JP2001235474A - Substrate for biochemical reaction detection chip and its manufacturing method, biochemical reaction detection chip, apparatus and method for executing biochemical reaction, and recording medium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a biochemical reaction detection chip and its substrate whereby a temperature of a biochemical reaction including hybridization can be adjusted for every reaction system on the same chip. SOLUTION: In this substrate for biochemical reaction detection chips, a plurality of islands of thermal conductors are formed on a thin film, which are separated from each other by spaces. Each of the islands is provided with a temperature regulator. This biochemical reaction chip has a probe fixed on the substrate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a biochemical reaction detection chip substrate and a method for producing the same, a biochemical reaction detection chip, an apparatus and method for performing a biochemical reaction, and a recording medium.

[0002]

2. Description of the Related Art As a technique for measuring the base sequence of a nucleic acid,
Using a polynucleotide detection chip in which a single-stranded oligonucleotide probe of a previously designed base sequence is divided and fixed for each type of base sequence, a single-stranded polynucleotide to be measured and a single-stranded oligonucleotide probe A method for detecting the presence or absence of a complementary strand bond (hybridization) is known. Examples of the polynucleotide detection chip include a polynucleotide detection chip for diagnosis in which DNA complementary to a specific mutant sequence of interest is arranged (Science Vol. 270, 467-470 (1995)), which can be present in a measurement object. An SBH (sequencing by hybridization) method for preparing an oligonucleotide probe that binds to a complementary strand to all base sequences and determining the base sequence of a measurement target is known (J. DNA Sequencing and Mapping, Vol. 1, 375- Three
88 (1991)).

[0003]

The thermal stability of the binding of the complementary strand between the oligonucleotide probe and the single-stranded polynucleotide varies from sequence to sequence. The reason is considered as follows. Binding of adenine (A) and thymine (T) or adenine (A)
The bond between uracil (U) and uracil (U) is two hydrogen bonds per base, whereas guanine (G) and cytosine (C)
Are three hydrogen bonds per base (FIG. 11).
As a result, a difference occurs in the bonding strength. GC bond is AT
Since the bonding strength is stronger than the bonding or the AU bonding (FIG. 12 (a)), the thermal stability is high. Therefore, when comparing the thermal stability at the same base length, the thermal stability of the double bond having only the AT bond or the AU bond is the lowest, and the thermal stability of the double bond having only the GC bond is the lowest. Thermal stability is highest. The thermal stability of complementary strand binding is generally indicated by the temperature (melting temperature) (Tm) at which binding and dissociation occur by 50% (FIG. 12 (b)).

Taking an octamer oligonucleotide DNA probe as an example, a double-stranded D consisting entirely of AT bonds
The Tm of NA is 15.2 ° C. (% GC method (Breslauer KJ, e
t.al.:"Predicting DNA duplexstability from the bas
e sequence ", Proc. Natl. Acad, Sci. USA83, 3746-3750)
In contrast, the Tm of a double-stranded DNA consisting entirely of GC bonds is 56.2 ° C., and the difference is 41.0 ° C. (FIG. 12 (c)).

As described above, when the Tm value of the complementary strand binding of the oligonucleotide probe changes greatly, it is necessary to perform hybridization at the temperature of the Tm value of the complementary strand binding of each probe. Under the condition of higher temperature than Tm, 1
The single-stranded polynucleotide hardly binds to the probe, and an effective effect cannot be obtained. On the other hand, under conditions lower than Tm,
Background noise due to mismatch binding increases, leading to a decrease in measurement resolution. Therefore, when a plurality of types of probes are immobilized on the polynucleotide detection chip, when the temperature on the polynucleotide detection chip is kept constant and the hybridization with the single-stranded polynucleotide sample to be tested is performed, the heat of each probe is increased. Since the stability is different, there is a problem that a difference occurs in the amount of complementary strand binding of the probe and a difference in mismatch probability.

In a conventional detection chip, the temperature at which hybridization is performed for all the probes on the detection chip is set to be constant, and the salt concentration of the solvent is adjusted, and the probe density and probe base length fixed to the detection chip are set for each type. Attempts have been made to eliminate the difference in the amount of complementary strand binding generated by the probe and the difference in the mismatch probability due to the difference in the thermal stability of each probe by using the changing method. However, Tm
The effects of the differences have not been fully resolved.

As an example for solving this problem, Japanese Patent Application Laid-Open
No. -127900 discloses a configuration in which a conductive heating track is provided around each analysis electrode, or each analysis electrode is heated by a laser. However, Japanese Patent Laid-Open No. 11-1
No. 27900 only heats the analysis electrode,
For example, it was not possible to perform control such as keeping the temperature of the analysis electrode constant.

Accordingly, an object of the present invention is to provide a biochemical reaction detection chip and a substrate thereof, which can control the temperature of a biochemical reaction including hybridization between an oligonucleotide probe and a polynucleotide. Another object of the present invention is to provide an apparatus and a method for simultaneously performing a biochemical reaction in a plurality of reaction systems by adjusting the temperature for each reaction system, and a recording medium.

[0009]

According to the present invention, a plurality of islands composed of a heat conductor are formed on a thin film,
The islands are separated from each other by a space, and
Each of the islands provides a substrate for a biochemical reaction detection chip provided with a temperature controller.

The thin film is preferably formed of a material having excellent insulating properties, heat insulating properties and strength. The electrical conductivity of the thin film material is 10
^The resistance is preferably ⁸ Ω · m or more, and more preferably 10 ¹⁰ Ω · m or more. The thermal conductivity of the thin film material is preferably 10 w / mk or less, and preferably 1 w / mk or less.

When the thin film is formed of a material having excellent insulating properties and heat insulating properties, the temperature of each island can be easily adjusted. Examples of the thin film include a thin film formed of at least one material selected from the group consisting of a Si nitride film, a Si oxide film, an Al oxide film, and Ta ₂ O ₅ ,
Examples include a thin film such as a Si nitride film, a Si oxide film, Al ₂ O ₃ , and Ta ₂ O ₅ , and a composite film thereof. Among these, a composite film of SiN and SiO ₂ is preferable. Si
Since N has alkali resistance, the probe can be fixed on the SiN film by silane coupling in an alkaline solution. Further, the SiN film can protect an electronic circuit formed thereunder for temperature control from a solution such as a sample solution.

[0012] The thickness of the thin film is preferably 1 to 500 µm, and more preferably 5 to 20 µm. It is preferable that a recess is provided in a region for fixing the probe on the thin film. The provision of the depression is advantageous because the sample solution can be held on the chip when the sample solution is brought into contact with the probe to perform a biochemical reaction.

Further, a resist film may be formed on a surface opposite to a surface on which the island is formed on the thin film. Examples of the resist film include photosensitive polyimide.

On the thin film, a plurality of islands made of a heat conductor are formed. “A plurality of islands” refers to at least two islands, and the number of islands is not particularly limited, but is preferably 10 to 1,000. The plurality of islands may be arranged linearly, or may be two-dimensionally arranged in a first direction (row) and a second direction (column).

The island is composed of a heat conductor. Examples of the heat conductor include metals such as crystalline Si, Ag, Au, and Cu, polysilicon, and amorphous silicon. Further, it is preferable that the heat conductor constituting the island is capable of electrically insulating the heat conductor from the temperature controller. Silicon is preferred as the heat conductor constituting the island, since it has good thermal conductivity and can be electrically insulated from the temperature controller.
By forming a pn junction in silicon, insulation between the heat conductor and the temperature controller can be ensured.

The islands are separated from each other by a space. The space between the islands acts as a thermal insulator, which facilitates independent temperature control of the individual islands. The size of the island is preferably 10 to 1000 μm ² , and more preferably 50 to 500 μm ² . The distance between the islands is preferably 50 to 1000 μm, and preferably 10 to 1000 μm.
It is 0 to 500 μm.

The shape of the island is not particularly limited. For example, when an unnecessary portion is removed by etching with KOH from a flat plate of Si crystal having a surface of 100 to form an island of Si crystal, in the manufacturing process, 11
One surface is exposed, and the shape of the island becomes like a quadrangular pyramid.

Each of the plurality of islands has a temperature controller. Specifically, a heating circuit and a temperature detection circuit may be formed in each of the islands. The heating circuit and the temperature detection circuit may be controlled to operate independently for each island, or may be controlled to operate independently for each group of islands.

Further, the plurality of islands are formed in the first direction and the second direction.
When two-dimensionally arranged in the directions, the islands may be controlled to operate independently for each of the islands arranged in either the first direction or the second direction. The size of the substrate for biochemical reaction detection chip may When it is 25 mm ² 100 cm ^2, preferably from 100mm ² ~14cm ^2.

By using a biochemical reaction detection chip manufactured by fixing a probe to the substrate for a biochemical reaction detection chip of the present invention, the influence of the temperature of the adjacent probe fixing surface (reaction system) is reduced, and individual A biochemical reaction can be performed at an appropriate temperature on the probe fixing surface (reaction system).

The biochemical reaction detection chip substrate of the present invention comprises:
A heat extruder for draining heat from the islands may be provided between the islands. The heat extruder may have a structure that does not directly contact the island (for example, a mesh structure). The heat outflow device may be provided in only one of the first direction and the second direction, or may be provided in both the first direction and the second direction. When the probes whose biochemical reactions have similar optimum temperatures are grouped and fixed to a thin film, a heat outlet may be provided for each region of the group.

The heat outlet is preferably made of a material having good heat conductivity, for example, a metal such as Si, Au, Ag, or Cu. Forming a heat sink between the islands allows the heat to escape before it travels from the adjacent island. The distance from the island to the heat sink is preferably between 10 and 500 μm, preferably between 10 and 250 μm
m.

The present invention also provides a method for producing a substrate for a biochemical reaction detection chip, comprising: (a) forming a thin film on one surface of a flat plate of a heat conductor; A method is provided for removing unwanted portions from the other surface side of a body slab to form thermal conductor islands. In the above method, a temperature controller may be formed on one surface of the flat plate of the heat conductor, and a thin film may be formed thereon.

As one embodiment of a method for manufacturing a substrate for a biochemical reaction detection chip, a mask having a desired pattern is provided on a surface opposite to a surface on which a flat thin film of a heat conductor is formed, and a mask is provided on the opposite surface. The surface on the side provided with the mask may be etched until the formed thin film is exposed, and the island of the heat conductor may be formed on the thin film in a pattern of the mask. The mask is
For example, a thin film of a Si nitride film may be used.

Further, the present invention provides a biochemical reaction detection chip in which a probe is fixed to the above substrate for a biochemical reaction detection chip. The surface on which the probe is fixed on the substrate is preferably the surface of the Si nitride thin film. In this case, the probe provided with the amino group can be fixed to the silanized surface of the Si nitride thin film by silane coupling.

The term "probe" refers to a substance capable of specifically detecting a specific substance, site, state, etc., and examples thereof include oligonucleotide DNA / RNA probes and protein probes such as antibodies. In the case of an oligonucleotide DNA / RNA probe, the number of bases is preferably 4 to 500 nt (nucleotide), and preferably 8 to 200 nt (nucleotide). The oligonucleotide probe may be single-stranded or double-stranded, but from the viewpoint of the binding efficiency between the probe and the measurement target,
It is preferably single-stranded.

The probe can be fixed on the thin film of the biochemical reaction detection chip substrate by a known method. For example, if the probe fixing surface of the thin film is silanized and an amino group is provided on the probe, the probe can be fixed on the thin film by silane coupling. An island exists below the probe fixing surface of the thin film.

It is preferable that the area other than the probe-immobilized surface on the thin film is coated with polylysine after the probe is immobilized, and the binding sites on the silane-coated surface not bonded to the probe are crushed. This allows sample DNA and RNA to be used at the time of use.
And the like can be prevented from non-specifically binding to the silane-coated surface.

The type of probe is not particularly limited, and may be one type or a plurality of types. For example, if a chip in which a plurality of types of probes are fixed to one chip substrate is used, a plurality of detection targets in one sample can be simultaneously detected. In addition, when a chip in which a large number of one type of probes are fixed to one chip substrate is used, one detection target in a plurality of samples can be simultaneously detected.

The detection chip of the present invention can be used for detecting biochemical reactions such as detection of DNA, cDNA, RNA, etc., detection of antigen-antibody reaction, detection of protein and the like. By using the biochemical reaction detection chip of the present invention, it is possible to reduce the influence of the temperature of the adjacent probe fixing surface (reaction system) and perform a biochemical reaction at an appropriate temperature on each probe fixing surface (reaction system). Can be.

Further, the present invention relates to an apparatus for performing a biochemical reaction by a plurality of reaction systems on a biochemical reaction detection chip, wherein the entire biochemical reaction detection chip is heated to a temperature higher than a temperature suitable for each biochemical reaction. And a temperature control device for controlling the temperature of each reaction system to a temperature suitable for each biochemical reaction. The temperature controller may adjust the temperature of each reaction system in a time sharing manner.

Furthermore, the present invention provides a computer
A device for performing a biochemical reaction using a plurality of reaction systems on a biochemical reaction detection chip, wherein the heating device heats the entire biochemical reaction detection chip to a temperature higher than a temperature suitable for each biochemical reaction; Provided is a computer-readable recording medium on which a program for causing a biochemical reaction device including a temperature control device for adjusting the temperature of each reaction system to a temperature suitable for a chemical reaction is recorded.

The present invention also relates to a method for simultaneously performing a plurality of biochemical reactions carried out in a plurality of reaction systems by controlling the temperature for each reaction system. (A) The method is suitable for the biochemical reactions of each reaction system. Heating all of the plurality of reaction systems to a temperature higher than the temperature, and (b) for each reaction system, lowering the temperature of the reaction system to a temperature suitable for a biochemical reaction, and reducing the temperature for a certain period of time. Provide a way to maintain.

The heating operation (a) is preferably performed in a constant temperature bath. For each reaction system of (b), the operation of lowering the temperature of the reaction system to a temperature suitable for a biochemical reaction may be performed by simply stopping the heating operation of (a) or by using a cooling device. .

In one embodiment of the method for simultaneously carrying out a plurality of biochemical reactions according to the present invention, the biochemical reaction is hybridization between a polynucleotide and an oligonucleotide, and the temperature suitable for the biochemical reaction is a temperature at which the oligonucleotide and its complement are complementary. It is the melting temperature of the double strand formed by the strand. The polynucleotide may be DNA in a sample, and the oligonucleotide may be an oligonucleotide probe of a biochemical reaction detection chip.

When the temperature suitable for the biochemical reaction is the melting temperature of the duplex formed by the oligonucleotide probe and its complementary strand, the temperature higher than the temperature suitable for the biochemical reaction is preferably The temperature at which the nucleotides of the strand are completely dissociated, for example, between 90C and 99C. The temperature suitable for the biochemical reaction may be a temperature around the melting temperature, for example, a temperature in the range of the melting temperature ± 2 ° C.

One embodiment of the method of the present invention will be described.
After dispensing the sample onto the biochemical reaction detection chip and covering the chip, the chip is placed in a thermostat and heated to the highest temperature (for example, 90 ° C.). A thermostat usually has a heater and a cooler, and can operate so that the inside may be at a set temperature. Thereafter, the temperature of the thermostat is lowered to the lowest temperature (for example, 15
° C), lower the temperature of all the reaction systems, and set the temperature of each reaction system (for example, the probe fixed surface) to the set temperature (for example, the melting temperature of the duplex formed by each probe and its complementary strand). Start the electric heater when
While maintaining the set temperature of each reaction system, a biochemical reaction is allowed to proceed (for example, 12 hours). After completion of the reaction, the reaction system (for example, the surface on which the probe is immobilized) is washed, and then a biochemical reaction is detected, and data processing of the detection result is performed.

For detection, a general method is to label a sample in advance with a fluorescent label, measure the amount of fluorescence of the label bound to the probe with a confocal microscope, and calculate the amount of bound sample from the amount of fluorescence. .

Usually, the biochemical reaction proceeds when the temperature of the reaction system reaches an appropriate temperature by heating the reaction system.
In this manner, the probe often binds to a substance other than the object to be detected, which becomes noise in the detection of the object to be detected. However, by raising the temperature of the reaction system once to a temperature higher than the appropriate temperature for the biochemical reaction and then lowering it to the appropriate temperature for the biochemical reaction, the probability that the probe will bind to a substance other than the target substance is reduced, and the target substance is detected. The noise in the detection of is reduced. For example, in the case of hybridization between an oligonucleotide probe and a polynucleotide, the appropriate temperature for binding a complementary strand is the melting temperature of the duplex formed by the probe and its complementary strand. When the reaction system is heated to the melting temperature and the reaction is allowed to proceed, the oligonucleotide probe may bind to nucleotides other than the nucleotide having the complementary strand (the target for detection) (so-called mismatch). It becomes noise in the detection of the detection target. However, once the temperature of the reaction system is raised to a temperature higher than the melting temperature and then lowered to the melting temperature, the probability that the probe binds to a nucleotide other than the nucleotide having the complementary strand is reduced, and noise in the detection of the detection target is reduced. Is reduced.

In the method of the present invention, first, the temperature of all the reaction systems is raised to a temperature higher than the appropriate temperature for the reaction, and then the temperature of each reaction system is reduced to the appropriate temperature for the reaction, and the temperature is maintained for a certain time. (For example, the necessary heat is supplied by a heater for a certain period of time).

As a result, not only does the total amount of heat supplied to the reaction system decrease, but also the temperature of each reaction system is individually raised to an appropriate temperature for the reaction and the temperature is maintained to advance the reaction. Temperature control of each reaction system is also facilitated. This method is advantageous when a large amount of biochemical reactions are performed in parallel. In addition, if the above method is applied to a plurality of reaction systems performing the same reaction, and a biochemical reaction is performed under different temperature conditions for each reaction system, the optimum temperature for the reaction can be determined.

The present invention relates to a method in which a computer simultaneously performs a plurality of biochemical reactions performed in a plurality of reaction systems by controlling the temperature for each reaction system. (A) The method is suitable for the biochemical reactions of each reaction system. Heating all of the reaction systems to a temperature higher than the temperature of the reaction system, and (b) reducing the temperature of the reaction system to a temperature suitable for biochemical reactions for each reaction system, and keeping the temperature constant. There is also provided a computer-readable recording medium on which a program for executing the time keeping method is recorded.

[0043]

Embodiments of the present invention will be described with reference to the drawings. Example 1 Configuration of DNA Chip Substrate FIG. 1 shows a substrate for a biochemical reaction detection chip for immobilizing oligonucleotide DNA (hereinafter, referred to as “DNA chip substrate”).
FIG. Note that a probe in which a probe is fixed to a DNA chip substrate is called a DNA chip.

FIG. 1A shows 10 rows in the horizontal direction and 10 rows in the vertical direction.
1 is a top view of a DNA chip substrate 1 having a total of 100 probe fixing surfaces 2 in a row. The longitudinal length hy and the lateral length hx of the DNA chip substrate 1 are respectively:
It is good to be 10-100 mm. 10 horizontally arranged from left to right
The distance Lx from the left end of the first probe fixing surface of the first probe fixing surface to the right end of the tenth probe fixing surface and the first L of the ten probe fixing surfaces arranged vertically from top to bottom. The distance Ly from the upper end of the probe fixing surface to the lower end of the tenth probe fixing surface is 5 to 100 m, respectively.
m is good.

FIG. 1B is an enlarged view of a region surrounded by the frame of FIG. 1A. The horizontal width X and the vertical width Y of the probe fixing surface for fixing each probe on the DNA chip substrate 1 are respectively
It is good to be 10 to 1000 μm. Probe spacing D is 5
The thickness is preferably from 0 to 1000 μm.

An island is formed below each probe fixing surface. FIG. 1C is a partially enlarged view of a region of the probe fixing surface 2 shown in FIG. 1B. On each probe fixing surface, a heater circuit 5 formed of an n-type diffusion layer and a temperature detection element 6 formed by a pn junction of a p-type diffusion layer and an n-type diffusion layer are formed. At both ends of the heater circuit 5, a heater terminal (+) 1001 and a heater terminal (-) 1002
Is formed. When a voltage is applied between both terminals so that the 1001 side becomes a positive electrode, a current flows through the heater circuit 5 formed of the n-type diffusion layer, and Joule heat is generated. The amount of Joule heat generated can be adjusted by controlling the applied voltage or by controlling the voltage application time. The temperature detection element 6 has a temperature detection terminal (+) 10
03. A temperature detection terminal (-) 1004 is formed in the n-type diffusion layer. The current-voltage characteristic at the pn junction of the temperature detecting element 6 greatly depends on the temperature of the pn junction. for that reason,
If the current-voltage characteristics between the elements are detected, the temperature of the pn junction can be determined. Further, since the island 4 is made of a heat conductor, the temperature of the pn junction and the temperature of the island 4 are almost equal. Therefore, by measuring the current-voltage characteristics between the pn junction elements, The temperature of a certain probe fixing surface 2 can be detected. Current between pn junctions-
The temperature dependence of the voltage characteristic is such that, for example, when the voltage is fixed in the forward bias direction with 1003 as the positive side, the flowing current varies exponentially with the temperature of the pn junction. If the current is fixed in the same forward bias direction, the temperature and the potential difference are almost one.
It can be approximated by the following function.

FIG. 18 shows another example of the shape of the heater circuit and the temperature detecting element on the probe fixing surface. In FIG. 18, an n-type substrate (n-sub) is used, and the heater circuit 5 and the temperature detecting element 6 on the probe fixing surface are separated by separate p-wells, so that the heater circuit side and the pn junction element are electrically connected. Independent. This is to prevent electrical interference between the heater circuit 5 formed of the n-type diffusion layer and the temperature detecting element 6. As shown in FIG. 18, the probe fixing surface is connected to a controller 105 including a heater power supply circuit 181 and a temperature detection circuit 182. The controller 105 is
This is an example of a circuit that detects the temperature of one probe fixing surface and controls heating by a heater. Heater power supply circuit 181
Are the heater power supply Vp, the output adjuster 111, and the switch 1
09, heater terminals 1001, 10 on the probe fixed surface
Connect to 02. By controlling the heater power supply Vpo and the output adjuster 111, the heater terminal 100 on the probe fixed surface is controlled.
By controlling the voltage and current between 1, 1002, Joule heat generated in the heater circuit 5 on the probe fixing surface can be controlled. The temperature detection circuit 182 includes a power supply Vc, a resistor R, and a voltmeter 110. The temperature detection terminal 1003 is set to zero potential, and 1004 is connected to negative potential. In this case, it is a forward bias.
When the circuit resistance R is set sufficiently large as compared with the resistance r between the pn junctions, the current flowing through the temperature detection circuit 182 is substantially determined by the power supply Vc and the resistance R, and can be approximated by the constant current condition of current I = Vc / R. . The potential difference v between the temperature detecting elements is
It can be measured by the voltmeter 110.

The principle of temperature detection will be described. FIG. 19 is a graph showing the relationship (see Table 1) between the temperature (t) of the probe fixing surface and the potential difference (v) between the temperature detection elements 6 in the temperature detection circuit of FIG. The condition in FIG.
= 800 (KΩ), Vc = 8 (V), I = 10 (μA)
The temperature of the pn junction was changed from 20 ° C. to 60 ° C.

[0049]

Table 1 Temperature of temperature detection element (° C.) Voltage difference of temperature detection element (mV) 20.0 537 25.0 524 30.0 512 35.0 500 40.0 489 45.0 477 50.0 465 55 0.0 453 60.0 442

From this experimental example, the relationship between the potential difference Vx and the temperature Tx can be approximated by a linear function having a slope of about −2.37 (mV / ° C.), and the following relational expression is obtained. Tx = 20 + (537−Vx) /2.37 By using the above expression, the temperature can be obtained from the measured value of the voltage difference in the temperature detecting element.

Next, the voltage applied to the working electrode and the timing of heating by the heater will be described with reference to FIG. Assuming that the set temperature of the island 4, that is, the target temperature of the pn junction is T ₀ , the potential difference v ₀ of the pn junction at that time becomes the target potential difference of the pn junction. As an initial condition, when the temperature is sufficiently high, the heater for heating the island is turned off, and when the temperature decreases, the potential difference at the pn junction increases according to the characteristics shown in FIG. heater is turned ON at the timing t1 when the potential difference of the pn junction exceeds the v _0, islands are heated. Thereby, the temperature of the island increases and the potential difference at the pn junction decreases. Heater is turned OFF to heat the island at time t ₂ falls below the target potential difference v _0, the heating of the island stops,
As the island temperature decreases, the potential difference at the pn junction increases again. While repeating such control, the potential difference v _{0 at} the pn junction of the island, that is, the target temperature T ₀ is maintained.

With the configuration described above, the temperature of the probe fixing surface 2 is measured, and the temperature can be controlled by adjusting the amount of generated Joule heat. FIG.
It is a figure explaining the shape of the island formed on the thin film. FIG. 2A is an enlarged rear view of the DNA chip substrate. Si island 21 is SiN / SiO ₂ thin film 22
Is formed on. Both the horizontal width and the vertical width of the Si island 21 are approximately 500 μm. The distance between the Si islands is substantially equal to the distance between the probe fixing surfaces.

FIG. 2B is a sectional view taken along line A-A of FIG.
It is a longitudinal cross-sectional view of A '. The height of the Si island 21 is 2
50 μm. The width of the bottom surface of Si island 21 is approximately 150 μm. The inclination angle of the inclined side surface of the Si island 21 is approximately 55 °. A heater circuit 5 formed of an n-type diffusion layer is incorporated in the Si island 21. S
The distance D between the i-islands is 500-700 μm. SiN / S
The thickness of the iO ₂ thin film 22 is approximately 5 μm in a region where an island is formed (that is, a region having a probe fixing surface). There is a 250 μm Si layer in the periphery (the region from 3 to 5 mm from the end of the thin film).

Si having a Si island 21 formed on the lower surface
A region on the upper surface of the N / SiO ₂ thin film 22 is a temperature setting region 24.
The probe 25 is fixed in this area. By silanizing the probe fixing surface (Si nitride film surface) on the thin film and providing the probe with an amino group, the probe can be fixed on the thin film by silane coupling. Sample solution 26
It may be added in an amount such that the thickness of the solution layer is 10 to 1000 μm. After adding the sample solution 26, a cover glass 27 is placed thereon.

FIG. 3 is a diagram showing an example of setting the temperature of the DNA chip. The temperature of each island is set at 15 to 90 ° C. As shown in FIG. 3, the setting temperature of the probe fixing surface is such that the probe may be arranged such that a probe having a high Tm is placed at the center of the DNA chip, and a probe having a higher Tm is placed at the periphery in the order of decreasing Tm. , A probe having a high Tm and a probe having a low Tm may be arranged from one side of the probe fixing surface toward the opposing surface.

With such an arrangement, the balance between the diffusion and supply of heat is improved, and the temperature is easily adjusted. FIG.
It is a figure explaining the shape and mesh structure of the island formed on the thin film. FIG. 4A is an enlarged back view of the DNA chip substrate. In addition to the Si islands 21, a Si mesh structure 41 is formed on the SiN / SiO ₂ thin film 22 between the islands.

FIG. 4B is a longitudinal sectional view taken along a broken line BB 'in FIG. 4A. The height of the peak of Si forming the mesh structure is approximately 250 μm, and the width is approximately 350 μm. In the present embodiment, the inclination angle of the inclined side surface of the peak of Si forming the mesh structure is approximately 55 °. The distance between the Si peak forming the mesh structure and the Si island 21 is approximately 75 μm.

By forming the heat conductor layer (mesh structure) 41 between the islands 21 as described above, the heat can be released using the mesh before the heat is transmitted from the adjacent island to the island. Become like That is, the mesh structure serves as a heat drain.

FIG. 5 is a diagram for explaining the effect of the mesh structure. The conditions are as follows. Si island
The width and height of 21 are both 500 μm. The height of the Si island 21 is 250 μm. One side of the bottom surface of the Si island 21 is 150 μm. The height of Si forming the mesh structure is approximately 250 μm, and the width is 350 μm.
The distance between the Si peak forming the mesh structure and the Si island 21 is approximately 75 μm. The thickness of the SiN / SiO ₂ thin film 22 is 5 μm
m. The thickness of the layer 51 of water (model of the sample solution) is 20
μm. The thickness of the acrylic plate 52 is 5 μm.

From point A (the center of the bottom of the Si island) to B
It is easy to transfer heat to the point (the center point of the bottom of the adjacent Si island), and it is 2m from point A to point C (the middle point M between points A and B).
m at a distance). From point A to point M, the easiness of conducting heat is the same, so the description is omitted. Heat transfer coefficient of thin film is 10, heat transfer of Si layer is 150,
Comparing the heat UMB conducted from point M to point B and the heat UMC conducted from point M to point C per unit time, UMB: UMC = 10 × (cross-sectional area of thin film) / 150: 150 × (mesh breakage) (Area) / 2000 = 10 x (5 x 500) / 150: 150 x (175 x 250) / 2000 = 1:20

As a result, the heat conduction from point M to point C is M
It can be seen that the heat conduction from point to point B is about 20 times. FIG. 6 is a diagram illustrating an embodiment in which a cooling function is provided on the outer peripheral portion of the mesh. For example, the DNA chip substrate 1 is fitted into a metal frame 61, and the metal frame 61 is connected to the Pellitier element. By providing the cooling function on the outer periphery of the mesh, the effect of waste heat is enhanced.

Embodiment 2 Chip Manufacturing Process FIGS.
9, a method for manufacturing a DNA chip substrate in which islands are formed in a mesh structure on a composite film of SiO ₂ and SiN will be described. FIG. 7 is a diagram illustrating a first process of manufacturing a DNA chip substrate. As the substrate, a 500 μm-thick n-type Si substrate (N-su
b) Use 71. After forming a p-well pattern on the surface with an SiO ₂ film 74, p-wells (1018 / cm ³ ) 72, 73 by B doping and diffusion
Is formed at a depth of 3 μm. This is to electrically insulate the temperature detecting element 6 and the heater circuit 5 which are manufactured later by n + diffusion. Si as element isolation and diffusion mask
An O ₂ film 74 is formed. A dope such as boric acid is used for p-well diffusion. Next, after forming a circuit pattern with an SiO ₂ oxide film, the high-concentration n-type diffusion layer 75,
76,77 (n +, 1020 / cm ³ ) and high-concentration p-type diffusion layers 78,79
(p +, 1020 / cm ³ ) was formed at a depth of 100 nm. The n + (n-type diffusion layer) 76 forms the temperature detecting element 6. The n + (n-type diffusion layer) 77 forms the heater circuit 5. n + (n-type diffusion layer) 75
Denotes a reference electrode terminal 75 of the n-type substrate. The potential of the substrate 71 is
The potential can be set at the reference electrode terminal 75, and the potentials of the p-well 72 and p-well 73 can be set at 78 and 79, respectively. As terminals for applying a p-well reference potential, p + (p-type diffusion layers) 78 and 79 are formed by diffusion of boron (high-concentration p-type impurity).

FIG. 8 shows a second example of the manufacture of the DNA chip substrate.
FIG. Subsequent to the first process, a first interlayer insulating film for protecting and insulating a surface circuit
(Eg, SiO ₂ film) 81 is formed. CVD oxide film (SiO ₂ ) 400nm
Is formed, and a BPSG film of 500 nm is laminated thereon.

Then, after forming holes for the respective terminals in the first interlayer insulating film 81, the first layer wirings 82 (801, 802, 803, 804) are formed of W as follows. 75 and 76 are electrically connected to form a sensor common wiring 801. Reference numeral 78 connects to the positive electrode 802 of the PN junction temperature sensor. The temperature of the substrate can be evaluated by detecting the amount of current flowing between 802 and 801 via the p-well. The n-type diffusion layer 77 constituting the heater circuit is connected to the two electrodes 803 and 804. 804 conducts with the p-type diffusion layer 79. 804 constitutes a common wiring of the heater circuit. When connected to a power supply with the positive electrode on the 803 side between 803 and 804, the heater current passes 77 and Joule heat is generated.

Next, a second interlayer insulating film is used as a wiring protection film.
(Eg, SiN film) 83 is formed. 83 is plasma CVD Si
It is a laminated film of an O ₂ film of 600 nm and a plasma CVD SiN film of 1200 nm. Then, after forming a connection hole for the first wiring in the second interlayer insulating film 83, a second-layer wiring (for example, Au) 84 is formed. 84
A sensor common electrode 805 connected to 801; a temperature sensor positive electrode (not shown) connected to 802; a heater positive electrode (not shown) connected to 803; a heater common electrode 806 connected to 804
Consists of Among them, 805 and 806 can be common among a plurality of islands.

Finally, an example of forming an island on the back surface will be described. FIG. 9 is a diagram illustrating a third process of manufacturing a DNA chip substrate. First, the back surface of the n-type substrate 71 is mechanically polished to reduce the thickness of the substrate from 500 μm to 250 μm. This is because the island thickness of 250 μm or less is sufficient, and a decrease in the thickness leads to a reduction in the time required for the etching step. After the polished surface is smoothed by chemical etching, an Si nitride film (Si ₃ N
₄ ) Using the 91 as an etching mask, 120 nm is laminated.

Using a double-sided aligner, align the back surface pattern (island pattern) corresponding to the devices on the front surface such as the heater and temperature sensor circuit previously prepared with the back surface.
The nitride film 91 is partially dry-etched.

Next, the remaining the Si ₃ N ₄ film 91 as a mask, SiO ₂ aqueous potassium hydroxide to dissolve the n-type substrate 71
The etching was performed until the film 74 was exposed, that is, until the film 74 reached the surface side Si oxide film 74. The Si nitride film 91 has extremely high resistance to an aqueous solution of potassium hydroxide, and the amount of shaving of the Si nitride film when etching the 250 μm Si substrate 71 is 10 nm or less. Further, the etching rate in the (111) plane direction of the Si substrate 71 is about 1/100 of that in the (100) plane direction.
Degree and slow. As a result, the chip is etched along the (111) plane from the protective surface of the Si nitride film 91, and the chip is formed with the (111) plane exposed as the side surface 901 of the island. The (111) plane is about 55 degrees with respect to the (100) plane. Therefore, when etching a 250 μm thick Si substrate, the slope is about 175
It can be obtained by spreading μm.

[Example 3: Probe immobilization process] A procedure for immobilizing an oligonucleotide probe on a prepared chip will be described. First, an OH group is formed on the Si nitride film on the chip surface. For this purpose, and H ₂ SO ₄ · H2O ₂ mixture, NaOH · H ₂ O
A method by hydrolysis using a mixture of _two or the like is generally used. It is also possible to simply leave the apparatus in a flooded state for a certain period of time.

Next, a silane coupling agent such as epoxy is injected into the chip surface, and the Si nitride film is silanized.
At this time, the OH group and the silane coupling agent are bonded.
This time, as a silane coupling agent, 3-glycidoxypropyltrimethoxysilane (glycidoxypropiltrimeth
oxy silane). The reaction conditions were room temperature reaction for 30 minutes and baking at 120 ° C. for 1 hour.

Next, an oligonucleotide probe having an amino group introduced into the terminal in advance is spotted on a predetermined probe-fixed surface. When the reaction is performed at 50 ° C. for 15 minutes under high humidity conditions to prevent drying, the probe is fixed to the chip surface by silane coupling. Next, excess polylysine is injected into the chip and reacted at 50 ° C. for 10 minutes under high humidity conditions to bind lysine to a functional group that is not bound to a probe. This treatment is effective in reducing the background due to non-specific adsorption when actually performing hybridization with the sample. Finally, the surface of the DNA chip is washed with Tris EDTA, and stored by drying.

[Embodiment 4: Temperature Control] An apparatus for controlling a DNA chip of the present invention will be described. FIG. 10 is a diagram illustrating a configuration of an example of a biochemical reaction detection device. DNA chip 1
01 is heater terminal (+) 100 for each probe fixed surface
1, heater terminal (-) 1002, temperature detection terminal (+) 100
3. It has a temperature detection terminal (-) 1004, and the wiring between these terminals and the controller 105 is the same as in FIG. The controller 105 includes a voltmeter 110, a temperature detection circuit 182 having a power supply Vc and a resistance R, a heater power supply Vpo, and an output controller 11.
The number of heater power supply circuits 181 each having 1 and the switch 109 is equal to the number of probe fixing surfaces whose temperature is to be independently set. FIG.
8, only one set is shown. The wiring to these terminals is connected to a holder 103 via a printed circuit board 102, and further from the holder 103 via a cable 104,
Connected to controller 105. For the sake of simplicity, the wiring of the terminal connected to the ground on the controller 105 side can be shared on a plurality of fixing surfaces.

The temperature is controlled by the method described above with reference to FIG. 18 independently for each probe fixing surface. The temperature of the probe fixing surface can be determined by measuring the potential difference of the incorporated temperature detecting element. Then, based on the measured temperature, the voltage connection to the heater is switched to the switch 109.
On / off control. Switch 109 and output adjuster 111
When the temperature obtained by the temperature detection circuit is lower than a preset value, the output of the heater power supply Vpo is adjusted to flow the heater current. The above control is independently controlled for each fixed surface.

The DNA chip 101, the printed circuit board 102 and the holder 103 are used in a thermostat 106, and are cooled by blowing air from below the DNA chip 101 by using a fan 107 as necessary, or by using a Peltier device 108. To cool the periphery of the DNA chip. In this embodiment, as in the embodiments described later, the temperature of the thermostatic layer is adjusted to the minimum value of each temperature set value required for the chip, and the temperature of the chip rises and the temperature distribution of each probe fixing surface in the vicinity is adjusted. Depending on the Peltier element 108
And the fan 107 is used.

The above-mentioned temperature control of the DNA chip may be controlled by a computer. In this case, the computer becomes a temperature control device according to the temperature control program, and this program is stored in a computer-readable recording medium. This recording medium is, for example, a RAM
Memory, ROM memory, magnetic disk, CD-ROM,
Any type of recording medium such as a magnetic tape and an IC card may be used.

[Example 5: Measurement] In this example, a case in which a DNA fragment having a length of 17 bases is measured using four types of probes having a length of 8 bases will be described. SEQ ID NO: 1 is an example of a DNA fragment having a length of 17 bases (hereinafter, referred to as sample DNA). TGACCGGCAGCAAAATG (SEQ ID NO: 1) The 17-base sample DNA is hybridized to the following four 8-base probes. CCGTCGTT (SEQ ID NO: 2) GCCGTCGT (SEQ ID NO: 3) GGCCGTCG (SEQ ID NO: 4) TGGCCGTC (SEQ ID NO: 5)

The probe of SEQ ID NO: 2 (hereinafter referred to as probe 2
Is a sequence complementary to the 6th to 13th bases of the sample DNA. Similarly, SEQ ID NO: 3 (hereinafter, referred to as
Probe 3) is the fifth to twelfth, SEQ ID NO: 4 (hereinafter, probe 4) is the fourth to eleventh, and SEQ ID NO: 5 (hereinafter, probe 5) is the third to tenth. , Respectively, are complementary sequences. Using the DNA chip of the present invention, the Tm temperature when these probes hybridized with the sample DNA was measured.

First, the four types of probes 2 to 5 were
The DNA chip of the present invention having 4 × 9 = 36 fixed surfaces fixed to the fixed surfaces is prepared. FIG. 13 is a schematic view of the chip viewed from above. A pre-labeled sample is injected into the chip and hybridized.
At this time, the hybridization temperature set value on each fixed surface in rows a to i in FIG.
Set at 5 ° C intervals up to 50 ° C (row a = 10, row b = 15,
row c = 20, row h = 45, row i = 50 ° C.).

The procedure of the reaction is as follows. FIG.
FIG. 3 is a diagram schematically showing, as an example, temperature transitions of fixed surfaces belonging to columns a, c, g, and i. First, the temperature of the thermostat was raised to 90 ° C. Next, the temperature of the thermostat is set to 10 ° C.
, The temperature of each fixed surface begins to drop. When the temperature of the fixed surface reaches about 50 ° C., the power is first turned on to the heater in row i, and the heater is heated so that the temperature does not become lower.
Similarly, when the heaters in each row detect the temperature of the preset hybridization temperature set value, they perform ON / OFF control of the heaters to maintain the temperature. As a result, the probe-fixed surfaces in each row maintain the set temperature and perform hybridization between the sample and the probe.
After a certain period of time, the DNA chip is washed with a washing solution to remove unhybridized samples and the like. The evaluation of hybridization is performed by scanning the chip surface using a laser fluorescence confocal microscope and measuring the amount of fluorescence emitted from each fixed surface.

FIG. 15 shows the temperature dependence of hybridization in the measurement using the sample DNA. Each temperature is a measurement result of each row. The amount of luminescence at a hybridization temperature of 10 ° C. was normalized as 1. It can be seen that the amount of hybridization decreases as the temperature increases. The temperature at 0.5 on the vertical axis is the Tm temperature. As a result, the Tm values of the probes 2 to 5 and the sample DNA are
The obtained temperatures are 25 ° C, 34 ° C, 42 ° C, and 31 ° C, respectively. In this way, this chip, for four types of probes,
Hybridization at 9 different temperature conditions
Each time, there is an advantage that the same sample can be competitively evaluated. Therefore, the measurement result does not include an error in sample preparation, and the measurement accuracy is significantly improved.

As described above, the optimum value of the hybridization temperature when one kind of sample DNA is evaluated with an 8-base probe differs for each probe. When measuring a sample DNA, it is necessary to select an optimum probe from probes 2 to 5. Therefore, the present DNA chip, which can evaluate the hybridization temperature characteristics of a plurality of types of probes at one time, is very effective in studying probes.

In the present chip, the temperature dependence of hybridization under the condition of single base mismatch can also be evaluated. Prepare the following two types of DNA fragments. TGACCGGGACAAATG (SEQ ID NO: 6) TGACCGGGAAGCAAATG (SEQ ID NO: 7)

These are two types of 17-mer single nucleotide polymorphism DNs which differ from SEQ ID NO: 1 only by the eighth base from the third end.
This is an example of an A fragment. For these single nucleotide polymorphism DNA fragments,
The temperature dependence was similarly measured using the above-mentioned chip. 16 and 17 show SEQ ID NO: 6 and SEQ ID NO: 7, respectively.
5 shows the results of the hybridization evaluation when the sample No. was used. These measurements are for a single base mismatch and ideally require no hybridization. However, as shown in the figure, some hybridization has occurred. In general, in a measurement using a DNA chip, it is necessary to be able to accurately determine such a single-base mismatch. 16 and FIG.
The same evaluation as in 7 is performed, and the probe to be used and its Tm temperature are determined from the characteristics. According to the present invention, it is possible to easily and accurately measure the hybridization temperature dependency of each probe.

[0084]

According to the present invention, there is provided a biochemical reaction detection chip and a substrate thereof, which can adjust the temperature of the biochemical reaction for each reaction system. Further, according to the present invention, there have been provided an apparatus and a method, and a recording medium for simultaneously performing a biochemical reaction in a plurality of reaction systems by controlling the temperature for each reaction system.

[0085]

[Sequence List] SEQUENCE LISTING <110> HITACHI, LTD. <120> Polynucleotide Detection Chips and Polynuc
leotide Detection Apparatus <130> H000230 <160> 7 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <223> fluorescently labeled DNA fragment <400> 1 tgaccggcag caaaatg 17 <210> 2 <211> 8 <212> DNA <213> Artificial Sequence <223> DNA probe hybridizing with DNA fragment <400> 2 ccgtcgtt 8 <210> 3 <211> 8 <212> DNA <213> Artificial Sequence <223> DNA probe hybridizing with DNA fragment <400> 3 gccgtcgt 8 <210> 4 <211> 8 <212> DNA <213> Artificial Sequence <223> DNA probe hybridizing with DNA fragment <400> 4 ggccgtcg 8 <210> 5 <211> 8 <212> DNA <213> Artificial Sequence <223> DNA probe hybridizing with DNA fragment <400> 5 tggccgtc 8 <210> 6 <211> 17 <212> DNA <213> Artificial Sequence <223> fluorescently labeled DNA fragment <400> 6 tgaccggtag caaaatg 17 <210> 7 <211> 17 <212> DNA <213> Artificial Sequence <223> flu orescently labeled DNA fragment <400> 7 tgaccggaag caaaatg 17

[0086]

[Sequence Listing Free Text] SEQ ID Nos. 1, 6 and 7
It is a nucleotide sequence of a 17-base synthetic DNA fragment. SEQ ID NOs: 2 to 5 are nucleotide sequences of an 8-base long probe.

[Brief description of the drawings]

FIG. 1 is a diagram showing an outline of a substrate for a biochemical reaction detection chip for immobilizing oligonucleotide DNA (hereinafter, referred to as a “DNA chip substrate”). FIG. FIG. 1 (b) is a top view of a DNA chip substrate 1 having a total of 100 probe fixing surfaces 2 in ten rows in a vertical direction.
Is a partially enlarged view of a region surrounded by a frame in FIG. 1 (a), and FIG. 1 (c)
FIG. 2 is a partially enlarged view of a region of a probe fixing surface 2 shown in FIG.

2A and 2B are diagrams for explaining the shape of an island formed on a thin film. FIG. 2A is an enlarged rear view of a DNA chip substrate, and FIG. 2B is a broken line portion in FIG. It is a longitudinal cross-sectional view of AA '.

FIG. 3 is a diagram showing an example of setting a temperature of a DNA chip.

4A and 4B are diagrams for explaining the shape and mesh structure of an island formed on a thin film. FIG. 4A is an enlarged rear view of a DNA chip substrate, and FIG. 4B is FIG. 1B. D
FIG. 3 is a longitudinal sectional view of a broken line AA ′ when a mesh structure is formed on the NA chip substrate 1.

FIG. 5 is a diagram for explaining the effect of the mesh structure.

FIG. 6 is a diagram illustrating an embodiment in which a cooling function is provided on an outer peripheral portion of a mesh.

FIG. 7 is a diagram illustrating a first process of manufacturing a DNA chip substrate.

FIG. 8 is a diagram illustrating a second process of manufacturing a DNA chip substrate.

FIG. 9 is a diagram illustrating a third process of manufacturing a DNA chip substrate.

FIG. 10 is a diagram illustrating an example of a configuration of a biochemical reaction detection device.

FIG. 11 is a diagram showing a binding mode between adenine (A) and thymine (T) and a binding mode between guanine (G) and cytosine (C). A dotted line connecting between O in the molecular structure and H in the molecular structure represents a hydrogen bond.

FIG. 12 is a diagram for explaining a phenomenon in which the Tm value of complementary strand binding varies depending on the type of a probe. FIG.
FIG. 12B shows that the binding force between the probe immobilized on the NA chip substrate and the polynucleotide in the sample solution differs depending on the probe sequence, and FIG. 12B shows a melting curve of complementary strand binding. The vertical axis is the degree of dissociation of the DNA double strand, and the horizontal axis is the temperature. FIG. 12 (C) shows the Tm of the 8-base probe in%.
It is a figure showing the result computed by the GC method.

FIG. 13 is a schematic view of a DNA chip viewed from above.

FIG. 14 is a diagram schematically showing the temperature transition of the fixed surface belonging to the columns a, c, g, and i of the DNA chip.

FIG. 15 is a diagram showing the temperature dependence of hybridization in the measurement using the sample DNA of SEQ ID NO: 1.

FIG. 16 is a diagram showing the temperature dependence of hybridization in the measurement using the sample DNA of SEQ ID NO: 6.

FIG. 17 is a diagram showing the temperature dependence of hybridization in the measurement using the sample DNA of SEQ ID NO: 7.

FIG. 18 is a diagram illustrating a shape example of a heater circuit and a temperature detection element on a probe fixing surface.

19 is a graph showing the relationship between the temperature (t) of the probe fixing surface and the potential difference (v) between the temperature detection elements in the temperature detection circuit of FIG.

FIG. 20 is a diagram showing a voltage applied to a working electrode and a heating timing by a heater.

[Explanation of symbols]

1 is a DNA chip substrate, 2 is a probe fixing surface, 4 is an island, 5 is a heater circuit, 6 is a pn-junction temperature detecting element, 1001 is a heater terminal (+), and 1002 is a heater terminal.
(-), 1003 is temperature detection terminal (+), 1004 is temperature detection terminal
(-), 21 is a Si island, 22 is SiN / SiO ₂
Thin film, 24 is a temperature setting area, 25 is a probe, 26 is a sample solution, 27 is a cover glass, 41 is a mesh structure (heat outflow device), 51 is a water layer, 52 is an acrylic plate, 61
Is a metal frame, 71 is an n-type substrate, 72 is a p-well, 7
3 is a p-well, 74 is an SiO ₂ film, 75 is an n-type diffusion layer, 76 is an n-type diffusion layer, 77 is an n-type diffusion layer, 78 is a p-type diffusion layer, 79 is a p-type diffusion layer, and 81 is a p-type diffusion layer. 1 interlayer insulating film, 8
2 is a first layer wiring, 83 is a second interlayer insulating film, 84 is a second layer wiring, 91 is a Si ₃ N ₄ film, 101 is a DNA chip,
2 is a printed circuit board, 103 is a holder, 104 is a cable, 105 is a controller, 106 is a thermostat, 107 is a fan, 108 is a Peltier element, 109 is a switch,
10 is a voltmeter, 111 is an output controller, Vpo is a heater power supply, Vc is a constant voltage power supply, 181 is a heater power supply circuit, 1
82 is a temperature detection circuit, 801 is a sensor common wiring, 80
2 is a positive electrode of the PN junction temperature sensor, 803 is an electrode, 804
Is an electrode, 805 is a sensor common electrode, and 806 is a heater
The common electrode 901 is the side of the island.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Katsuji Murakawa 1-280 Higashi Koigakubo, Kokubunji-shi, Tokyo F-term in Central Research Laboratory, Hitachi, Ltd. 4B024 AA19 AA20 CA01 CA04 CA11 HA12 HA19 4B029 AA07 AA23 CC02 CC08 FA12

Claims (22)

[Claims] 1. An insulative thin film substrate, a plurality of islands provided on one surface of the thin film substrate, and a probe for detecting a biochemical reaction at a position opposed to the island with the thin film substrate interposed therebetween. A probe fixing surface, a heater for heating the island, a temperature detection unit for detecting the temperature of the island, and a temperature detection terminal for outputting the detection result of the temperature detection unit to the outside. A heater terminal for controlling the heating of the heater, wherein heating control of the island is performed from the outside through the heater terminal in accordance with an output result of the temperature detecting terminal. Chemical reaction detection chip. 2. The thin film substrate according to claim 1, wherein the thin film substrate is formed of at least one material selected from the group consisting of a Si nitride film, a Si oxide film, an Al oxide film, and Ta ₂ O _5. The biochemical reaction detection chip according to claim 1, wherein the chip is a composite membrane of a selected material. 3. The biochemical reaction detection chip according to claim 1, wherein the plurality of islands provided on the thin film substrate are independently temperature-controlled. 4. The heater includes an n-type diffusion layer, and the temperature detection unit includes a p-type diffusion layer and a p-type diffusion layer.
2. The semiconductor device according to claim 1, wherein said n-type junction is formed.
Biochemical reaction detection chip. 5. The biochemical reaction detection chip according to claim 1, wherein the probe fixing surface is depressed in a direction of a corresponding island. 6. The biochemical reaction detection chip according to claim 1, wherein when the thin film substrate is an n-type substrate, a p-type diffusion layer is provided between the heater and the temperature detection unit. 7. A probe having a plurality of islands whose temperature can be individually controlled on lattice points on one surface of a substrate, and a probe fixing surface for fixing a probe at a position opposite to the island on the other surface of the substrate. On each of the fixed surfaces, a biochemical reaction detection chip to which a probe is fixed, in which the Tm of the probe is increased from high to low in order from the center of the chip to the outside or from one side of the chip to the other side. A biochemical reaction detection chip characterized by being placed facing away. 8. The method according to claim 8, wherein the probe is an oligonucleotide D
The biochemical reaction detection chip according to any one of claims 1 to 7, wherein the chip is selected from the group consisting of an NA probe, an oligonucleotide RNA probe, and a protein probe. 9. The biochemical reaction detection chip according to claim 1, wherein a heat conductor is provided between the adjacent islands. 10. The biochemical reaction detection chip according to claim 1, wherein a metal frame is provided on an outer periphery of the thin film substrate. 11. A method for producing a substrate for a biochemical reaction detection chip, comprising: (a) forming a thin film on one surface of a flat plate of a heat conductor; and (b) forming a thin film on the flat plate of a heat conductor. A method of removing unnecessary portions from the other surface side to form thermal conductive islands. 12. The method according to claim 11, wherein a temperature controller is formed on one surface of the flat plate of the heat conductor, and a thin film is formed thereon. 13. A mask having a desired pattern is provided on a surface opposite to a surface on which a flat thin film of a heat conductor is formed,
13. The method of claim 12, wherein the masked side is etched until the thin film formed on the opposite side is exposed, thereby forming thermal conductor islands in the pattern of the mask on the thin film. 14. The method according to claim 13, wherein the mask is a thin film of a Si nitride film. 15. An apparatus for performing a biochemical reaction by a plurality of reaction systems on a biochemical reaction detection chip, wherein the heating device heats the entire biochemical reaction detection chip to a temperature higher than a temperature suitable for each biochemical reaction. A biochemical reaction device comprising: a device; and a temperature control device that controls the temperature of each reaction system to a temperature suitable for each biochemical reaction. 16. The biochemical reaction device according to claim 15, wherein the temperature control device adjusts the temperature of each reaction system in a time sharing manner. 17. An apparatus for performing a biochemical reaction on a biochemical reaction detection chip by a plurality of reaction systems on a computer, wherein the entire biochemical reaction detection chip is heated to a temperature higher than a temperature suitable for each biochemical reaction. A computer-readable recording medium storing a program for causing a biochemical reaction device to include a heating device for heating and a temperature control device for adjusting the temperature of each reaction system to a temperature suitable for each biochemical reaction. 18. A method for simultaneously performing a biochemical reaction with a plurality of reaction systems by controlling the temperature for each reaction system, wherein (a) the temperature is higher than a temperature suitable for the biochemical reaction of each reaction system. Heating all of the plurality of reaction systems, and (b) for each reaction system, lowering the temperature of the reaction system to a temperature suitable for a biochemical reaction and maintaining the temperature for a certain period of time. 19. The method according to claim 17, wherein the heating operation (a) is performed in a constant temperature bath. 20. The biochemical reaction is a hybridization between a polynucleotide and an oligonucleotide, and a temperature suitable for the biochemical reaction is a melting temperature of a duplex formed by the oligonucleotide and a complementary strand thereof. 18
The described method. 21. The method according to claim 19, wherein the polynucleotide is DN in the sample.
20. The method according to claim 19, wherein A is the oligonucleotide probe of the biochemical reaction detection chip. 22. A method in which a computer performs a biochemical reaction simultaneously by adjusting the temperature of each reaction system by a plurality of reaction systems, wherein (a) a temperature higher than a temperature suitable for the biochemical reaction of each reaction system. Until all the reaction systems are heated, and (b) for each reaction system, reduce the temperature of the reaction system to a temperature suitable for biochemical reactions and maintain the temperature for a certain period of time A computer-readable recording medium on which a program for causing a computer to execute is recorded.