample separation / extraction function by changing the amount, size, shape, or the like of the spacer 5. In FIGS. 5 and 6, the spacer 5 has a cylindrical shape. However, the shape of the spacer 5 is a spherical shape used in a conventional liquid crystal display device as long as the above functions are exhibited. It may be cylindrical, prismatic, or other shapes.

  The spacer 5 may be arranged at any position between the substrates 1a1b. However, as will be described later, when the substrate 1a1b is an active matrix substrate and sample detection (impedance detection) is performed between the pixel electrodes 4, the spacer 5 is provided so as to avoid the pixel electrode 4. preferable. Thereby, a sample can be accurately detected between the opposing pixel electrodes 4.

  When the sample is a protein, a spacer is not essential in order to simply perform two-dimensional electrophoresis. However, if electrophoresis is performed on a gel plate in which spacers 5 are distributed before performing isoelectric focusing, the spacers are sieved, and proteins can be separated and purified on the same principle as column chromatography. . That is, the spacer 5 can also perform purification of the protein in the sample (separation of the protein from the sample).

  FIGS. 8 and 9 are graphs showing the results of one-dimensional electrophoresis of a 100 bp DNA standard sample (100 bp DNA Ladder (3407A) manufactured by Takara Bio Inc.), with separation and resolution depending on the difference in spacer density and size. The result of investigating the difference is shown. The migration medium 2 was 0.7% hydroxypropyl methylcellulose, the migration distance was 30 mm, the sample usage was 0.5 μg / μL, and the migration voltage was 700 volts. FIG. 8B shows the result of electrophoresis when a spacer is provided. FIG. 8A, FIG. 9A, and FIG. 9B show the migration results when a cylindrical spacer is provided. The size (radius) and density of the spacer are shown in FIG. In FIG. 9A, the radius is 200 nm and the pitch is 790 nm. In FIG. 9A, the radius is 2.3 μm and the pitch is 12.8 μm. In FIG. 9B, the radius is 2.3 μm and the pitch is 9.1 μm.

  As described above, it was confirmed that by providing a steric obstacle such as a spacer in the migration path, the fluorescence height was increased, and the resolution and accuracy of each peak were improved. That is, by providing the spacer, it is possible to perform a high-accuracy analysis based on the molecular size (difference in molecular weight) of the sample. Therefore, by comparing the migration results of the standard sample (various markers) and the sample to be examined and examining the presence or absence of a peak, gene identification, disease determination, and the like can be performed.

  In the present electrophoresis apparatus, the substrate 1a1b is not particularly limited as long as it has insulating properties, but the substrate 1a1b must be transparent or translucent in order to visually recognize the electrophoresis result in real time. Is preferred. Examples of the substrate 1a1b include inorganic substances such as glass and quartz, and organic substances such as polymers (resins) such as polycarbonate, polymethyl methacrylate, and polyethylene terephthalate. Moreover, the board | substrate used for a liquid crystal display device may be sufficient.

  Moreover, the space | interval of board | substrate 1a1b should just be set suitably according to a use, and is not specifically limited. In order to analyze a small amount of sample (especially bio-related substances (gene (nucleic acid), amino acid, peptide, protein, sugar chain, etc.)) and reduce the amount of medium 2 for electrophoresis, this interval should be reduced. Thereby, a thin film of the electrophoresis medium 2 can be formed, and a thin-film flat plate type electrophoresis apparatus can be obtained.

  Moreover, as the electrophoresis medium 2, it is possible to use an electrophoretic solution (solution) or gel-like substance that is naturally used for electrophoresis. Examples thereof include cellulose derivatives such as methyl cellulose, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), and hydroxypropylmethyl cellulose (HPMC), polymer solutions of polyethylene glycol, polyethylene oxide, polyacrylamide, agarose, and acrylamide derivatives. For example, a liquid crystal material such as ester, biphenyl, or cyclohexane may be used as the migration medium 2. In addition, the electrophoresis medium 2 may be used alone or as a mixture of a plurality of types. For example, when a mixture of a normal electrophoretic solution and a liquid crystal material is used as the electrophoresis medium 2, it can be separated not only by the charged state of the sample but also by the degree of adsorption of the sample to the liquid crystal molecules. For this reason, the separation accuracy is further improved.

  In the present electrophoresis apparatus, it is preferable that the sample to be subjected to electrophoresis contains a biological substance such as nucleic acid, amino acid, peptide, protein, sugar chain. As the sample containing such a biological substance, for example, a sample prepared by separation / extraction from blood, urine, saliva, and each cell (tissue) according to a conventional method may be used.

  In the current proteome analysis, a large gel plate is prepared and two-dimensional electrophoresis of proteins is performed. Thereafter, structural analysis is performed by mass spectrometry or the like. However, there are few proteins that cause disease and proteins that serve as markers for clinical examinations. In ordinary plate electrophoresis, a large amount of sample is required; the amount of reagents to be used is inevitably increased because of the long migration time; and so on, and is not suitable for protein analysis. Therefore, in protein analysis, it is very important to separate a very small amount of protein present in a sample in a short time.

  Since this electrophoresis device can be a thin-film flat-plate electrophoresis device, it can be used particularly well for genome analysis and proteome analysis, to elucidate the causes of various diseases, perform structural analysis of receptors, and develop new drugs. Very useful above.

  Moreover, in this electrophoresis apparatus, sample sampling can be performed as follows, for example.

  For example, when the sample is a nucleic acid, a sample inlet is provided on the side of the electrophoresis medium, the sample is sampled using a syringe or the like, and two-dimensional electrophoresis is performed. Thus, for example, separation can be performed by grouping samples of approximately the same molecular weight in the first dimension, and the samples having the same molecular weight can be further subdivided in the second dimension. Separation is possible.

  Further, as in normal gel electrophoresis, a large number of sample inlets may be provided, and a sample may be sampled at each inlet.

  In particular, when the sample is a protein, first, isoelectric focusing (using IPG-gel or the like) is performed in the first dimension, and normal electrophoresis based on molecular weight is performed in the second dimension. For this reason, in order to perform isoelectric focusing, a region for electrophoresis medium having a gradient in pH and a medium for electrophoresis (SDS-PAGE, etc.) for electrophoresis based on molecular weight should be provided in the electrophoresis medium. Is preferred.

  Since nucleic acids and proteins have a three-dimensional structure (rounded structure), it is preferable to loosen the structure by applying a pulse as a pretreatment step for performing normal electrophoresis.

  In the present electrophoresis apparatus, the sample may be modified in order to detect the sample optically in order to recognize the sample migration state (electrophoresis result). Here, “modification” refers to, for example, adding a reagent (modifying reagent) such that the sample absorbs light (fluorescence, chemiluminescence, bioluminescence), isotope labeling, light (ultraviolet light, etc.) Indicates that the sample is guided to a derivative. Note that such a reagent may be added to the electrophoresis medium 2 and modified while the sample is migrated.

  However, such a reagent, particularly a fluorescent reagent for causing fluorescence emission, may exhibit carcinogenicity. For this reason, the sample after use (after electrophoresis) needs to be handled as industrial waste. Therefore, if detection is possible without a fluorescent dye, it is not necessary to collect the fluorescent dye, and a highly practical disposable panel can be provided.

  Therefore, in the present electrophoresis apparatus, for example, in the case of FIG. 3, regardless of the presence or absence of the common electrode 3, the sample is changed by the impedance change when the sample is migrated between the pixel electrodes 4 and 4 provided on the substrate 1 b. In the case where the common electrode 3 is provided as shown in FIG. 3, the sample can also be detected by the impedance change between the pixel electrode 4 and the common electrode 3. However, as shown in FIGS. 4 and 5, it is preferable that the two opposing substrates 1a1b are both active matrix substrates. For example, the sample is detected by a change in impedance when the sample is migrated between the opposing pixel electrodes 4 and 4 provided on the two active matrix substrates 1a1b. According to this, since the distance between the electrodes can be shortened as compared with FIG. 3, the electric field strength is strong and the detection area is small, so that the portion where the sample concentration is high can be detected, so that the detection accuracy can be improved.

  There is no particular limitation on the method of measuring the impedance when the impedance is measured between the opposing pixel electrodes 4 and 4 to detect the sample. For example, a sample (DNA or the like) can be detected by applying an alternating current between the opposing pixel electrodes 4 and 4 and detecting a dielectric constant (capacitance) between the electrodes. Specifically, when only the migration medium 2 flows between the opposing pixel electrodes 4 and 4 (the sample is not migrated), the dielectric constant due to the dipole of the migration medium 2 is between the pixel electrodes 4 and 4. Is detected. On the other hand, when a sample is migrated between the pixel electrodes 4 and 4, the dielectric constant is changed between the pixel electrodes 4 and 4 by adding a dipole of the sample (DNA) (average motion of the dipole). Changes). As a result, for example, the dielectric constant in the low frequency region is increased as compared with the case where there is no sample. This is a case where the dielectric constant of the sample (DNA) is higher than the dielectric constant of the migration medium 2, and therefore the dielectric constant may be reduced depending on the migration medium or sample used.

  In this way, a sample between the pixel electrodes 4 and 4 can be detected by applying a pulse between the pixel electrodes 4 and 4 and scanning the dielectric constant. Thereby, a gene can be detected without using a fluorescent dye that is a carcinogenic substance.

  In addition to detecting capacitance, the Agilent Technologies Impedance Measurement Handbook (HYPERLINK "http://cp.literature.agilent.com/litweb/pdf/5950-3000EN.pdf" http: //cp.literature.agilent .com / litweb / pdf / 5950-3000EN.pdf), measuring the impedance between the counter electrodes with reference to the contents (circuit configuration, etc.) disclosed in Agilent Technology Co., Ltd. and Japanese Patent Laid-Open No. 4-230851 You can also

  When detecting the impedance between the pixel electrodes 4 and 4, it is preferable that there is no spacer 5 between the pixel electrodes 4 and 4. As a result, accurate impedance detection is possible.

  In this way, if the two opposing substrates 1a1b are both active matrix type substrates, electrical detection of the sample migrated between the opposing pixel electrodes 4 and 4 provided on the two substrates 1a1b is performed. Is possible. Accordingly, it is not necessary to modify the sample (fluorescence, chemiluminescence, bioluminescence, ultraviolet absorption, isotope labeling, etc.) as in normal electrophoresis. For this reason, it is not necessary to use a carcinogenic substance such as a fluorescent light-emitting reagent. Therefore, it is not necessary to collect the luminescent reagent after the completion of electrophoresis, so that a highly practical (disposable) electrophoresis apparatus can be provided.

  As described above, this electrophoresis apparatus is, for example, a thin-film flat-plate electrophoresis apparatus in which an electrophoresis medium is sandwiched between two substrates that are very narrow (on the order of μm or less). That is, the present electrophoresis apparatus includes two opposing planar substrates and a plurality of electrodes for applying a voltage to the electrophoresis medium sandwiched between the planar substrates. In other words, the electrophoretic apparatus is characterized in that a plurality of electrophoresiss are performed between them. In addition, the present electrophoresis device can be used as a liquid crystal layer in a liquid crystal display device by using a pixel electrode or a transparent electrode for migration as an electrode in a liquid crystal display device and diverting an electrophoresis medium with a liquid crystal material. Thus, a large number of electrodes for applying a voltage to the electrophoresis medium 2 are arranged throughout the electrophoresis medium 2. For this reason, it is possible to perform electrophoresis by selecting an electrode to which a voltage is applied according to the purpose and application. That is, the migration direction and migration distance can be set arbitrarily. For this reason, complicated operations such as processing of channels (flow paths) of microcapillary electrophoresis are not necessary. Therefore, it is possible to provide an electrophoresis apparatus that can be easily manufactured and is suitable for high-throughput analysis.

  In the above description, the most basic configuration has been described between the substrates 1a1b as the electrophoresis apparatus. However, other layers (for example, color filter layers, etc.) provided in a normal liquid crystal display device are provided between the substrates 1a1b. An alignment film or the like may be further provided. In other words, this electrophoresis apparatus normally uses a so-called electrophoresis buffer (such as cellulose or acrylamide) instead of a liquid crystal such as ester, biphenyl, or cyclohexane that fills the space formed through the spacer 5. Electrophoresis medium 2) or a mixture of the liquid crystal and the electrophoresis buffer solution is filled, and the electrode (pixel electrode 4) or the transparent electrode 3 formed on the sealing end of the filling material is electrically connected. It is used as a voltage application terminal for electrophoresis, and can be implemented together with light emission of each RGB pixel, which is a basic function of a conventional liquid crystal display device. Therefore, an electrophoresis apparatus having a liquid crystal display function and a sample separation function by electrophoresis can be provided.

  In addition, this electrophoresis apparatus uses the spacer 5 as a molecular sieve of a sample (biologically related substance or the like) corresponding to an arbitrary molecular weight by changing the amount of the spacer 5 used. Furthermore, this electrophoresis apparatus performs execution of electrophoretic separation of a sample (biologically related substance, etc.), data processing of the detection result of electrophoretic separation, and display of the result (electrophoresis result) on the same substrate. A simple, inexpensive, and high-performance electrophoresis apparatus having an interface is provided. The separation between electrophoresis separation (sample detection) and display of the result on the liquid crystal screen can be performed by, for example, a touch panel switch provided in the liquid crystal layer.

  When the state of electrophoresis is displayed on the liquid crystal screen in real time, for example, in order to display in real time, a backlight is provided on the back side of the substrate and excitation light (ultraviolet light) for causing the sample to emit light does not pass. A filter for passing the emitted fluorescence is provided separately (because the wavelengths of the excitation light and the detection wavelength (fluorescence wavelength) are different). For example, in the case of DNA, a filter that passes fluorescence for four bases is provided. Thus, the electrophoresis result can be displayed in real time while performing electrophoresis. Furthermore, it is preferable that real-time electrophoresis can be recorded.

  In addition, the electrophoresis apparatus can be provided with functions such as separation and concentration of biologically related substances such as DNA by filling an appropriate amount of spacers 5 between the substrates and distributing the spacers at high density. It is.

  In addition, the electrophoresis apparatus emits light using an excitation light source such as a laser or LED, and a sample (such as a DNA fragment) so that the sample can be detected by a light receiving element such as a photodiode, phototransistor, cooled CCD, or photomultiplier. It is preferable that the biological substance is modified with a fluorescent reagent in advance, or the fluorescent reagent is added to the electrophoresis medium 2 (electrophoresis buffer solution).

  Further, it is preferable that a microlens is formed on the substrate side on which the color filter is formed, and a sample (biologically related substance such as a DNA fragment) that is electrophoresed while emitting fluorescence can be directly observed by a liquid crystal display device. .

  Furthermore, part of the function of the liquid crystal display device is to drive a thin film transistor at an arbitrary position corresponding to a pixel connected to an S • G electrode (source electrode / gate electrode (X • Y electrode)). A voltage is applied between the transparent electrodes corresponding to. As a result, the liquid crystal orientation is changed and light is transmitted through the RGB filters to form an image.

  In the present electrophoresis apparatus, in addition to the function of such a normal liquid crystal display device, it is possible to apply a voltage between an upper electrode and a lower electrode that do not directly correspond to a pixel. Thereby, it is possible to use from an arbitrary position of the liquid crystal display device to an arbitrary position as an electrophoresis channel. In addition, since the electrode is provided on the substrate, it is not necessary to provide a hole for the electrode as in the conventional electrophoresis microchip. In addition, when liquid crystal is used as a migration medium, a conventional electrophoretic channel that uses a conventional liquid crystal display function at the same time is provided by providing a display area for performing normal liquid crystal display and an electrophoretic area for performing electrophoresis. Visualization (real-time display) is possible by displaying the image itself in the display area. Therefore, it is preferable that the sample inlets are periodically arranged in the display-side substrate 1a or 1b of the liquid crystal display device for electrophoresis from an arbitrary position.

  In addition, in this electrophoresis device, each pixel electrode of RGB is made up of thin film transistors so that it is possible to perform electrophoretic separation of samples containing biological materials such as DNA fragments and observe the actual movement state. It is preferable that the function of turning on and off the voltage between the electrodes and the voltage application between the pair of electrodes for electrophoresis can be performed while alternately switching.

  In addition, this electrophoresis device is in a single substrate except for a driver circuit, a data processing arithmetic circuit, a memory circuit, etc. formed by a thin film transistor of a liquid crystal display device that cannot be used for unique functions. The dedicated area for the liquid crystal display function, the dedicated area for the electrophoretic separation, and the separation / extraction area for the biological material may be separated or combined as a shared area.

  By the way, although tailor-made medicine has recently attracted attention, many channels for electrophoresis (multi-channel) are required for practical use (for example, performing mad cow disease, virus testing, infectious disease testing, etc.). The current microcapillary type array chip has the largest number of 384 channels. It is said that about 0.1% (3 million bases) determine that the DNA is about 3 billion bases present in humans. Therefore, in tailor-made medicine, 3 million bases are used. However, in order to analyze 3 million bases, several months and tens of millions of hours and costs are required. Therefore, at present, it is impossible to achieve tailor-made medicine. In order to realize this, high throughput and cost reduction are essential by 10 minutes to 100,000 yen.

  In the present electrophoresis apparatus, since the entire plate migration medium can be used as a channel, a large number of channels capable of applying a voltage to the migration medium can be formed by using an active matrix substrate. For this reason, if a gel screen medium having a large screen, such as a liquid crystal display panel, is used for the present electrophoresis apparatus, ultra-multi-channeling is possible, and 10,000 to 100,000 samples are migrated on the same plate. be able to.

  In addition, this electrophoresis apparatus is applicable to the following uses and is highly useful.

  (1) For example, different labels (e.g., different emission colors) are applied to samples that have developed disease and samples that have not developed disease. Next, a sample obtained by mixing these samples is used as a sample for electrophoresis on this electrophoresis substrate.

  Furthermore, a clinical test (diagnosis of disease onset (or possibility of onset)) can be performed by using a plot that appears only in one of the samples as an index. The present invention can also be used as a clinical examination instrument (diagnostic instrument).

  (2) Utilizing the fact that the base sequences of genes of various agricultural products differ depending on the production area, it is possible to identify the production brand name of agricultural products and the like and determine infectious diseases by genetic analysis.

  (3) Recently, the development of artificial biomaterials such as artificial organs, artificial bones, and artificial muscles has been activated, and the number of cases of transplanting them into patients has been increasing. However, when an artificial biomaterial is transplanted, it becomes stress and the gene changes. In the present invention, such a change in gene sequence can be followed up.

  The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the embodiments obtained by appropriately combining the technical means disclosed in the claims are also included in the present invention. It is included in the technical scope of the invention.

  The electrophoresis apparatus (sample detection apparatus) according to the present invention can efficiently separate and detect biologically related substances (biopolymers) such as nucleic acids and proteins such as DNA and RNA, and many other polymers. . For this reason, in the aspect of isolating and detecting genes and proteins that cause various diseases, it is particularly rapid in medical tests, pharmaceutical industries, food industries, such as clinical examinations and identification of the diagnosis origin of diseases. Use in fields that require analysis is expected.

1 is a plan view of an electrophoresis apparatus according to an embodiment of the present invention. FIG. 2 is a diagram showing a configuration that is a simple matrix type substrate in an electrophoresis apparatus according to an embodiment of the present invention, FIG. 2A to FIG. 2D are plan views, and FIG. It is AA sectional drawing of Fig.2 (a). FIG. 3 is a diagram showing a configuration in which one substrate is an active matrix type in an electrophoresis apparatus according to an embodiment of the present invention, FIGS. 3A to 3D are plan views, and FIG. e) is a sectional view taken along line AA of FIG. FIG. 4A is a plan view showing a configuration in which two substrates are both active matrix type substrates in an electrophoresis apparatus according to an embodiment of the present invention. FIG. FIG. 4E is a cross-sectional view taken along line AA in FIG. FIG. 5 is a diagram showing a configuration in which a spacer is provided in the electrophoresis apparatus of FIG. 4, FIG. 5 (a) is a plan view, and FIG. 5 (b) is a cross-sectional view taken along line AA of FIG. In the electrophoresis device concerning one embodiment of the present invention, it is a mimetic diagram showing composition which provided a spacer on a substrate. 1 is a plan view of an electrophoresis apparatus according to an embodiment of the present invention. In the electrophoresis apparatus which concerns on one Embodiment of this invention, the electrophoresis result by the presence or absence of a spacer is a graph, When FIG. 7 (a) provides a spacer, FIG.7 (b) is a case where a spacer is not provided. It is a graph. It is a graph which shows the electrophoresis result at the time of providing the spacer in the electrophoresis apparatus which concerns on one Embodiment of this invention.

Explanation of symbols

1a and 1b Substrate 2 Electrophoresis medium 3 Common electrode 4, 4a to 4l Pixel electrode 5 Spacer 6 Sample 7 Electrode X1 to X4 X electrode Y1 to Y4 Y electrode S1 to S4, S′1 to S′4 Source electrode G1 G4, G'1 to G'4 Gate electrode

Claims (12)

Two opposing substrates, and a plurality of electrodes for applying a voltage to the electrophoretic medium sandwiched between the substrates,
The electrophoresis medium is in a thin film form,
An electrophoresis apparatus, wherein a plurality of samples are migrated between the electrodes.   An electrophoresis apparatus, wherein a sample is migrated between electrodes arbitrarily selected from the plurality of electrodes.   The electrophoretic device according to claim 1, wherein the substrate is a matrix type substrate in which the plurality of electrodes are arranged in a matrix on the opposing surface of the substrate.   The electrophoresis apparatus according to claim 3, wherein the matrix type substrate is a simple matrix type substrate.   The electrophoresis apparatus according to claim 3, wherein the matrix type substrate is an active matrix type substrate.   6. The electrophoresis apparatus according to claim 5, wherein the two opposing substrates are both active matrix type substrates.   7. The electrophoresis apparatus according to claim 5, wherein the opposing electrodes of the active matrix substrate are for detecting the impedance of the sample.   The electrophoresis apparatus according to claim 1, wherein a spacer is provided between the substrates.   9. The electrophoresis apparatus according to claim 8, wherein the spacer is provided at a density corresponding to the molecular weight of the sample.   The electrophoresis apparatus according to claim 1, wherein the sample contains a biological substance.   The electrophoresis apparatus according to claim 10, wherein the biological substance is a gene. The electrophoresis apparatus according to claim 10, wherein the biological substance is a protein.

Images