JP2008275333A - Gene detection chip, nucleic acid sequence accumulation method, and gene detection apparatus using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To achieve high sensitivity and to provide a form for performing PCR. <P>SOLUTION: In a gene detection chip, a surface to which a nucleic acid probe is formed in a solid phase is a surface which is temperature-controllable and which totally reflects light. Since evanescent light which has oozed out due to incident light onto the surface therefore excites a fluorescent substance, it is possible to highly sensitively detect genes by performing solid-phase PCR including fluorescent-labeled nucleic acids in a microarray in an evanescent region. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a gene detection chip for detecting genes on a microarray quickly and with high sensitivity, a nucleic acid sequence integration method using the same, and a gene detection apparatus.

  In recent years, a technique for detecting a plurality of genes all at once using a microarray in which nucleic acid sequences such as DNA are immobilized on a substrate in a microarray form has been widely used.

  Along with this, microarray technology is required to be more rapid, sensitive detection, and cost reduction.

  As a conventional technique that meets such a requirement, for example, there is one in which hybridization time is reduced by performing PCR (Polymerase Chain Reaction) on the surface of a solid phase (Patent Document 1). On the other hand, there is a method disclosed in Patent Document 2 as a method for detecting molecules with high sensitivity on the surface of a solid phase.

  The prior art disclosed in Patent Documents 1 and 2 will be described.

  In FIG. 10 of Patent Document 1, a single nucleotide polymorphism or gene polymorphism detection method is given as an example. Using a PCR reaction mixture containing a die terminator containing double-stranded DNA as a template, the nucleic acid probe formed on the bottom surface is heated with a heater formed on the bottom surface to perform PCR on the solid surface. At this time, along with the PCR proceeding in the container, a chain elongation reaction occurs with a nucleic acid probe complementary to the template, and a die terminator is incorporated on the 3 ′ end side of the nucleic acid probe. After a predetermined number of PCR cycles, unreacted dye terminators are washed away, fluorescence images are acquired with a microarray scanner, and analysis evaluation is performed.

FIG. 1 of Patent Document 2 shows a method for quickly and quantitatively measuring the interaction between an immobilized protein or nucleic acid molecule and a target molecule by detecting a labeling substance. Here, single molecule imaging of intermolecular interaction is possible using evanescent light.
JP 2005-304445 A JP 2002-253240 A

  However, in Patent Document 1, since the target application is for detection of single nucleotide polymorphisms and does not require high sensitivity, it is difficult to cope with the high-sensitivity application that is the subject of the present invention.

  On the other hand, in Patent Document 2, high sensitivity is achieved and detection of intermolecular interaction is possible at the molecular level. However, the reaction mode simply forms a complex on the surface of the solid phase, such as an antigen-antibody reaction, and the two dissociation required for PCR (95 ° C.), primer annealing (˜63 ° C.), enzyme extension ( 72 ° C.) is not required.

  For example, in order to detect a very small amount of a virus, a bacterium, or the like in the early stage of infection, a higher sensitivity is desirable, and at the same time, a form of performing PCR is required.

  Accordingly, an object of the present invention is to provide a gene detection chip that realizes a form of performing PCR at the same time as further increasing sensitivity. Further, when the sensitivity is increased, there is a problem that the amount of the detection substance contained in the sample is small with respect to the amount of the sample. Therefore, another object of the present invention is to provide a nucleic acid sequence integration method and gene detection apparatus that can perform PCR even with a small amount of reagent used.

  To achieve the above object, the gene detection chip of the present invention is a gene detection chip having a surface capable of capturing a gene, the surface having a plurality of nucleic acid probes complementary to the nucleic acid sequence to be detected, The total reflection surface is capable of controlling the temperature and totally reflects incident light.

  In addition, the gene detection chip includes a transparent electrode disposed at a position facing the liquid sandwiching the liquid.

  Further, the gene detection chip is characterized in that the surface is arranged in a flow path capable of transporting a liquid containing the nucleic acid sequence to be detected.

  Further, the gene detection chip is characterized in that the surface is arranged in a container containing a liquid containing the nucleic acid sequence to be detected.

  In order to achieve the above object, the nucleic acid sequence integration method according to one aspect of the present invention is a nucleic acid sequence integration method for integrating the nucleic acid sequence to be detected on the surface of the gene detection chip. A first step of transporting a liquid containing a nucleic acid sequence by a liquid amount passing through the transparent electrode or a liquid amount less than the liquid amount; a second step of applying a voltage to the transported liquid; A third step of heating the nucleic acid sequence to be detected that has migrated to the surface in step 2 so that the double strands are separated into single strands, and the first to third steps These steps are sequentially repeated to accumulate the nucleic acid sequence to be detected on the surface.

  In order to achieve the above object, a nucleic acid sequence integration method according to another aspect of the present invention is a nucleic acid sequence integration method for integrating the nucleic acid sequence to be detected on the surface of the gene detection chip. A first step of applying a voltage to a liquid containing a nucleic acid sequence to be detected, and a double strand dissociated into a single strand with respect to the nucleic acid sequence to be detected migrated to the surface in the first step A second step of performing heating for the purpose, and the first to second steps are sequentially repeated to accumulate the nucleic acid sequence to be detected on the surface.

  In order to achieve the above object, the gene detection device of the present invention is a gene detection device using the gene detection chip, comprising: illumination means for irradiating light for forming an evanescent field on the surface; and the illumination means. Imaging means for imaging light that has oozed from the surface by irradiation, and heating means for heating the nucleic acid sequence to be detected that has moved to the surface so that double strands are separated into single strands. It is characterized by having.

  Further, the gene detection device applies a voltage to the liquid transporting means for transporting the liquid containing the nucleic acid sequence to be detected and the transported liquid, and transfers the nucleic acid sequence to be detected to the surface. Voltage applying means for causing electrophoresis.

  In the gene detection chip of the present invention, the surface of the gene detection chip can be temperature-controlled and has a light total reflection surface, so that PCR including a fluorescently labeled nucleic acid can be performed on a surface on which a diffusion probe that is an evanescent region is solid-phased.

  For this reason, PCR on the solid phase surface can be detected with higher sensitivity. At the same time, since the surface also serves as a total reflection surface of the laser, the number of parts and the number of manufacturing processes can be reduced. Moreover, since the sensitivity is increased, a small amount of assay can be performed, so that the amount of blood collected is reduced, the burden on the patient is reduced, and the amount of reagent used is reduced, which helps to reduce the cost. In addition, since the amount of nucleic acid sequence to be detected in the sample is very small, the nucleic acid sequence to be detected can be accumulated on the surface of the solid phase on the flow path even when a large amount of sample solution is required. Useful for assaying, reducing reagent usage, and reducing costs. Further, since the B / F separation is not required by the laser evanescent illumination, it is not necessary to discharge a large amount of cleaning liquid, and there is no concern about waste liquid processing problems and infections associated with the cleaning work, which helps to make the inspection safe. At the same time, since real-time observation can be performed by detecting the fluorescent substance, quantitative PCR measurement can be performed. In addition, since the sensitivity is increased, the number of PCR cycles can be reduced, which helps speed up the examination. At the same time, the nucleic acid sequences are accumulated by electrophoresis, so that they can be accumulated faster than simple diffusion, which helps to speed up the examination.

  The best mode for carrying out the present invention will be described below with reference to the drawings.

  Referring to FIG. 1, in the gene detection chip of this embodiment, the surface 1 capable of capturing a gene has a plurality of nucleic acid sequences 5 complementary to the nucleic acid sequence 3 to be detected, and the nucleic acid sequence 5 is a nucleic acid probe. As a solid phase.

  The surface 1 is a total reflection surface that can be heated and controlled to a temperature required to cause PCR and totally reflects incident light for forming an evanescent field. Such a total reflection surface can be formed on the substrate 2 on which the laser beam 6 can be incident, for example, the transparent substrate 2. The evanescent light oozing out from the surface 1 excites the fluorescent substance 4 in the vicinity of the surface contained in the reaction solution 7, so that the presence of the fluorescent substance 4 can be detected by a photodetector. When the nucleic acid sequence 3 to be detected is present in the sample, the presence of the nucleic acid sequence 3 can be detected by performing PCR including a fluorescently labeled nucleic acid on the solid phase surface.

Hereinafter, preferred embodiments will be described.
(Regarding surface 1-1)
The surface 1 has a thin film that forms a total reflection surface of light. The film thickness of the thin film, including the probe length of the nucleic acid probe to be solid-phased, can be in a range not exceeding the oozing of evanescent light. Assuming that the depth of the bleeding is the distance d (nm) from the interface where the light intensity is 1 / e, the following can be summarized.

d = λ / 4π [n ₁ ² · sin ² θ−n ₂ ² ] ^1/2 (1)
λ (nm): wavelength of the laser beam 6
n ₁ : Refractive index of substrate 2 with a thin film attached
n ₂ : Refractive index of specimen
θ: Incident angle (a value larger than the critical angle)
For example, when a general green laser such as He—Ne is used as a laser for irradiating the laser beam 6, the value of d can be estimated as about 150 nm. In the case of observation at a single molecule level, if irradiation is performed with light having an excitation intensity of about 0.5 μW / μm ² , the irradiation area is S (μm ² ) and 0.5 S · e (where e = 2. 71) The laser output is μW. According to this equation, the irradiation area of 1 mm ² 1.35 W, the 135W at irradiation area 10 mm ^2. This value corresponds to the power of an available commercial laser.

  The irradiation area S has to be irradiated on the entire surface of the microarray that is solid-phased, but is not limited if the solid-phase surface can be scanned.

  Further, for the laser, an optical element such as a linear deflection through a beam expander or a circular deflection can be used in order to make the irradiation as uniform as possible.

The wavelength of the laser beam 6 can be 532 nm of SHG if the fluorescent dye is Cy3 or rhodamine, 473 nm of SHG if SYBR Green I or FITC, and 635 nm of LD excitation if Cy5.
(Regarding surface 1-2)
The surface 1 can be temperature controlled as described above. When electrically heated, the surface has an area with a resistance value that can be driven and a shape pattern.

  First, the area of the surface 1 will be described.

In a temperature range of 0 ° C. <T <99 ° C. to be considered in the PCR cycle, the resistance value at 0 ° C. is represented by R ₀ , the resistance value at T ° C. is represented by R _T , and the temperature coefficient of resistance is represented by linear approximation. Then, it becomes as follows.

R _T = R ₀ [1 + αT] (2)
The bulk resistance value of a typical metal can be referred to, for example, in a thin film handbook (Japan Society for the Promotion of Science Thin Film 131st Committee / Edition, published by Ohmsha). Moreover, what is necessary is just to measure a bulk resistance value with a resistivity meter with any substance. When the bulk resistance value is R _bulk (μΩcm), the sheet resistance is
R _sheet = R _bulk / thickness (μΩ) (3)
Can be expressed as Accordingly, the resistance value R _S of the sheet having the length L and the width W is as follows.

R _S = R _sheet · L / W (4)
Since the value of R _S at this time changes R _T (from T = primer annealing temperature to T = template deigner temperature), a current source and a voltage source that can drive the resistance value R _S are sufficient. As an example, taking a metal Au having a low resistance value and a large burden for driving as an example, since Au has a bulk resistance value of 2.2 μΩcm, when the thickness is 10 nm (= 10 ⁻⁶ cm), the resistance value R _S is ,
R _S = 2.2Ω · L / W (5)
It becomes. Since Au is α = 0.0040 / ° C. (room temperature), it can be estimated from the formula (2) that the resistance value increases about 1.4 times at 100 ° C. Therefore, the resistance value R _S is as follows: Become.

R _S = 3.1Ω · L / W (6)
Here, when L: W = 10: 1, the resistance value R _S can take several tens of Ω, and can be driven by a general circuit such as an audio circuit. As an example, if L = 10 mm, W = 1 mm, and the solid phase area is 1 mm ² , 10 × 10 = 100 microarray dots can be configured when the pitch of the microarray dots is 100 μm. This number can almost cover general bacterial identification test items.

  In the above description, the area of the surface 1 that is a simple rectangle having a length L and a width W has been described.

  Next, the shape pattern of the surface 1 will be described.

FIG. 2A shows an H-shaped heater pattern 49 obtained by patterning a single-layer thin film on the substrate 2. By thickening both ends to which the voltage is applied and making the solid phase region 54 narrow, the solid phase region 54 can be heated by single layer patterning. FIG. 2B shows two-layer patterning. By patterning a metal thin film with good conductivity, here, for example, gold electrodes 15a and 15b on both ends of the heater pattern 49, the thick square heater pattern 49 can be heated uniformly. At this time, the square heater pattern 49 desirably has a high resistance value. In addition, by patterning the solid phase spot 55 with gold, a form suitable for binding a nucleic acid probe having an SH group at its end is obtained.
(Regarding surface 1-part 3)
The surface 1 has a plurality of nucleic acid probes. A general method may be used as the solid phase method of the nucleic acid probe on the surface 1. For example, a method may be used in which a biotinylated oligonucleotide probe is spotted on a solid phase surface in which avidin is added to a photoresponsive polymer, irradiated with UV, and avidin is immobilized in advance. A commercially available apparatus can be used for spot work. In the case of this method, solid phase is possible regardless of the surface material. Further, not only gold but also an oligonucleotide probe having an SH group can be used for a noble metal thin film.
(About thin film material on surface 1)
The thin film material of the surface 1 thin film includes noble metals such as gold, silver and platinum, metals such as iron, nickel, manganese, chromium, zinc, aluminum and titanium, and oxides thereof, and solid solutions of these metals and oxides thereof. Can be used. In addition, a conductive polymer or a polymer resin blended with carbon black or a metal filler can be used. Further, glass or transparent resin can be used for the substrate 2 on which the laser 6 is incident. As the transparent resin, it is possible to use a resin material having a glass transition temperature that is approximately the same as the denature temperature required for the PCR cycle. For example, Poly (vinyl alcohol), Poly (vinyl chloride), Poly (p-methoxy styrene), Polystyrene, Cellulose Acetate, Poly (p-methyl styrene) can be used. In addition, there are materials that can be used to prevent overheating by breaking electrical contact due to expansion in an overheated state (95 ° C. or higher).
(Regarding temperature control method of surface 1)
The temperature of the surface 1 can be controlled by the bridge circuit shown in FIG. The resistance indicated as R _S among the four resistances constituting the bridge 13 is the resistance of the gene detection chip. R _n is a known resistance. A resistor 10 surrounded by a broken-line square is a D / A converter or a ladder resistor. The resistor 10 is connected to the bus line 11 of the microphone processor, and an arbitrary resistance ratio can be selected. If the resistance value of R _S corresponding to the temperature to be controlled is known, the current flowing through the transistor 9 connected to the power supply voltage Vc is controlled so that the output of the differential amplifier 12 becomes zero. Can be obtained.

An example of other temperature control is shown in FIG. The thin film 14 on the surface 1 can be heated, and electrodes 15 c to 15 f are provided at four locations around the thin film 14. When the current i flows from the electrode 15c to the electrode 15d, the voltage V between the electrode 15e and the electrode 15f may be led to an A / D converter of a one-chip microcomputer and converted into a temperature. As another temperature control example, when a part of the laser 6 in FIG. 1 is guided to a two-division photodiode sensor and the refractive index of the substrate 2 changes with temperature, the temperature is determined based on this refractive index information. You may control.
(Method for collecting nucleic acid sequences to be detected-when the surface is arranged in a flow path)
FIG. 5 is a diagram for explaining a method for collecting nucleic acid sequences in the case where the surface 1 is arranged in a flow path 25 capable of transporting a specimen (liquid). On the solid phase surface of the channel 25, nucleic acid sequences 28 a, 28 b, 28 c that are at least complementary to the DNA 29 are solid phased as nucleic acid probes on the thin film 14 that can be heated. In addition, a transparent electrode 30 is disposed at a position opposite to the sample that contacts the thin film 14. ITO can be used for the transparent electrode 30. A method for accumulating DNA 29 will be described.

  (1) in FIG. 5 shows the conveyance of the specimen (first step). At this time, the specimen is transported by the amount of liquid that passes through the transparent electrode 30 or less.

  Next, by applying a voltage to the transported specimen, the DNA 29 is electrophoresed toward the solid phase ((2) in FIG. 5, second step). After the DNA 29 has sufficiently moved to the solid surface, a heater on the solid surface is driven to heat the DNA 29 sufficiently to dissociate from double strands to single strands (third step). At this time, if a complementary nucleic acid sequence is present on the solid phase side, a complementary strand is formed, so that DNA 29 can be captured and accumulated on the surface of the solid phase.

  Although DNA 29 does not exist in (3) of FIG. 5, the sample is transported in the same manner. The above steps are sequentially repeated until all the specimen passes through the solid surface.

The above process is represented by a flowchart as shown in FIG. 6 and can be controlled by the software of the gene detection apparatus of this embodiment. The sample trap subroutine 26 is repeated until it is determined that the sample transport end determination 27 is finished. In the sample trap subroutine 26, first, the sample is transported by driving the pump, and a voltage is applied between the terminal 32 (+) and the terminal 34 (−). In general, the migration speed of a substance is proportional to the electric field strength with the mobility of the substance as a coefficient. Since the mobility of the anion is about 10 ⁻³ (cm ² / Vs), when the distance between the terminals is 100 μm, assuming that 10 V is applied, the speed can be estimated as 1 cm / s. Therefore, the applied voltage may be less than 100V and the application time may be less than 1 second. After the voltage is applied, the switch 31 is tilted to the heater heating side, and a heater heating current is passed through the terminal 33.
(Method for collecting nucleic acid sequences to be detected-when the surface is placed in a container)
FIG. 7 is a diagram for explaining a method for accumulating nucleic acid sequences in the case where the surface 1 is arranged in a container 8 that contains a specimen. Since FIG. 7 is a simple container, the sample cannot be transported as shown in FIG. However, as shown in FIG. 5, since the transparent electrode 30 is arranged at the upper facing position, the DNA 29 can be electrophoresed (first step). Further, after electrophoresis, the switch 31 can be tilted to the heater heating side, and a heater heating current can be passed through the terminal 33 (second step). By sequentially repeating the above two steps, the DNA 29 can be captured and accumulated on the solid surface. Note that substances having different polarities, such as proteins other than DNP (DNA binding protein), are electrophoresed in the direction of the transparent electrode 30, so that impurities that hinder the reaction can be kept away. The transparent electrode 30 is preferably connected to the ground potential, but may be opened.

Note that a normal PCR buffer, TAE, TBE buffer, or PBS buffer can be used as a diluted solution for diluting the specimen.
(About gene detection reaction)
The reaction for detecting a gene is an enzymatic reaction involving chain extension or repair. For such enzyme reaction, φ29 polymerase, T4 DNA polymerase, DNA polymerase I, E. coli DNA ligase, T4 ligase, reverse transcriptase, heat-resistant polymerase, etc. can be used. In particular, it is suitable to use a heat-resistant polymerase. As the nucleic acid serving as a substrate, deoxynucleotide triphosphates such as A, C, T, G, and U, fluorescent labels thereof, dideoxynucleotides that cause extension termination, and labels thereof can be used. The nucleic acid may be a nucleic acid analog. In addition, it is necessary to add a primer required for the heat-resistant polymerase reaction to the reaction solution when the asymmetric PCR can be performed because the nucleic acid chain existing on the solid phase side also serves as a primer. Absent. However, if it is desired to increase the amplification factor associated with the reaction, it is desirable to provide a primer that becomes a complementary sequence in the solution to achieve symmetric PCR. The gene detection chip of this embodiment is in the form of a disposable chip because the fluorescent label is incorporated into the nucleic acid chain on the solid phase side. However, when detecting the double strand formation of the nucleic acid strand to be detected and the nucleic acid strand on the solid phase side with an intercalator such as Cyber Green, the gene detection is performed by removing the nucleic acid strand to be detected by the deigner operation. The chip can be reused. At this time, it is desirable that the material used as the substrate 2 avoids glass in order to reduce dye adsorption.

  Hereinafter, the present invention will be described more specifically with reference to examples.

[Example 1]
Fig.8 (a) is a perspective view which shows the gene detection chip | tip of Example 1 of this invention. The solid phase surface of this gene detection chip is arranged in a container. As shown in FIGS. 8B and 8C, the container is configured by bonding two non-fluorescent glass substrates 17 for a microscope and an acrylic resin 19 together. As shown in FIG. 8B, chromium is patterned on the non-fluorescent glass substrate 17 as the gold electrodes 15a and 15b, and titanium is formed as the Pt heater thin film 16 serving as a solid surface and a heater surface therebetween. The molded transparent acrylic resin 19 has sample injection holes 18a and 18b, and by sealing the sample injection holes 18a and 18b with a rubber cap 20, evaporation during the reaction can be prevented. After spotting the nucleic acid probe with a commercially available spotter device, the two pieces are bonded together with an adhesive.

[Example 2]
FIG. 9 is a perspective view showing the gene detection chip of Example 2 of the present invention. The solid phase surface of this gene detection chip is disposed in the flow path 25. The gene detection chip has a specimen injection hole 21, a buffer injection hole 22, a PCR reaction liquid injection hole 23, and a waste liquid hole 24, each having a liquid reservoir. Since the nucleic acid sequence to be detected in the sample is very small, the sample may be required enormously. Considering such cases, take a large sample liquid reservoir, capture the nucleic acid sequence only on the solid phase surface, wash it off with buffer solution, and use a PCR reaction solution for a small area of the flow path height x solid phase area. , The reaction can proceed. In addition, a transparent electrode (not shown) is provided on the solid phase surface on the flow path 25 and at a position opposite to the heater surface, and a nucleic acid sequence to be detected can be captured on the solid phase surface by electrophoresis.

  Hereinafter, a procedure for producing the gene detection chip of FIG. 9 will be described with reference to FIG.

  A 25 × 76 mm non-fluorescent glass substrate 51 having a thickness of 1.2 mm is spin-coated with a resist, the heater pattern 49 is exposed and developed, and a Ti film is formed by a sputtering method with reference to the film formation rate. A Pt film is deposited to about 50 nm. Next, the resist and unnecessary films are removed by a lift-off process. The heater pattern 49 after the lift-off process is as shown in FIG.

  Further, an ITO film is formed to a thickness of about 100 nm on a PET (polyethylene terephthalate) substrate 52 in which the specimen injection hole 21, the buffer injection hole 22, the PCR reaction liquid injection hole 23, and the waste liquid hole 24 are previously formed. Next, the transparent electrode pattern 50 is protected with a resist, and unnecessary portions are removed with an ITO etching solution.

  Further, a PDMS (polydimethylsiloxane) sheet 53 having a 21 × 72 mm thickness of 100 nm is prepared by hollowing out the mold of the flow path 25. Further, the surface of the non-fluorescent glass substrate 51 is cleaned by immersing it in sulfuric acid-hydrogen peroxide solution at 70 ° C. for about 1 hour or by UV ozone irradiation.

  Next, the PDMS sheet 53 is overlaid on the fluorescent glass substrate 51. A nucleic acid probe is immobilized on the solid phase region 54 where the heater pattern 49 of the non-fluorescent glass substrate 51 and the mold part of the flow path 25 of the PDMS sheet 53 intersect using a commercially available spotter device.

In this embodiment, the transparent electrode pattern 50 and the heater pattern 49 are 1 mm × 25 mm, and the flow path width is 1 mm so that the area of the solid phase region 54 is 1 mm × 1 mm. Moreover, the nucleic acid probe is for detecting the following 10 bacterial species, and the nucleic acid probe disclosed in JP-A-2004-313181 is used. In addition, an SH (thiol) group is bonded to the 5 ′ end.
1. Staphylococcus aureus gAACCgCATggTTCAAAAgTgAAAgA
2. Staphylococcus epidermidis TAgTgAAAgACggTTTTgCTgTCACT
3. E. coli ATACCTTTgCTCATTgACgTTACCCg
4). K. pneumoniae ggTCTgTCAAgTCggATgTgAAATCC
5. Pseudomonas aeruginosa TCAgATgAgCCTAggTCggATTAgC
6). Serratia AggTggTgAgCTTAATACgCTCATC
7). Streptococcus pneumoniae CTTgCATCACTACCAgATggACCTg
8). Haemophilus influenzae CTTgggAATgTACTgACgCTCATgTg
9. Enterobacter cloacae ggTgTTgTggTTAATAACCACAgCAA
10. Enterococcus faecalis AACACgTgggTAACCTACCCATCAg
Thereafter, when the fixation of the nucleic acid probe is completed, the PET substrate 52 is overlaid and fixed as shown in FIG.

[Example 3]
A gene detection apparatus using the gene detection chip of Example 2 will be described with reference to FIG.

  A laser beam for forming an evanescent field on the surface 1 is emitted from a laser 35 which is an illuminating means, and is controlled by a laser power controller 48. The laser beam is guided to the beam expander 36, and enters the gene detection chip 38 of the present invention through the lens 37. The evanescent light that oozes from the surface 1 of the gene detection chip 38 is imaged by a cooled CCD camera 39 that is an imaging means. On the other hand, the diaphragm micropumps 41, 42, and 43 are connected to the sample injection hole 21, the buffer injection hole 22, and the PCR reaction liquid injection hole 23, respectively, via tubing. The diaphragm type micropump 40 is a liquid feeding means that is connected to the waste liquid hole 24 and transports the liquid by sucking air that fills the flow path 25. Control of the diaphragm type micropumps 40 to 43 is controlled by the PC controller 47 through the I / O 44 and the data / control bus 45. The control state can be confirmed on the display 46.

[Example 4]
The gene of the pathogen is detected using the gene detection chip of Example 2.
(Sample adjustment)
First, a crude fraction containing pathogen DNA is obtained from a sample collected with a vacuum blood collection tube containing EDTA. Next, 10 μl of pure water is added to 10 μl of blood and incubated at 95 ° C. for 10 minutes. Next, this is centrifuged at 8000 × g for 10 minutes, and the supernatant is discarded. Next, 10 μl of an enzyme cocktail consisting of N-acetylmuramidase (0.625 mg / ml) and beta-1,3-glucanase (Zymolyase) (0.25 mg / ml) is added thereto. Further, 10 μl of enzyme cocktail consisting of lysozyme (100 mg / ml) and lysostaphin (10 mg / ml) is added. This is then incubated at 37 ° C. for 10 minutes. Next, this is centrifuged at 8000 × g for 10 minutes, the supernatant is discarded, and the volume is made up to about 10 μl using the PCR buffer attached to the PCR kit (Invitrogen Platinum Taq DNA Polymerase) (solution A). Similarly, the PCR premix solution in the PCR kit is diluted so that the enzyme becomes 3 Units, and a fluorescent dye is added thereto so that Cy3-dUTP becomes 10 pmol / μl (solution B).
(Dispensing to gene detection chip)
Dispense 10 μl of the liquid A into the sample injection hole 21. 7 μl of diluted PCR buffer is dispensed into the buffer injection hole 22. Into the PCR reaction solution injection hole 23, 3 μl of the previous solution B is dispensed. This is incorporated in the gene detection apparatus of Example 3 as a gene detection chip 38, and tubing is connected.
(Gene capture and accumulation)
The prepared sample (liquid A) connected to the diaphragm micropump 41 is pushed out at a speed of 0.1 μl / sec. After passing through the flow path intersection with another solution, the diaphragm micropump 40 is driven at the same speed to stabilize the liquid feeding. After the specimen reaches the solid phase region 54, a voltage of 5 V is applied between the transparent electrode pattern 50 and the solid surface by a voltage application unit (not shown) for 50 msec. Next, a voltage is applied to both ends of the heater on the solid phase surface, which is a heating means, and the temperature of the solid phase surface is controlled to be 93 ° C. In the control method, the bridge 13 performs feedback control so that the voltage difference between both ends of the bridge is eliminated. The correspondence with the temperature is preliminarily calibrated with the relationship with the resistance value of the heater. The heating time is 1 second after reaching 93 ° C. The above steps are repeated until 0.1 μl of solution is delivered and a total of 10 μl is reached.
(PCR)
The PCR buffer connected to the diaphragm micropump 42 is pushed out at a speed of 0.1 μl / sec. After passing through the flow path intersection with another solution, the diaphragm micropump 40 is driven at the same speed to stabilize the liquid feeding. The solution feeding is continued until the solution reaches the solid phase region 54 and reaches a total of 7 μl. Next, the PCR reaction solution connected to the diaphragm micropump 43 is pushed out at a speed of 0.1 μl / sec. The solution reaches the solid phase region 54 and PCR is started. The temperature cycle is set to 25 times with a holding time of 93 ° C. for 1 second, 63 ° C. for 1 second, and 72 ° C. for 0.5 second.
(Detection of genes by PCR)
The laser beam is guided to the total reflection surface of the gene detection chip 38, and imaging is started by the cooled CCD camera 39 simultaneously with the start of PCR. The captured image is subjected to image processing in the PC controller 47, and the portion of the nucleic acid probe when PCR occurs is converted into relative fluorescence brightness in real time for each number of PCR reactions. The pathogen is identified from the detected spot information.

  The present invention can be used for genetic diagnosis that requires highly sensitive detection as described above. However, it may be used for other industries. The present invention has a surface that is a temperature-controllable total reflection surface of light, and controls the temperature in the vicinity of light so that all chemical reactions that can occur in the vicinity at that temperature It provides a reaction field. Therefore, the present invention can be used for detection of reactants and analysis of reaction temperature if the light that exudes interacts with a chemical substance and can be detected by a photodetector.

It is a figure which shows the gene detection chip | tip of one Embodiment of this invention. It is a figure which shows an example of the heater pattern of the gene detection chip | tip shown in FIG. It is a figure which shows the bridge circuit used for an example of temperature control of the gene detection chip | tip shown in FIG. It is a figure which shows the other example of temperature control of the gene detection chip | tip shown in FIG. It is a figure explaining an example (when the surface is arrange | positioned in the flow path) of integrating the nucleic acid sequence which should be detected on the gene detection chip | tip shown in FIG. It is a flowchart explaining the nucleic acid sequence integration method shown in FIG. It is a figure explaining the other example (when the surface is arrange | positioned to the container) of the method of integrating the nucleic acid sequence which should be detected on the gene detection chip | tip shown in FIG. It is a figure which shows the gene detection chip | tip of Example 1 of this invention. It is a figure which shows the gene detection chip | tip of Example 2 of this invention. It is a figure explaining an example of the preparation methods of the gene detection chip | tip shown in FIG. It is a figure which shows an example of the gene detection apparatus using the gene detection chip | tip shown in FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Surface 2 Substrate 3 Nucleic acid sequence to be detected 4 Fluorescent substance 5 Complementary nucleic acid sequence 6 Laser 7 Reaction liquid 8 Container 9 Transistor 10 Resistance 11 Microprocessor bus line 12 Differential amplifier 13 Bridge 14 Heatable thin film 15a, 15b Gold electrode 15c, 15d, 15e, 15f Electrode 16 Pt heater thin film 17 Non-fluorescent glass substrate 18a, 18b Sample injection hole 19 Acrylic resin 20 Rubber cap 21 Specimen injection hole 22 Buffer injection hole 23 PCR reaction liquid injection hole 24 Waste liquid hole 25 Flow Path 26 Specimen trap subroutine 27 Specimen transport end determination 28a, 28b, 28c Complementary nucleic acid sequence 29 DNA
30 Transparent electrode 31 Switch 32, 33, 34 Terminal 35 Laser light source 36 Beam expander 37 Lens 38 Gene detection chip 39 Cooling CCD camera 40, 41, 42, 43 Diaphragm micropump 44 I / O
45 Data / Control Bus 46 Display 47 PC Controller 48 Laser Power Controller 49 Heater Pattern 50 Transparent Electrode Pattern 51 Non-Fluorescent Glass Substrate 52 PET Substrate 53 PDMS Sheet 54 Solid Phase Region 55 Solid Phase Spot

Claims (8)

A gene detection chip having a surface capable of capturing a gene,
The gene detection chip, wherein the surface has a plurality of nucleic acid probes complementary to a nucleic acid sequence to be detected, is temperature-controllable, and is a total reflection surface that totally reflects incident light. The gene detection chip according to claim 1,
A gene detection chip comprising a transparent electrode disposed at a position facing the liquid in contact with the surface. The gene detection chip according to claim 2,
The gene detection chip, wherein the surface is disposed in a flow path capable of carrying a liquid containing the nucleic acid sequence to be detected. The gene detection chip according to claim 2,
The gene detection chip, wherein the surface is arranged in a container that contains a liquid containing the nucleic acid sequence to be detected. A nucleic acid sequence integration method for integrating the nucleic acid sequences to be detected on the surface of the gene detection chip according to claim 3,
A first step of transporting the liquid containing the nucleic acid sequence to be detected by a liquid amount passing through the transparent electrode or a liquid amount less than that,
A second step of applying a voltage to the conveyed liquid;
A third step of heating the nucleic acid sequence to be detected that has migrated to the surface in the second step so that double strands are separated into single strands;
A nucleic acid sequence accumulation method, wherein the nucleic acid sequences to be detected are accumulated on the surface by sequentially repeating the first to third steps. A nucleic acid sequence integration method for integrating the nucleic acid sequence to be detected on the surface of the gene detection chip according to claim 4,
A first step of applying a voltage to the liquid containing the nucleic acid sequence to be detected;
A second step of heating the nucleic acid sequence to be detected, which has migrated to the surface in the first step, so that double strands are separated into single strands,
A nucleic acid sequence accumulation method, wherein the nucleic acid sequences to be detected are accumulated on the surface by sequentially repeating the first to second steps. A gene detection device using the gene detection chip according to any one of claims 1 to 4,
Illumination means for irradiating light for forming an evanescent field on the surface;
Imaging means for imaging light oozing from the surface by irradiation by the illumination means;
A gene detection apparatus, comprising: a heating unit that heats the nucleic acid sequence to be detected that has moved to the surface to cause a double strand to dissociate into a single strand. The gene detection apparatus according to claim 7,
A liquid-feeding means for transporting a liquid containing the nucleic acid sequence to be detected;
And a voltage applying means for applying a voltage to the transported liquid and causing the nucleic acid sequence to be detected to be electrophoresed on the surface.