WO2005045409A1

WO2005045409A1 - Electrical resistivity sensor and sensing method

Info

Publication number: WO2005045409A1
Application number: PCT/JP2004/006583
Authority: WO
Inventors: Tsutomu Nagaoka; Hiroshi Shiigi
Original assignee: Osaka Prefecture
Priority date: 2003-11-07
Filing date: 2004-05-10
Publication date: 2005-05-19
Also published as: JPWO2005045409A1; JP4389088B2

Abstract

An electrical resistivity sensor characterized in that a set of electrodes are arranged oppositely on the electrically insulated surface of a substrate and a film of conductive fine particles modified with probe is formed on and/or between the electrodes.

Description

Description Electric resistance type detection sensor and detection method

The present invention relates to an electric resistance type detection sensor and a detection method for detecting and confirming a target substance such as a nucleic acid such as DNA or RNA or a protein such as an antigen or an antibody. Background art

In recent years, various DNA chips have been disclosed in Japanese Patent Application Laid-Open No. 2003-287538 (Patent Document 1) and Japanese Patent Application Laid-Open No. 2003-250088 (Patent Document 2). However, with a conventional DNA chip, for example, a single-stranded DNA sample is first labeled with a fluorescent substance. Then, the sample is sprayed on a chip having the target DNA bound to the surface, and the target DNA in the sample and the DNA bound to the chip surface are hybridized. The fluorescent substance labeled with the sample emits light, and the emitted light is read using a microscope or laser fluorescence scanner to detect the presence of the target DNA. However, with this technique, it cannot be ignored that DNA is non-specifically adsorbed on a substrate, and a method for immobilizing an oligonucleotide probe on a substrate is not sufficiently established. Furthermore, methods for controlling the amount of probes immobilized on a substrate have not been well established. In addition, detection of the presence of DNA in a sample requires the use of appropriate fluorescent labeling agents and intercalators, as well as equipment such as a laser fluorescence scanner. Therefore, the detection was costly and the operation was complicated.

In Japanese Patent Application Laid-Open No. 2003-514224 (Patent Document 3), detection of the presence or absence of a target DNA is performed by using a change in the refractive index of light caused by surface plasmon resonance (SPR) occurring on the chip surface. Is disclosed.

Furthermore, Japanese Patent Application Laid-Open No. 2002-533698 (Patent Document 4) discloses a DNA chip having, on a substrate, a region in which a DNA complementary to a target DNA is bound. The publication Then, the DNA in the sample is first modified with conductive particles, and the obtained sample is sprayed on the above-mentioned region, and the DNA binding to the region and the DNA present in the sample are compared with the DNA present in the sample. Hybridize with complementary DNA. As a result, in the hybridized DNA, a current flows in the region through the conductive particles that have modified the DNA, and that fact is used to detect the presence or absence of the target DNA.

That is, in the above method, while the complementary binding partner (6) is immobilized on the conductive particles (62), the specific binding partner (5) needs to be immobilized between the two electrodes on the substrate. The immobilization to the conductive particles in (6) is achieved by the SH-modified oligonucleotide. On the other hand, (5) is immobilized on a substrate by a silane coating method using APTES and the like, and the oligonucleotide is immobilized using this as a linker.

In such a detection method, it is necessary to add an electron transfer mediator (redox, conductive substance) in order to improve the electrical conductivity between the two electrodes. In other words, the target substance (or probe) is immobilized on the conductive particles, deposited between the electrodes by the probe (or target substance) previously immobilized on the substrate, and the conductivity is increased by adding a mediator or the like. Must be detected.

Therefore, in the above-described detection method, the sample must be modified with conductive particles every time the detection is performed, and the detection is troublesome and costly. Disclosure of the invention

Accordingly, the present invention provides a simpler, cheaper, more accurate, and more accurate method for reducing target substances such as nucleic acids such as DNA and RNA or proteins such as antigens and antibodies. Provided are an electric resistance type detection sensor and an electric resistance type detection method which can be detected and confirmed at a low cost, and which can be inexpensively and easily used repeatedly.

Thus, according to a first aspect of the invention,

A pair of electrodes are arranged facing each other on an electrically insulated substrate surface, and a film of conductive fine particles modified with a probe is formed on or between the electrodes. An electric resistance type detection sensor is provided. Further, according to a second aspect of the present invention,

A concave portion is provided on the surface of the electrically insulated substrate, and a pair of electrodes are arranged in the concave portion so as to face each other, and a film of conductive fine particles modified with a probe is provided on and / or between the electrodes. An electric resistance type detection sensor characterized by being formed is provided. Further, according to a third aspect of the present invention,

A substrate having a plurality of fine concave portions formed on the surface thereof; a film of conductive fine particles formed on the inner surface of each concave portion; and a first conductive film formed to be electrically connected to the film of conductive fine particles. And a second electrode,

Provided is an electric resistance detection sensor characterized in that a film of conductive fine particles is modified with a probe. Further, according to a fourth aspect of the present invention,

An electric resistance type detection sensor characterized in that a first electrode is formed on a surface of a substrate, a second electrode is formed inside a recess, and a film of conductive fine particles is modified with a probe. I do. Further, according to a fifth aspect of the present invention,

Modifying a film of conductive fine particles formed on the surface of the electrically insulated substrate with a probe, and spraying a measurement sample containing a specimen on the modified film;

By measuring the electrical resistance value between two points of the obtained film of conductive fine particles,

Provided is an electric resistance type detection method for detecting the presence or absence of a target substance that reacts with a probe. Further according to a sixth aspect of the present invention,

Prepare a measurement sample containing a sample and a probe in advance,

By spraying the sample on a film of conductive fine particles formed on the surface of an electrically insulated substrate and measuring the electrical resistance value between two points of the obtained film of conductive fine particles,

Provided is an electric resistance type detection method for detecting the presence or absence of a target substance that reacts with a probe. Brief Description of Drawings

FIG. 1 is a diagram showing the selectivity of the electric resistance type detection sensor of the present invention when single-stranded DNA is used as a probe. Arrows indicate when the target substance was dropped.

Figure 2 shows the change in the electrical resistance of the DNA before and after the DNA present on the membrane of the DNA nanoparticles modified with DNA (probe) was degraded by the DNAse enzyme to illustrate the present invention. It is. The arrow shows the case where the target substance was dropped at the position 1 and the DNA degrading enzyme was dropped at the position of arrow 2.

FIG. 3 is a diagram showing a change in the electric resistance value of the electric resistance type detection sensor of the present invention when a single-stranded DNA having both ends thiolated is used as a probe. The arrow indicates when the target substance has been dropped.

FIG. 4 is a diagram showing a change in the electric resistance value of the electric resistance type detection sensor of the present invention when a single-stranded DNA having both ends thiolated is used as a probe and the conductive fine particle film does not contain a binder. It is. Arrows indicate when the target substance was dropped.

FIG. 5 shows a sample of the present invention in which a single-stranded DNA having both ends thiolated as a probe and a measurement sample were prepared in advance, and the resulting sample was sprayed on a membrane consisting of only conductive fine particles containing a binder. It is a figure showing change of an electric resistance value of an electric resistance type detection sensor.

FIG. 6 shows the change in the resistance value of the gold nanoparticle membrane when a rabbit anti-mouse IgG antibody was used as a probe and a mouse IgG antigen was used as a target substance to illustrate the present invention. FIG. The target substance was dropped at the position of the arrow.

FIG. 7 (a) is a block diagram showing a configuration of an electric resistance type detection sensor according to Embodiment 6 of the present invention. (B) is a side sectional view of a main part showing a state of a concave portion of the electric resistance type detection sensor. is there.

FIG. 8 is a block diagram illustrating a configuration of a detection sensor according to Embodiment 6 of the present invention.

FIG. 9 is a block diagram illustrating a configuration of a lock-in amplifier circuit according to Embodiment 6 of the present invention. '

(A) of FIG. 10 is a block diagram showing a configuration of a detection sensor according to Embodiment 7 of the present invention, and (b) is a side sectional view of a main part showing a state of a concave portion of the detection sensor. BEST MODE FOR CARRYING OUT THE INVENTION

In the electric resistance type detection sensor according to the first embodiment of the present invention, one set of electrodes is arranged on the substrate surface.

As the “substrate” of the present invention, an electrically insulating substrate can be suitably used. Specific examples of the material include glass, plastic, quartz, and silicon.

As the material used for the "electrode" of the present invention, the material used for the electrode of a normal sensor is sufficient, and specifically, Au, Pt, Cu, Al, Ni, Ti, etc. In addition to the above metals or alloys thereof, polymers such as polypyrrole, polyaniline and polyacene may be mentioned. The shape of the electrode is not particularly limited, and examples thereof include a comb electrode having a comb shape. Further, the electrodes may be coated with an insulating material.

Then, a film of conductive fine particles is formed on the electrodes and / or between the electrodes. The “film of conductive fine particles” of the present invention refers to a film formed so that the conductive fine particles and the electrode are in direct contact with each other, as well as close enough that the conductive fine particles and the electrode can be electrically connected. Nano-gap state. According to one aspect, the film of conductive fine particles can be expressed as a layer of conductive fine particles.

In the case where the film of the conductive fine particles described later contains a binder, the conductive fine particles and the electrode only need to be electrically conductive through the binder.

In addition, the “conductive fine particles” of the present invention include a substance having conductivity and capable of directly and / or indirectly binding to a probe described later. Examples include particles made of materials such as irene, platinum, aluminum, gold, and silver. Among them, particles composed of metals such as gold and silver are preferable, and particles composed of gold are more preferable.

Further, the size of the conductive fine particles may be appropriately selected according to the material of the particles and the probe. Among them, the average particle size of the conductive fine particles is preferably nano-sized, and more preferably 50 to 100 nm.

Further, the film of the conductive fine particles can be formed by using a known method. For example, when gold nanoparticles are used as the electroconductive fine particles, a gold nanoparticle film can be formed by bringing a gold colloid solution in which the gold nanoparticles are suspended in an appropriate solvent into contact with a substrate. . In this case, examples of the solvent used include water and alcohols such as methanol and ethanol.

It is also preferable that the film of the conductive fine particles contains a binder.

The “binder” may be appropriately selected according to the type of the conductive fine particles and the probe. Specifically, when a metal such as gold or silver is used as the material of the conductive fine particles, dithiols having an SH group such as 1,10-decanedithiol or NH such as 1,10-diaminodecane can be used.

Examples of the binder include diamines having _two groups. Of these, dithiols are preferred, and 1,10-decanedithiol is particularly preferred.

The film of the conductive fine particles containing the binder can be formed using a known method. For example, when gold nanoparticles are used as conductive fine particles and the above dithiols and diamines are used as a binder, the binder and the gold nanoparticles are suspended in an appropriate solvent, and the suspension is applied to a substrate. The contact makes it possible to form a film of conductive fine particles containing a binder. In this case, the solvent to be used is water or an alcohol such as methanol or ethanol, as described above.

Further, the film of the conductive fine particles with / without the binder of the present embodiment is decorated with a probe.

“Modifying the conductive fine particle film of the present invention with a probe” means not only that at least a part of the probe is in direct contact with the conductive fine particle film, but also that the probe and the conductive fine particle This includes the case where the film is in a nanogap state that is close enough to allow electrical conduction with the film.

When a probe described later is modified with a specific group or the like, the probe may be modified on the film of the conductive fine particles via the group.

Further, the “probe” of the present invention includes those capable of changing the electric resistance between the conductive fine particles by reacting the probe with the target substance. Specifically, a nucleic acid such as DNA or RNA, or a protein such as an antigen or an antibody is used as a probe. Further, the probe used may be a natural one or an artificial one. Further, the probes need not necessarily be 100% identical, and may contain other substances as long as they do not interfere with detection.

When single-stranded DNA is used as a probe, first, double-stranded DNA is collected using a known method, and the double-stranded DNA is suspended in distilled water or the like. After heating for about 10 minutes, transfer to ice and cool rapidly to obtain single-stranded DNA. Further, the “reaction” of the present invention includes not only a chemical reaction but also a physical interaction. As an example of the reaction, single-stranded DNA is used as a probe, and DNA that is complementary to the single-stranded DNA is used as a target substance.Hybridization between the DNAs, an antibody is used as a probe, Examples of such binding include those occurring when an antigen is used as a target substance.

When DNA is used as a probe, the length of the DNA used as a probe is at least 2 to 3000 bp, preferably 4 to 100 bp, and more preferably 10 to 12 bp.

When an antibody is used as a target substance, examples of the probe include an antibody or an antigen capable of reacting with a target antigen or an antibody. Further, it is also preferable to activate the probe with a specific group or the like.

In particular, when gold nanoparticles are used as the conductive fine particles and DNA or antibody is used as the probe, the probe is preferably activated with a specific group such as an SH group or NH ₂ group. At this time, the sites to be activated include sites involved in the reaction (for example, if the target substance is DN

In the case of A, it is preferable to use a site other than the site containing the target sequence, or the site where the antigen is used as the target substance). Among them, the part to be activated is preferably at the end of the probe, and more preferably at both ends of the probe.

Further, as long as the detection of the target substance is not hindered, the site to be activated may include a part of the site involved in the reaction.

The method of activating the probe may be appropriately selected from known methods depending on the type of the probe and the conductive fine particles to be used. For example, when DNA is used as the probe, the probe can be treated with a thiol having an SH group such as 1,10-decanedithiol or a diamine having an NH ₂ group such as 1,10-diaminodecane. Then, the probe can be activated.

In addition, when an antibody is used as a probe and gold nanoparticles are used as conductive fine particles, for example, mercaptopropionic acid having an SH group that binds to the surface of the gold nanoparticles and having a lipoxyl group at the terminal, etc. And treated with N-hydroxysuccinimide, triethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride, etc. to form an activated site capable of binding to the antibody. Then, the probe having the NH ₂ group can be immobilized on the gold nanoparticle, the electrode, or the like via the peptide bond.

Further, the electric resistance type detection sensor shown in the second embodiment has a concave portion formed on the substrate surface of the first embodiment, and a film of the conductive fine particles modified with the probe is formed on the inner surface of the concave portion. It is formed on the surface. Otherwise, the same sensor as the electric resistance type detection sensor described in the first embodiment can be applied.

Specifically, the “recess” of the present invention has such a size that a probe and a sample can react in the recess and a film of conductive fine particles can be formed in the recess. And those having a shape.

Therefore, the shape of the concave portion may be a round or polygonal cylindrical shape or a mortar shape. Especially, it is preferable that it is a mortar shape. In other words, it is desirable that the concave portion has a smaller area at the bottom than the opening. Furthermore, the number of recesses present on one substrate surface can be appropriately selected according to the application of the sensor. For example, when the size of the substrate and 1 cm ^² ~ 3 cm ^2, the number of recesses, 1 0 0-3 0 0 0, preferably 5 0 0 or more, 1, 0 0 0 or more, More than 1,500, less than 2,000, less than 2,500.

Further, it is preferable that the electrodes are provided so as to be in contact with the film of the conductive fine particles in each concave portion and not to directly contact each other.

Further, the portion where the electrode is formed is not limited to the surface of the substrate, but may be the inside of the recess, the shoulder or the bottom.

The method for forming the concave portion can be selected from known methods according to the material of the substrate and the size and shape of the concave portion. For example, when a glass substrate or a plastic substrate is used, a mask having a pattern of a predetermined shape is formed on the substrate surface, and a predetermined concave portion is formed on the substrate surface by using a laser beam using a chemical etching agent. Can be formed.

Note that the concave portion may be formed on the surface of the substrate by attaching the sheet having the concave portion to the substrate by lamination or the like.

As a method for forming an electrode, a known method can be used. For example, an electrode can be formed by forming a mask having a predetermined pattern on the substrate surface and depositing a metal film thereon.

Either the formation of the concave portion or the formation of the electrode may be performed first! /.

Further, the binder described above may be contained in the gold nanoparticle film of the present invention. Further, as a third embodiment,

There is also an electric resistance type detection sensor characterized in that a film of conductive fine particles is modified with a probe.

A plurality of fine concave portions are formed on the surface of the substrate according to the present embodiment.

Further, the first and second electrodes of the electric resistance type detection sensor according to the present embodiment include one or more It can be connected via a multiplexer, preferably an analog multiplexer. The multiplexer here functions as a demultiplexer. The multiplexer includes an element having a function of switching a target sensor based on an external address signal.For example, a multiplexer having an input terminal, an output terminal, and an address input terminal for inputting an address signal is used. be able to. It is preferable that a control unit such as a microphone computer that outputs an address signal is connected to the address input terminal.

The first and second electrodes connected via one or more multiplexers are connected to an electrical device such as a voltmeter, current meter, or resistance meter that measures electrical characteristics such as voltage, current, or resistance between electrodes. It is preferable to be connected via a characteristic measuring device.

Further, the first electrode and the second electrode may be connected via a lock-in circuit instead of the electrical property measuring device. Further, the electrical characteristic measuring device may be connected to an output terminal of the lock-in amplifier circuit. In this case, by detecting the synchronous component of the periodic voltage change by the lock-in amplifier, noise characteristics generated in the measurement environment can be eliminated or reduced, and the sensitivity of the electric signal (voltage) can be increased.

The electrical characteristics measuring instrument preferably has an output terminal that outputs current, voltage, etc. according to the magnitude of the measured electrical characteristics, and a control unit is connected to this output terminal. Is preferred. When a lock-in amplifier circuit is used instead of the electrical characteristic measuring device, the control unit is preferably connected to an output terminal of the lock-in amplifier circuit. It is preferable that the control unit has a storage unit such as a memory and stores the output of the electrical characteristic measuring device.

The control unit is preferably further connected to an output device such as a monitor or a printer, and is preferably configured to output the electrical characteristics stored in the storage unit to the output device. Other than the above, those described in the first and second embodiments can be used. Further, as a fourth embodiment,

A substrate having a plurality of fine concave portions formed on the surface thereof; a film of conductive fine particles formed on the inner surface of each concave portion; and a first conductive film formed to be electrically connected to the film of conductive fine particles. And a second electrode, By using an electric resistance type detection sensor in which a film of conductive fine particles is modified with a probe and connected to at least one electrode and / or another conductive fine particle, a separate probe is provided in each concave portion. After binding, the target substance in the sample can be easily detected and confirmed easily and in a short time.

The first or second electrode corresponding to each recess may be electrically connected to each other. In this case, with the same configuration as in the third embodiment, it is possible to measure the sample introduced into each recess.

“The first electrode is formed on the substrate surface” includes the case where the first electrode formed on the substrate surface is covered with an insulating material or the like. That is, the portion where the first electrode is formed is not limited to the surface of the substrate, but may be the inside of the recess or the shoulder.

The second electrode may be exposed on the back surface. When the second electrode is exposed on the back surface, the second electrode may be entirely or partially covered with an insulating material or the like.

The second electrode can be formed by forming a plurality of grooves that do not cross each other, preferably extend in parallel, on the back surface of the substrate, and fill the grooves with a conductor such as platinum. In addition, a second substrate having a plurality of electrodes that do not intersect each other, and preferably has a plurality of electrodes extending in parallel, is formed on the back surface of the first substrate by forming a through hole in the first substrate. An electrode may be formed.

Further, it is preferable that the plurality of recesses are arranged in a matrix of a plurality of rows and columns, and the first electrode of each row and the second electrode of each column are electrically connected to each other. The rows and columns of the matrix preferably intersect at right angles, but may intersect at any desired angle. The rows and columns may be linear or curved.

Each column of the first electrode and each row of the second electrode can be connected to a multiplexer, and by sequentially changing an address signal applied to the multiplexer, the output of the sensor is obtained for each concave portion arranged in a matrix. Can be measured.

For the multiplexer, control unit, electrical characteristic measuring device, lock-in amplifier circuit, output device, etc., those described above can be applied. With such a configuration, there is an advantage that many measurements can be easily performed in a small space.

A method for detecting a target substance using the electric resistance type detection sensor described in the first to fourth embodiments will be described below.

In the present invention, first, a measurement sample is sprayed on a film of conductive fine particles modified with a probe. “Spraying on the conductive fine particle film” in the present invention means that the measurement sample is brought into contact with the conductive fine particle film. Specifically, for example, the measurement can be performed by dropping a measurement sample onto a film of conductive fine particles.

Further, the “measurement sample” of the present invention is a sample in which the presence or absence of a target substance is detected so that the measurement is not hindered. Specifically, for example, the sample is diluted with an appropriate solvent so that the concentration of the sample becomes an appropriate concentration for detection, or the obtained double-stranded DNA is converted into a single-stranded DNA in a form suitable for detection. And those treated with strand DNA.

The amount of the measurement sample is not particularly limited as long as it can contact at least the film of the conductive fine particles in the concave portion on the substrate.

Furthermore, the conditions under which the probe reacts with the measurement sample can be appropriately selected according to the probe and the measurement sample used.

Then, by using a known method, the electric resistance between the obtained films of the conductive fine particles is measured, and the electric resistance of the film before and after the measurement sample is sprayed is measured to determine the presence or absence of the target substance. Can be detected and confirmed.

That is, usually, the current between the electrodes flows through the film of the conductive fine particles, but when the target substance is present in the measurement sample and the target substance reacts with the probe, the current flows through the probe. As a result, a difference occurs in the electric resistance value between the electrodes. By utilizing this fact, the presence or absence of the target substance can be electrically detected and confirmed.

Furthermore, the fifth embodiment is not limited to the electric resistance type detection sensors shown in the first to fourth embodiments,

Modifying a film of conductive fine particles formed on the surface of the electrically insulated substrate with a probe, and spraying a measurement sample containing a specimen on the modified film; By measuring the electrical resistance value between two points of the obtained film of conductive fine particles,

The presence / absence of a target substance that reacts with the probe can be detected, confirmed, and confirmed.

Further, as a detection method of the sixth embodiment,

Prepare a measurement sample containing a sample and a probe in advance,

The sample is sprayed on a film of conductive fine particles formed on the surface of an electrically insulated substrate, and the electrical resistance between two points on the obtained film of conductive fine particles is measured to react with the probe. The presence or absence of the target substance to be detected can be detected and confirmed.

In this detection method, a sample prepared in advance containing a sample and a probe is sprayed on a film of conductive fine particles that have not been modified with a probe on the substrate. It differs from the above detection method in that it measures.

That is, the above detection method does not require a step of modifying the conductive fine particle film with the probe. The above-mentioned “preparing a measurement sample” includes exposing the probe and the measurement sample to conditions under which the target substance and the probe can react.

Specifically, for example, when a single-stranded DNA of about 12 bp is used as the probe, the measurement sample can be prepared by leaving the probe and the measurement sample to be detected at room temperature for 30 minutes.

Otherwise, those described above can be applied. Example 1

Example 1 Example 1 examined the selectivity of the electric resistance type detection sensor of the present invention. In this example, 200 ml of an aqueous solution containing 6 ml of a 1% aqueous solution of tetrachloro base (III) acid tetrahydrate (Wako Pure Chemical) and 10 ml of a 3% aqueous solution of citrate (Katayama Chemical) was used. The aqueous solution was stirred at 80 ° C for 20 minutes to prepare a colloidal gold solution. Then, “comb-shaped electrode platinum” (manufactured by BAS Co., Ltd.), which is formed by depositing platinum on a glass substrate (lcm X lcm) so that the gap between the electrodes is 5 zm, is applied to 1,10_decanedithiol Z By dipping in an ethanol solution and then in a gold colloid solution containing gold nanoparticles, a film of gold nanoparticles was formed on the electrodes and between the electrodes. Then, the 5 'end that serves as a probe is activated with an SH group on the obtained membrane. 100] MW) DNA (SEQ 1: 5'-TCTCAACTCGTA-3 ') aqueous solution of DNA was dropped 51 and allowed to stand for 30 minutes to form a film of gold nanoparticles on the electrodes and between the electrodes. Was modified with a probe. .

Next, TE buffer solution (1 OmM Tris_HCl, 1 mM EDTA, 1 M NaCl) was added dropwise to the resulting membrane to wet the gold nanoparticle membrane, and the electrical resistance of the membrane was stabilized. It was left until. Then, a TE buffer containing 100 / zM of DNA having the sequence of SEQ2 to 5 was prepared as a measurement sample, and the prepared solution 5 was dropped on the membrane obtained above. FIG. 1 shows a DNA having a sequence of SEQ 1 consisting of 12 bp as a probe, and a DNA having a sequence of SEQ 2 (single-stranded DNA having 2: 1 bp complementary) and a sequence of SEQ3 having a sequence of SEQ 2 as a measurement sample. DNA (3: 8 bp complementary single-stranded DNA), SEQ ID NO: 4 having a sequence of SEQ 4 (4:11 bp complementary single-stranded DNA), DNA having a sequence of SEQ 5 This figure shows the change in the resistance value of the membrane before and after the detection when each measurement sample was used using (5: 12 b ρ all single-stranded DNAs are complementary). As a result, when the measurement sample was dropped on the DNA-modified gold nanoparticle film, the electric resistance of the film decreased, and the resistance value of the film stabilized in about 1 minute thereafter. At that time, when using the DNA of SEQ5 as a measurement sample (5), the change in the electrical resistance value before and after detection was greatest (5.16X10- ² Qcm). On the other hand, in the case of using any other DNA as a measurement sample, the change in the electrical resistance of the film, 2.40X10-2 □ cm (4), 1.44X10- 2 Qcm (3), 1.39Xl (T 2 Qcm In other words, the change in the electrical resistance of the film before and after the detection was l.OlXlO-cmZbase in (4) and (2), whereas (4) and (5) between) and 2.76X10- ² Ω cm / base, are use (between 4) and (5), the most significant changes were observed. this electrical resistive sensor of the present invention This shows that even a difference of 1 bp from the target DNA can be detected efficiently, indicating that the electric resistance detection sensor of the present invention has excellent selectivity. Then, as described above, after modifying the gold nanoparticle membrane with DNA having the sequence of SEQ 1 as a probe, 1 μl of TE buffer was dropped on the obtained gold nanoparticle membrane. And gold nanoparticles Damp film evenly. Then, the electric resistance of the film of the gold nanoparticles to stable Left until the Then, about 100 seconds after stabilization, TE buffer 51 containing DNA having the sequence of SEQ5 was dropped onto the gold nanoparticle film, and again until the electrical resistance of the gold nanoparticle film was stabilized. I left it. Then, the DNA-degrading enzyme DNase I (Wako Pure Chemical,

^ 11) was added dropwise and left at room temperature for about 1 hour to decompose the DNA bound to the gold nanoparticle film and return the film to the state before modification with the probe. Figure 2 shows the electrical resistance of the DNA (probe) -modified gold nanoparticle film before and after the DNA is degraded by the DNAse. As a result, the electrical resistance of the gold nanoparticle film after decomposing the DNA modified with the gold nanoparticle film is almost the same as the electrical resistance value of the gold nanoparticle film before the DNA modification (624.36Ω). Became. This indicates that the electric resistance type detection sensor of the present invention can be used repeatedly. Example 2

Same as Example 1 except that DNA having the sequence of SEQ5 was used as the measurement sample, and DNA having the sequence of SEQ1 (manufactured by Nisshinbo) whose both ends (3 and 5 ′) were thiolated was used as the probe The electrical resistance of the film was measured by various methods.

As shown in Fig. 3, the change in the electrical resistance of the membrane before and after the detection was 2.9 compared to the case where only one (5 ') end of the probe DNA was thiolated (Example 1 (5)). (Example 1: 0.30Ω, Example 2: 0.87Ω). This indicates that the detection sensitivity increases as the number of probe modification sites increases. Example 3

1.5 ml of colloidal gold solution containing gold nanoparticles and 5 μl of an aqueous solution containing 50 μl of DNA having the sequence of SEQ 1 with thiolated terminal (3 ′ and 5 ′) were mixed for 30 minutes After standing, the gold nanoparticles were modified with the probe DNA (SEQ1). Then, the solution was centrifuged (600 O rpm, 10 minutes), and the solution was re-dispersed with 1.5 ml of water. This operation was repeated twice, and finally, the solution was redispersed with 0.5 ml of water. Then, 30 μl of the obtained solution is dropped on a comb-shaped electrode platinum (manufactured by BS Co., Ltd.), and the solution is placed on the electrode. The electrical resistance was measured in the same manner as in Example 2 except that a film of gold nanoparticles containing no binder was prepared between the electrodes.

As a result, as shown in FIG. 4, the change in the electrical resistance of the film before and after the detection was 0.57Ω. This indicates that the electrical resistance type detection sensor of the present invention can detect a target substance without including a binder in the conductive fine particle film. Example 4

The same glass substrate as used in Example 1 and a comb-shaped electrode platinum were used, and as a probe.5 μl of a buffer solution containing DNA100 /] ^ having a sequence of 5 EQS-thiolated SEQU 1 was used as a probe. Then, as a measurement sample, a 5 μl buffer solution containing 100 μl of DNA having the sequence of SEQ 5 is mixed in a microphone opening tube (TreffLab, manufactured by Treff AG, Switzerland) in advance and prepared. Then, the electrode was immersed in a 1,10-decanedithiol / ethanol solution and then immersed in a colloidal gold solution to form a film of gold nanoparticles on the electrode and between the electrodes. Then, the gold nanoparticle film was moistened with a 1/1 TE buffer, the above prepared solution was dropped on the gold nanoparticle film, and the electric resistance value of the film before and after the drop was measured. Figure 5 shows the results.

Increase as a result, as compared with the case of using a DNA thiolated only 5 'end as probes as described in Example 1 (0. ³ 0Ω), the electrical resistance of the film before and after detection 2. sevenfold (0.81Ω). This indicates that the detection method described in the sixth embodiment of the present invention is useful. Example 5

Next, the case where an antibody is used as a probe will be described below.

Using the same glass substrate and comb-shaped electrode (manufactured by BAS Inc.) as used in Example 1, immerse them in a 1,10-decanedithiol ethanol solution and then immerse them in a colloidal gold solution. As a result, a film of gold nanoparticles was formed on the electrodes and between the electrodes. Then, the electrode on which the gold nanoparticle film was formed was connected to 1 OmM mercaptopropionic acid (Tokyo It was immersed in a (chemical formation) / ethanol solution for 30 minutes, and the gold nanoparticle film was modified with mercaptopropionic acid. Then, the obtained membrane was rinsed with ultrapure water and ultrasonically washed in ethanol for 5 minutes. Next, the gold nanoparticle film was rinsed with ultrapure water and dried. Next, the film of the gold nanoparticles is brought into contact with 20 μl of an aqueous solution of 10 OmgZ 1 of N-hydroxysuccinimide (Wako Pure Chemical), and further, 10 Omg // x 1 of 1-ethyl-3- [3- [Dimethylaminopropyl] carbodiimide hydrochloride (WSC) (Dojindo) aqueous solution 201 and then allowed to stand at room temperature.

Then, the obtained gold nanoparticle film is rinsed with ultrapure water, further washed with 0.1 M Tris-monohydrochloride buffer (ρΗ8), and 10 μl of 0.1 μm is added to the gold nanoparticle film. 1M Tris-hydrochloride buffer was added dropwise and left at room temperature for 100 seconds. An antibody solution of rabbit anti-mouse IgG (Wako Pure Chemical Industries, Ltd.) diluted 100-fold with 0.1 M Tris-hydrochloride buffer was added dropwise to the obtained membrane, and the mixture was allowed to stand at room temperature for 30 minutes. .

Thereafter, the obtained membrane is rinsed with Tris-hydrochloride buffer, and then 0.1 1 of 0.1 M Tris-hydrochloride buffer is dropped thereon, followed by a drop of an aqueous solution of 201 ethanolamine (Wako Pure Chemical Industries). After allowing to stand at room temperature for 1 hour, the activated site where the antibody was not immobilized was masked. Then, the antibody-modified gold nanoparticle membrane was washed with ultrapure water, further washed with a tris-hydrochloride buffer, and the gold nanoparticle membrane was exposed to 10 μl of tris-hydrochloride buffer. . After allowing the mixture to stand for 100 seconds, an antigen solution of 100 / zg mouse IgG (Upstate BioTechnology) / tris-hydrochloride buffer solution (1 iL) was added dropwise to the obtained membrane at 10 / z1. Immediately after dropping, the electrical resistance of the film was observed, and the results are shown in Figure 6.

The results in FIG. 6 indicate that the electric resistance type detection sensor of the present invention can be applied to the detection of an antigen. Example 6

FIG. 7 shows an electric resistance type detection sensor 51 according to Embodiment 6 of the present invention. The electric resistance detection sensor 51 according to the second embodiment of the present invention includes a plurality of recesses 53 formed on the surface of a substrate 54, and a gold nanoparticle film 57 is formed on the inner surface of each recess 53. ing. Also, The first and second electrodes 55 and 56 are formed so as to be electrically connected to the gold nanoparticle film 57 in each recess 53. The electric resistance type detection sensor 51 is manufactured by the following method. Formation and cleaning of electrodes

First, a plurality of platinum electrodes extending parallel to each other are formed on a substrate. The platinum electrode can be formed, for example, by depositing platinum while masking a place other than the place where the platinum electrode is formed. Next, as shown in FIG. 7A, a recess is formed at the center of each electrode so as to bisect the platinum electrode. The platinum electrode is divided by the recess, and the first and second electrodes are formed. After the formation of the concave portion, the platinum electrode is washed by the following method.

First, the platinum electrode was swept in 0.1 M H ₂ SO ₄ using Ag / Ag C 1 as a reference electrode and a platinum coil (manufactured by Niraco) as a counter electrode. Wash 25 to 10 times in a range of 1.3 V by repeating the sweep 50 times. The electrochemical cleaning is performed using a potentiostat (263A-1 manufactured by Seiko EG & G).

Formation of gold nanoparticle film

Next, a method of forming a gold nanoparticle film will be described.

First, 6 ml of an aqueous solution of 1% tetrachloro base (III) acid tetrahydrate (Wako Pure Chemical) and a 20 ml of an aqueous solution containing 10 ml of a 3% aqueous solution of citrate (Katayama Chemical) at 80 ° C for 20 minutes Stir to prepare colloidal gold solution.

Next, inject the ethanol solution containing 5 mM 1,10-decanedithiol into the upper part, and after the ethanol evaporates, lightly rinse with ethanol. Next, a gold nanoparticle film is formed on the surface of the concave portion by pouring a colloidal gold solution into the concave portion.

By forming the gold nanoparticle film in this manner, the first and second electrodes 55 and 56 are electrically connected to the gold nanoparticle film 57 in each recess 53.

Modification with DNA probes

First, DNA (Nisshinbo) having a thiolated 5 ′ end was used as a probe. Next, fill the recess with 1 μl (recess volume 1 mm ³ ) of buffer containing 10 μM of the above DNA (10 mM Tris_HC1, 1 mM EDTA, 1M NaC1) for 30 min. put. This modifies the gold nanoparticles with the probe DNA. Next, the surface of the gold nanoparticles was washed with TE buffer to remove excess thiolated DNA, and further removed with 1 μl of TE buffer to remove the effect of interfacial resistance. Wet the particle surface.

Peripheral equipment

Next, peripheral devices and the like connected to the electric resistance type detection sensor 51 will be described.

As shown in FIG. 7, the first electrode 55 corresponding to each recess 53 is electrically connected to the input terminal 61 of the multiplexer 60. Further, the output terminal 62 of the multiplexer 60 and the second electrode 56 corresponding to each of the concave portions 53 are electrically connected via the electric resistance measuring device 63. The electric resistance measuring device 63 outputs a voltage corresponding to the measured electric resistance from its output terminal 64. Further, a microcomputer 65 is connected to an address input terminal 66 of the multiplexer 60 and an output terminal 64 of the electric resistance measuring instrument 63. Here, since the multiplexer 60 functions as a demultiplexer, a plurality of outputs from each recess 53 are input to the input terminal 61 and output from a single output terminal 62.

The microcomputer 65 sequentially changes the output address and stores the output of the electric resistance measuring device 63 for each recess 53. The microcomputer 65 is connected to an output device 67 such as a printer or a monitor, and is configured to output stored data to the output device 67. With such a configuration, many target DNAs can be easily detected at one time.

Detection of target DNA

Next, a method for detecting a target DNA using the electric resistance type detection sensor 51 of the present invention and its peripheral devices will be described.

First, a 50 / i 1 TE buffer solution containing a 100 M measurement sample is dripped evenly over the surface of the previously prepared gold nanoparticles into the recess 53 of the electric resistance detection sensor, and left for 3 minutes. . Then, using a digital multimeter (type 34401A, manufactured by HEWLETT PACKARD) 63, measure the electrical resistance at both ends of the electrode at 22 ± 1 ° C. Example 7 FIG. 8 is a block diagram illustrating a configuration of the electric resistance type detection sensor according to the seventh embodiment. In the electric resistance type detection sensor according to the seventh embodiment, the output terminal 62 of the multiplexer 60 and the second electrode 56 corresponding to each recess 53 are electrically connected to the second electrode 56 via the lock-in amplifier circuit 68. It is. The output terminal 69 of the mouth-in amplifier 68 is connected to the microcomputer 65. Other configurations and the method of forming the gold nanoparticles are the same as those in the sixth embodiment.

FIG. 9 is a block diagram illustrating a configuration of the lock-in circuit 68 according to the seventh embodiment. A is an adder, B is an electric resistance type detection sensor indicated by a dotted line in FIG. 3, C is a current-voltage converter, D is a lock-in amplifier, E is a bias voltage, and F is a synchronization signal. The noise generated in the measurement environment can be eliminated or reduced by selectively detecting the output component synchronized with the signal F with the lock-in amplifier. Example 8

FIG. 10 shows an electric resistance type detection sensor 71 provided with a plurality of the electric resistance type detection sensors on a substrate according to Embodiment 7 of the present invention. The electric resistance type detection sensor 71 includes a plurality of concave portions 73 arranged in a matrix composed of a plurality of rows X and a plurality of columns Y.

Further, a film 77 of gold nanoparticles is formed on the inner surface of each recess 73. In addition, the first and second electrodes 75, 76 1 are formed so as to be electrically connected to the gold nanoparticle film 77 in each recess 73. Further, the electrode 75 may be formed on the surface of the substrate 74 in the concave portion 73 in a ring or a similar shape.

The first electrode 75 is formed on the surface of the substrate 74, and the second electrode 76 is formed inside the concave portion 73 and is exposed on the back surface of the substrate 74. The first electrode 75 of each row X and the second electrode 76 of each column Y are electrically connected to each other.

Electrode and gold nanoparticle film formation

Next, a method of forming the electrodes 75 and 76 and a gold nanoparticle film 77 electrically connected thereto will be described.

First, a plurality of grooves extending parallel to each other are formed on the back surface of the substrate, and a second electrode 76 is formed of platinum so as to fill the grooves. Next, the first electrode 75 is formed in a shape as shown in FIG. 10 (a).

Next, a plurality of concave portions 73 arranged in a matrix are formed from the surface of the substrate 74 so as to face the second electrode 76. The concave portion 73 is formed at a depth where the second electrode 76 is exposed on the surface side of the substrate 74.

Finally, a gold nanoparticle film 77 is formed on the inner surface of the concave portion 73 by the same method as that used in Example 6, and the formation of the electrode and the gold nanoparticle film is completed.

Modification with DNA probe

Next, the gold nanoparticle film 77 is modified with a DNA probe in the same manner as that used in Example 6.

Peripheral equipment

Next, peripheral devices connected to the electric resistance type detection sensor 71 will be described.

As shown in FIG. 10, each column Y of the first electrode 75 and each row X of the second electrode 76 are connected to the input terminals 82, 83 of the multiplexers 80, 81, respectively. The output terminals 84, 85 of the multiplexers 80, 81 are electrically connected to each other via an electric resistance measuring device 86. The electric resistance measuring device may use a lock-in amplifier circuit 68 as shown in FIG. The electric resistance measuring device 86 outputs a voltage corresponding to the measured electric resistance from its output terminal 87. A microcomputer 88 is connected to address input terminals 89, 90 of the multiplexers 80, 81 and an output terminal 87 of the electric resistance measuring device 86.

Further, the microcomputer 88 sequentially changes and outputs the output address to each of the multiplexers 80 and 81, scans the concave portions 73 arranged in a two-dimensional array, and measures the electric resistance of each concave portion 73. 8 Store the output of 6. The microcomputer 88 is connected to an output device 91 such as a printer or a monitor, and has a structure for outputting stored data to the output device 91.

With such a configuration, more target DNAs can be detected at one time. In addition, by adopting a structure in which the first electrode is arranged on the front surface of the substrate and the second electrode is arranged on the back surface of the substrate, the sensors can be arranged at a high density, and the space of the device can be saved. . Detection of target DNA

Using the electrical resistance type detection sensor 1 of Example 9 and its peripheral devices, the target DNA can be detected in the same manner as that used in Example 6. Industrial potential

By using the above-described electric resistance type detection sensor of the present invention, it is easier, faster, cheaper and more compact than before, without using special reagents such as fluorescent substances or complicated devices. The presence or absence of the target substance can be accurately detected and confirmed accurately.

Further, the electric resistance type detection sensor of the present invention can be used more easily, quickly, and inexpensively than before, and can be used repeatedly.

Further, by forming a concave portion in the substrate, forming a film of conductive fine particles modified with a probe in the concave portion, and reacting the measurement sample with the probe in the concave portion, the space required for the reaction can be reduced.

In addition, by forming a plurality of parts on the same substrate, it is possible to electrically detect, confirm, and confirm a variety of measurement samples and Z or target substances at once.

Furthermore, by using gold nanoparticles as conductive fine particles and using a DNA or an antibody activated with an SH group or NH ₂ group as a probe, a target substance can be detected and confirmed with higher accuracy.

Further, by activating one end of the probe, the target substance can be detected and confirmed more accurately.

In addition, by activating both ends of the probe, the target substance can be detected and confirmed more accurately.

In addition, by making the shape of the concave portion into a mortar shape, detection can be performed more efficiently. In addition, by using the above-described electric resistance type detection method of the present invention, it is easier, faster, and cheaper than before, without using a special reagent such as a fluorescent substance or a complicated device. The target substance can be electrically detected and confirmed accurately and compactly.

Also, by using the electric resistance type detection method of the present invention, it is easier, faster, and easier than before. And the target substance can be detected and confirmed repeatedly at low cost.

Claims

The scope of the claims

1. A pair of electrodes are placed facing each other on the surface of an electrically insulated substrate and placed on the electrodes.

An electric resistance type detection sensor characterized in that a film of conductive fine particles modified with a probe is formed between Z or an electrode.

2. A concave portion is formed on the surface of the electrically insulated substrate, and a pair of electrodes are arranged in the concave portion so as to face each other. An electric resistance type detection sensor characterized by being formed.

3. The electric resistance detection sensor according to claim 1, wherein the film of the conductive fine particles contains a binder.

4. The electrical resistance type detection sensor according to claim 1, wherein the probe is DNA or an antibody.

5. The conductive particles are gold nanoparticles. The electrical resistance type detection sensor according to any one of claims 1 to 4.

6. The electric resistance type detection sensor according to claim 5, wherein the binder is 1,10-decanedithiol.

7. The electric resistance type detection sensor according to claim 5, wherein the DNA or the antibody is activated by an SH group or an NH ₂ group.

8. The electrical resistance type detection sensor according to any one of claims 5 to 7, wherein at least one end of the DNA or the antibody is activated by an SH group or an NH ₂ group.

9. The electrical resistance type detection sensor according to any one of claims 5 to 8, wherein both ends of the DNA or the antibody are activated by an SH group or an NH ₂ group.

10. A substrate having a plurality of fine concave portions formed on the surface, a film of conductive fine particles formed on the inner surface of each concave portion, and a second conductive film formed to be electrically connected to the conductive fine particle film. A first electrode and a second electrode,

An electric resistance type detection sensor characterized in that a film of conductive fine particles is modified with a probe.

11. A substrate having a plurality of fine concave portions formed on the surface, a film of conductive fine particles formed on the inner surface of each concave portion, and a second conductive film formed to be electrically connected to the conductive fine particle film.

A first electrode and a second electrode,

An electric resistance type detection sensor, wherein a first electrode is formed on a surface of a substrate, a second electrode is formed inside four parts, and a film of conductive fine particles is modified with a probe.

12. The electric resistance type detection sensor according to claim 10, wherein one of the first and second electrodes S is electrically connected to each other.

13. The method according to claim 11, wherein the plurality of recesses are arranged in a matrix comprising a plurality of rows and columns, and the first electrode in each row and the second electrode in each column are electrically connected to each other. Electric resistance type detection sensor.

14. The electric resistance type detection sensor according to any one of claims 10 to 13, wherein the recess has a mortar shape.

15. The electric resistance type detection sensor according to any one of claims 10 to 14, wherein the film of the conductive fine particles contains a binder.

16. The electric resistance type detection sensor according to any one of claims 10 to 15, wherein the probe is a DNA or an antibody.

17. The electric resistance detection sensor according to any one of claims 10 to 16, wherein the conductive particles are gold nanoparticles.

18. The electric resistance type detection sensor according to claim 17, wherein the binder is 1,10-decanedithiol.

19. The electric resistance type detection sensor according to claim 17, wherein the DNA or the antibody is activated by an SH group or an NH ₂ group.

20. The electric resistance type detection sensor according to any one of claims 17 to 19, wherein at least one end of the DNA or the antibody is activated by an SH group or an NH ₂ group.

21. The electric resistance type detection sensor according to any one of claims 17 to 20, wherein both ends of the DNA or the antibody are activated by SH groups or NH ₂ groups.

2 2. Modify the conductive fine particle film formed on the electrically insulated substrate surface with a probe,

Spray the measurement sample containing the analyte on the modified membrane,

An electric resistance type detection method for detecting the presence or absence of a target substance that reacts with a probe.

2 3. Prepare a measurement sample containing a sample and a probe in advance,

24. The electric resistance type detection method according to claim 22, wherein the probe is a DNA or an antibody.