JP2012196189A - Cell detection device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell detection device detecting presence/absence of a target cell type with high accuracy.SOLUTION: This cell detection device includes a substrate 1, and a capacitance detection element disposed on the substrate 1. The capacitance detection element comprises: a first electrode P1 disposed in a state that one portion thereof contacts with the substrate 1, that the other portion is separated from the substrate 1, and that an end part on the opposite side to the one portion that is an end part of the other portion contacts with the substrate 1; and a second electrode P2 disposed in a state that the second electrode P2 at least partially faces to the separated portion of the first electrode P1 and that the second electrode P2 contacts with the substrate 1. The cell detection device includes a flow passage for cells formed between the separated portion of the first electrode P1 and the substrate 1.

Description

  The present invention relates to a cell detection device. More specifically, the present invention relates to a device that senses the presence of each cell size, or a device that measures the number of cells for each cell size.

  Conventionally, a technique for measuring a capacitance between two electrodes and a frequency characteristic thereof has been used to sense a target substance. Specifically, there is a technique disclosed in Patent Document 1. In this technique, a groove is formed in the substrate, and an electrode parallel to the wall surface of the groove is formed. By placing probe molecules that bind specifically to the target substance at the bottom of the groove, the target substance is retained between the electrodes, the capacitance change between the electrodes caused by the presence of the target substance is detected, and the target substance is detected. Yes.

  Moreover, there exists a technique shown by patent document 2 as a technique which sifts a cell for every magnitude | size. In this technique, channels having different sizes are formed, and a cell-containing liquid is poured into the channel, so that the cells flow into the channel according to the size, and as a result, the cells are separated for each cell size. You can make a picture.

JP-T 2009-529683 JP-A-8-23967

  Of the two technologies described above, in the technique of Patent Document 1, when the target substance is spherical, point contact occurs when the target substance and the electrode or the target substance and the bottom of the groove are in contact with each other. There are few probe molecules to capture. As a result, the target substance is likely to be released between the electrodes. Therefore, it becomes difficult to sense the capacitance change between the electrodes, and the target substance detection capability of the apparatus becomes small. In the technique of Patent Document 2, fractionation can be performed for each cell size contained in a sample, but the presence or absence of a target cell type cannot be detected.

  Accordingly, an object of the present invention is to provide a cell detection device capable of detecting the presence or absence of a target cell type with high accuracy.

  The inventor of the present application has intensively studied to solve the above problems, and as a result, is a cell detection device including a substrate and a capacitance detection element disposed on the substrate, wherein the capacitance detection element is relative to the substrate. A first electrode disposed in contact with the substrate, a part of which is in contact with and part of the other part is spaced apart and an end of the other part opposite to the part; and A cell formed between the spaced apart portion of the first electrode and the substrate, the second electrode disposed opposite the spaced apart portion of the first electrode and in contact with the substrate; The present invention has been completed by finding that the above-mentioned problems can be solved by a cell detection device characterized by comprising the above-mentioned flow path.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to provide the cell detection apparatus which can detect the presence or absence of the target cell type with high precision.

It is the perspective view (a) and side view (b) which show roughly one Example of the specific structure of the cell detection apparatus concerning this invention. It is a side view which shows roughly the various modifications of the cross section or side surface shape of an electrode pair in the cell detection apparatus concerning this invention. It is a figure showing the flow of the formation method of the electrode pair used for the cell detection apparatus which concerns on this invention. It is a figure showing the flow of the formation method of the various electrode pair from which the shape of a 1st electrode differs following the flow represented by FIG. It is a figure explaining the target specific mechanism with which the cell detection device concerning the present invention is provided. It is a side view showing the diagnostic apparatus which is one application example of the cell detection apparatus concerning this invention.

  Hereinafter, an embodiment of a cell detection device according to the present invention will be described. The cell detection device according to the present invention includes a substrate made of an arbitrary material and a capacitance detection element arranged on the substrate. The capacitance detection element is arranged such that a part thereof is in contact with the substrate and the other part is separated and an end of the other part is in contact with the substrate. And a second electrode disposed so as to be in contact with the substrate and at least a part of the first electrode spaced apart from the first electrode. Hereinafter, the first electrode and the second electrode may be referred to as an electrode pair. The cell detection device according to the present invention includes a cell flow path formed between the separated portion of the first electrode and the substrate, and at least a part of the second electrode exists in the cell flow path.

  FIG. 1 shows an embodiment of a specific structure of a cell detection apparatus according to the present invention. In FIG. 1, the substrate 1 and the first electrode (P1) having a flat part and a curved part are sandwiched between the second electrode (P2). In the example shown in FIG. 1, the cross section or the side surface of the cell flow path formed by the first electrode (P1) and the substrate 1 has an arc shape. The shape is not limited. It may be a shape having a corner instead of a curved shape, and may be, for example, a rectangle as shown in FIG. 2A or a triangle as shown in FIG.

  In the present invention, the cell flow path means a space in which a solution containing cells can enter. That is, a cell flow path is formed by the presence of a portion (hereinafter referred to as an opening) that communicates the space between the first electrode P1 and the substrate 1 with other spaces. There may be at least one opening, and preferably two or more openings. That is, it is preferable from the viewpoint of detection efficiency that it has two or more openings and the solution flows from one opening to another. At this time, it is more preferable that the solution always flows from one opening to another opening.

On the surface of the first electrode and / or the second electrode, as shown in FIGS. 1 and 2, the probe molecule 4 for retaining the target substance between the first electrode P1 and the second electrode P2 is provided. May be fixed. Specific examples of the probe molecule 4 include non-functional molecules, single functional molecules, bifunctional molecules, multifunctional molecules, oligomers, polymers, catalysts, cells, bacteria, viruses, enzymes, proteins, heptanes and saccharides. , Lipids, glycogen, enzyme inhibitors, enzyme substrates, neurotransmitters, hormones, antigens, antibodies, DNA, RNA. Specific examples of the material of the substrate 1 include semiconductor materials such as Si and GaAs, oxides such as SiO ₂ , SiN, and TiO ₂ , and organic structures such as nitride insulators and acrylic plates. Structure.

  For cell detection, a time-dependent voltage is applied to at least one of the first electrode P1 and the second electrode P2, and a current generated by the voltage application is measured. As a result, the current amplitude and phase relative to the voltage amplitude and phase can be measured. By performing such measurement, the capacity of the electrode pair can be measured. At this time, if cells exist between the electrode pairs, the capacitance of the electrode pair changes. Cells can be detected by measuring this change in volume.

  Next, an example of a method for forming the first electrode P1 and the second electrode P2 will be described with reference to FIGS. First, as shown in FIG. 3, in order to form the second electrode (P2) on the substrate 1 using a photoresist, patterning is performed by primary development. Subsequently, after depositing the metals 6a and 6b by using a metal deposition method such as vapor deposition or sputtering, the lower electrode is deposited, and then the excess metal 6a and the photoresist 2 are removed from the substrate 1 by lift-off or etching. Thus, the second electrode 6b (P2) is formed. Subsequently, the photoresist 3 is applied again.

  After applying the photoresist 3, the process proceeds to the flow of FIG. 4 to perform patterning of the photoresist 3. At this time, an operation for determining the shape of the first electrode P1 is performed. That is, the size of the first electrode can be determined by the pattern size of the photoresist 3, and the spatial shape of the cell flow path can be determined by the three-dimensional structure of the pattern of the photoresist 3.

  For example, when the first electrode P1 is curved and the cross-sectional shape or side surface shape of the cell channel is an arc shape, the heat treatment is performed after the photoresist patterning according to the flow shown in FIG. Thus, the patterning shape can be a desired arc shape.

  Further, in the case of patterning the first electrode P12 having a triangular cross-sectional shape or side surface shape of the cell flow path, the irradiation amount of the exposure light beam 7 that irradiates the photoresist according to the flow shown in FIG. The desired patterning shape of the triangle can be obtained by giving a change to the above and minimizing the exposure amount of the portion desired to be the vertex of the triangle.

  After a desired patterning shape is obtained, after forming a photoresist pattern for forming the first electrodes P1 and P12, vapor deposition or sputtering is performed in the same manner as when the second electrode 6b (P2) is formed. The first electrodes P1 and P12 having arbitrary shapes are formed by laminating metals using a metal film forming method such as the above, and removing excess metal and photoresist from the substrate 1 by lift-off or etching. When the probe molecule 4 is formed on the surface of the first electrode P1, P12 and / or the second electrode 6b (P2), the probe molecule 4 is further patterned so that an arbitrary part of the electrode is exposed. What is necessary is just to fix to the surface.

  The mechanism at the time of using the cell detection apparatus of this invention is demonstrated using FIG. For example, when eosinophils in the blood are used as detection target cells (targets), the eosinophil antibody 8 that specifically binds the eosinophils to the probe molecule 4 on the surface of the electrode pair is used as an eosinophil. The sphere 9 can selectively stay between the electrode pair for a long time. This effect is further enhanced by the structure of the first electrode P1, which is the curved upper electrode shown in FIG. That is, by setting the curvature radius of the curved first electrode P1 to 15 μm which is the diameter of the eosinophil 9, cells having a diameter of 15 μm or more cannot enter between the electrode pairs. Therefore, with the structure of the present invention, a size limit can be imposed on a substance that can enter between electrode pairs. As a result, the specificity for the target is improved. Further, since the structure of the curved first electrode P1 shown in FIG. 5 (a) is an electrode structure along the surface of the eosinophil 9 which is spherical, the first electrode shown in FIG. 5 (b). Compared to the case where P13 is parallel to the second electrode P2, the number of antibodies on the electrode that can bind to the eosinophil 9 increases. As a result, the target eosinophil 9 can be held very strongly between the electrode pairs, and the sensing ability for eosinophil cells can be significantly improved.

  The electrode pairs as described above can be applied to cell selection and screening by changing the size, shape and probe molecules and arranging them vertically and horizontally. As a specific example, FIG. 6 shows an example of a diagnostic apparatus that can be used for a disease diagnostic test using blood.

  Blood tests are frequently used in health care and disease diagnosis. As pathogenic cells that can be contained in the blood, for example, hepatitis virus 13, Mycobacterium tuberculosis 17, and cancer cell 21 have diameters of 40 nm, 400 nm, and 5 μm, respectively. Three electrode pairs each having curved first electrodes P1a, P1b, and P1c having a radius of curvature comparable to these sizes are formed on one substrate 1, and each electrode surface corresponds to each pathogenic cell. By binding probe molecules such as antibodies 12, 16, and 20 and arranging these electrode pairs vertically and horizontally, it is possible to sense pathogenic cells contained in the blood with high accuracy from a small amount of blood. . Therefore, it is possible to configure a diagnostic apparatus that can easily and accurately perform disease diagnosis on pathogenic cells of other items from a small amount of blood using the cell detection apparatus according to the present invention.

1: Substrate 2, 3: Photoresist 4: Probe molecule 6a, 6b: Metal 7: Exposure light 8: Anti-eosinophil antibody 9: Eosinophil 12: Anti-hepatitis virus antibody 13: Hepatitis virus 16: Anti-tuberculosis antibody 17: Mycobacterium tuberculosis 20: Anti-cancer cell antibody 21: Cancer cells P1, P11, P1a, P1b, P1c, P12, P13: First electrode P2, P2a, P2b, P2c: Second electrode

Claims (2)

At least a portion of the second electrode is present in the flow path of the cell;
A cell detection device comprising a substrate and a capacitance detection element disposed on the substrate,
A part of the capacitance detecting element is in contact with the substrate and another part is separated from the substrate, and an end of the other part is in contact with the substrate. And a second electrode disposed at least partially opposite to the spaced apart portion of the first electrode and in contact with the substrate,
A cell detection device comprising a cell channel formed between the spaced apart portion of the first electrode and the substrate. Two or more capacitance detection elements are disposed on the substrate;
The shape of the other part of the first electrode included in each capacitance detection element is different, whereby two or more flow paths of the cells are provided in different shapes. The cell detection apparatus according to 1.

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