JP2002325566A - Nucleic acid sensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleic acid sensor having a small size and excellent in operating property. SOLUTION: This nucleic acid sensor equipped with an electrode zone in which a nucleic acid probe is fixed, is to detect the presence of an objective nucleic acid by measuring a generated potential change caused by a double chain formation between the objective nucleic acid and the fixed nucleic acid probe. The nucleic acid sensor comprises a semiconductor base substrate, an insulating film arranged on the above base substrate, and as necessary a protective film arranged on the above insulating film, and is equipped with a means for holding a liquid specimen arranged on the sensor base substrate; a functioning electrode arranged on the above semiconductor base substrate; and the above insulating film or protective film, in which the means for holding the liquid specimen prescribes the electrode zone on the sensor base substrate, and the electrode zone is equipped with the means for fixing the nucleic acid probe. The sensor takes out the generated potential change caused by the double chain formation between the nucleic acid probe and the objective nucleic acid in the liquid specimen corresponding to an irradiation of a light, as a corresponding signal from the functioning electrode.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a nucleic acid sensor for detecting a nucleic acid having a specific sequence of interest and a nucleic acid detecting device using the same.

[0002]

2. Description of the Related Art In recent years, technology utilizing genetic information has been actively developed. In the medical field, analysis of disease-related genes has made it possible to treat diseases at the molecular level. In addition, tailor-made medical treatment corresponding to each patient has become possible by genetic diagnosis. In the pharmaceutical field, genetic information is used to identify protein molecules such as antibodies and hormones, and use them as drugs. Many other products using genetic information have also been produced in the agricultural and food fields.

In order to obtain such genetic information, conventionally,
Analysis methods such as the Southern hybridization method have been used, but instead, DNA chips or DNA
Microarrays have come into wide use. These integrated DNA sensors can process tens of thousands to hundreds of thousands of DNA fragments at one time, but one problem is how to analyze a large amount of obtained data.

On the other hand, as the integration of DNA sensors progresses, the price of one sensor becomes extremely high. Also, in general, in the integration of a DNA sensor, the sensitivity of the sensor tends to be sacrificed. Therefore, a step of amplifying DNA is required before the measurement, for example, by using a PCR method or the like, and the apparatus is complicated and large, and the time required for the measurement is long.

[0005] From such a viewpoint, a nucleic acid sensor using an electrochemical system has been developed (Japanese Patent Application Laid-Open No. 9-5033).
07). In a nucleic acid sensor using such an electrochemical method, it is expected that a nucleic acid sensor with higher sensitivity and lower cost can be obtained as compared with a fluorescent method. However, according to this method, wiring between the electrode and the terminal is required. For example, FIG. 5 shows a nucleic acid sensor disclosed in Japanese Patent Laid-Open Publication No. 9-503307. As shown,
In this nucleic acid sensor, many metal wirings, for example, made of aluminum or the like are required between the electrodes and the terminals. Further, in order to realize a high density of electrodes, it is necessary to increase the density and precision of the wiring, and it is difficult to integrate the wiring.

[0006] As an example of a small and highly operable device used for diagnosis in the medical field, an L-type device using a semiconductor substrate is used.
APS (Light-Addressable Pot)
entometric Sensor: see Molecular Devices, Inc., U.S. Pat. Nos. 4,758,786 and 4,963,815).
FIG. 6 shows the structure of LAPS. As shown in FIG. 6, the LAPS includes a semiconductor silicon substrate 101 and a sensor substrate including an oxide film 102 and a nitride film 103 formed thereon. It is used as a sensor for measuring the pH of the (electrolyte solution) 104. The principle of measuring the pH of LAPS is as follows.

As shown in the figure, an electrolytic solution (Electr)
EIS composed of an insulating film, an insulator, and a semiconductor.
A DC bias voltage is applied to the structure by potentiostat 105. In this state, when light modulated at a predetermined frequency is irradiated from the back surface of the EIS structure, an optical alternating current flows as shown in FIG. At this time, it is known that the IV curve indicating the relationship between the DC bias voltage V and the optical AC current I shifts in the horizontal axis (DC bias voltage) direction as shown in FIG. 7 according to the pH of the solution. Have been. Therefore, it is possible to measure the pH of the electrolyte by detecting the photo-current AC while a predetermined DC bias voltage is being applied.

The reason why the IV curve shifts according to the pH of the solution is considered as follows. When a voltage is applied to the EIS structure, an energy band bends at the interface between the semiconductor and the insulating film, and this bend also depends on the pH of the solution in contact with the insulating film. That is, a silanol group (Si-OH) and an amino group (Si-NH) are formed on the surface of the insulating film.
_{Although 2} ) is formed, these functional groups are selectively bonded to protons (H ⁺ ), and the equilibrium between the protons present in the solution and the bonded protons is maintained.
Therefore, when the pH of the solution changes, the charge on the insulating film changes, and accordingly, the bending of the energy band also changes.
As a result, the width of the depletion layer existing at the interface between the semiconductor and the insulating film, that is, the capacitance of the depletion layer changes, and the flowing optical AC current also changes. The LAPS also utilizes the photoconduction phenomenon of a semiconductor whose electric conductivity is increased by light irradiation.

[0009]

Most of the nucleic acid sensors that have been developed at present use a method of detecting a probe labeled with a fluorescent dye for detection, and thus emit fluorescence and measure fluorescence. And has the drawback that the device is bulky. In general, in gene diagnosis and the like in the medical field, there is a strong demand for a small and highly operable device for detecting such hybridization between nucleic acid molecules so that it can be used as a general-purpose detection device. I have. Further, in order to omit the nucleic acid amplification step by the PCR method, a highly sensitive detection device capable of detecting a trace amount of nucleic acid is desired. In addition, there is a need for low cost detection devices due to their ease of use at many medical institutions.

[0010]

SUMMARY OF THE INVENTION The present invention relates to a nucleic acid sensor provided with an electrode region on which a nucleic acid probe is immobilized. The presence of the nucleic acid of interest is detected by measuring the change in potential generated due to the formation of the main strand.

The present invention also provides a sensor substrate including a semiconductor substrate and an insulating film disposed on the substrate; a working electrode disposed on the surface of the semiconductor substrate; and a liquid sample disposed on the insulating film. The nucleic acid sensor has a means for holding the liquid sample, the means for holding the liquid sample defines an electrode region on the sensor substrate, and the electrode region has means for fixing the nucleic acid probe. Then, a change in potential generated due to double-strand formation between the nucleic acid probe and the target nucleic acid in the liquid sample is extracted from the working electrode as a corresponding signal in response to light irradiation.

Preferably, the semiconductor device further includes a protective film on the insulating film, the semiconductor substrate includes Si, and the insulating film includes S
It contains iO ₂ and the protective film contains Si ₃ N ₄ .

According to one aspect of the present invention, there is provided a nucleic acid sensor, a laser device for irradiating the nucleic acid sensor with a laser beam, a DC power supply for applying a DC bias voltage to the nucleic acid sensor, and a signal from the nucleic acid sensor. The present invention relates to a nucleic acid detection device provided with a processing unit.

[0014] Preferably, the nucleic acid detecting apparatus further comprises means for driving the laser device at a high frequency, and the means for processing the signal detects a change in the amplitude of the optical alternating current.

[0015] Preferably, the nucleic acid detection device further includes means for scanning a predetermined region of the nucleic acid sensor with a laser beam emitted from the laser device.

Preferably, the laser device is a laser array including a plurality of laser light sources arranged two-dimensionally in a matrix.

Preferably, the nucleic acid detection device further includes a means for controlling a position of the nucleic acid sensor in a horizontal direction and a vertical direction.

Preferably, the means for controlling is XY
The stage.

The present invention, in one aspect, relates to a method for detecting a nucleic acid of interest, the method comprising the steps of: immobilizing a nucleic acid probe on at least one of electrode regions on a sensor substrate; Contacting the nucleic acid probe with a sample suspected of containing the nucleic acid of interest under conditions such that the nucleic acid probe and the nucleic acid of interest hybridize to form a double strand; Measuring the potential change caused by the formation of the nucleic acid; and determining whether or not the target nucleic acid is present based on the presence or absence of the potential change.

The conditions under which the above-mentioned nucleic acid probe and the above-mentioned target nucleic acid hybridize to form a double strand can be usually achieved by denaturing the target nucleic acid into a single strand prior to the reaction. .

Preferably, the sensor substrate includes a semiconductor and an insulating film, and the step of measuring the potential change includes: applying a DC bias voltage to the solution including the sample, the insulating film, and the structure including the semiconductor. Irradiating at least one region in the electrode region with a laser beam; and measuring an optical alternating current passing through a depletion layer.

Preferably, the step of irradiating the laser beam includes modulating the laser beam.

Preferably, the change in potential is amplified by a substance that specifically binds to a double-stranded nucleic acid. Preferably, the substance that specifically binds to the double-stranded nucleic acid is ethidium bromide, acridine orange, Hoechst 332
Metal complexes such as Tris (phenanthrone) cobalt and tris (bipyridyl) ruthenium in addition to 58 (Molecular Probes) are used as intercalators. The intercalator is not limited to these.

Preferably, the potential change is amplified by a substance capable of labeling a nucleic acid.

[0025]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiments of the present invention will be described in detail.

According to the present invention, there is provided a sensor substrate including a semiconductor substrate, an insulating film disposed on the substrate, a working electrode disposed on a surface of the semiconductor substrate, and a sensor electrode disposed on the insulating film.
A nucleic acid sensor comprising means for holding a liquid sample, wherein the means for holding the liquid sample defines an electrode region on a sensor substrate, the electrode region fixing the nucleic acid probe. A potential change generated due to double-strand formation between the nucleic acid probe and the target nucleic acid in the liquid sample is extracted from the working electrode as a corresponding signal in response to light irradiation.

Preferably, the semiconductor device further includes a protective film on the insulating film, the semiconductor substrate includes Si, and the insulating film includes S
It contains iO ₂ and the protective film contains Si ₃ N ₄ .

The nucleic acid probe is immobilized on the electrode region according to procedures known in the art, such as ionic bonding and covalent bonding. Immobilization of a nucleic acid probe by ionic bonding can be achieved by coating a polycation such as polylysine, polyornithine, or polyethyleneimine on the surface of a sensor substrate and bringing the polycation into contact with the nucleic acid probe. Immobilization of nucleic acid probe by covalent bond, hydroxysilane, aminoalkyl trialkoxysilane and other silanes, polycarbodiimide and the like by coating the sensor surface, to introduce a hydroxyl group, a functional group such as an amino group on the sensor substrate surface, Thereafter, it can be achieved by contacting with a nucleic acid probe. The contact between the nucleic acid probe and the sensor substrate can be performed, for example, by spotting a solution containing the nucleic acid probe on at least one predetermined position in the electrode region of the sensor substrate. At least one predetermined position in the electrode is appropriately selected depending on the type of the nucleic acid probe to be used, its concentration, and the like. For example, as shown by black circles in FIG. Spot. FIG. 4 shows 69 nucleic acid probe-immobilized regions formed by spotting nucleic acid probes in the electrode region. These nucleic acid probe-immobilized regions, depending on the purpose of measurement, may be immobilized nucleic acid probes of the same type,
Alternatively, one or more types of nucleic acid probes may be immobilized. This configuration enables high-sensitivity detection of a target nucleic acid by using various nucleic acid probes arranged at high density.

The means for holding the liquid sample may be, for example, in the form of an enclosure for accommodating a measuring solution containing a target nucleic acid and a reference electrode. The nucleic acid sensor of the present invention,
The potential change caused by the double-strand formation between the probe nucleic acid fixed to the electrode region defined by the enclosure and the target nucleic acid is directly measured, thereby reducing the size and sensitivity of the nucleic acid detection device. To achieve.

In the nucleic acid sensor of the present invention, unlike the pH sensor using LAPS, a potential is not generated on the surface of the insulating film due to the bond between the proton and the silanol group and the amino group formed on the surface of the insulating film. The potential of the probe nucleic acid in contact with the insulating film surface is directly generated on the insulating film surface due to the double-strand formation with the target nucleic acid, and thereby the width of the depletion layer at the interface between the semiconductor and the insulating film, Therefore, the capacitance changes. By utilizing the fact that the electric conductivity of the light-irradiated portion is further increased, a signal corresponding to the potential change of the light-irradiated portion is mainly extracted from the working electrode.

A nucleic acid detection apparatus using a nucleic acid sensor according to the present invention comprises a device having a light source for irradiating a light beam on the back surface of a sensor substrate, a working electrode on the back surface of the sensor substrate, and a reference electrode disposed on the front surface. The apparatus may include a DC power supply for applying a DC bias voltage between the electrodes, and processing means for processing a signal obtained between the electrodes.

Preferably, the light source may be a laser light source, and the device including the light source may be a laser device. The laser light source can accurately position the irradiation spot and narrow the spot diameter, thereby increasing the sensitivity of the nucleic acid detection device.

The device including the light source may include, for example, light source driving means for driving the laser light source at high frequency to emit high-frequency modulated light, whereby the means for processing the signal is provided between the working electrode and the reference electrode. It is possible to detect a change in the amplitude of the optical alternating current flowing through the optical path. That is, as described above, the depletion layer at the interface between the semiconductor and the insulating film, which is caused by a potential change caused by the formation of a double strand between the probe nucleic acid immobilized on the insulating film surface and the target nucleic acid, as described above. A change in the width (capacity) is detected as a change in the amplitude of the optical alternating current.

Further, the nucleic acid detecting apparatus of the present invention is provided with a means for scanning a laser beam emitted from a laser device in a predetermined area on the back surface of the sensor, whereby the nucleic acid is detected at a plurality of locations in the predetermined area. Can be measured almost simultaneously.

The laser device may include a plurality of laser light sources. The laser beams emitted from these laser light sources can be applied almost perpendicularly to the back surface of the nucleic acid sensor. The plurality of laser light sources may be a laser array arranged in a two-dimensional matrix in a laser device. A plurality of laser light sources in this laser array are
Driving in a predetermined order or manner, depending on the time, allows for a rapid detection of the potential change.

Alternatively, the nucleic acid detecting device of the present invention may include a means for moving the nucleic acid sensor in the horizontal direction and the vertical direction, whereby the probe-immobilized region can be moved without moving the laser beam. Can scan.
The means for moving the nucleic acid sensor may be, for example, an XY stage on which the nucleic acid sensor is mounted.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.

FIG. 1 shows a cross-sectional view (FIG. 1A) and a plan view (FIG. 1B) of a nucleic acid sensor according to the present invention.

As shown in FIG. 1A, the nucleic acid detecting device of the present invention acts on the back side of the sensor substrate on the Si side of the sensor substrate composed of three layers of Si, SiO ₂ and Si ₃ N _4. A gold antimony thin film 1a for an electrode is vapor-deposited, and an enclosure 1b for accommodating a measurement liquid and a reference electrode is provided on the sensor substrate surface on the Si ₃ N side. The cross-sectional view is Yes exaggerated in the thickness direction, actually about 200μm thickness of the whole, of which, SiO ₂ of about 50nm or less thick, Si ₃ shown in FIG. 1 (a) The N ₄ film has a thickness of 100 nm or less.
The silicon (Si) substrate used was an N-type semiconductor having a thickness of about 200 μm and a resistivity of 10 ohm cm, and whose front and rear surfaces were mirror-polished. A 500 ° gold antimony thin film 1a was formed on the back surface of the sensor substrate by vapor deposition, and then alloyed by heat treatment to form an ohmic contact with the semiconductor. The enclosure 1b for accommodating the measurement liquid or the like is a polycarbonate cylindrical body having an inner diameter of about 26 mm, and is adhered to the surface of the Si ₃ N ₄ film using, for example, an adhesive.

FIG. 2 shows the configuration of one embodiment of the nucleic acid detection device employing the nucleic acid sensor of the present invention.

As shown in FIG. 2, the nucleic acid detecting device includes a reference electrode (RE) disposed in an enclosure disposed on the front surface of the sensor substrate 1 and a working electrode disposed on the back surface of the sensor substrate. And a potentiostat 8 for applying a DC bias voltage between them. Note that, in the enclosure of the nucleic acid sensor 1, a counter electrode (CE) connected to the same potentiostat 8 as the reference electrode (RE) immersed in the measurement solution is provided. A current signal flowing between the reference electrode and the working electrode is input to a computer 10 as processing means via an amplifier 9. The computer 10 is equipped with a 16-bit A / D converter.

On the back surface of the nucleic acid sensor 1, there are provided a laser device 11 for irradiating the nucleic acid sensor 1 with a laser beam and a driving device 12 therefor. The laser beam emitted from the laser device 11 is condensed to a beam diameter of about several μm by an optical system including a mirror and a lens (for example, an objective lens of an inverted microscope can be used), and is irradiated on the back surface of the nucleic acid sensor 1. Is done. Further, the laser driving device 12 generates a laser beam of several k
A modulator that modulates at a high frequency of about 10 Hz to 10 kHz is included.

Further, the nucleic acid detecting device comprises a nucleic acid sensor 1
An XY stage 13 for displacing the nucleic acid sensor 1 in the horizontal direction together with the incubator unit 3 is provided as means for changing the area on the back surface of the substrate irradiated with the laser beam. The XY stage 13 is driven by a step motor in accordance with a command from the computer 10,
The position where the laser beam of the nucleic acid sensor 1 is irradiated is 1 μm
It can be displaced in the XY directions in units.

As described above, in the present embodiment, the position of the laser beam is fixed, and the nucleic acid sensor 1 is displaced in the XY directions. Alternatively, high-speed scanning (scanning) can be realized by fixing the position of the nucleic acid sensor 1 and scanning the laser beam. This means that, for example, X
This can be realized by using a Y galvanometer mirror. Alternatively, as the laser device, a laser array in which a plurality of laser light sources that irradiate laser light substantially perpendicularly to the back surface of the nucleic acid sensor can be used is two-dimensionally arranged in a matrix, and the plurality of laser light sources are arranged in a predetermined order. And in a time-sharing manner to achieve high-speed scanning.

As described above, the laser of the nucleic acid sensor 1
A hole-electron pair is generated in the part irradiated with light,
DC bias voltage applied between the electrode and the working electrode
A photocurrent flows according to the pressure. The surface of the nucleic acid sensor 1 is
Edge film (SiO_TwoAnd Si_ThreeN _Four) Is formed
No DC current flows, but the laser beam is high as described above.
An alternating current flows due to the frequency modulation.

Here, the capacitance of the insulating film (hereinafter simply referred to as capacitance) is Ci, the capacitance of the depletion layer at the interface between the semiconductor and the insulating film is Cd, and the depletion layer is generated in the depletion layer by photoexcitation with a high-frequency modulated laser beam. Assuming that the photo alternating current to be detected is i _P , the nucleic acid detection device is replaced by the equivalent circuit shown in FIG. 3, and the photo alternating current i flowing between the externally detectable reference electrode and the working electrode is as follows. It is represented by equation (1).

I = Ci · i _P / (Ci + Cd) (1) A double strand is formed between the nucleic acid probe immobilized on the insulating film surface and the target nucleic acid, and the insulating film surface , A bending of the energy band occurs near the interface between the semiconductor and the insulating film, and as a result, the width of the depletion layer at the interface between the semiconductor and the insulating film, that is, the capacitance Cd of the depletion layer changes. Then, according to the above equation (1), the optical alternating current i detected outside also changes.

In the case of a sensor using an N-type semiconductor as in this embodiment, if the potential generated on the surface of the insulating film is positive, the capacitance Cd of the depletion layer increases and the optical alternating current i decreases.
Conversely, if the potential generated on the surface of the insulating film is negative, the capacitance Cd of the depletion layer decreases and the photo-current AC increases.

[0049]

EXAMPLES (Example 1) Hereinafter, an example of an experiment in which a nucleic acid is detected using a nucleic acid detection device employing the nucleic acid device of the present invention will be described.

As a nucleic acid probe, an oligonucleotide probe consisting of 12 bases (5'-GCC-ACC-A
GC-TCC-3 ′) was immobilized in the electrode region inside the above-described nucleic acid sensor enclosure. In summary, the nucleic acid probe solution is
It was immobilized by contact with the surface of a nucleic acid sensor treated with polylysine as follows.

-Polylysine treatment of sensor surface- First, the surface of the sensor substrate was dissolved in an alkaline solution (50 g of sodium hydroxide in 150 ml of distilled water and 95%
Using 200 ml of ethanol).
It was immersed for hours. After rinsing the sensor substrate surface at least three times with distilled water, the sensor substrate was further rinsed with a polylysine diluent (70 ml of a poly-L-lysine solution (P892).
0, Sigma)) for 1 hour. Finally, the poly-L was centrifuged at 500 rpm for 1 minute.
-Remove the lysine diluent from the sensor substrate and
Dried for minutes. After drying, it was left at room temperature for 2 hours before use.

The sensor substrate on which the above operation has been performed is referred to as a sensor sample 1.

After washing the sensor sample 1 with pure water,
10 μM oligonucleotide ligand (5′-GGA
-GCT-GGT-GGC-3 ′) / TE buffer solution (50 μl) was added thereto, followed by immersion treatment for 1 hour, and hybridization was performed to form a double-stranded nucleic acid on the sensor substrate surface. The sensor substrate on which the operations up to this point have been performed is referred to as a sensor sample 2.

Table 1 shows the values of the optical alternating current for each sample detected by the above measuring apparatus. The numerical values shown in Table 1 are standardized by setting the value of the optical alternating current in the sensor sample 1 to 1.00. In addition, as a measuring solution, 50 mM
A potassium chloride solution was used. As shown in Table 1, in the sensor sample 2 in which the double strand was formed, the value of the optical alternating current was increased by 5%, indicating that the potential of the insulating film surface was decreased.

[0055]

[Table 1] From this, it is considered that a negative potential was generated on the surface of the insulating film due to the formation of the double strand of the nucleic acid at the position irradiated with the laser beam. If the laser beam irradiation position is moved to a place other than the nucleic acid probe fixing place,
No change in the potential was observed between the sensor sample 1 and the sensor sample 2.

(Example 2) As in Example 1, the surface of the sensor substrate was treated with polylysine. Two types of nucleic acid probe solutions were prepared, and each nucleic acid probe was applied to the electrode region of the sensor substrate using a spotter made by Japan Laser Electronics Co., Ltd. for about 1 hour.
Four spots were spotted at a spot diameter of 00 μm in the same manner as the matrix indicated by the black circles in FIG. Of the two types of nucleic acid probes, a single-stranded nucleic acid solution having a base sequence complementary to only one of the nucleic acid probes (hereinafter referred to as a complementary probe) is added to the sensor substrate, and immersion treatment is performed for 1 hour to perform hybridization. I let you. Next, using a device having the same configuration as the nucleic acid detection device described in Example 1, the photo-current was measured for each spot.
The results showed that the value of the photo-current at the spot where the complementary probe was immobilized was larger than at the spot of the other nucleic acid probe, and therefore the potential was lower.

(Example 3) In order to obtain a sensor sample 2,
Hoechst 33 to 1 μM as intercalator
The potential change of the region where the nucleic acid probe was immobilized was measured in the same manner as in Example 1 except that 258 was added.

Table 2 shows the values of the photo alternating current detected for each sensor sample using the above-described nucleic acid detector.
The values in Table 2 are standardized with the value of the optical alternating current in the sensor sample 1 being 1.00. As shown in Table 2, in the sensor sample 2 in which the duplex was formed in combination with the intercalator, the value of the optical alternating current was reduced by 10%, and the potential of the insulating film surface was increased. Indicated. From this, it was considered that a positive potential was generated on the surface of the insulating film by the intercalator at the portion irradiated with the laser beam. When the irradiation site of the laser beam was moved to a position other than the immobilized region of the nucleic acid probe, no change in the potential was observed in the sensor sample 1 and the sensor sample 2.

[0059]

[Table 2]

[0060]

According to the present invention, there is provided a nucleic acid sensor provided with an electrode region on which a nucleic acid probe is immobilized, wherein the nucleic acid probe comprises a target nucleic acid and a nucleic acid probe immobilized on at least one region within the electrode region. By measuring the change in potential generated due to the formation of the main strand, a small-sized nucleic acid sensor excellent in operability, which detects the presence of the target nucleic acid, is provided. This nucleic acid sensor provides a nucleic acid sensor capable of arbitrarily and accurately adjusting the position of a region (measurement electrode) to be irradiated with a laser beam without providing complicated wiring.

[Brief description of the drawings]

FIG. 1 is a schematic cross-sectional view and a plan view of a nucleic acid sensor of the present invention.

FIG. 2 is a diagram schematically showing a nucleic acid detection device of the present invention.

FIG. 3 is a diagram showing an equivalent circuit of the nucleic acid sensor of the present invention.

FIG. 4 is a plan view of a modified example of the nucleic acid sensor of the present invention.

FIG. 5 is a plan view of a conventional nucleic acid sensor.

FIG. 6 is a diagram showing a pH measurement circuit using LAPS.

FIG. 7 is obtained from a circuit diagram of pH measurement by LAPS,
The figure which shows the relationship of an optical alternating current vs. DC bias voltage characteristic.

[Explanation of symbols]

 Reference Signs List 1 nucleic acid sensor 1a working electrode 1b enclosure 2 sample (nucleic acid) 3 incubator unit 4 temperature control unit 5 heater / fan unit 6 flow meter 7 solenoid valve 8 potentiostat 9 amplifier 10 computer 11 laser light source 12 laser drive device 13 XY stage

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/566 G01N 27/46 U (72) Inventor Ozaki Tadahiko 1006 Odakadoma, Kadoma City, Osaka Matsushita Electric Within Sangyo Co., Ltd. (72) Inventor Hirokazu Sugihara 1006 Kazuma, Kadoma, Osaka Prefecture Matsushita Electric Industrial Co., Ltd. F-term (reference) 4B029 AA07 AA23 BB20 CC03 CC08 FA12 4B063 QA01 QQ42 QR32 QR55 QS34

Claims (14)

[Claims] 1. A nucleic acid sensor having an electrode region on which a nucleic acid probe is immobilized, wherein the potential change occurs due to double-strand formation between a target nucleic acid and the immobilized nucleic acid probe. A nucleic acid sensor for detecting the presence of the target nucleic acid by measuring the nucleic acid. 2. A sensor substrate including a semiconductor substrate and an insulating film disposed on the substrate; a working electrode disposed on a surface of the semiconductor substrate; and a liquid sample disposed on the insulating film. A nucleic acid sensor, wherein the means for holding the liquid sample defines an electrode region on the sensor substrate, and the electrode region includes means for fixing the nucleic acid probe, A nucleic acid sensor for extracting a potential change generated due to double-strand formation with a target nucleic acid in the liquid sample from the working electrode as a corresponding signal in response to light irradiation. 3. The method according to claim 1, further comprising a protective film on the insulating film,
It said semiconductor substrate comprises Si, the insulating film comprises SiO _2, and the protective film comprises Si ₃ N _4, the nucleic acid sensor according to claim 2. 4. The nucleic acid sensor according to claim 1 or 2;
A nucleic acid detection device, comprising: a laser device that irradiates a laser beam to the nucleic acid sensor; a DC power supply that applies a DC bias voltage to the nucleic acid sensor; and a unit that processes a signal from the nucleic acid sensor. 5. The nucleic acid detection apparatus according to claim 4, further comprising a unit for driving the laser device at a high frequency, wherein the unit for processing the signal detects a change in the amplitude of the photo-current. 6. The nucleic acid detecting apparatus according to claim 4, further comprising: means for scanning a predetermined region of the nucleic acid sensor with a laser beam emitted from the laser device. 7. The nucleic acid detection device according to claim 4, wherein the laser device is a laser array including a plurality of laser light sources arranged two-dimensionally in a matrix. 8. The apparatus further comprises means for controlling the position of the nucleic acid sensor in a horizontal direction and a vertical direction. The nucleic acid detection device according to claim 4. 9. The nucleic acid detection device according to claim 8, wherein the control unit is an XY stage. 10. A method for detecting a target nucleic acid, comprising the steps of: immobilizing a nucleic acid probe on at least one region within an electrode region on a sensor substrate; Contacting a sample suspected of containing under conditions such that the nucleic acid probe hybridizes with the target nucleic acid to form a double strand; a potential generated due to the formation of the double strand Measuring the change; and determining whether or not the nucleic acid of interest is present based on the presence or absence of the potential change. 11. The sensor substrate includes a semiconductor and an insulating film, wherein the step of measuring the potential change includes: a solution containing the sample,
Applying a DC bias voltage to the insulating film and the structure including the semiconductor; irradiating at least one region in the electrode region with a laser beam; and measuring a photo-AC current passing through a depletion layer. The method of claim 10, wherein 12. The step of irradiating the laser beam,
The method of claim 11, comprising modulating a laser beam. 13. The method according to claim 10, wherein the change in potential is amplified by a substance that specifically binds to a double-stranded nucleic acid. 14. The method according to claim 10, wherein the potential change is amplified by a substance capable of labeling a nucleic acid.