JP3842600B2 - Charged mass transfer device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a charged substance transfer device for transferring charged substances such as biological substances such as genes and proteins, and more particularly to a charged substance transfer apparatus suitable for a biological substance detection apparatus.
[0002]
[Prior art]
In recent years, development of detection systems for biological substances such as genes and proteins has been promoted. For example, gene detection is used for predicting therapeutic effects by interferon. A conventional biological substance detection technique will be described with an example of prediction of therapeutic effect by interferon.
[0003]
Infection with hepatitis C is known to progress to liver cancer through cirrhosis, and one of the treatment methods is to use interferon. However, in Japan, interferon irradiation is effective only for about 20-30% of people, and even if it is effective, very strong side effects have been reported. Therefore, tailor-made medical treatment that predicts the therapeutic effect of interferon in advance and uses it only when the effect can be expected has recently started to attract attention.
[0004]
In order to predict the therapeutic effect of interferon, methods for examining the virus type and viral load at the gene level are known. This is less effective for type 1b, which is common in Japanese, but is effective for type 2a and is considered to be less effective when the viral load is as high as 106 copies / mL or more. In actual diagnosis, these combinations are often used, and prediction is sometimes difficult. Recently, a method using a single nucleotide polymorphism (SNP) present in the promoter region of a gene encoding an MxA protein as an index has been reported as a method for predicting the therapeutic effect of interferon. This is because the effect of interferon is low in the G / G type, and conversely it works effectively in the G / T type and the T / T type.
[0005]
Thus, it is becoming possible to predict the therapeutic effect of interferon by analysis at the gene level, but until now it has been a method that uses all the complicated and expensive conventional techniques (electrophoresis, microplate + EIA, etc.) to detect. There is a need for a simpler technique when conducting a clinical test.
[0006]
Against this background, genetic testing technology using a biological substance detection element called a DNA chip has recently attracted attention (Beattie et al. 1993, Fodor et al. 1991, Khrapko et al. 1989, Southern et al 1994). A DNA chip is made of a glass or silicon chip of several centimeters square with a plurality of types of DNA probes having different sequences immobilized thereon, and a sample gene labeled with a fluorescent dye or a radioisotope (RI) on the chip, or A mixture of unlabeled sample gene and labeled oligonucleotide is reacted. When a sequence complementary to the DNA probe on the chip is present in the sample, a signal derived from the label is obtained at a specific site on the chip. If the sequence and position of the immobilized DNA probe are known in advance, the base sequence present in the sample gene can be easily examined. Such a DNA chip can be used as a clinical diagnostic technique because a lot of information about the base sequence can be obtained in one test (Pease et al. 1994, Parinov et al. 1996).
[0007]
[Problems to be solved by the invention]
In the biological material detection apparatus as described above, a biological material is used to react a ligand such as a DNA probe formed on an electrode on a biological material detection element such as a DNA chip with a biological material to be detected in a sample solution. It is necessary to move the biological material in the sample liquid on the detection element. However, in the past, since the sample solution was simply flowed on the biological material detection element, there was a problem that the biological material could not necessarily be reacted with the corresponding ligand.
[0008]
In general, particularly when the detection target is a gene, since the gene concentration in the sample solution is low, it is necessary to amplify the target gene in advance by a gene amplification method such as the PCR method.
[0009]
The present invention has been made to solve such problems, and moves a charged substance so that a charged substance such as a biological substance reacts with each ligand on the charged substance detection element efficiently. It is an object of the present invention to provide a charged mass transfer device.
[0010]
It is another object of the present invention to provide a charged substance transfer device that can also concentrate a charged substance to be detected in a sample and can improve detection sensitivity when used in a biological substance detection apparatus.
[0011]
[Means for Solving the Problems]
In order to solve the above problems, the present invention moves a substrate, a plurality of electrodes arranged along a predetermined arrangement direction on the substrate, and a charged substance in a sample solution along the arrangement direction on the electrodes. A drive circuit for driving the electrode, the drive circuit applies a voltage having a polarity opposite to the charged polarity of the charged substance to a part of the plurality of electrodes, and the voltage having the opposite polarity is applied to the electrode. The position of the electrode to be applied is sequentially changed in the electrode arrangement direction. When applied to a gene detection apparatus, a ligand that reacts with a predetermined charged substance is immobilized on each of the plurality of electrodes.
[0012]
As described above, since the charged substance sequentially moves along the arrangement direction on the electrode, when applied to detection of a biological substance, it is possible to efficiently react the biological substance and each ligand immobilized on the electrode. Become.
[0013]
In the present invention, the drive circuit applies a voltage having the same polarity as the charge polarity to at least one electrode adjacent to the electrode in the arrangement direction of the electrodes with respect to the electrode to which a voltage having a polarity opposite to the charge polarity of the charged substance is applied. May be applied. When the voltage is applied to the electrode in this way, the charged substance trapped by the electrostatic attraction force on the predetermined electrode is confined on the predetermined electrode by the electrostatic repulsion force from the adjacent electrode, thereby concentrating the charged substance. Therefore, when used for biological substance detection, the detection sensitivity is improved.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments applied to the biological material detection apparatus of the present invention will be described below with reference to the drawings.
FIG. 1 is a cross-sectional view showing a configuration of a biological material detection apparatus according to an embodiment of the present invention. The base 1 has a protruding element mounting portion 2 for mounting a biological material detection element 5 to be described in detail later in the center upper part, further a sample liquid passage hole 3 on the left side in the figure, and a sample liquid passage in the lower center part. Each has a sample solution discharge port 4 communicating with the hole 3.
[0015]
On the base 1, the biological material detection element 5 mounted on the element mounting portion 2 is held from both the upper and lower sides, the sample liquid is guided to the element 5, and the sample liquid that has passed through the element 5 is sampled. An upper holder 6 and a lower holder 7 for mainly guiding to the passage hole 3 are arranged.
[0016]
A sample liquid introduction section 8 is provided at the center of the upper holder 6, and a sample liquid supply pipe 10 is connected to the sample liquid introduction section 8. The sample solution introduced from the sample solution supply pipe 10 into the sample solution introduction unit 8 is guided onto the biological material detection element 5 and used for detection of the biological material, and then the lower holder 6 and the upper holder 7 The sample liquid guide duct 9 and the sample liquid passage hole 3 formed in the base 1 are discharged to the outside from the sample liquid discharge port 4.
[0017]
A rectangular through hole 11 is formed in the upper holder 6, and the tip of the contact electrode 15 functions as a working electrode on the biological material detection element 5 by inserting the contact electrode 15 through the through hole 11. It is possible to contact the electrode. By using the contact electrode 15, the biological material detection signal can be extracted as an electrical signal such as current or potential.
[0018]
FIG. 2 is a plan view showing the configuration of the biological material detection element 5. A sample liquid receiving part 21 made of a concave portion is provided at a position directly below the sample liquid introducing part 8 shown in FIG. Further, a spiral sample liquid guide groove 22 having one end communicating with the sample liquid receiving portion 21 is formed on the element substrate 20, and a plurality of circles functioning as working electrodes along the spiral at the bottom of the guide groove 22. Shaped electrodes 23 are arranged. A sample liquid discharge portion 24 is provided at the other end of the guide groove 22 of the element substrate 20, and this sample liquid discharge portion 24 communicates with the sample liquid guide duct 9 shown in FIG.
[0019]
At least one specific detection ligand is immobilized on the electrode 23. That is, the electrode 23 also serves as a ligand immobilization part. The ligand immobilized on each of the electrodes 23 is selected from, for example, a gene, a gene probe, a protein, a protein fragment, a coenzyme, a receptor, and a sugar chain, depending on the biological material to be detected.
[0020]
If different ligands are immobilized on each of the electrodes 23, a plurality of biological substances can be detected at one time. In addition, by immobilizing the same ligand on each of the electrodes 23, it is possible to detect a large number of biological substances at once. If a large number of electrodes 23 (ligand immobilization parts) are patterned in advance on the element substrate 20 using photolithography, the mass productivity of the biological material detection element 5 is improved.
[0021]
The electrodes 23 are connected to the electrode pads 25 by, for example, multilayer wiring formed on the element substrate 20. A drive circuit 26 that drives the electrode 23 by applying a predetermined voltage to the electrode 23 is connected to the electrode pad 25. The operation of the drive circuit 26 will be described in detail later.
[0022]
Thus, the sample solution supplied from the sample solution supply pipe 10 is guided by the upper holder 6 and the lower holder 7 and introduced into the biological material detection element 5 from the center, and the ligand provided in the periphery of the element 5. After being uniformly supplied onto the immobilization section, it is discharged from below. Therefore, it is possible to detect the biological material in the sample solution under uniform conditions.
[0023]
When the detection target biological material is a gene, a DNA probe is immobilized on the electrode 23 as a ligand. As is well known, a DNA probe is a single-stranded gene that reacts with a specific gene. When the gene in the sample solution is in a single-stranded state, only a gene having a specific sequence corresponding to the DNA probe fixed to the electrode 23 is trapped by the electrode 23, and the DNA probe and its gene are eventually Complementary binding is performed (hybridization).
[0024]
More specifically, the substrate material used for the element substrate 20 is not particularly limited. For example, glass, quartz glass, alumina, sapphire, forsterite, silicon carbide, silicon oxide, nitride Inorganic green materials such as silicon can be used. In addition, polyethylene, ethylene, polypropylene, polyisobutylene, polymethyl methacrylate, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, Organic materials such as polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, polyphenylene oxide, polysulfone, etc. are used as substrate materials. It can also be used. Furthermore, if a biological material is detected by an optical method to be described later, a fiber thin film such as nylon or cellulose can be applied as a substrate material.
[0025]
The electrode material used for the electrode 23 is not particularly limited, but for an electrode including a ligand-immobilized spot, when detecting biological material electrochemically, for example, gold, gold alloy, silver , Platinum, mercury, nickel, palladium, silicon, germanium, gallium, tungsten and other simple metals and alloys containing at least two of these metals, carbon such as graphite and glassy carbon, and oxides and compounds thereof Alternatively, it is possible to use a semiconductor compound such as silicon oxide, or various semiconductor devices such as CCD, FET, and CMOS.
[0026]
As a manufacturing method of the electrode 23, plating, printing, sputtering, vapor deposition, or the like can be used. As the vapor deposition method, any one of a resistance heating method, a high frequency heating method, and an electron beam heating method can be used. As the sputtering method, any one of direct current bipolar sputtering, bias sputtering, asymmetrical alternating current sputtering, getter sputtering, and high frequency sputtering can be used. Further, an electrolytic polymer film such as polypyrrole or polyaniline or a conductive polymer can be used as the electrode.
[0027]
The insulating material used for the insulating thin film covering the surface of the electrode 23 is not particularly limited, but for example, a photopolymer or a photoresist material is preferable. As the photoresist material, a photoexposure photoresist, a far ultraviolet photoresist, an X-ray photoresist, and an electron beam photoresist are used. Photoresist for photoexposure includes cyclized rubber, polycinnamic acid, and novolak resin as main raw materials. For the deep ultraviolet photoresist, cyclized rubber, phenol resin, polymethylisopropenyl ketone (PMIPK), polymethyl methacrylate (PMMA), or the like is used. In addition, for the X-ray resist, COP, metal acrylate, and other substances described in the Thin Film Handbook (Ohm) can be used. For the electron beam resist, it is possible to use substances described in a thin film handbook (Ohm) such as PMMA. The thickness of the resist used here is desirably 10 nm or more and 1 mm or less.
[0028]
By covering the electrode 23 serving as the working electrode with a photoresist and performing lithography, the area of the electrode 23 can be made constant. As a result, the amount of ligand immobilized on the DNA probe or the like becomes uniform between the electrodes 23, and biological material detection with excellent reproducibility is possible. Conventionally, the resist material is generally finally removed. However, when the electrode 23 is used for gene detection with a DNA probe fixed thereto, the resist material is removed as a part of the electrode 23 without removing the resist material. It is also possible to use it. In this case, it is necessary to use a substance having high water resistance for the resist material to be used.
[0029]
It is possible to use a material other than the photoresist material for the green thin film formed on the electrode 23. For example, oxides, nitrides, carbides, and other alloys such as Si, Ti, Al, Zn, Pb, Cd, W, Mo, Cr, Ta, and Ni can be used. After these materials are formed into a thin film by sputtering, vapor deposition, CVD or the like, the electrode exposed portion is patterned by photolithography to control the area to be constant.
[0030]
Next, the driving operation of the driving circuit 26 will be described with reference to FIGS. The drive circuit 26 sequentially applies the voltage of a predetermined polarity to the electrode 23 to sequentially detect the detection target biological materials in the sample solution received in the sample solution receiving unit 21 provided in the central portion on the biological material detection element 5. The electrode 23 is moved in the arrangement direction.
[0031]
When the detection target biological material is a gene, a sample solution that is an aqueous solution of the gene is supplied from the sample solution supply pipe 10 to the sample solution receiving unit 21 on the biological material detection element 5 through the sample solution introducing unit 8 and guided. 22 is guided on the electrode 23. The charge polarity of the gene is negative, and FIGS. 3 to 6 show driving examples when the detection target biological material is a gene. 3 to 6 all show the temporal transition of the voltage application state with respect to the electrode 23, and the voltage application state (voltage polarity) changes in the order of (a) → (b) → (c). To do.
[0032]
The driving example of FIG. 3 will be described. First, as shown in FIG. 3A, a positive voltage having a polarity opposite to the charge polarity of the gene is applied to the electrode 23-1 closest to the sample liquid receiving unit 21. A negative voltage is applied to the electrode 23-2 adjacent to the electrode 23-1. Therefore, the sample solution supplied to the sample solution receiving unit 21 moves onto the electrode 23-1 by an electrostatic attraction force by the electrode 23-1 to which a voltage having a polarity opposite to the charge polarity of the gene is applied. At this time, if a slight negative pressure is applied to the sample solution, the sample solution can move more quickly. Since a voltage having the same polarity as the charge polarity of the gene is applied to the electrode 23-2 adjacent to the electrode 23-1, the gene is trapped on the electrode 23-1.
[0033]
Next, as shown in FIG. 3B, the polarity of the voltage applied to the electrode 23-1 is inverted to make it negative, and the polarity of the voltage applied to the adjacent electrode 23-2 is also inverted to make it positive. To do. At this time, the gene in the sample solution receives an electrostatic repulsive force from the electrode 23-1 to which a voltage having the same polarity as the charged polarity is applied, and from the electrode 23-2 to which a voltage having a polarity opposite to the charged polarity is applied. Since it receives an electrostatic attraction force, it moves from the electrode 23-1 to the electrode 23-2 and is trapped on the electrode 23-2.
[0034]
Next, as shown in FIG. 3 (c), the polarity of the voltage applied to the electrode 23-2 is reversed to be negative, and the polarity of the voltage applied to the adjacent electrode 23-3 is also reversed to be positive. Thus, the gene in the sample solution is moved from the electrode 23-2 to the electrode 23-3 and trapped.
[0035]
Similarly, the genes are sequentially adjacent by sequentially shifting the position of the electrode to which the voltage of the same polarity as the charged polarity of the gene in the sample solution and the position of the electrode to which the voltage having the opposite polarity to the charged polarity are applied. It can be moved onto the electrode. In the course of this movement, if a specific gene held on a certain electrode has a sequence complementary to the ligand immobilized on the electrode, that gene and the ligand will react and bind. become. That is, hybridization occurs.
[0036]
In this way, the gene sequentially moves on the electrode 23 along the arrangement direction in the biological material detection element 5, and is discharged from the sample liquid discharge unit 24 to the outside of the element 5.
[0037]
FIG. 4 shows another driving example of the driving circuit 26 in the present embodiment. In the driving example shown in FIG. 3, for example, referring to FIG. Shi Only the electrode 23-1 on the side opposite to the direction of gene movement of the electrode 23-2 of the electrode is the same polarity as the charge polarity of the gene negative A polarity voltage was applied. Show The electrode 23-3 on the moving direction side negative A polarity voltage may be applied. FIGS. 4A, 4B, and 4C show voltage application states at the same timing as FIGS. 3A, 3B, and 3C, respectively. The initial state shown in FIG. 3 (a), but in FIGS. 4 (b) and 4 (c), negative polarity is applied to the electrodes before and after the gene movement direction (on both sides in the arrangement direction of the electrodes 23) as viewed from the electrode to which a positive voltage is applied. Is applied.
[0038]
In this way, the negative polarity voltage is applied to both electrodes before and after the gene transfer direction with respect to the electrode to which the positive polarity voltage that traps the gene is applied. Genes can be concentrated because the electrostatic repulsion by the electrodes traps the genes on the electrodes trapping the genes. Therefore, the gene can be detected efficiently, and it is not necessary to amplify the gene in advance or the degree of amplification is reduced.
[0039]
Next, in the driving example of FIG. 5, an operation in which a positive voltage is applied to two adjacent electrodes among the electrodes 23 and a voltage having an opposite polarity is applied to an electrode adjacent to the set of the two electrodes. Are shifted by one electrode in the arrangement direction of the electrodes 23.
[0040]
Further, FIG. 6 shows a driving example in which a voltage is simultaneously applied to all the electrodes 23. Similarly to FIG. 5, a positive voltage is applied to two adjacent electrodes among the electrodes 23, and the two electrodes 23 The operation of applying a voltage of the opposite polarity to the electrodes adjacent to the set is performed by shifting one electrode at a time in the arrangement direction of the electrodes 23.
[0041]
Note that, among the electrodes 23, the number n of electrodes to which a positive polarity voltage having a polarity opposite to the charge polarity of the gene is applied, the number m of electrodes to which a negative polarity voltage having the same polarity is applied, and the polarity of the applied voltage are switched. Where n, m, and p can all be selected to an arbitrary number of 1 or more. In the simplest case, n = m = p = 1 may be used. In this case, when attention is paid to one of the electrodes 23, the polarity of the applied voltage is periodically switched alternately between positive polarity and negative polarity.
FIG. 7 shows an electrode configuration and a driving example in a biological material detection element according to another embodiment of the present invention. As shown in the figure, in this embodiment, a guide groove 32 is formed on the circumference centering on the sample liquid receiving portion 31 disposed in the central portion of the element substrate in the biological material detection element. An electrode 33 in which a ligand is solidified is arranged at the bottom of 32. The sample liquid supplied to the sample liquid receiving unit 31 is guided into the guide groove 32 and reaches the electrode 33.
[0042]
In such a configuration, as shown in FIGS. 7 (a), 7 (b), and 7 (c) by a drive circuit (not shown), a voltage is applied to the electrode 33 in the same manner as in the drive example shown in FIG. It is possible to move the detection target biological material such as the above gene 33 on the electrode 33 along the circumference.
[0043]
That is, first, as shown in FIG. 7A, a positive voltage is applied to a set of two adjacent electrodes surrounded by a dotted line in the electrode 33, and a negative voltage is applied to an electrode adjacent to this set of electrodes. Apply. Next, after a predetermined unit time, as shown in FIG. 7B, the position of the pair of two electrodes to which the positive voltage is applied is shifted by one electrode, and accordingly, the pair of two electrodes is changed. The position of the electrode to which the negative voltage adjacent is applied is also shifted by one electrode. Further, after a predetermined unit time has elapsed, as shown in FIG. 7C, the position of the pair of two electrodes to which the positive voltage is applied and the position of the electrode to which the polarity voltage is applied are shifted by one electrode. . Hereinafter, by switching the polarity of the applied voltage every unit time, that is, at a predetermined cycle, the detection target biological material (for example, gene) in the sample liquid is arranged in the circumferential direction on the adjacent electrode 33 array. It can move and react uniformly and efficiently with the ligand immobilized on each of the electrodes 33.
[0044]
In this way, it is possible to sequentially move on the electrode 33 while concentrating the detection target biological material in the sample liquid introduced into the biological material detection element. That is, according to this embodiment, since the detection target biological material is concentrated on the electrode 33 on which the ligand is immobilized, the detection target biological material is not amplified in advance by a gene amplification method such as the PCR method as in the past. The biological substance to be detected and the ligand can be reacted efficiently, and the detection efficiency is improved.
[0045]
8A and 8B are a plan view and a cross-sectional view showing a configuration of a biological material detection device according to another embodiment of the present invention.
In the biological material detection device of the present embodiment, a sample liquid receiving part 41, a recess 42 formed around the sample liquid receiving part 41 at the center on the protrusion 40A formed in the center of the base 40, and A plurality of fan-shaped electrodes 43 formed radially around the sample liquid receiving portion 41 are provided in the recess 42. A ligand is immobilized on the electrode 43. Further, the base 40 is provided with a sample liquid passage hole 44 for guiding the sample liquid that has passed through the biological material detection element to the sample liquid discharge port.
[0046]
Even in such a biological material detection device, the electrode 43 is driven in the same manner as described with reference to FIG. 7 by a drive circuit (not shown), thereby concentrating the detection target biological material in the circumferential direction in which the electrodes 43 are arranged. By sequentially moving, the detection target biological substance and the ligand can be reacted efficiently.
[0047]
FIG. 9 shows a configuration of a biological material detection element according to another embodiment of the present invention.
In the vicinity of two opposing corners on the element substrate 50, a sample liquid receiving part 51 and a sample liquid discharging part 54 are arranged. Further, a zigzag sample liquid guide groove 52 is formed between the sample liquid receiving part 51 and the sample liquid discharge part 54, and an electrode 53 with a ligand immobilized thereon is disposed in the sample liquid guide groove 52. Yes.
[0048]
Even when the biological material detection element having such a configuration is used, the electrodes 53 are arranged by arranging the detection target biological materials by driving the electrodes 53 in the same manner as described with reference to FIGS. By sequentially moving in the circumferential direction, and further concentrating the detection target biological material during the movement, the ligand can be reacted efficiently.
[0049]
FIG. 10 shows a schematic configuration of a biological material treatment apparatus having a plurality of reaction steps as still another embodiment of the present invention.
In the present embodiment, a plurality of reaction chambers 61 </ b> A to 61 </ b> F are disposed on the substrate 60, and these reaction chambers 61 </ b> A to 61 </ b> F are appropriately communicated with each other through a sample liquid guide groove 62. An electrode 63 is arranged in the sample liquid guide groove 62. For example, a ligand may be immobilized on the electrode 63.
[0050]
Also in such a biological material processing apparatus, the electrode 63 is driven by a drive circuit (not shown) in the same manner as described with reference to FIGS. 3 to 6 so that the biological material to be processed is sequentially placed in the circumferential direction in which the electrodes 53 are arranged. By moving and further concentrating the treatment target biological material during the movement, the treatment target biological material can be efficiently reacted in the reaction chambers 61A to 61F.
[0051]
Next, a biological material processing apparatus according to still another embodiment of the present invention will be described with reference to FIG. The present embodiment is an apparatus used for processing a biological material having no electric charge, and has a micelle structure using, for example, a surfactant.
[0052]
The micelle structure is a structure in which an uncharged substance is forcibly charged by coating with a micelle. In FIG. 11, the biological substance is negatively charged. As described above, even when the biological material that is forcibly charged by the micelle structure is moved, the biological material moving device and the biological material processing device having the configurations described in the above-described embodiments can be used.
[0053]
The specimen for biological material detection using the biological material transfer device according to the present invention or the biological material detection device is not particularly limited, and examples thereof include blood, serum, white blood cells, urine, stool, semen, Saliva, tissue, cultured cells, sputum and the like can be used. Here, when the detection target substance is a gene, for example, a gene is extracted from these sample specimens. The extraction method is not particularly limited, and a liquid one-liquid extraction method such as a phenol-chloroform method or a solid-liquid extraction method using a carrier can be used. Further, a commercially available nucleic acid extraction method QIAamp (manufactured by QIAGEN), Sumitest (manufactured by Sumitomo Metals), or the like can also be used.
[0054]
Next, the gene sample solution thus extracted is introduced onto the biological material detection element (DNA chip) described in the above embodiment, and a hybridization reaction is performed on an electrode on which a DNA probe as a ligand is immobilized. . The reaction solution is, for example, a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10. In this solution, dextran sulfate which is a hybridization accelerator, salmon sperm DNA, bovine thymus DNA, EDTA, surfactant and the like can be appropriately added. It is necessary to add the sample gene extracted here and heat denature at 90 ° C. or higher before introducing it into the biological material detection element. Unreacted sample genes are collected from the sample solution outlet 4 and can be reintroduced into the element for detecting a biological material as necessary.
[0055]
The extracted gene is previously labeled with a fluorescent dye such as FITC, Cy3, Cy5, rhodamine, an enzyme such as biotin, hapten, oxidase or phosphatase, or an electrochemically active substance such as ferrocene or quinones, or Detection is possible by using a second probe labeled with these substances. When labeled with a fluorescent dye, optical detection is possible.
[0056]
When detection is performed using an electrochemically active DNA binding substance, detection is performed according to the following procedure.
Electrochemical measurement is performed by allowing a DNA binding substance to selectively bind to the double-stranded DNA portion formed on the surface of the electrode (working electrode) on which the DNA probe is immobilized. Although the DNA binding substance used here is not particularly limited, for example, Hoechst 33258, acridine orange, quinacrine, dounomycin, metallointercalator, bisintercalator such as bisacridine, trisintercalator, polyintercalator, etc. It is possible to use. Furthermore, these intercalators can be modified with an electrochemically active metal complex such as ferrocene or viologen.
[0057]
The concentration of the DNA-binding substance varies depending on the type, but is generally used in the range of 1 ng / ml to 1 mg / ml. At this time, a buffer solution having an ionic strength of 0.001 to 5 and a pH of 5 to 10 is used.
[0058]
After the electrode as the working electrode is reacted with the DNA binding substance, it is washed and subjected to electrochemical measurement. The electrochemical measurement is performed with a three-electrode type, that is, a reference electrode, a counter electrode, and a working electrode, or with a two-electrode type, that is, a counter electrode and a working electrode. In the measurement, a potential higher than the potential at which the DNA binding substance reacts electrochemically is applied, and the reaction current value derived from the DNA binding substance is measured. At this time, the potential can be swept at a constant speed, applied with a pulse, or a constant potential can be applied. For the measurement, current and voltage are controlled by using a potentiostat, a digital multimeter, a function generator, or the like.
[0059]
[Example 1]
The biological substance detection element described in FIG. 2 was configured as an interferon therapeutic effect prediction DNA chip, and the following experiment was performed.
First, chromosomal DNA was collected from human leukocytes, and a fragment of about 100 bp in the MxA gene portion was PCR amplified using an appropriate primer. After amplification, the heat-denatured product was introduced into a DNA chip. A DNA probe related to SNP (single nucleotide polymorphism) present in the MxA gene is immobilized on the electrode 33 of the DNA chip in advance. After the introduction of the sample solution, the electrode 23 is driven, and finally the excess sample solution is observed. Then, the sample 23 is washed with a buffer solution, and a DNA binding substance (Hoechst 33258) is applied. It was.
[0060]
Furthermore, in this case, as described with reference to FIG. 6, by switching the polarity of the voltage applied to the electrode 23, the introduced gene is moved to the periphery while being concentrated, and the gene is trapped on the electrode 23. It was found that the therapeutic effect can be predicted with high accuracy without performing PCR amplification by switching the polarity of the voltage applied to each electrode 23 and moving the gene on the electrode 23 array.
[0061]
[Example 2]
The biomaterial detection element described in FIG. 9 was configured as a tumor marker detection chip, and the following experiment was performed.
Antibodies for various human tumor markers were immobilized on the surface of the electrode 53 in order to constitute a tumor marker detection chip. In order to prevent non-specific adsorption, 1% bovine serum albumin was allowed to act. In the same manner as in Example 1, the charge of the electrodes was changed in order, and when tumor markers were detected using human serum as a sample, it was shown that detection was possible with high sensitivity and reproducibility up to the order of 0.1 ng / mL. . In this case, the second antibody labeled with peroxidase derived from radish was used, and the substrate for the luminescence detection system was flowed last.
[0062]
In the above-described embodiment, the biological material transfer device has been described. However, the present invention is not limited to the biological material transfer device, and can be applied to all charged material transfer devices having a predetermined charge polarity. .
[0063]
【The invention's effect】
As described above, according to the present invention, the biological material can be moved so that the biological material reacts with each ligand on the biological material detection element efficiently.
[0064]
Furthermore, according to the present invention, it is possible to provide a biological material transfer device capable of concentrating the detection target biological material in the sample and achieving an improvement in detection sensitivity when used in the biological material detection device.
[Brief description of the drawings]
FIG. 1 is a cross-sectional view showing a configuration of a biological material detection apparatus according to an embodiment of the present invention.
FIG. 2 is a plan view showing a schematic configuration of a biological substance detection element according to the embodiment.
FIG. 3 is a view showing a first driving example of the biological material detection element according to the embodiment;
FIG. 4 is a view showing a second driving example of the biological material detection element according to the embodiment;
FIG. 5 is a view showing a third driving example of the biological material detection element according to the embodiment;
FIG. 6 is a view showing a fourth driving example of the biological material detection element according to the embodiment;
FIG. 7 is a diagram for explaining an electrode configuration of a biological material detection element according to another embodiment of the present invention and a concentration and movement operation of a detection target biological material in the biological material detection element;
FIGS. 8A and 8B are a plan view and a cross-sectional view showing a configuration of a biological material detection device according to another embodiment of the invention. FIGS.
FIG. 9 is a plan view showing a configuration of a biological material detection element according to another embodiment of the present invention.
FIG. 10 is a plan view showing a schematic configuration of a biological material treatment apparatus according to an embodiment of the present invention.
FIG. 11 is a plan view showing a schematic configuration of a biological material treatment apparatus according to still another embodiment of the present invention.
[Explanation of symbols]
1 ... Base
2 ... Element placement part
3 ... Sample solution passage hole
4 ... Sample solution outlet
5 ... Element for detecting biological material
6 ... Upper holder
7 ... Lower holder
8 ... Sample solution introduction part
9 ... Sample liquid guide duct
20 ... element substrate
21 ... Sample liquid receiving part
22 ... Sample liquid guide groove
23 ... Electrode
24. Sample liquid discharge part
25 ... Electrode pad
26 ... Drive circuit
31 ... Sample liquid receiving part
32 ... Sample liquid guide groove
33 ... Electrode
40 ... Base
41 ... Sample liquid receiving part
42 ... recess
43 ... Electrode
44 ... Sample solution discharge part
50: Element substrate
51. Sample liquid receiving part
52 ... Sample liquid guide groove
53 ... Electrode
54 ... Sample liquid discharge part
60 ... Board
61A-61F ... Reaction chamber
62 ... Sample liquid guide groove
63 ... Electrode

Claims (3)

In an apparatus for moving a charged substance having a predetermined charge polarity,
A substrate having guide grooves ;
A plurality of electrodes arranged along a predetermined arrangement direction at the bottom of the guide groove ;
Applying a voltage having a reverse polarity to the charge polarity of the charged substance to a part of the plurality of electrodes and applying the reverse polarity voltage to the electrodes on both sides adjacent to each other in the arrangement direction. The charged substance is arranged on the plurality of electrodes by performing a driving operation in which a voltage having the same polarity as the charge polarity is applied and a position of the electrode to which the reverse polarity voltage is applied is sequentially changed in the arrangement direction. charged mass transfer apparatus comprising a drive circuit for moving along the direction. The charged mass transfer apparatus according to claim 1, wherein the guide groove has a spiral shape .   In an apparatus for moving a charged substance having a predetermined charge polarity,
  A substrate having a recess centered around the charged substance receiving portion;
  A plurality of fan-shaped electrodes formed radially in the concave portion around the receiving portion and arranged along a predetermined arrangement direction;
  A voltage having a polarity opposite to the charge polarity of the charged substance is applied to some of the plurality of electrodes, and the electrodes on both sides adjacent to each other in the arrangement direction with respect to the electrode to which the voltage having the opposite polarity is applied The charged substance is arranged on the plurality of electrodes by performing a driving operation in which a voltage having the same polarity as the charge polarity is applied and a position of the electrode to which the reverse polarity voltage is applied is sequentially changed in the arrangement direction. A charged substance moving device comprising a drive circuit for moving along a direction.

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