JP2008026017A - Method and apparatus for detecting hybridization - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a hybridization detection method capable of expanding the dynamic range of a measurable current value to perform precise detection. <P>SOLUTION: In the detection method of a current of hybridization in a plurality of a microreaction chips to which a probe for capturing a physiologically related substance is fixed, a current detection circuit 11 is constituted of an integration circuit 12 and a plurality of integration condensers 122a-122d mutually different in volume are added to the integration circuit 12. The integration condenser of volume necessary for measuring precision and a measuring range is selected from a plurality of the integration condensers 122a-122d by selection switches 123a-123d to measure a current value. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method and apparatus for detecting hybridization of a biological substance such as DNA (deoxyribonucleic acid) using, for example, a micro reaction chip.

In recent years, technology development for efficiently analyzing various gene functions of living organisms has been promoted. For the analysis of gene expression and nucleotide sequences, for example, microreactions such as DNA chips and DNA microarrays. A chip is used. This micro reaction chip consists of a solid substrate on which different probe nucleic acids are arranged and fixed at a high density. A single-stranded target nucleic acid prepared separately is supplied to the probe nucleic acid, and the sequence of the probe nucleic acid is adjusted. By detecting the presence or absence of binding due to complementarity, the gene expression and base sequence of the target nucleic acid are analyzed.
Here, as a method for detecting the hybridization between the probe nucleic acid fragment on the DNA chip and the target nucleic acid fragment, a double-stranded nucleic acid fragment label that specifically binds to the double-stranded nucleic acid fragment and is electrochemically active. There are a method of detecting using a body and a method of detecting hybridization with a target nucleic acid by electrochemical measurement by labeling an oxidoreductase.

  As a method for arranging and fixing such probe nucleic acid, a method of binding and fixing a probe nucleic acid fragment prepared in advance on the surface of an electrode and a method of directly synthesizing an oligonucleotide on the surface of the electrode are known. As a method of directly synthesizing an oligonucleotide on the surface of an electrode, a combination of the use of a protective group that is selectively removed by light irradiation, a photolithography technique and a solid-phase synthesis technique used in semiconductor manufacturing, A method of selectively synthesizing oligonucleotides in a matrix region is a typical method, but a method of selectively synthesizing oligonucleotides on the surface of an electrode by an electrochemical reaction has also been proposed (for example, Patent Document 1). reference).

As shown in FIGS. 6 (A) and 6 (B), such a DNA chip is provided with a probe-immobilized electrode 52 provided at a plurality of locations on the surface of the insulating substrate 51 to which probe nucleic acid fragments are bound and fixed. Each immobilization electrode 52 is provided with a counter electrode 53 arranged to face each other with a probe nucleic acid fragment interposed therebetween. Each probe-immobilized electrode 52 is connected to, for example, a CMOS (Complimentary Metal Oxide Semiconductor) circuit that amplifies the current detected through the probe-immobilized electrode 52. Then, a signal current flowing between the probe fixed electrode 52 and the counter electrode 53 and a potential difference between the probe fixed electrode 52 and the counter electrode 53 are measured.
Here, in order to detect hybridization using the above-described oxidoreductase as a label, an oxidation-reduction potential of 50 mV to 500 mV is applied between the probe-immobilized electrode 52 and the counter electrode 53, depending on the substrate used. Has been. The interval between the respective fixed electrodes 52 is, for example, 0.05 mm to 0.5 mm, and the distance between the probe fixed electrode 52 and the counter electrode 53 is, for example, 0.05 to 1.0 mm. .

The following problems remain in the conventional hybridization detection methods as described above. That is, in order to increase the measurement accuracy, a plurality of electrodes to which the same probe is fixed are provided, but the dynamic range of the measurable current value is insufficient.
Conventionally, as a method for obtaining a dynamic range, a method of preparing several types of electrode areas for fixing a probe has been proposed (see, for example, Patent Document 2).
Special Table 2000-514802 JP 2004-309462 A

  However, in the conventional method as described above, the hybridization efficiency varies depending on the area of the probe-immobilized electrode on which the probe is immobilized, and there is an array in which the current does not increase even when the area is increased. There was found. Therefore, there is a problem in terms of measurement accuracy with probe-immobilized electrodes having different areas of probe-immobilized electrodes. Further, when about four types of probes having large to small probe-immobilized electrodes are prepared, there is a problem that the arrangement area becomes large and the chip size cannot be reduced.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a hybridization detection method and apparatus capable of expanding the dynamic range of measurable current values and performing accurate detection.

  In order to achieve the above object, the present invention provides a plurality of probe-immobilized electrodes to which probes having nucleic acids complementary to a target nucleic acid are immobilized, and is arranged on each probe-immobilized electrode via the probes. A method for detecting hybridization between a nucleic acid of a probe and a target nucleic acid by applying a voltage between the probe-immobilized electrode and the counter electrode, having a microreaction chip provided with a counter electrode formed And a detection circuit for detecting a current flowing due to a hybridization reaction between the nucleic acid of the probe and the target nucleic acid, wherein the detection circuit obtains an output signal proportional to the time integral of the current And the integration circuit includes an operational amplifier having one input terminal connected to the probe-immobilized electrode; A plurality of integrating capacitors having different capacities connected in parallel via separate selection switches between one input end and an output end of the operational amplifier, and measuring accuracy from among the plurality of integrating capacitors The integration capacitor having the capacity required for the measurement range is selected by the selection switch, and the integration output of the current is measured with the selected integration capacitor connected between one input terminal and the output terminal of the operational amplifier. It is characterized by doing.

  The present invention also provides a plurality of probe-immobilized electrodes to which probes having nucleic acids complementary to the target nucleic acid are immobilized, and counter electrodes arranged on the probe-immobilized electrodes via the probes. A method for detecting hybridization between a nucleic acid of the probe and the target nucleic acid by applying a voltage between the probe-immobilized electrode and the counter electrode, the microreaction chip provided; A detection circuit for detecting a current flowing due to a hybridization reaction between a nucleic acid of a probe and the target nucleic acid, the detection circuit having an integration circuit for obtaining an output signal proportional to a time integral of the current; The integration circuit includes an operational amplifier in which one input terminal is connected to the probe fixed electrode, and one of the operational amplifiers. An integration capacitor connected in parallel between the input terminal and the output terminal, and an integration time control means for setting the integration time of the integration capacitor due to the current to a plurality of different times, and measuring accuracy and measurement The integral time required for the range is set by the integral time control means, and the integral output of the current is measured.

  The present invention also provides a plurality of probe-immobilized electrodes to which probes having nucleic acids complementary to the target nucleic acid are immobilized, and counter electrodes arranged on the probe-immobilized electrodes via the probes. An apparatus for detecting hybridization between a nucleic acid of the probe and the target nucleic acid by applying a voltage between the probe-immobilized electrode and the counter electrode, the microreaction chip provided; A detection circuit that detects a current that flows due to a hybridization reaction between a nucleic acid of a probe and the target nucleic acid, and the detection circuit includes an integration circuit that obtains an output signal proportional to a time integral of the current; The circuit includes an operational amplifier having one input terminal connected to the probe-immobilized electrode, and one of the operational amplifiers. And having different and a plurality of integrating capacitors to one another capacitor connected in parallel via separate selection switch between the input terminal and the output terminal.

  The present invention also provides a plurality of probe-immobilized electrodes to which probes having nucleic acids complementary to the target nucleic acid are immobilized, and counter electrodes arranged on the probe-immobilized electrodes via the probes. An apparatus for detecting hybridization between a nucleic acid of the probe and the target nucleic acid by applying a voltage between the probe-immobilized electrode and the counter electrode, the microreaction chip provided; A detection circuit that detects a current that flows due to a hybridization reaction between a nucleic acid of a probe and the target nucleic acid, the detection circuit having an integration circuit that obtains an output signal proportional to a time integral of the current; The circuit includes an operational amplifier having one input terminal connected to the probe-immobilized electrode, and one of the operational amplifiers. An integration capacitor connected in parallel between the input terminal and the output terminal of the first and second output terminals, and an integration time control means for setting the integration time of the integration capacitor by the current to a plurality of different times. .

  According to the hybridization detection method and apparatus of the present invention, a plurality of integration capacitors having different capacities are added to the integration circuit constituting the current detection circuit connected to the probe-immobilized electrode, and the plurality of integration capacitors are selected from the plurality of integration capacitors. Since the integration capacitor of the capacity required for measurement accuracy and measurement range is selected by the selection switch and the integrated output of the current is measured, the measurement current value overlaps the capacity of each integration capacitor and the measurement range is expanded. Thus, the measurement accuracy can be improved and the measurement range can be expanded.

  According to the hybridization detection method and apparatus of the present invention, the time for charging the integration capacitor of the integration circuit constituting the current detection circuit connected to the probe fixed electrode is set to a plurality of different times by the integration time control means. Since the integration time control means sets the integration time required for the measurement accuracy and measurement range and measures the integral output of the current, the measurement current values overlap and the measurement range is The charging time can be extended, thereby improving the measurement accuracy and extending the measurement range.

(First embodiment)
Embodiments of a hybridization detection method and apparatus according to the present invention will be described below with reference to the drawings. The hybridization detection method and apparatus according to the present invention are not limited to the embodiments described below.

FIG. 1A is a schematic diagram of a DNA chip (microreaction chip) provided with a hybridization detection apparatus according to the present invention, and FIG. 1B is a schematic configuration diagram of a hybridization detection unit in the DNA chip (microreaction chip). It is.
As shown in FIG. 1A, the DNA chip 1 includes a substrate 1a such as a glass substrate or a ceramic substrate, and a detection array portion 1b formed by aligning single-stranded DNA fragments on the substrate 1a. Have. As shown in FIG. 1 (B), the detection array section 1b is connected to a detection probe-immobilized electrode 23 and the probe-immobilized electrode 23 via a wiring 21, and hybridizes a probe nucleic acid and a target nucleic acid. And a detection circuit 11 for detecting a current caused by the desorption.

The configuration of the detection circuit 11 will be described with reference to FIG.
In FIG. 2, a detection circuit 11 detects a current that flows due to a hybridization reaction between a probe nucleic acid and a target nucleic acid that is a sample, and includes an integration circuit 12 that obtains an output signal proportional to the time integration of the current. Have. The integrating circuit 12 includes an operational amplifier 121 having one input terminal connected to the probe-immobilized electrode 23 and a parallel connection between one input terminal 121a and the output terminal 121c of the operational amplifier 121. A plurality of integrating capacitors 122a to 122d having different capacities, and each of the integrating capacitors 122a to 122d are connected in series, and one of the integrating capacitors 122a to 122d is connected to one input terminal 121a and an output terminal 121c of the operational amplifier 121. A plurality of selection switches 123a to 123d that are selectively connected to each other, a reset switch 124 that discharges charges accumulated in the integration capacitors 122a to 122d and resets the integration capacitors 122a to 122d, and a probe-immobilized electrode 23 and one input terminal 121a of the operational amplifier 121 They are connected in series, and a integration time control switch 125 for setting the integration time of the integration capacitor 122a to 122d.

The capacitances of the integrating capacitors 122a to 122d are, for example, “C” for the integrating capacitor 122a, “2C” for the integrating capacitor 122b, “5C” for the integrating capacitor 122c, and “5C” for the integrating capacitor 122d. 10C "is set to a value divided into a plurality of stages from a small capacity to a large capacity. Further, the integration capacitors 122a to 122d are selected and selected to have the capacity required for the measurement accuracy and the measurement range by turning on one of the selection switches 123a to 123d separately connected thereto. The integrating capacitor is connected between one input terminal 121a and the output terminal 121c of the operational amplifier, and the integrated output of the current is measured in this state.
The selection switches 123a to 123d, the reset switch 124, and the integration time control switch 125 are turned on / off by an external signal. Further, a reference power supply is supplied to the other input terminal 121 b of the operational amplifier 121. A voltage (integrated output) proportional to the current is output from the output terminal 121c of the operational amplifier 121 as a result of integration of the current value and current-voltage conversion.

3A is an enlarged plan view showing a part of the DNA chip including the probe-immobilized electrode, and FIG. 3B is a cross-sectional view taken along the line BB in FIG. 3A.
As shown in FIG. 3, the DNA chip 1 has a silicon substrate 15 as a base, and a CMOS transistor for constituting an operational amplifier 121 of the integration circuit 12 in the detection circuit 11 is formed on the upper surface region of the silicon substrate 15. In addition, a MOS type capacitance element that constitutes the integration capacitors 122a to 122d, and a MOS type switching element that constitutes the selection switches 123a to 123d, the reset switch 124, and the integration time control switch 125 are formed by a standard CMOS process. ing. An input / output electrode 16 for connecting the detection circuit 11 and the outside is formed on the upper surface of the silicon substrate 15. The upper surface of the silicon substrate 15 including the input / output electrode 16 is covered with a protective layer 17. Yes.

The detection circuit 11 independently applies a voltage to the probe fixing electrode 23 and the surrounding electrode 24 for detection, and amplifies a current detected on each fixed surface 23a described later to a potential measurement unit (not shown). It is configured to output to.
The input / output electrode 16 is made of a conductor such as Al (aluminum), for example, and is exposed to the outside from a contact hole 17 a formed in the protective layer 17. The protective layer 17 has a film thickness of 2 μm, for example, and is formed of an insulator such as SiN (silicon nitride) / SiO ₂ (silicon dioxide).

The detection electrode unit 12 including the probe-immobilized electrode 23 and the surrounding electrode 24 includes a connection electrode unit 21 connected to the input / output electrode 16, a first insulating layer 22 sealing the connection electrode unit 21, and the first insulation. A probe-immobilized electrode 23 and an surrounding electrode portion 24 formed on the layer 22 and connected to the connection electrode portion 21; a probe-immobilized electrode 23, the surrounding electrode 24, and a second insulating layer 25 covering the first insulating layer 22; It has.
The connection electrode portion 21 is formed of, for example, Al (aluminum), fills the contact hole 17a, and a part thereof is laminated on the upper surface of the protective layer 17 in the vicinity of the contact hole 17a. The connection electrode portion 21 is formed to have a thickness of, for example, 3 μm so as to fill the contact hole 17a, and is then flattened so that the protruding amount from the upper surface of the protective layer 17 is 0.5 μm or less. .

The first insulating layer 22 is made of an insulator such as SiN or SiO ₂ and is formed so as to cover the connection electrode portion 21. The first insulating layer 22 is flattened on the upper surface of the protective layer 17 or the connection electrode portion 21 using, for example, a plasma CVD (Chemical Vapor Deposition) method so as to have a thickness of 0.5 μm, for example. Formed.
In addition, the first insulating layer 22 is provided with a contact hole 22a that reaches the connection electrode portion 21 at a position that does not coincide with the contact hole 17a formed in the protective layer 17 in plan view. The contact hole 22a is formed by dry etching using a mask pattern having a predetermined opening shape.

The probe-immobilized electrode 23 is made of, for example, Pt (platinum) and has a substantially circular shape in plan view. A part of the probe-immobilized electrode 23 is connected to the connection electrode portion 21 by being formed in the contact hole 22a. The probe-immobilized electrode 23 is formed on the first insulating layer 22 with a thickness of, for example, 0.2 to 0.3 μm by a sputtering method using a mask pattern having a predetermined opening shape. The probe-immobilized electrode 23 is formed with a fixing surface 23a that is exposed to the outside through a through hole 25a formed in the second insulating layer 25 and on which the probe nucleic acid is immobilized. The fixed surface 23a is substantially circular in a plan view, and the diameter is, for example, 10 μm to 100 μm.
Note that a plurality of fixing surfaces 23a of the probe fixing electrode 23 are provided at equal intervals in two directions on the first insulating layer 22, and a microarray is formed by forming a plurality of fixing surfaces 23a.

The second insulating layer 25 is made of, for example, SiN, and a circular through hole 25a is formed at a position that overlaps the probe fixed electrode 23 and does not coincide with the contact hole 22a in a plan view. The second insulating layer 25 is formed with an annular through hole 25b at a position that overlaps with the surrounding electrode 24 in a plan view and does not coincide with the contact hole 22a. By this through hole 25b, the exposed surface 24a is configured to surround the periphery of the fixed surface 23a.
The second insulating layer 25 is formed on the probe fixed electrode 23 or the first insulating layer 22 to a thickness of, for example, 0.7 μm to 0.9 μm using a plasma CVD method. The through holes 25a and 25b are formed by dry etching using a mask pattern having a predetermined opening shape.

In addition, the voltage applied at the time of actual hybridization is a voltage applied between the voltage of the electrode 23 and the counter electrode 33 installed facing it.
The probe nucleic acid is a nucleic acid that captures a target nucleic acid that is an ecologically related substance on the surface, and is bound and fixed to the fixing surface 23a by electrochemical synthesis. Here, the same type of probe nucleic acid is bonded and fixed to one fixed surface 23a, and various probe nucleic acids are bonded and fixed to each of the plurality of fixed surfaces 23a.
In the present embodiment, “nucleic acid” refers to DNA, RNA (ribonucleic acid), nucleic acid analogues, and the like represented by general base sequences, and such nucleic acids are naturally present. It may be present or artificially synthesized or modified.

A method for detecting hybridization using the DNA chip 1 configured as described above will be described. In the present embodiment, the DNA chip 1 has a fixing surface 23a of 11 rows × 30 columns.
When detecting hybridization in the present embodiment, an integration capacitor having a capacity required for measurement accuracy and measurement range is selected from a plurality of integration capacitors 122a to 122d, and a corresponding selection switch is turned on by an external signal. The selected integration capacitor is connected between one input terminal 121a and the output terminal 121c of the operational amplifier 121. Further, the ON time of the integration time control switch 125, that is, the integration time is set by an external signal. Then, a probe that captures a biological substance is fixed to the fixed surface 23a of the probe-immobilized electrode 23, and a sample solution that is a target nucleic acid that is complementary to the probe nucleic acid is poured into the space 40 sandwiched between the counter electrodes. In this state, a predetermined voltage is applied between the probe-immobilized electrode 23 and the counter electrode 33. As a result, a voltage (integrated output) proportional to the current flowing due to the hybridization reaction between the probe nucleic acid and the target nucleic acid is output from the output terminal 121c of the operational amplifier 121 as a result of the integration of the current value and the current-voltage conversion. Is output.

  Therefore, in this embodiment, the capacities of the integrating capacitors 22a to 122d of the current detection circuit connected to the electrodes of the plurality of fixed surfaces 23a are different from each other. The capacity can be expanded. Therefore, for example, if the measurement range covered by the integration capacitor 122a having the capacity “C” is 0 to 30, the measurement range covered by the integration capacitor 122b having the capacity “2C” is 20 to 50, and the capacity “5C” is set. The measurement range covered by the integration capacitor 122c is 40 to 70, and the measurement range covered by the integration capacitor 122d having the capacity “10C” is 60 to 90.

In this way, the amount of overlap of each integrating capacitor can be varied depending on the required measurement accuracy and the required measurement range, and when measurement accuracy is required. For example, if the measurement range covered by the integration capacitor 122a is 0 to 30, the measurement range covered by the integration capacitor 122b is 15 to 45, the measurement range covered by the integration capacitor 122c is 30 to 60, and the integration capacitor The accuracy can be increased by increasing the overlap so that the measurement range covered by 122d is 45 to 75.
When a measurement range is required, for example, the measurement range covered by the integration capacitor 122a is 0 to 30, the measurement range covered by the integration capacitor 122b is 25 to 55, and the integration capacitor 122c is covered. The measurement range to be performed is 50 to 80, and the measurement range covered by the integrating capacitor 122d is 75 to 105. Thereby, a measurement range can be expanded.
The present invention is not limited to the above numerical values.

(Second Embodiment)
Next, another embodiment of the hybridization detection apparatus according to the present invention will be described with reference to FIG.
FIG. 4 shows an example of the configuration of a detection circuit 41 in another embodiment. This detection circuit 41 detects a current that flows due to a hybridization reaction between a probe nucleic acid and a target nucleic acid that is a sample. And an integration circuit 42 for obtaining an output signal proportional to the time integration of the current. This integrating circuit 42 is connected in parallel between an operational amplifier 421 in which one input terminal 421a is connected to the probe fixing electrode 23, and one input terminal 421a and output terminal 421c of this operational amplifier 421. An integration capacitor 422, a reset switch 423 for discharging the charge accumulated in the integration capacitor 422 to reset the integration capacitor 422, and the probe fixing electrode 23 and one input terminal 421a of the operational amplifier 421 are connected in series. And an integration time control switch 424 that sets the integration time of the integration capacitor 422 from a small value to a large value in a plurality of stages.
Further, a reference power is supplied to the other input terminal 421b of the operational amplifier 421. Further, a voltage (integrated output) proportional to the current is output from the output terminal 421c of the operational amplifier 421 as a result of integration of the current value and current-voltage conversion.

An example in which the integration time (charging time) of the integrating capacitor is set in a plurality of stages will be described with reference to FIG.
In FIG. 5, the integration time of the integration circuit applied to the detection circuit of the DNA chip 1A is set to “T”, the integration time of the integration circuit applied to the detection circuit of the DNA chip 1B is set to “2T”, The integration time of the integration circuit applied to the detection circuit of the DNA chip 1C is set to “5T”, and the integration time of the integration circuit applied to the detection circuit of the DNA chip 1D is set to “15T”.

  In such another embodiment, the integration time of the current charged in the integration capacitor of the current detection circuit 41 connected to the fixed surface of the plurality of probe fixing electrodes 23 is different as shown in FIG. Each integration time can be an integration time in which the measurement current values overlap and the measurement range is expanded. Thus, for example, if the measurement range covered by the integration time “T” is 0 to 30, the measurement range covered by the integration time “2T” is 20 to 50 and the measurement range is covered by the integration time “5T”. 40 to 70, and the measurement range covered by the integration time “15T” is 60 to 90.

The amount of measurement range overlap can vary depending on the accuracy of the measurement required for the measurement and the size of the measurement range required. If the measurement range covered by time “T” is 0 to 30, the measurement range covered by integration time “2T” is 15 to 45, the measurement range covered by integration time “5T” is 30 to 60, and integration The accuracy can be increased by increasing the overlap so that the measurement range covered by time “5T” is 45 to 75.
When a measurement range is required, for example, the measurement range covered by the integration time “T” is set from 0 to 30, and the measurement range covered by the integration time “2T” is set from 25 to 55. The measurement range covered by time “5T” is 50 to 80, and the measurement range covered by integration time “15T” is 75 to 105.
As a result, the measurement accuracy can be improved and the measurement range can be expanded. In addition, this invention is not limited to the numerical value mentioned above.
Then, gene expression and base sequence of the probe nucleic acid immobilized on the immobilization surface 23a of the probe immobilization electrode 23 are analyzed from the supplied target nucleic acid and the acquired electric signal.
As described above, according to the hybridization detection method of the present embodiment, current measurement with a wide dynamic range is possible and measurement can be performed with high accuracy.

In addition, this invention is not limited to the said embodiment, A various change can be added in the range which does not deviate from the meaning of this invention.
Further, one end of the probe nucleic acid is directly immobilized on the immobilization surface 23a, but it may be immobilized via a linker or via a polymer network. Here, examples of the linker include aryl acetylene, ethylene glycol oligomer having 2 to 10 monomer units, diamine, diacid, amino acid, or a combination thereof.
Moreover, although the target nucleic acid is applied as a biological substance to be captured by the probe nucleic acid, other biological substances such as proteins may be captured. Furthermore, other biological materials such as proteins may be applied as probes.

Moreover, although the fixed surface 23a has a circular shape in plan view, it may have another shape such as a rectangle.
Moreover, although the exposed surface 24a is formed in the position spaced apart from the periphery of the fixed surface 23a at equal intervals, you may change suitably according to design.
Alternatively, the surrounding electrode portions 24 may be electrically connected to have the same potential.
Further, one probe-immobilized electrode 23 and the surrounding electrode portion 24 are connected to the connection electrode portion 21 in the contact hole 22a. However, the probe-immobilized electrode 23 and the surrounding electrode portion 24 are directly sputtered on the connection electrode portion 21. It is good also as a structure formed by these. Moreover, it is good also as a structure which does not provide the surrounding electrode part 24. FIG.
Further, the voltage applied to each probe fixed electrode 23 is controlled by the detection circuit 11. However, the detection circuit 11 is not limited to one manufactured from a CMOS process, and other semiconductor devices may be used to control each probe fixed electrode 23. Voltage control may be performed.

(A) is a schematic diagram of a DNA chip provided with a hybridization detection apparatus according to the present invention, and (B) is a schematic configuration diagram of a hybridization detection unit in the DNA chip. It is a block diagram of the detection circuit in one embodiment of the present invention. (A) is a top view which expands and shows a part of DNA chip containing a probe fixed electrode, (B) is sectional drawing which follows the BB line of (A). It is a block diagram of the detection circuit in other embodiment of this invention. It is explanatory drawing which shows the example of a setting of the integration time of the integration circuit in other embodiment of this invention. (A) is a schematic plan view which shows the conventional micro reaction chip, (B) is a schematic sectional drawing which follows the CC line of (A).

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... DNA chip, 1a ... Board | substrate, 1b ... Detection array part, 11 ... Detection circuit, 12 ... Integration circuit, 121 ... Operational amplifier, 122a-122d ... Integration capacitor, 123a-123d ... Selection switch 124... Reset switch 125. Integration time control switch 23... Probe immobilization electrode 23 a... Fixed surface 41... Detection circuit 42. ... Integral capacitor, 423 ... Reset switch, 424 ... Integral time control switch.

Claims (10)

A micro reaction chip in which a plurality of probe-immobilized electrodes to which probes having nucleic acids complementary to a target nucleic acid are immobilized are provided, and each of the probe-immobilized electrodes is provided with a counter electrode arranged via the probe A method of detecting hybridization between the nucleic acid of the probe and the target nucleic acid by applying a voltage between the probe-immobilized electrode and the counter electrode,
A detection circuit for detecting a current flowing due to a hybridization reaction between the nucleic acid of the probe and the target nucleic acid;
The detection circuit has an integration circuit for obtaining an output signal proportional to the time integration of the current,
The integrating circuit is connected in parallel via an operational amplifier in which one input terminal is connected to the probe fixing electrode, and a separate selection switch between one input terminal and the output terminal of the operational amplifier. A plurality of integrating capacitors having different capacities,
An integration capacitor having a capacity required for measurement accuracy and measurement range is selected from the plurality of integration capacitors by the selection switch, and the selected integration capacitor is placed between one input terminal and an output terminal of the operational amplifier. A method for detecting hybridization, comprising measuring an integrated output of the current in a connected state.   2. The hybridization detection method according to claim 1, wherein the plurality of integrating capacitors are composed of a plurality of capacitors divided into a plurality of stages from a small capacitance value to a large capacitance value.   2. The hybridization detection method according to claim 1, further comprising integration time control means for setting an integration time of the integration capacitor by the current. A micro reaction chip in which a plurality of probe-immobilized electrodes to which probes having nucleic acids complementary to a target nucleic acid are immobilized are provided, and each of the probe-immobilized electrodes is provided with a counter electrode arranged via the probe A method of detecting hybridization between the nucleic acid of the probe and the target nucleic acid by applying a voltage between the probe-immobilized electrode and the counter electrode,
A detection circuit for detecting a current flowing due to a hybridization reaction between the nucleic acid of the probe and the target nucleic acid;
The detection circuit has an integration circuit for obtaining an output signal proportional to the time integration of the current,
The integration circuit has an operational amplifier in which one input terminal is connected to the probe fixing electrode, and an integration capacitor connected in parallel between one input terminal and an output terminal of the operational amplifier,
Integration time control means for setting the integration time of the integration capacitor by the current to a plurality of different times;
A method for detecting hybridization, wherein an integration time necessary for measurement accuracy and a measurement range is set by the integration time control means, and an integrated output of the current is measured.   4. The hybridization detection method according to claim 3, wherein the plurality of integration times set by the integration time control means are times that change in a plurality of steps from a small value to a large value. A micro reaction chip in which a plurality of probe-immobilized electrodes to which probes having nucleic acids complementary to a target nucleic acid are immobilized are provided, and each of the probe-immobilized electrodes is provided with a counter electrode arranged via the probe An apparatus for detecting hybridization between the nucleic acid of the probe and the target nucleic acid by applying a voltage between the probe-immobilized electrode and the counter electrode,
A detection circuit for detecting a current flowing due to a hybridization reaction between the nucleic acid of the probe and the target nucleic acid;
The detection circuit has an integration circuit for obtaining an output signal proportional to the time integration of the current,
The integrating circuit is connected in parallel via an operational amplifier in which one input terminal is connected to the probe fixing electrode, and a separate selection switch between one input terminal and the output terminal of the operational amplifier. A plurality of integrating capacitors having different capacities,
A hybridization detection apparatus characterized by the above.   7. The hybridization detection apparatus according to claim 6, wherein the plurality of integrating capacitors are composed of a plurality of capacitors divided into a plurality of stages from a small capacitance value to a large capacitance value.   7. The hybridization detection apparatus according to claim 6, further comprising integration time control means for setting an integration time of the integration capacitor by the current. A micro reaction chip in which a plurality of probe-immobilized electrodes to which probes having nucleic acids complementary to a target nucleic acid are immobilized are provided, and each of the probe-immobilized electrodes is provided with a counter electrode arranged via the probe An apparatus for detecting hybridization between the nucleic acid of the probe and the target nucleic acid by applying a voltage between the probe-immobilized electrode and the counter electrode,
A detection circuit for detecting a current flowing due to a hybridization reaction between the nucleic acid of the probe and the target nucleic acid;
The detection circuit has an integration circuit for obtaining an output signal proportional to the time integration of the current,
The integration circuit includes an operational amplifier in which one input terminal is connected to the probe fixing electrode, and an integration capacitor connected in parallel between one input terminal and an output terminal of the operational amplifier. ,
Integration time control means for setting the integration time of the integration capacitor by the current to a plurality of different times;
A hybridization detection apparatus characterized by the above. The hybridization detection apparatus according to claim 9, wherein the plurality of integration times set by the integration time control means are times that change in a plurality of steps from a small value to a large value.

Images