JP2002300871A - Detecting chip for gene and detector - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a detecting chip designed so as to enable the detection of a large amount of genes with a high sensitivity. SOLUTION: This detecting chip is characterized by installing a plurality of pins 12 which are measuring electrodes, common electrodes 22 that are counter electrodes thereof and a platy member 14 having a plurality of pin holes 15 for respectively inserting the plurality of pins 12. The hole diameter of the pin holes 15 may be reduced in a tapered form in the direction to insert each pin 12 and composed so as to hold the pin 12 in a part where the hole diameter of the pin holes 15 is minimized.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a single nucleotide substitution SNP (single nucleotide) of gene DNA in addition to the nucleotide sequence of a gene.
The present invention relates to a gene detection chip and a detection device capable of detecting and analyzing gene abnormalities such as nucleotide polymorphism (a variant in a human genetic code), several base substitutions, point mutations, and gene deletion.

[0002]

2. Description of the Related Art As one of means for detecting a base sequence of gene DNA, a method of immobilizing a probe DNA on an electrode, hybridizing the probe DNA with a sample DNA, and electrochemically detecting a hybrid formed product. (JP-A-9-288080 and Proceedings of the 57th Symposium on Analytical Chemistry, P137-13)
8, 1996). This method makes it possible to detect hybrids with extremely high sensitivity.

[0003]

However, the number of single nucleotide substitution SNPs and gene mutations in genes is enormous. For example, in the case of humans, a single nucleotide substitution SNP map with a density (resolution) of 15 KB is required. Must identify at least 2 million single nucleotide substitution SNPs. Also, the number of point mutations in genes associated with known diseases is extremely large. Therefore, there is a need for a means capable of realizing comprehensive analysis of single nucleotide substitution and point mutation.

[0004] In view of the above, the present invention provides a gene detection chip capable of detecting a large amount of genes, that is, capable of high-throughput (high-speed large-volume) processing and capable of highly sensitive detection and analysis. , A detection device.

[0005]

The gene detecting apparatus of the present invention comprises a plurality of pins serving as measurement electrodes and a common electrode serving as a counter electrode thereof, wherein the pins have at least a part of their surfaces coated with a resin. Is what is being done. The “detection device” here includes a so-called detection chip. Further, the “detection device” also includes a device in which a detection chip is attached to a measurement device.

[0006] By coating at least a part of the surface of the pin with a resin, it is possible to fix the gene only to the uncovered and exposed portion, to keep the exposed area constant and to control the amount of the immobilized probe. Therefore, detection can be performed with higher sensitivity. In particular, it is possible to cover the side surface of the pin with a resin and fix the gene only to the exposed portion opposite to the base end supported and fixed to the support member of the pin. Is easily determined. As the resin to be coated, a thermoplastic resin such as PEEK (polyether ether ketone) or a fluororesin or an epoxy resin is suitably used in view of chemical resistance.

[0007] In the above, the pin is in contact with or implanted on the surface of the support member, and the side surface of the pin and the portion of the surface of the support member where the pin is not in contact or implanted. , Respectively, may be coated with a resin. In this case, the resin is preferably a fluororesin. Particularly, a copolymer of tetrafluoroethylene and hexafluoropropylene is preferable.

In the above, a plurality of pin holes into which the plurality of pins are inserted are provided, and a plate-like member made of resin is provided, and a part of the surface of the pin is coated with the plate-like member. Is also good.

The pins are inserted into and held in the pin holes by using a single plate member having a plurality of pin holes formed at positions corresponding to the positions of the plurality of pins. And it can be held firmly.

By making the hole diameter of the pin equal to or slightly smaller than the outer diameter of the pin, when the pin is inserted into the pin hole, each pin is inserted into the pin hole without any gap. That is, each pin is held in an airtight manner by the plate-shaped member, so that the solution used in the detection process can be prevented from passing through the contact portion between the pin and the pin hole.

It is preferable that the diameter of the pin hole is tapered toward the direction in which the pin is inserted, and the pin is held at a portion where the hole diameter of the pin hole is minimized.

[0012] The diameter of the pin hole is tapered,
Since the pin is inserted from the side with the larger hole diameter and the pin is held by the minimum hole diameter portion, the positioning when inserting the pin into the pin hole becomes easy, and the work of attaching the plate member to the pin becomes easy. In addition, by making the minimum hole diameter the same as or slightly smaller than the pin diameter, when the pins are inserted into the pin holes, each pin is held airtight by the plate-like member, and is used in the detection process. The solution to be passed can be prevented from passing through the contact portion between the pin and the pin hole.

It is preferable that the pin is in contact with or implanted on the surface of the support member, and the plate-like member is in close contact with the surface of the support member.

Since the plate-like member holds the pin while being in close contact with the support member, it is possible to prevent the solution used in the detection process from entering the contact portion between the plate-like member and the support member. Note that the support member may be a circuit board having an electric circuit provided therein.

[0015] In the above, it is preferable that the plate-like member is composed mainly of a thermoplastic resin. Specifically, fluororesins such as PTFE (polytetrafluoroethylene), PEEK (polyetheretherketone),
Are preferred. By using a plate-shaped member made of a thermoplastic resin, it is possible to provide a gene detection chip having excellent resistance to chemicals such as alkalis and acids used in the pretreatment for detection and also having excellent heat resistance. . Since it is excellent in chemical resistance and heat resistance, after detecting once, the sample DNA is removed, another sample DNA is hybridized again, and the gene detection chip is repeatedly used or the probe gene is removed. Then, another probe gene is fixed to a pin again, and the method is suitable for repeatedly using a gene detection chip.

Further, a thermoplastic resin (eg, PEEK)
By utilizing the property of heat shrinkage, the hole diameter of the pin hole of the plate member is formed slightly larger, and after the pin is inserted into the pin hole, the plate member is heat-treated to be thermally contracted, and the hole diameter of the pin hole is reduced. The present detection device may be configured such that the pin is contracted and the pin is tightened by the pin hole. With this configuration, since the hole diameter of the pin hole is larger than the outer diameter of the pin, the positioning when inserting the pin into the pin hole becomes easy, and the work of attaching the plate member to the pin becomes easy, The pin can be held airtight in a state where the pin and the pin hole are in close contact with each other.

It is particularly preferable that the plate-like member is made of PTFE (polytetrafluoroethylene). PTFE
Has excellent flexibility and is suitable for keeping the pin airtight in the pin hole. Further, PTFE is excellent in flexibility, and thus is suitable for bringing a plate-shaped member into close contact with a ceramic support member.

In the above, the pins are Fe (iron),
An Au (gold) film is formed on the surface of an alloy mainly composed of Ni (nickel) and Co (cobalt).

The use of a pin having an Au film formed on the surface of an Fe--Ni--Co alloy is suitable and preferable for mass production. The alloy preferably has a coefficient of linear expansion of 5.0 × 10 ⁻⁶ / ° C. or more and 9.0 × 10 ⁻⁶ / ° C. or less, because it is easy to match the expansion of the ceramic and is preferable. An alloy having such a linear expansion coefficient contains, for example, 15 to 20% by weight of a Ni component,
25-30% by weight of Co component and 50-6% of Fe component
It is obtained by containing 0% by weight. The alloy may contain impurities of 1.0% by weight or less.

The thickness of the Au film is preferably 20 μm or less from the viewpoint of sufficiently covering the base and increasing the thickness more than necessary so as not to increase the cost. The Au film is preferably an Au plating film formed by a known electroplating method. Note that, before plating Au, Ni plating may be performed as a base treatment, and Au may be plated thereon.

In the present invention, since the above pins are used,
Genes can be detected electrochemically with high sensitivity.

If the pins are arranged in an array, a plurality of genes can be analyzed simultaneously. Here, the array means a state in which a large number of pins are arranged so as to project from a predetermined surface so as to be parallel to each other.

The apparatus for detecting a gene of the present invention preferably includes a support member for supporting and fixing one end of the pin, and the support member is preferably composed of ceramic as a main component. Since the support member contains ceramics as a main component, it has excellent chemical resistance and strength. As ceramics, alumina (Al ₂ O ₃ ), silicon carbide (SiC), silicon nitride (Si ₃ N ₄ ), zirconia (Z
rO ₂ ), beryllia, and the like are preferably mentioned in terms of chemical resistance. In particular, alumina is preferred. This ceramic has a coefficient of linear expansion of at least 6.0 × 10 ⁻⁶ / ° C. and 11.0
When the temperature is at most 10 ⁶ / ° C, joining with a pin is facilitated, which is preferable. It is preferable that the supporting member contains 90% by weight or more of ceramics.

In the above description, the pins include a plurality of PCR products, oligonucleotides, mRNA, cDNA, PNA (peptidic nuclei) having the same or different gene sequences.
c acid) or LNA (locked nucleic acid; Prol
igo LLC (trademark) is preferably fixed.

The above-described gene detecting apparatus is useful for detecting, for example, the nucleotide sequence of a gene, single nucleotide substitution SNP, several nucleotide substitutions, point mutation, translocation, deletion, amplification, or triplet repeat. By performing a genetic diagnosis using the detection device of the present invention, muscular dystrophy, hemophilia,
Monogenic diseases such as phenylketonuria, diabetes, cancer,
Diagnose whether or not you have genes related to multifactorial diseases such as hypertension, myocardial infarction, and obesity, diagnose genes before onset, and use them as a basis for selecting appropriate treatments and drugs be able to.

The present invention also provides a chip for detecting a gene, comprising a plurality of pins serving as measurement electrodes, wherein the pins are at least partially coated with a resin. The gene detection chip may further include a common electrode that is a counter electrode of the plurality of pins.
That is, the detection chip of the present invention may have both a pin electrode and a common electrode, or a solution tank having only a pin electrode without a common electrode and having a common electrode. The measurement may be performed by attaching the detection chip to the device.

The pin is contacted or implanted on the surface of the support member, and the side surface of the pin and the portion of the surface of the support member on which the pin is not contacted or implanted are respectively made of resin. It may be coated.

A plate-like member having a plurality of pin holes into which the plurality of pins are respectively inserted and made of resin;
A part of the surface of the pin may be coated with the plate-like member.

The pins may be formed by forming an Au film on the surface of an alloy containing Fe, Ni and Co as main components.

The present invention also provides a gene detection device having the above-described gene detection chip and a measurement device capable of inserting and removing the detection chip.

According to the present invention, there are provided a plurality of pins serving as measurement electrodes,
A common electrode which is the counter electrode thereof,
e, Ni, and Co on the surface of an alloy containing
Provided is a chip for detecting a gene having a u film formed thereon.

The pins are preferably in contact with or implanted on the surface of a support member, and the support member is preferably formed of ceramic as a main component.

The pins are preferably in contact with or implanted on the surface of a supporting member, and the supporting member is preferably formed of alumina as a main component.

As described above, by using the above-mentioned chip to bind an electrochemically active molecule after hybridization, or to perform hybridization in the presence of the electrochemically active molecule, a large amount of genes can be simultaneously obtained. In addition, it can be detected with extremely high sensitivity and simple operation.

[0035]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiments of a gene detection chip and a detection device according to the present invention will be described below with reference to the drawings. It should be noted that the drawings are merely embodiments of the present invention, and the present invention is not limited thereto.

(Detection Chip According to First Embodiment)
FIGS. 1A and 1B show a chip for gene detection according to a first embodiment of the present invention, wherein FIG.
FIG. 4 is a view of the main body viewed from a side provided with pins.

As shown in FIGS. 1 (a) and 1 (b), the gene detecting chip 10 is configured as a card-shaped or cassette-shaped chip, and includes a main body 1 and a frame detachably mounted thereon. And part 2.

The main body 1 includes a substrate 11 as a support member,
A plurality of pins 12 supported and fixed on one surface of the substrate 11,
A coating resin 13 for covering the side surface of the pin 12 and a plate-like member 14 having a pin hole 15 for inserting the pin 12
And

As shown in FIG. 1B, a plurality of pins 12
Are evenly arranged in parallel with each other so as to protrude from one surface of the substrate 11, and form an array. An electric circuit is provided inside the substrate 11, and a number of pins 12 serving as measurement electrodes are each independently connected to each wiring of the electric circuit. The other end of the wiring is connected to a terminal terminal for a pin and a terminal terminal for a common electrode.
Each wiring connected to the pin may be connected one by one corresponding to the pin, and may be connected to the terminal terminal for each pin. However, an active matrix type TFT liquid crystal display device provided with a switching element composed of a TFT, etc. As in the matrix electrode described above, a configuration may be adopted in which the conductors and pins of the matrix wiring are connected via FETs (field effect transistors) at the intersections of the matrix wiring composed of a number of vertical and horizontal conductors. With such a configuration, the conductive line of the matrix wiring is scanned, the selected TFT is turned on, and a specific pin is electrically connected to the terminal terminal.

As shown in FIG. 2, the base end 17 of the pin 12 is supported and fixed (preferably fixed by silver brazing) on the substrate 11. The side surface of the pin 12 is covered with a coating resin 13. The coating resin is preferably made of PTFE (polytetrafluoroethylene) or epoxy resin. A pin hole 1 having a tapered surface formed by changing the diameter of the pin hole into a tapered shape.
The pin coated with the coating resin 13 is inserted into the pin 8. The minimum hole diameter of the pin hole 18 is slightly smaller than the outer diameter D1 of the pin covered with the coating resin 13, and by inserting the pin 12 into the pin hole 18, the minimum hole diameter portion is covered with the pin 12. Pressed by the side. In this way, the covered pin 12 is inserted into the pin hole 18.
Is inserted airtightly. Further, the plate-like member 14 is
It is fixed in close contact with.

As described above, the solution in the depression 21 may be displaced between the coating resin 13 and the plate-like member 14 or the plate-like member 14.
And the substrate 11 can be prevented from entering.

The pin 12 is made of an alloy containing Fe, Ni and Co as main components, and its surface is plated with Au. This alloy contains 15-20% by weight of Ni component, 25-30% by weight of Co component, and 50-60% by weight of Fe component.
It is preferred to contain.

The tip 16 of the pin is
3 is exposed without being covered with the oligonucleotide. An SH-modified oligonucleotide in which an oligonucleotide, which is a PCR product, is thiolated and an SH group is introduced at the 5 ′ end thereof is immobilized on the exposed portion. This SH-oligonucleotide has a length including 20 to 50 bases, and is immobilized on a portion where Au is exposed via the SH group. The immobilization of the SH-oligonucleotide is performed by inserting the pin 12 into each of the microplate-containing microcavities having the DNA microcavities arranged at the same pitch as the pins 12, thereby modifying the same or different types of DNA into the respective micropins. Do it. In addition, a technique for immobilizing the SH-oligonucleotide on the Au via the SH group is known.

Regarding a pretreatment method before Au plating on the surface of the pin electrode and SH-Au bonding, for example, J. Am.
m.Soc 111 No. P321-1989 C.I. D. Bain, Anal.
Chem. No. 70, p. 2396 to 1998, described by JJ. Gooding. The removal of the probe gene was performed according to J. Am. Chem. Soc 1
No. 11, P321-1989 D. This is performed by the method described by Bain.

It is preferable that the plate-like member 14 is composed mainly of PTFE.

The substrate 11 is preferably composed mainly of alumina.

The frame 2 is made of a solution (sample DNA,
A recess 21 that can be filled with a sewn intercalator, a cleaning liquid, or the like is provided at a portion corresponding to the pin 12. The frame 2 is formed of ceramics, resin, or the like.

A common electrode 22 is provided in the recess 21, and the common electrode 22 is connected to a terminal terminal for a common electrode (not shown). The common electrode 22 is disposed on a part (for example, a peripheral portion) or the entire bottom surface of the depression 21 or on an inner peripheral side surface near the bottom surface of the depression 21 at a position not in contact with the pin. A seal may be provided near the periphery of the depression 21.

The frame 2 can be detachably connected to the main body 1 by, for example, forming irregularities (not shown) which elastically fit each other on the contact surfaces between the main body and the frame. It is. Alternatively, the main body 1 and the frame 2 may be stopped by a clip or a clamp, or may be attracted to each other by a magnet.

The measurement is performed by detecting a current flowing between the common electrode and each pin when a voltage is applied between the common electrode terminal terminal and each pin electrode terminal terminal. As described in detail in JP-A-9-288080, an electrolyte solution containing electrochemically active molecules may be used for the detection.

By using the above detection chip,
Genes can be detected with high sensitivity and high speed.

FIG. 3 is a perspective view for explaining the overall configuration of a detection device provided with the detection chip according to the first embodiment. is there.

In FIG. 3, a gene detecting apparatus 100 according to the present invention comprises a detecting chip 10 and a detecting chip 1.
Measuring apparatus 10 having insertion slot 103 into which 0 can be inserted
2 and a personal computer 101.

After immobilizing the probe gene on the tip 16 of the pin 12, a solution containing the target gene is injected into the depression 21. Next, the detection chip 10 in which the main body 1 is mounted on the frame 2 is inserted into the insertion opening 103, and the temperature is controlled to a hybridization temperature condition by a temperature control device including a Peltier device provided in the measurement device 102. Then, the probe gene and the target gene are hybridized. Then, the detection chip 10 is withdrawn from the measurement device 102, and after washing the inside of the depression 21, an intercalator is introduced into the double strand formed by hybridization, and the detection chip 1 is inserted into the insertion slot 103. At this time, the terminal terminal for the common electrode and the terminal terminal for the pin are respectively connected to terminals in the measuring device 102. When a voltage is applied between the common electrode 22 and the pin 12, a weak current flows between the double strand generated by hybridization and the pin 12 via the common electrode. The target gene can be detected by controlling the temperature with a Peltier device provided in the measuring device 102 and measuring current values at different temperatures.

(Detection Chip According to Second Embodiment)
FIG. 4 is an enlarged cross-sectional view showing a relationship among pins, a substrate, and a plate-like member of a chip for gene detection according to a second embodiment of the present invention.

In the present embodiment, only the point that the base end side of the pin 12 is changed to be held in the pin hole 18 is the first
Is different from the embodiment of the present invention.

That is, as shown in FIG. 4, the pin 12 is inserted into a pin hole 18 formed in the plate member. The pin hole 18 has a tapered surface, the minimum hole diameter of which is slightly smaller than the outer diameter D2 of the pin. By inserting the pin 12 into the pin hole 18 of the plate member 14, the minimum hole diameter portion is reduced. Pressed by the side surface of the pin 12, the pin is airtightly fitted. In the present embodiment, after the plate member 14 is provided, the side surface of the portion where the pin 12 protrudes from the plate member 14 is coated with the coating resin 13. The plate member 14 is made of PTFE and is in close contact with the substrate 11.

Thus, the solution is transferred between the pin 12 and the plate-like member 1.
4 or between the plate-like member 14 and the substrate 11 can be prevented.

By using the above detection chip,
Genes can be detected with high sensitivity and high speed.

(Detection Chip According to Third Embodiment)
FIG. 5 is an enlarged cross-sectional view showing the relationship among the pins, the substrate, and the plate-like member of the gene detection chip according to the third embodiment of the present invention.

This embodiment is different from the first embodiment only in that the pin hole 18 of the plate member 14 does not have a tapered surface.

That is, as shown in FIG. 5, the pin 12 is inserted into a pin hole 18 having a constant hole diameter. Pin hole 18
Is the same as the outer diameter D3 of the pin covered with the coating resin 13,
Is pressed against the pin hole 18 so that the covered pin 12 is fitted into the pin hole 18 in an airtight manner. Plate member 14
Is made of PTFE, and keeps the pins airtight while being in close contact with the substrate 11. As a result, the solution becomes the coating resin 1
It is possible to prevent entry between the plate member 3 and the plate member 14 or between the plate member 14 and the substrate 11.

By using the above detection chip,
Genes can be detected with high sensitivity and high speed.

(Detection Chip According to Fourth Embodiment)
FIG. 6 is an enlarged cross-sectional view showing a relationship among pins, a substrate, and a plate-like member of a chip for gene detection according to a fourth embodiment of the present invention.

In the present embodiment, the only difference is that the pin hole diameter of the plate-like member 14 changes stepwise, and the coating resin 13 on the base end side of the pin 12 is changed so as to be held in the pin hole 18a. This is different from the first embodiment.

As shown in FIG. 6, in the present embodiment, the pin hole 18 of the plate-like member 14 has a portion 18a having a hole diameter of D5.
And a portion 18b having a hole diameter of D6. Pin 1
The side surface of No. 2 is coated with a coating resin 13, and the outer diameter of the coated pin is D5. By inserting the covered pin 12 into the pin hole 18, the coating resin 13 and the portion 18a having a hole diameter of D5 abut,
The coated pin 12 is kept airtight. Plate member 14
Is made of PTFE and is in close contact with the substrate 11. Thereby, it is possible to prevent the solution from entering between the coating resin 13 and the plate member 14 or between the plate member 14 and the substrate 11.

By using the above detection chip,
Genes can be detected with high sensitivity and high speed.

(Detection Chip According to Fifth Embodiment)
FIG. 7 is an enlarged cross-sectional view showing a relationship among pins, a substrate, and a plate-like member of a gene detection chip according to a fifth embodiment of the present invention.

In the present embodiment, the only difference from the first embodiment is that the pin hole diameter of the plate-like member 14 changes stepwise, and the base end side of the pin 12 is changed to be held in the pin hole 18a. It is different from the form.

That is, the pin hole 18 of the plate member 14 is
Portion 18a having a hole diameter of D7 and portion 1 having a hole diameter of D6
8b. By inserting the pin 12 having an outer diameter of D7 into the pin hole 18a of the plate member 14, the pin hole 18a is pressed by the side surface of the pin 12, and the pin 12 is held in the pin hole 18a in an airtight manner. In the present embodiment,
After the plate member 14 is provided, the plate member 1 of the pin 12 is
4 is coated with a coating resin 13. The plate-like member 14 is made of PEEK (polypenco; trademark of Japan Polypenco) and is in close contact with the substrate 11. Thereby, it is possible to prevent the solution from entering between the pin 12 and the plate member 14 or between the plate member 14 and the substrate 11.

By using the above detection chip,
Genes can be detected with high sensitivity and high speed.

The detection chips according to the second to fifth embodiments can be applied to the detection device and detected similarly to the detection chips according to the first embodiment.

Note that the above is an example of the embodiment of the present invention, and the alloy constituting the pin, the coating resin coating the pin, the substrate, and the components of the plate member are not limited to the above.

In the above description, the case where the coating resin is provided on the side surface of the outermost surface of the pin has been described. However, the portion to be covered is not limited to the side surface as long as it is a part of the outermost surface of the pin.

In the above description, the case where one end of the pin is fixedly supported on the substrate has been described, but one end of the pin may be implanted in the substrate.

In the above description, the case where 25 pins are provided has been described, but the number of pins may be changed as appropriate.

(Detection Chip According to Sixth Embodiment)
FIG. 8 is an enlarged cross-sectional view showing a relationship among pins, a support member, and a coating portion of a gene detection chip according to a sixth embodiment of the present invention.

In the present embodiment, the first embodiment is characterized in that the side surface of the pin 12 and the portion of the surface of the support member 11 where the pin 12 is not fixed are coated with the resin 130, respectively. It is different from the form.

As shown in FIG. 8, the base end 17 of the pin 12 is supported and fixed on the support member 11. This resin is preferably made of a copolymer of tetrafluoroethylene and hexafluoropropylene.

As described above, the solution can be extremely effectively prevented from entering the support member 11 side. Examples Hereinafter, examples will be described, but it goes without saying that the present invention is not limited to the examples.

(Example 1) [Sample 1] In order to operate only the tip of the Au-plated pin as an electrode, the side surface of the pin was coated with polytetrafluoroethylene as in the sixth embodiment. The coating was performed by a known spraying method.

[0082] Then, only the tip portion of the pin protruding from the substrate, and 1hr boiled immersed in 2M NaOH, concentrated HNO ₃
The sample was immersed in the sample for 0.5 hours to perform pretreatment, thereby obtaining Sample 1.

[Sample 2] Instead of polytetrafluoroethylene, the side surfaces of the pins were coated in the same manner as above using ethylene copolymerized tetrafluoroethylene, followed by pretreatment to obtain Sample 2.

[Sample 3] Instead of polytetrafluoroethylene, the side surfaces of the pins were coated with a tetrafluoroethylene / hexafluoropropylene copolymer in the same manner as described above, and then a pretreatment was performed. Obtained.

[Visual Observation of Deterioration Status] With respect to the obtained Samples 1 to 3, the degradation status of the coating resin before and after the pretreatment was visually observed. In the visual observation, in the samples 1 and 3, the deterioration of the resin was hardly observed. In sample 2, the resin was slightly deteriorated by the pretreatment.

[Evaluation of Electrochemical Characteristics] Samples 1 and 3 were evaluated for electrochemical characteristics. ECA CH
DPV (differential pulse voltammetry) was measured using IPREADER TGE 1000 (manufactured by TUM Laboratories).
All measurements were performed at room temperature. More specifically, sample 1
For 3 and 3, the change in multi-electrode response was 500
0.2M Phosphate Buffer containing μM ferrocenecarboxylic acid (pH
During 7.0), the DPV was measured, and the peak current value ipa of ferrocenecarboxylic acid, the DPV voltammogram waveform, and the like were evaluated.

The pre-processing is repeated three times, and D
PV was measured. In samples 1 and 3, the peak current value ipa (μA) for each pin (25 pins) was determined and
When the average value of the peak current value ipa at the second time was set to 100 (%), the relative value of the average value at the second and third times was obtained. In addition, the standard deviation and the coefficient of variation were determined. The results are as follows.

Sample 1: First time Second time Third time Average value (relative value) 100.0 94.7 164.7 Standard deviation 9.0 20.3 22.4 Coefficient of variation 9.0 21.4 13.6 samples 3: First time Second time Third time Average (relative value) 100.0 98.8 91.2 Standard deviation 9.4 12.6 11.7 Coefficient of variation 9.4 12.7 12.9 For samples 1 and 3 , Each of the above averages (relative values)
Is shown in FIG.

From the above results, it was found that Samples 1 and 3 were particularly preferable. In particular, Sample 3 was found to be most suitable for multiple uses.

Further, all of the samples 1 to 3 were made of PTFE.
Since the coating is performed using a system resin, it is easy to reduce the thickness of the coating, and it is possible to realize the micro integration of the electrodes. Further, by coating with a PTFE-based resin, there is also an advantage that perfect sealing to the base portion (welded portion) of the pin electrode on the substrate side is easy.

(Example 2) [Evaluation of uniformity of effective surface area] As in the first embodiment, the side surfaces of the pins are coated with PEEK (polyether ether ketone), and a plate-like member made of polytetrafluoroethylene is formed. A sample 4 was obtained. The coating was performed by covering the pin with a tube-shaped PEEK and then performing a heat treatment (thermal shrinkage).

The measurement was performed with a 500 μM ferrocenecarboxylic acid system, and the peak current value ipa proportional to the effective surface area of the electrode was
I asked. The measurement was performed in the same manner as in Example 1. FIG. 10 shows the peak current value ipa measured at each pin. Average value 1064.
An almost uniform effective surface area of 1 nA, standard deviation of 73.6 nA, and a coefficient of variation of 6.9% was achieved, indicating that the pin side surface was completely sealed with resin. Since there was no significant change in this tendency even after six consecutive uses, it was found that the resin did not expand and the Au plating surface did not deteriorate due to the pretreatment.

Further, a plurality of BAS Au electrodes (effective surface area 2
mm ² ), a similar measurement was attempted. The results are shown in FIG.
Since the average value is 2.428 μA, the standard deviation is 0.110 μA, and the variation coefficient is 4.5%, which is not much different from the above values, the uniformity of the effective surface area of Sample 4 can be seen.

Finally, the simple surface area calculated from the diameter and ip
The relationship between a and the previously acquired pad array (effective surface area 0.2
FIG. 12 also shows the result of mm ² ). It can be seen that a very good correlation is established between the two. That is, the electrode activity per unit area of Sample 4 is the same as that of commercially available Au.
It shows that it is at the same level as that of the electrode, which indicates that it is excellent in electrochemical multi-sensing of DNA.

With the above configuration, it is easier to reduce the coating film thickness.
Perfect sealing is possible up to the base portion (welded portion) of the pin electrode on the substrate side.

[0096]

As described above, the gene detection apparatus and the detection chip according to the present invention can detect a gene in a large amount with high sensitivity, at high speed, and easily. In addition, if such a detection chip is applied to a measuring device that can be inserted and removed, it is possible to provide a detection system capable of analyzing a gene in a high-sensitivity, high-speed, large-scale and simple manner.

As described above, the detection chip and the detection apparatus according to the present invention, which can perform high-sensitivity, high-throughput (high-speed, large-volume) processing, are effective for analyzing correlations between genes and phenotypes in the fields of biology and medicine. Means. Drug metabolizing enzymes,
A specific gene such as a tumor suppressor gene, the nucleotide sequence of the gene according to the present invention, a single nucleotide substitution SNP, several nucleotide substitution, point mutation, translocation, deletion, amplification, or a triplet repeat detection and analysis device, The analysis can be used for genetic diagnosis.

For example, since the detection apparatus according to the present invention can perform high-sensitivity, high-throughput (high-speed, large-volume) processing, data on Japanese genes is collected and genes associated with the onset of disease are identified. Can be used to predict and prevent future onset.

[0099] Diagnosis of the gene can be useful in selecting an appropriate treatment method or a drug with few side effects.

Furthermore, a new drug can be developed using the results of the genetic analysis of a disease without repeating clinical experiments and the like.

[Brief description of the drawings]

FIG. 1 relates to a gene detection chip according to a first embodiment of the present invention, in which (a) is a partial cross-sectional view and (b) is a view of a main body viewed from a side provided with pins. It is.

FIG. 2 is a partially enlarged view of FIG.

FIG. 3 is a perspective view illustrating an overall configuration of a detection device including the detection chip according to the first embodiment of the present invention.

FIG. 4 is an enlarged cross-sectional view of a chip for gene detection according to a second embodiment of the present invention.

FIG. 5 is an enlarged sectional view of a gene detection chip according to a third embodiment of the present invention.

FIG. 6 is an enlarged sectional view of a gene detection chip according to a fourth embodiment of the present invention.

FIG. 7 is an enlarged sectional view of a gene detection chip according to a fifth embodiment of the present invention.

FIG. 8 is an enlarged sectional view of a gene detection chip according to a sixth embodiment of the present invention.

FIG. 9 is a graph showing measurement results of DPV of Samples 1 and 3.

FIG. 10 is a graph showing a peak current value ipa at each pin of sample 3.

FIG. 11 shows a peak current value ipa using a BAS Au electrode.
(Control).

FIG. 12 is a graph showing a relationship between a simple surface area and ipa.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Main body part 2 Frame part 10 Detection chip 11 Substrate 12 Pin 13 Coating resin 14 Plate member 15 Pin hole 16 Pin tip 17 Pin base end 18 Pin hole 22 Common electrode

Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) G01N 37/00 102 G01N 27/46 336M // C12Q 1/68 C12N 15/00 F (72) Inventor Kazuhiro Naka Kagoshima 1-1, Yamashita-cho, Kokubu-shi Kyocera Corporation Kagoshima Kokubu Plant (72) Inventor Shingo Sato 1-1, Yamashita-cho, Kokubu-shi, Kagoshima Kyocera Corporation Kagoshima Kokubu Plant (72) Inventor Hiroyoshi Miyahara Mihama-ku, Chiba Chiba 2-6 Nakase F-term in TMU Laboratories, Inc. (reference) 4B024 AA11 AA19 CA01 CA04 CA09 CA12 HA12 4B029 AA07 AA23 BB20 CC03 FA15 4B063 QA01 QQ41 QR32 QR36 QR55 QR81 QS34 QS36 QS39 QX04

Claims (24)

[Claims] An apparatus for detecting a gene, comprising: a plurality of pins as measurement electrodes; and a common electrode as a counter electrode, wherein the pins have at least a part of their surface coated with a resin. Detection device. 2. The pin is in contact with or implanted on a surface of a support member, and a side surface of the pin and a portion of the surface of the support member where the pin is not in contact or implanted,
The gene detection device according to claim 1, wherein each of the genes is coated with a resin. 3. The gene detection device according to claim 1, wherein the resin is PTFE. 4. The gene detection device according to claim 2, wherein the support member is mainly composed of ceramics. 5. The gene detection device according to claim 4, wherein the support member is composed mainly of alumina. 6. A plate-like member having a plurality of pin holes into which the plurality of pins are inserted, and made of resin,
The gene detection device according to claim 1, wherein a part of the surface of the pin is coated with the plate-shaped member. 7. The pin hole diameter of the pin hole is tapered toward the direction in which the pin is inserted, and the pin hole is held at a portion where the hole diameter of the pin hole becomes minimum. Gene detection device. 8. The gene detecting device according to claim 6, wherein the pin is in contact with or implanted on a surface of the support member, and the plate-shaped member is in close contact with the surface of the support member. 9. The gene detection device according to claim 6, wherein the support member is mainly composed of ceramics. 10. The gene detection device according to claim 9, wherein the support member is constituted mainly of alumina. 11. The gene detecting device according to claim 6, wherein the plate-like member is mainly composed of a thermoplastic resin. 12. The gene detection device according to claim 6, wherein the plate-like member is mainly composed of a fluororesin. 13. The method according to claim 1, wherein the pins are Fe, Ni, and Co.
The gene detection device according to claim 1, wherein an Au film is formed on a surface of an alloy containing as a main component. 14. The gene detecting apparatus according to claim 13, wherein the support member is mainly composed of ceramics. 15. The gene detecting device according to claim 14, wherein the support member is constituted mainly of alumina. 16. A gene detection chip, comprising: a plurality of pins serving as measurement electrodes, wherein at least a part of a surface of the pin is coated with a resin. 17. The gene detection chip according to claim 16, further comprising a common electrode which is a counter electrode of said plurality of pins. 18. The pin is contacted or implanted on a surface of a support member, and a side surface of the pin and a portion of the surface of the support member where the pin is not contacted or implanted, respectively. The gene detection chip according to claim 16 or 17, which is coated with a resin. 19. A plurality of pin holes into which the plurality of pins are respectively inserted, and a plate-like member made of resin is provided, and a part of the surface of the pin is coated with the plate-like member. 18. A chip for detecting a gene according to 16 or 17. 20. The method according to claim 19, wherein the pins include Fe, Ni, and Co.
18. The gene detection chip according to claim 16 or 17, wherein an Au film is formed on a surface of an alloy containing as a main component. 21. An apparatus for detecting a gene, comprising: the chip for detecting a gene according to claim 16; and a measuring apparatus capable of inserting and removing the chip for detection. 22. A chip for detecting a gene, comprising: a plurality of pins serving as measurement electrodes; and a common electrode serving as a counter electrode thereof, wherein the pins are made of an alloy containing Fe, Ni, and Co as main components. A gene detection chip comprising an Au film formed on the surface. 23. The gene detection chip according to claim 22, wherein the pins are in contact with or implanted on a surface of a support member, and the support member is mainly composed of ceramics. 24. The gene detection chip according to claim 23, wherein the pin is in contact with or implanted on a surface of a support member, and the support member is mainly composed of alumina.