JP2010008256A - Detection chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detection chip of a simple structure with its cost reduced for detecting a measuring object even where sample solution is little. <P>SOLUTION: In this detection chip, a liquid-permeable spacer 2 with molecules 1 having a molecule recognizing function fixed thereto is patterned on a substrate 3 so as to serve as a flow path 6 for a liquid sample. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a detection chip, and more particularly to a detection chip that can be measured with a small amount of sample solution and has a simple structure.

Bovine mastitis develops when microorganisms such as bacteria invade the breast of a milking cow. In milking cows infected with this causative mastitis causative agent, a decrease in milk yield and quality is observed, and when it becomes serious, not only milk is not produced at all, but the cow itself may die. It is particularly important to deal with mastitis in milking cows at the very early stages of onset, since it is very difficult to cure once they are affected. Therefore, establishment of an efficient early diagnosis method is strongly desired.
Conventionally, a culture test that has been conducted as a search for microorganisms (causative bacteria) that cause bovine mastitis requires at least several days. In addition, since the optimal therapeutic drug and coping method differ depending on the type of causative bacteria, development of a method capable of detecting specific bacteria quickly and quantitatively from a plurality of types of bacteria has been awaited.

  Regarding identification of causative bacteria, in addition to conventional identification methods using culture, identification methods by staining, and methods for measuring substances related to bacterial metabolism such as ATP, SPR (Surface using surface plasmon resonance method) Bacterial identification methods have been developed by immunoassay. In particular, the SPR immunoassay is being applied in various forms because it can be measured quickly with high sensitivity using a small amount of measurement sample.

  In SPR immunoassay, a sample solution is transferred to the surface of a thin film on which an antibody is immobilized, and a measurement object is detected by using a change in the refractive index of the thin film surface due to an antigen-antibody reaction. At this time, in order to transfer the sample solution to the region where the antibody is immobilized, a flow path composed of minute grooves and pipes formed on the surface of the substrate is used. At this time, in order to transfer the sample solution, power of a pump or the like is necessary, and more sample solution is required to arrange the pipe or the like.

However, when an antigen-antibody reaction is used for identification of causative bacteria, etc., multiple measurements may be required, and it has been desired to develop a detection chip that can be detected with a smaller amount of sample solution. For example, in Patent Document 1, a detection chip in which a minute flow path is formed using a semiconductor processing technique has been developed. In this method, since a minute flow path can be formed on the substrate, the object to be measured can be detected with a small amount of sample solution.
JP 2005-24483 A

  However, when the flow path is formed on the substrate using the semiconductor processing technique, the operation is complicated and a special apparatus is required, resulting in an increase in manufacturing cost.

  The present invention has been made in view of the above circumstances, and can detect an object to be measured even when the amount of sample solution is small, and can detect costs with a simple structure. The purpose is to provide a chip.

The detection chip according to claim 1 of the present invention is characterized in that a liquid permeable spacer to which a molecule having a molecular recognition function is fixed is patterned on a substrate so as to be a flow path for a liquid sample. To do.
A detection chip according to a second aspect of the present invention is the detection chip according to the first aspect, wherein the spacer is a bead.
The detection chip according to a third aspect of the present invention is the detection chip according to the first aspect, wherein the spacer is a fiber.
A detection chip according to a fourth aspect of the present invention is the detection chip according to any one of the first to third aspects, wherein the substrate has flexibility.
A detection chip according to a fifth aspect of the present invention is the detection chip according to any one of the first to fourth aspects, wherein the substrate is transparent.
A detection chip according to a sixth aspect of the present invention is the detection chip according to any one of the first to fifth aspects, characterized in that a gold thin film is disposed on the part to be measured of the substrate.
A detection chip according to a seventh aspect of the present invention is the detection chip according to the sixth aspect, wherein the spacers are arranged in a straight line.

  According to the detection chip of the present invention, the liquid sample penetrates onto the liquid-permeable spacer on which molecules having a molecular recognition function are arranged. Therefore, the amount of sample required for measurement can be greatly reduced, and the liquid sample can be reduced. Even when there are few, it becomes possible to measure sufficiently by SPR etc. In addition, since the liquid-permeable spacer in which molecules having a molecular recognition function are arranged on the substrate is simply arranged in a desired pattern, a simple structure can be achieved and cost can be reduced.

  Hereinafter, the present invention will be described in detail with reference to the drawings. However, the present invention is not limited thereto, and various modifications can be made without departing from the gist of the present invention.

<First Embodiment>
FIG. 1 is a diagram schematically showing a detection chip according to the first embodiment of the present invention. FIG. 1A is a perspective view, and FIG. 1B is an LL cross-sectional view in FIG. In the detection chip 10A (10) of the present invention, a liquid permeable spacer 2 on which molecules 1 having a molecular recognition function are fixed is arranged on a substrate 3 in a patterned manner. Hereinafter, each will be described in detail.

  The molecule 1 having a molecular recognition function is a molecule that recognizes and specifically binds to a specific molecule. For example, an antibody, an enzyme, a nucleic acid, a membrane protein, a biomolecule-binding molecule (for example, a compound such as a drug, Sugar chain, aptamer, molecular imprint compound, etc.) and lectin. These may be biological samples, cultures obtained from biological samples, those extracted from these samples, or synthesized by known methods. These extraction methods are not particularly limited, and can be performed by a conventionally known method.

  The liquid permeable spacer 2 is coupled with the molecule 1 having a molecular recognition function, and can transfer the sample solution added to the detection chip 10A. In this embodiment, the case where the bead 2a is used as the liquid-permeable spacer 2 is illustrated.

  The beads 2a are arranged on the substrate 3 at a predetermined interval, and molecules 1 having a molecule recognition function are arranged on the surface thereof. The bead 2a is composed of, for example, agarose gel, polystyrene, metal fine particles alone or a mixture thereof, and the diameter thereof is, for example, 1 μm or more and 100 μm or less. If it is less than 1 μm, the flow rate is remarkably slow. However, in the case of a mixture of metal fine particles and polystyrene, the metal fine particles may be 20 nm or more and the diameter of polystyrene or less. By arranging the beads 2a on the substrate 3 at a predetermined interval, the sample solution added to the detection chip 10A can permeate through the beads 2a due to surface tension, and acts as the flow path 6. The interval at which the beads 2a are arranged can be appropriately adjusted according to the viscosity and flow rate of the sample solution, the size of the beads 2a, etc., for example, at intervals not less than 10 times the diameter of the beads. It can be provided on the substrate 3. Further, the molecule 1 having a molecular recognition function may be directly bonded to the bead 2a, or the surface of the bead 2a may be coated with, for example, a gel such as agarose, polyethylene glycol having an amino group introduced into a side chain, and the like. Molecules 1 having a molecular recognition function may be bound to the beads 2a through the coating layer.

  By arraying the molecules 1 having different types of molecular recognition functions to the beads 2a and patterning them on the substrate 3, it can be easily arrayed. For example, in FIG. 1, FIG. 2 and FIG. 3 (a), the beads 2aA in the A row are arranged with an antibody against a kind of causative bacteria causing bovine mastitis, and the beads 2aB in the B row are An antibody against another kind of causative bacteria that causes bovine mastitis is arranged, and an antibody against another kind of causative bacteria that causes bovine mastitis is arranged on the beads 2aC in the row C, for example, in SPR. By detecting the antigen-antibody reaction, it is possible to easily identify multiple types of causative bacteria at once.

  The substrate 3 has a liquid permeable spacer 2 (beads 2a) in which molecules 1 are arranged on one surface 3a thereof arranged at a predetermined interval, and a flow path 6 is formed. The substrate 3 is not denatured or deactivated by the molecule 1 having a molecular recognition function, and does not inhibit the interaction when the molecule 1 having a molecular recognition function interacts with a target molecule. For example, a glass substrate, a plastic substrate, a plastic film, a plastic sheet and the like can be used without particular limitation.

  The substrate 3 preferably has flexibility. In particular, if it is in the form of a film, it can be wound and stored in a state where the beads 2 a are arranged on the substrate 3. Therefore, it can be stored in a space-saving manner and can be manufactured while winding up the substrate 3 on which the beads 2a are arranged, so that mass productivity can be improved. Examples of such a substrate 3 include a PET film, a PVDF film, and polyester.

  According to the detection chip 10A of the present embodiment, the sample solution added on the detection chip 10A is transferred to the flow path 6 by the surface tension of the beads 2a or the substrate 3. Therefore, a power source is not required for transferring the sample solution, and the sample solution is disposed only in the vicinity of the beads 2a, so that the sample solution can be measured in a very small amount. The channel 6 may be connected to a material that absorbs the liquid sample.

The substrate 3 is preferably transparent. In addition, as shown in FIG. 2, it is preferable that a gold thin film 4 is disposed on at least a portion to be measured of the substrate 3. Thus, the detection chip 10A of the present invention can be used for SPR measurement. 2A is a perspective view, and FIG. 2B is an LL cross-sectional view in FIG. 2A.
In the SPR measurement, light is incident at a critical angle or more from a surface 3b opposite to the surface 3a on which the gold thin film 4 is disposed, and the evanescent wave that has exuded to the surface 4a of the gold thin film 4 and the surface plasmon resonate. Measure the angle. Therefore, the thickness of the gold thin film 4 is preferably 40 nm or more and 50 nm or less so that an evanescent wave can be observed.

  Further, when the detection chip 10A of the present invention is used for SPR measurement, it is preferable that the liquid-permeable spacer 2 (bead 2a) in the measurement target portion on the gold thin film 4 is arranged linearly. Since the evanescent wave is used for the measurement by SPR as described above, if the liquid permeable spacer 2 is not arranged in a straight line, the measurement by the SPR becomes difficult.

  In the present embodiment, as shown in FIG. 3A, the beads 2 a may be arranged so as to be sandwiched between the first substrate 3 (substrate 3) and the second substrate 5. The sample solution added to the detection chip 10A can be more efficiently transferred through the flow path 6 made of the beads 2a due to the surface tension between the beads 2a and the substrates 3 and 5. At this time, as shown in FIG. 3B, the flow path 6 of the sample solution can be formed by arranging the beads 2a in two rows.

  When measuring the sample solution using the detection chip 10 </ b> A of the present embodiment, first, the sample solution is added to the vicinity of the beads 2 a disposed on the end portion of the substrate 3, for example. The added sample solution permeates between the beads 2 a due to the surface tension of the beads 2 a and is transferred to the entire flow path 6. Thereafter, the reaction is performed at a predetermined temperature for a predetermined time. The predetermined temperature and time may be appropriately adjusted according to the reaction between the molecule 1 having a molecular recognition function and the molecule interacting with the molecule 1 having the molecular recognition function. When molecule 1 is an antibody and the interacting molecule is an antigen, it is sufficient to carry out the reaction at 37 ° C. for 15 minutes. Thereafter, when performing ELIAS measurement or the like, washing is performed as appropriate, and the secondary antibody is fed. In the case of SPR measurement, the presence or absence of a reaction with the molecule 1 having a molecular recognition function and the intensity of the reaction can be measured by performing the measurement as it is.

Second Embodiment
FIG. 4 is a diagram schematically showing a detection chip 10B according to the second embodiment of the present invention. 4A is a perspective view, and FIG. 4B is an LL cross-sectional view in FIG. 4A. The detection chip 10B of the present embodiment is different from the detection chip 10A of the first embodiment in that fibers 2b are used as the liquid permeable spacer 2.

  The fiber 2b is not particularly limited as long as it is hydrophilic and can distribute the molecule 1 having a molecular recognition function. For example, vegetable cellulose fibers made of cellulose, hemicellulose, etc., chitin And the like, carbon fibers, glass fibers, rayon fibers, fibers composed of natural polyamino acids, fibers composed of polyamide bonds, and the like. Further, the molecule 1 having a molecular recognition function may be directly bonded to the fiber 2b, or the surface of the fiber 2b is coated with, for example, a gel such as agarose, polyethylene glycol having an amino group introduced into a side chain, and the like. Molecules 1 having a molecular recognition function may be bonded to the fibers 2b through the coating layer.

  By binding molecules 1 having different types of molecular recognition functions to the fibers 2b and patterning them on the substrate 3, it can be easily arrayed. For example, in FIGS. 4 to 5, the fiber 2bD in the region D is arranged with an antibody against one of the causative bacteria causing bovine mastitis, and the fiber 2bE in the region E has bovine mastitis. An antibody against another causative causative fungus is arranged, and an antibody against another causal causative fungus causing bovine mastitis is arranged on the fiber 2bF in the region F, for example, an antigen antibody by SPR. By detecting the reaction, it is possible to easily identify a plurality of types of causative bacteria at once. In addition, fibers on which antibodies against the respective causative bacteria are arranged may be prepared and arranged on the same substrate.

  As shown in the present embodiment, by using the fibers 2b as the spacers 2, the molecules 1 having a molecular recognition function can be simply and highly arranged on the substrate 3 through the fibers 2b. Further, since the sample solution travels through the fiber 2b, there is no need for a sample solution flow path comprising a power source and grooves or pipes as in the prior art, and the object to be measured can be detected with a minimum amount of sample solution. . Furthermore, by using the fiber 2b as the liquid-permeable spacer 2, the fiber 2b combined with the molecule 1 having a molecular recognition function can be wound and stored, so that it can be stored in a small space and mass productivity can be improved.

  Also in the present embodiment, when the detection chip 10B is used for measurement by SPR, the substrate 3 is transparent as in the first embodiment, and at least the portion to be measured of the substrate 3 is as shown in FIG. It is preferable that the gold thin film 4 is disposed on the surface. Further, in the portion to be measured on the gold thin film 4, the fibers 2b are preferably arranged in a straight line. 5A is a perspective view, and FIG. 5B is an LL cross-sectional view in FIG. 5A.

  Further, as shown in FIG. 6, the fibers 2 b may be arranged so as to be sandwiched between the first substrate 3 (substrate 3) and the second substrate 5. Due to the surface tension between the fiber 2b and the substrates 3 and 5, the sample solution can be more efficiently transmitted through the fiber 2b and diffused.

  When measuring the sample solution using the detection chip 10 </ b> B of the present embodiment, first, the sample solution is added to the vicinity of the fiber 2 b disposed on the end portion of the substrate 3, for example. The added sample solution permeates the fibers 2b and is transferred to the entire flow path 6. Thereafter, as with the detection chip 10A of the first embodiment, the reaction is performed at a predetermined temperature for a predetermined time. Thereafter, washing is performed as appropriate, and for example, by performing SPR or ELIAS, the presence or absence of reaction with the molecule 1 having a molecular recognition function and the intensity of the reaction can be measured.

"Detection chip fabrication method"
<First Embodiment>
Next, a manufacturing method of the detection chip 10A according to the first embodiment of the present invention will be described.
First, the molecule 1 having a molecular recognition function such as an antibody is fixed to the bead 2a. The immobilization method is not particularly limited and can be performed by a conventionally known method such as a physical adsorption method, a chemical adsorption method, a covalent bond method using carbodiimide or glutaraldehyde.
Next, the beads 2a on which the molecules 1 having a molecular recognition function are fixed are patterned on the substrate 3 by sputtering or screen printing. When the detection chip 10A is used for SPR measurement, the gold thin film 4 is formed in advance on the surface 3a including the region to be the measurement target portion of the substrate 3 by sputtering or the like.
With the above, the detection chip 10A according to the first embodiment of the present invention can be manufactured. Since the detection chip 10A of the present invention does not require a special manufacturing apparatus, material, or the like at the time of manufacture, the cost can be reduced.

  In the case where a flexible substrate, particularly a film-like substrate is used as the substrate 3, it is possible to manufacture the detection chip 10A while winding the substrate 3A. Therefore, mass productivity is improved and space can be saved during storage.

Second Embodiment
Next, a manufacturing method of the detection chip 10B according to the second embodiment of the present invention will be described.
First, the molecule 1 having a molecular recognition function such as an antibody is fixed to the fiber 2b. The immobilization method is not particularly limited and can be performed by a conventionally known method such as a physical adsorption method, a chemical adsorption method, a covalent bond method using carbodiimide or glutaraldehyde.
In addition, it is also possible to preserve | save in the state which fixed the molecule | numerator 1 which has a molecule recognition function to the fiber 2b. At this time, since the fiber 2b can be stored in a wound state, the space can be saved.

Next, the fiber 2b on which the molecule 1 having a molecular recognition function is fixed is arranged at a desired position on the substrate 3.
As described above, the detection chip 10B according to the second embodiment of the present invention can be manufactured. When the detection chip 10B is used for SPR measurement, the gold thin film 4 is formed in advance on the surface 3a including the region to be measured of the substrate 3 by sputtering or the like, as in the first embodiment described above. Keep it.
Since the detection chip 10B of the present invention does not require a special manufacturing apparatus, material, or the like when manufactured, the cost can be reduced.

  When a flexible substrate, particularly a film-like substrate, is used as the substrate 3, it is possible to manufacture the detection chip 10B while winding the detection chip 10B. Therefore, mass productivity is improved and space can be saved during storage.

  The present invention can be applied to a detection chip used for SPR.

It is the figure which showed typically an example of the detection chip which concerns on 1st Embodiment of this invention. It is the figure which showed typically another example of the detection chip which concerns on 1st Embodiment of this invention. It is the figure which showed typically another example of the detection chip which concerns on 1st Embodiment of this invention. It is the figure which showed typically an example of the detection chip which concerns on 2nd Embodiment of this invention. It is the figure which showed typically another example of the detection chip which concerns on 2nd Embodiment of this invention. It is the figure which showed typically another example of the detection chip which concerns on 2nd Embodiment of this invention.

Explanation of symbols

  1 molecule having molecular recognition function, 2 liquid permeable spacer, 2a bead, 2b fiber, 3 substrate, 4 gold thin film, 5 second substrate, 6 channel, 10 (10A, 10B) detection chip.

Claims (7)

  A detection chip, wherein a liquid-permeable spacer to which molecules having a molecular recognition function are fixed is patterned on a substrate so as to be a flow path for a liquid sample.   The detection chip according to claim 1, wherein the spacer is a bead.   The detection chip according to claim 1, wherein the spacer is a fiber.   The detection chip according to claim 1, wherein the substrate has flexibility.   The detection chip according to claim 1, wherein the substrate is transparent.   The detection chip according to claim 1, wherein a gold thin film is disposed on a portion to be measured of the substrate.   The detection chip according to claim 6, wherein the spacers are linearly arranged.

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