JP4018672B2 - Nucleic acid detection apparatus and nucleic acid detection method - Google Patents

Description

  The present invention relates to a nucleic acid detection apparatus and a nucleic acid detection method for detecting a target nucleic acid molecule contained in a specimen.

Conventionally, there has been a nucleic acid sensor for detecting a nucleic acid molecule that detects whether or not a target nucleic acid molecule is contained in a sample using a field-effect transistor (FET) (see, for example, Non-Patent Document 1). Similarly, there are nucleic acid sensors that detect the presence or absence of a nucleic acid molecule by measuring the magnitude of a redox current associated with oxidation of a double-stranded recognizer.
Toshiya Sakata et al., “Detection of DNA hybridization using gene transistors”, Proc. 1179 (2003)

  However, when the sensor opening area is reduced to improve sensitivity and integration, the redox current resulting from the chemical reaction on the sensor electrode decreases in proportion to the area. The limit point where the current is detected by the instrument may be reached, and the current may not be detected by the measuring instrument.

  In view of the prior art, the present invention provides a nucleic acid detection device and a nucleic acid capable of measuring a signal for capturing a chemical change occurring on the sensor when the sensor is miniaturized and detecting the nucleic acid with high accuracy. An object is to provide a detection method.

According to the nucleic acid detection device of the present invention, in a nucleic acid detection device that detects a target nucleic acid molecule having a specific sequence contained in a specimen based on the intensity of characteristic modulation of a MOSFET (metal-oxide semiconductor field-effect transistor). A sensor electrode in which a probe nucleic acid molecule capable of hybridizing with the target nucleic acid molecule is immobilized on an electrode connected to the gate electrode of the MOSFET or the gate electrode of the MOSFET, and the potential of the sensor electrode is controlled A control circuit having a counter electrode for generating a chemical reaction of a double-stranded recognizer on the sensor electrode, and whether the chemical reaction is generated by short-circuiting the gate electrode to a ground point or a reference voltage source. A switch for switching whether or not, a measuring means for measuring a physical quantity indicating the electrical characteristics of the MOSFET before and after the chemical reaction, It is characterized by comprising determination means for determining whether or not the difference in physical quantity indicating the electrical characteristics of the MOSFET generated before and after the chemical reaction is larger than a predetermined value.

  According to the nucleic acid detection method of the present invention, in the nucleic acid detection method for detecting a target nucleic acid molecule having a specific sequence contained in a specimen based on the intensity of characteristic modulation of a MOSFET (metal-oxide semiconductor field-effect transistor). The first physical quantity indicating the electrical characteristics of the MOSFET is measured, a sample containing the target nucleic acid molecule is introduced onto the sensor electrode on which the probe nucleic acid molecule is immobilized in advance, and the target nucleic acid molecule and the sensor electrode are A hybridization reaction is caused to the probe nucleic acid molecule, a reagent containing a double strand recognition body is introduced onto the sensor electrode, and a binding reaction between the double strand recognition body and the hybridized nucleic acid molecule is caused to occur. The potential of the electrode is controlled to cause a chemical reaction of the double-stranded recognizer, a second physical quantity indicating the electrical characteristics of the MOSFET is measured, and the first physical A difference between a quantity and the second physical quantity is calculated, and it is determined whether or not the magnitude of the difference is larger than a predetermined value.

  Moreover, according to the nucleic acid detection method of the present invention, nucleic acid detection for detecting a target nucleic acid molecule having a specific sequence contained in a specimen based on the intensity of characteristic modulation of a MOSFET (metal-oxide semiconductor field-effect transistor) In the method, a sample containing a target nucleic acid molecule is introduced onto a sensor electrode on which a probe nucleic acid molecule is immobilized in advance, and a hybridization reaction is caused between the target nucleic acid molecule and the probe nucleic acid molecule on the sensor electrode, whereby the MOSFET Measuring a first physical quantity exhibiting the electrical characteristics of, introducing a reagent containing a double-stranded recognizer on the sensor electrode, causing a binding reaction between the double-stranded recognizer and a hybridized nucleic acid molecule, The potential of the sensor electrode is controlled to cause a chemical reaction of a double-stranded recognizer, a second physical quantity indicating the electrical characteristics of the MOSFET is measured, and the first The difference between the physical quantity of the second and the second physical quantity is calculated, and it is determined whether or not the magnitude of the difference is larger than a predetermined value.

  Furthermore, according to the nucleic acid detection method of the present invention, nucleic acid detection for detecting a target nucleic acid molecule having a specific sequence contained in a specimen based on the intensity of characteristic modulation of a MOSFET (metal-oxide semiconductor field-effect transistor) In the method, a sample containing a target nucleic acid molecule is introduced onto a sensor electrode on which a probe nucleic acid molecule has been immobilized in advance, and a hybridization reaction is caused between the target nucleic acid molecule and the probe nucleic acid molecule on the sensor electrode, whereby the sensor Introducing a reagent containing a double-stranded recognizer onto the electrode, causing a binding reaction between the double-stranded recognizer and the hybridized nucleic acid molecule, and measuring a first physical quantity indicating the electrical characteristics of the MOSFET; Controlling the potential of the sensor electrode, causing a chemical reaction of a double-stranded recognizer, measuring a second physical quantity indicating the electrical characteristics of the MOSFET, The difference between the physical quantity of 1 and the second physical quantity is calculated, and it is determined whether or not the magnitude of the difference is larger than a predetermined value.

  Still further, according to the nucleic acid detection method of the present invention, a nucleic acid for detecting a target nucleic acid molecule having a specific sequence contained in a specimen based on the intensity of characteristic modulation of a metal-oxide semiconductor field-effect transistor (MOSFET). In the detection method, a sample containing a target nucleic acid molecule is introduced onto a sensor electrode on which a probe nucleic acid molecule is immobilized in advance, and a hybridization reaction is caused between the target nucleic acid molecule and the probe nucleic acid molecule on the sensor electrode. A reagent containing a double-stranded recognizer is introduced onto the sensor electrode, a binding reaction between the double-stranded recognizer and the hybridized nucleic acid molecule is caused, the potential of the sensor electrode is controlled, and the double-stranded recognizer A chemical reaction is caused, the first physical quantity indicating the electrical characteristics of the MOSFET is measured, the potential of the sensor electrode is controlled again, and the chemical reaction of the double-stranded recognizer is performed. Measure the second physical quantity indicating the electrical characteristics of the MOSFET, calculate the difference between the first physical quantity and the second physical quantity, and the magnitude of the difference is greater than a predetermined value It is characterized by determining whether it is large.

  According to the nucleic acid detection device and the nucleic acid detection method of the present invention, it is possible to measure a signal for capturing a chemical change occurring on the sensor when the sensor is miniaturized, and to detect the nucleic acid with high accuracy.

Hereinafter, a nucleic acid detection device and a nucleic acid detection method according to embodiments of the present invention will be described in detail with reference to the drawings.
As shown in FIG. 1, the nucleic acid detection apparatus of the present embodiment includes a nucleic acid detection FET 10, an amplification circuit 20 for amplifying the characteristics of the nucleic acid detection FET 10, and a control circuit 30 for controlling the potential of the sensor electrode WE. And a voltmeter 22 for measuring a voltage drop generated at the drain terminal of the nucleic acid detection FET. The nucleic acid detection FET 10 includes a sensor electrode WE11 as a gate electrode.
The amplification circuit 20 includes a signal amplification current source circuit 21, a substrate bias voltage source 23, a power supply Vdd24 set to a higher value than Vss, and a power supply Vss25 set to a value lower than Vdd. The control circuit 30 also includes a chemical reaction switch 31 on the sensor electrode, a counter electrode CE32, a reference electrode RE33, a chemical reaction tank 34 on the sensor electrode, operational amplifiers 35 and 36, a resistor 37, and a sweep voltage generation. A container 38 is provided. This control circuit 30 is a well-known circuit in the field of electrochemistry called a potentiostat circuit.
The signal amplification current source circuit 21 generates a reference current Ids necessary for reading the characteristics of the nucleic acid detection FET 10. The voltmeter 22 is for measuring a voltage drop generated at the drain terminal D of the nucleic acid detection FET 10. The substrate bias voltage source 23 is a voltage source applied to the substrate terminal B of the nucleic acid detection FET 10. The power supply Vdd24 is a voltage source applied to the drain terminal D of the nucleic acid detection FET 10, and this voltage value is set to be positive. The power supply Vss25 is a power supply applied to the source terminal S of the nucleic acid detection FET 10. The voltage value of the power supply Vss25 applied to the source terminal S may be smaller than the voltage value of the power supply Vdd24, and the voltage value of the power supply Vss25 may be set negative. The measurement of the output voltage value Vds of the amplifier circuit 20 by the voltmeter 22 including the function of an A / D converter (analog-to-digital converter) or a block having the equivalent function is performed twice, and is performed by a computer or the like. A determination is made as to whether or not a nucleic acid molecule has been detected by obtaining the difference between the measurements. Of course, this function may be realized on a chip. Further, by adjusting the reference current Ids and the voltage value generated by the substrate bias voltage source 23, the change in the output voltage value Vds measured by the voltmeter 22 can be increased. This adjustment will be described later with reference to FIG.
The amplifier circuit 20 shown in FIG. 1 is a well-known n-channel metal-oxide semiconductor (nMOS) inverter circuit. The amplifier circuit 20 shown in FIG. 1 is merely an example, and any circuit configuration may be used instead of the amplifier circuit 20 as long as it is a circuit for amplifying the characteristics of the nucleic acid detection FET 10. For example, instead of the amplifier circuit 20, a circuit used for reading out a flash memory or the like can be used. That is, any circuit may be used as long as it is a secondary physical quantity reflecting the physical quantity of the threshold voltage of the MOSFET and can be obtained as a signal having a large value.

  A plurality of nucleic acid detection FETs 10 and amplification circuits 20 may be set. Furthermore, there may be a plurality of the nucleic acid detection FETs 10, and these may be arranged in an array.

The sweep voltage generator 38 generates a predetermined voltage or voltage pattern. The operational amplifier 36 converts the voltage or the voltage value indicated by the voltage pattern into an inverted voltage, and applies the inverted voltage to the counter electrode CE32. Then, by the operation of the control circuit, the potential of the sensor electrode WE11 with respect to the reference electrode RE33 is relatively set to the potential generated by the sweep voltage generator 38 with respect to the reference potential.
The chemical reaction switch 31 is for causing a chemical reaction to occur on the sensor electrode WE11 attached to the nucleic acid detection FET 10. When the chemical reaction switch 31 is turned on after the potential of the sensor electrode WE11 with respect to the reference electrode RE33 is set to the potential generated by the sensor electrode WE11 with respect to the reference potential, an oxidation-reduction current flows through the chemical reaction switch 31. .
The reference electrode RE33, the operational amplifiers 35 and 36, and the resistor 37 are for maintaining the potential in the vicinity of the sensor electrode WE11 at the output voltage of the sweep voltage generator 38. It is possible to cause a chemical reaction even when the counter electrode CE32, the sensor electrode WE and the sweep voltage generator 38 are combined. However, when an oxidation-reduction current is passed through the chemical reaction switch 31, the nucleic acid is reduced due to the voltage drop in the solution. The potential in the vicinity of the sensor electrode WE11, which is a working electrode used as the sensor electrode, deviates from the predetermined potential generated by the predetermined sweep voltage generator 38. The reference electrode RE33 measures the potential in the vicinity of the sensor electrode WE11 with the reference electrode RE33, and a negative feedback circuit including an operational amplifier 35, a resistor 37, and an operational amplifier 36 that form a voltage follower topology is provided in the vicinity of the sensor electrode WE11. The potential is controlled to the output voltage of the sweep voltage generator 38. If the potential near the sensor electrode WE11 is controlled by introducing the reference electrode RE33 in this way, the potential near the sensor electrode WE11 can be set to an accurate potential as compared with the case where the reference electrode RE33 is not introduced.
Further, a plurality of control circuits 30 shown in FIG. 1 may be installed. For example, when there are a plurality of nucleic acid detection FETs 10, one control circuit 30 may be provided for each nucleic acid detection FET 10. Further, one control circuit 30 may control the potential of the sensor electrode WE11 included in each of the plurality of nucleic acid detection FETs 10.

Next, the nucleic acid detection FET 10 and the sensor electrode WE11 will be described with reference to FIG. FIG. 2 is a diagram schematically showing a cross-sectional structure of the nucleic acid detection FET 10.
The nucleic acid detection FET 10 includes a gate electrode, a gate insulating film 12, and a silicon substrate 13, and has the same structure as a normal metal-oxide semiconductor (MOS). In the nucleic acid detection FET 10, a gate insulating film 12 is formed on a silicon substrate 13, and a gate electrode is formed on the gate insulating film 12. Unlike the normal MOS, the nucleic acid detection FET 10 of the present embodiment uses a gold thin film as the gate electrode. This gate electrode is used as the sensor electrode WE11. That is, the sensor electrode WE11 binds the probe nucleic acid molecule 14 modified with a thiol group to the sensor electrode WE11. The probe nucleic acid molecule 14 is a single-stranded nucleic acid molecule that reacts with a specific nucleic acid molecule. Only the target nucleic acid molecule having a specific sequence can be specifically adsorbed from the sample nucleic acid molecule in a single-stranded state by the probe nucleic acid molecule 14.

Next, the operation of the nucleic acid detection device of this embodiment will be described with reference to FIG.
First, a sample nucleic acid molecule containing a target nucleic acid molecule is introduced into a chemical reaction tank 34 on a sensor on which the probe nucleic acid molecule is immobilized in advance, and the probe nucleic acid molecule and the target nucleic acid molecule are hybridized on the sensor electrode WE11. After the double strand formation), the double strand recognition body is introduced into the chemical reaction tank 34. Subsequently, the chemical reaction switch 31 is opened (step S1). The double-stranded recognizer will be described in detail later with reference to FIG.
The sweep voltage generator 38 generates a predetermined voltage value Va, and the voltage drop generated at the drain terminal D of the nucleic acid detection FET 10 is read by the voltmeter 22 (step S2). Of course, the voltage drop measured by the voltmeter 22 in step S2 may be set to automatically store the value in a storage device (not shown).
Subsequently, the chemical reaction switch 31 is closed (step S3).
The sweep voltage generator 38 generates a predetermined voltage value Vb, and performs a chemical reaction on the sensor electrode (step S4). The voltage value Vb is set to a voltage value necessary for performing a chemical reaction.
The chemical reaction switch 31 is opened (step S5).
A change in chemical characteristics on the sensor electrode WE11 due to a chemical reaction on the sensor electrode WE11 is examined. That is, the sweep voltage generator 38 applies the predetermined voltage value Va again and reads the voltage drop with the voltmeter 22 (step S6). This voltage drop may be set to automatically store the value in the storage device as described above.
Finally, the magnitude of the difference between the inverter output voltage Vds data obtained in step S2 and the inverter output voltage Vds data obtained in step S6 is calculated (step S7).

It is determined whether or not the magnitude of the difference obtained in step S7 is larger than the reference value determined in advance during calibration, and when the magnitude of the difference obtained in step S7 is larger than the reference value, It is determined that a nucleic acid molecule has been detected.
According to the nucleic acid detection device of the present embodiment, the chemical change of the substance occurring on the sensor electrode WE11 can be indirectly observed with the MOSFET. In addition, according to the nucleic acid detection device of the present embodiment, the sensor electrode WE11 using the nucleic acid detection FET 10 can release the oxidation-reduction current on the sensor electrode WE11 and the chemical reaction switch 31 on the sensor electrode WE11. A counter electrode CE32 and a reference electrode RE33 that supply an oxidation-reduction current for causing a chemical reaction are provided, and a chemical change on the sensor electrode WE11 can be actively caused.
Furthermore, according to the nucleic acid detection device of the present embodiment, the source and drain that are modulated by fluctuations in the threshold voltage of the MOSFET that is less dependent on the size of the sensor electrode WE11 than the redox current on the sensor electrode WE11. The presence of nucleic acid molecules can be detected from the intercurrent, and the sensor can be further miniaturized. Moreover, the amount of the target nucleic acid molecule bound to the probe nucleic acid molecule can be known more reliably by performing a chemical reaction of the substance on the sensor on the sensor electrode.

Next, whether or not hybridization has occurred between the sample nucleic acid molecule and the probe nucleic acid molecule 14 in the chemical reaction tank 34 will be described with reference to FIG.
Since the double strand recognizer specifically adsorbs to the double strand of the nucleic acid molecule, a difference appears in the conduction characteristics of the nucleic acid detection FET 10 depending on the presence or absence of hybridization. Furthermore, since the properties of the double-stranded recognizer change by performing a chemical reaction on the sensor electrode WE11, the conduction characteristics of the nucleic acid detection FET 10 before and after oxidation reduction (that is, before and after step S3) are also considered. A difference appears. According to FIG. 4, the magnitude of the difference in the inverter output voltage Vds obtained in step S7 is greater when there is hybridization than when there is no hybridization. Therefore, the presence or absence of hybridization can be determined based on whether or not the magnitude of the difference of the inverter output voltage Vds obtained in step S7 is larger than a certain reference value determined in advance by calibration.
In addition, a signal can be observed at a higher level by selecting a double-stranded recognizer that greatly changes the conduction characteristics of the nucleic acid detection FET 10 before and after oxidation / reduction of the double-stranded recognizer.

In the present embodiment, the “double-stranded recognizer” refers to a substance that recognizes and specifically binds to a double-stranded nucleic acid molecule. Examples of such substances include intercalating agents and biopolymers that recognize double-stranded nucleic acid molecules.
The intercalator used in this embodiment is preferably a substance that exhibits an oxidation or reduction reaction electrochemically, but is not particularly limited. For example, ethidium, ethidium bromide, acridine, aminoacridine, acridine orange, pro Flavin, elibutisin, actinomycin D, donomycin, mitomycin C and the like can be used. Other usable intercalating agents include those described in JP-A-62-2282599.

  In addition to the intercalating agent described above, a metal complex containing a substance that exhibits an oxidation or reduction reaction electrochemically as a central metal, that is, a metallointercalator can be used. For example, a zinc complex, a ruthenium complex, a cobalt complex, and a platinum complex can be mentioned, but it is not limited to these.

  By the way, apart from the intercalating agent described above, there are substances in the biopolymer that specifically recognize and bind to double-stranded nucleic acid molecules. Therefore, in this embodiment, an electrochemically active substance can be used among such biopolymers. In addition, an electrochemically active substance can be labeled and used on these biopolymers or substances that recognize the biopolymers. Examples of such biopolymers include, but are not limited to, an anti-DNA antibody, a clot protein, a DNA-binding protein, and an enzyme such as RNase H whose catalytic activity has been deactivated. The biopolymer may be derived from a living body or obtained by synthesis.

(Modification)
Although the operation of the nucleic acid detection apparatus has been described with reference to FIG. 3, there are various variations in which the inverter output voltage Vds is measured at two points. Hereinafter, this modification will be described.
The nucleic acid detection device operates to detect nucleic acid molecules according to the following procedure.
(Step S11) A sample containing the target nucleic acid molecule is introduced onto the sensor on which the probe nucleic acid molecule is immobilized in advance, and the target nucleic acid molecule undergoes a hybridization reaction with the probe nucleic acid molecule on the sensor (before step S1).
(Step S12) In order to wash the target nucleic acid molecule adsorbed nonspecifically on the portion other than the probe nucleic acid molecule, the target nucleic acid molecule is replaced with an electrolyte solution not containing the nucleic acid molecule (in some cases, unnecessary).
(Step S13) A reagent containing a double-stranded recognizer is introduced onto the sensor, and a binding reaction occurs with the nucleic acid molecule to which the double-stranded recognizer has hybridized.
(Step S14) In order to wash the double-stranded recognizer that is non-specifically bound to a portion other than the nucleic acid molecule and the double-stranded recognizer remaining in the solution, the double-stranded recognizer is included. Replace with a non-electrolyte solution (not required in some cases).
(Step S15) The potential of the sensor electrode WE11 is controlled to cause a chemical reaction of the double-stranded recognizer.
The voltmeter 22 measures the inverter output voltage Vds when the reference current Ids is supplied to the nucleic acid detection FET 10 for nucleic acid molecule detection. As shown in FIG. 3 and FIG. 4, this measurement is performed, for example, by measuring the first output voltage “before the double-strand recognizer oxidation reaction” and then “after the double-strand recognizer chemical reaction”. Measurement is performed at two points, such as measurement of the second output voltage, and the presence / absence of hybridization is determined based on the difference between the inverter output voltages Vds.

  This "before voltage change" measurement may be performed at any point before step S11, after step S11, after step S12, after step S13, or after step S14. The ideal two measurement points are the two points that have the largest voltage change, little variation, and are easy to measure in a comprehensive manner, depending on the characteristics of the double-stranded recognizer used. The measurement of the second output voltage is performed after step S15.

Next, the result of measuring the inverter output voltage Vds generated at the drain terminal D of the nucleic acid detection FET 10 before hybridization, after hybridization, after addition of the double strand recognizer, and after chemical reaction of the double strand recognizer Will be described with reference to FIG. FIG. 5 shows the history of the inverter output voltage Vds in two cases of whether or not there is hybridization. The horizontal axis of FIG. 5 shows the time change at each step, the time is later as it goes to the right, and the vertical axis shows the inverter output voltage Vds generated at the drain terminal D of the nucleic acid detection FET 10.
The inverter output voltage Vds “before hybridization” shown in FIG. 5 is a value measured by the voltmeter 22 before the above step S11. The inverter output voltage Vds “after hybridization” shown in FIG. 5 is a value measured by the voltmeter 22 after step S12 and before step S13. Furthermore, the inverter output voltage Vds “after the addition of a double-stranded recognizer” shown in FIG. 5 is a value measured by the voltmeter 22 after step S14 and before step S15.
Further, “after chemical reaction” shown in FIG. 5 is a value measured by the voltmeter 22 after the above step S15.

  In the example shown in FIG. 5, the inverter output voltage Vds after the chemical reaction and the inverter output voltage Vds in any one of the cases before the hybridization, after the hybridization, and after the addition of the double strand recognizer. The magnitude of the difference is calculated, and it is determined whether or not the magnitude of the difference is greater than a predetermined value. If it is greater, it is determined that a nucleic acid molecule has been detected.

  In the example described with reference to FIG. 3, step S <b> 2 corresponds to “after double strand recognizer addition”, and step S <b> 6 corresponds to “after chemical reaction”. As shown in FIG. 5, when the inverter output voltage Vds changes, the difference in the inverter output voltage Vds between the two points “after hybridization” and “after chemical reaction” becomes the largest. It is preferable to measure the inverter output voltage Vds generated at the drain terminal D of the detection FET 10.

  Further, in the example such as the graph showing the inverter output voltage Vds shown in FIG. 5, the difference in the inverter output voltage Vds due to the presence or absence of hybridization after the chemical reaction is higher than before the addition of the double-stranded recognizer. It is several times larger than the difference in inverter output voltage Vds due to the presence or absence of hybridization. Therefore, measuring the magnitude of the difference in the inverter output voltage Vds at two points of the chemical reaction and others is measuring the magnitude of the difference in the inverter output voltage Vds without referring to the inverter output voltage Vds after the chemical reaction. Sensitivity is dramatically increased compared to doing.

  Also, instead of measuring at two points, it is possible to always monitor the change in the inverter output voltage and determine the presence or absence of hybridization based on the fluctuation speed of the output voltage. That is, as can be seen from FIG. 5, the change in the inverter output voltage Vds when there is hybridization is larger than the change in the inverter output voltage Vds when there is no hybridization, so it is higher than the preset voltage value. The presence or absence of hybridization can be determined based on whether or not the inverter output voltage Vds in a certain unit time is large. This preset voltage value is determined in advance by calibration.

Depending on the nature of the double-stranded recognizer, the first voltage drop may be measured after step S15. Two typical examples in this case will be described with reference to FIGS. 6 (A) and 6 (B). The arrows shown on the curves in FIGS. 6A and 6B indicate the direction of time.
The double-stranded recognizer includes a double-stranded recognizer that causes a larger chemical reaction again even after step S15 when the sweep voltage generator 38 increases the potential difference of the sensor electrode WE11 with respect to the reference electrode RE33. For example, in a double-stranded recognizer showing a curve as shown in FIG. 6A, there is a larger chemical reaction (γ) after the first chemical reaction (α) corresponding to step S15. In this case, since it is preferable to measure the inverter output voltage Vds before and after this larger chemical reaction, paying attention to this larger chemical reaction (γ), the measurement of the first inverter output voltage Vds is “attention. The second inverter output voltage Vds is measured at δ “after the chemical reaction of interest”. Thus, if the inverter output voltage Vds is measured before and after a larger chemical reaction, a larger difference in the inverter output voltage Vds can be obtained, and the sensitivity of nucleic acid molecule detection can be increased.

  In addition, the sweep voltage generator 38 raises the potential of the sensor electrode WE11 with respect to the reference electrode RE33 to generate a chemical reaction, further raises this potential to end this chemical reaction, and further lowers this potential again. There are also double-stranded recognizers in which a chemical reaction occurs and the chemical reaction ends when this potential is further lowered. For example, in a double-stranded recognizer showing a curve as shown in FIG. 6B, there is a chemical reaction (η) after the first chemical reaction (ε) corresponding to step S15. In this case as well, as in the example of FIG. 6A, paying attention to the chemical reaction (η), the measurement of the first inverter output voltage Vds is ζ “before the chemical reaction of interest”. The measurement of the second inverter output voltage Vds may be performed at θ after “the chemical reaction of interest”.

Next, a calibration method for the voltmeter 22 to measure the change in the inverter output voltage Vds with the conduction characteristic of the nucleic acid detection FET 10 depending on the surface state of the sensor electrode WE11 with high sensitivity will be described with reference to FIG.
In order for the voltmeter 22 to easily measure the inverter output voltage Vds generated at the drain terminal D of the nucleic acid detection FET 10, the reference current generated by the signal amplification current source circuit 21 and the voltage generated by the substrate bias voltage source 23 Adjust. That is, as shown in FIG. 7, the magnitude of the current generated by the signal amplification current source circuit 21 and the magnitude of the voltage generated by the substrate bias voltage source 23 are set so that the nucleic acid detection FET 10 operates in the saturation region. Adjust to. Then, the magnitude | size of a change can be enlarged before and after the chemical reaction of Vds which the voltmeter 22 measures.

  According to the embodiment described above, the inverter output voltage Vds generated at the drain terminal of the nucleic acid detection FET before and after the chemical reaction is measured, the magnitude of the difference between these inverter output voltages Vds is obtained, and the magnitude of this difference is obtained. By detecting that a nucleic acid molecule is detected when the value is larger than a predetermined value, it is possible to measure a signal for capturing a chemical change that occurs on the sensor when the sensor is miniaturized and detect the nucleic acid with high accuracy. can do.

  Note that the present invention is not limited to the above-described embodiment as it is, and can be embodied by modifying the components without departing from the scope of the invention in the implementation stage. In addition, various inventions can be formed by appropriately combining a plurality of components disclosed in the embodiment. For example, some components may be deleted from all the components shown in the embodiment. Furthermore, constituent elements over different embodiments may be appropriately combined.

1 is a block diagram of a nucleic acid detection device according to an embodiment of the present invention. Sectional drawing which shows the cross-section of the FET 10 for nucleic acid detection of FIG. The flowchart which shows operation | movement of the nucleic acid detection apparatus of FIG. The figure which shows the difference in the magnitude | size of the difference of the inverter output voltage Vds by the presence or absence of hybridization. The figure which shows the value in each step of the inverter output voltage Vds by the presence or absence of hybridization. (A) is a figure which shows the 1st example in the case of measuring the first voltage drop after a chemical reaction, (B) shows the 2nd example in the case of measuring the first voltage drop after a chemical reaction. Figure. The figure for demonstrating the calibration for measuring the inverter output voltage Vds with sufficient sensitivity.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... Nucleic acid detection FET, 11 ... Sensor electrode WE, 12 ... Gate insulating film, 13 ... Silicon substrate, 14 ... Probe nucleic acid molecule, 20 ... Amplification circuit, 21 ... Current source circuit for signal amplification, 22 ... Voltmeter, 23 ... Voltage source for substrate bias, 24 ... Power supply Vdd, 25 ... Power supply Vss, 30 ... Control circuit, 31 ... Chemical reaction switch 32... Counter electrode CE 33 Reference electrode RE 34 Chemical reaction tank 35 36 Operation amplifier 37 Resistor 38 Sweep voltage generator

Claims (8)

In a nucleic acid detection apparatus for detecting a target nucleic acid molecule having a specific sequence contained in a specimen based on the intensity of characteristic modulation of a MOSFET (metal-oxide semiconductor field-effect transistor),
A sensor electrode in which a probe nucleic acid molecule capable of hybridizing with the target nucleic acid molecule is immobilized on an electrode connected to the gate electrode of the MOSFET or the gate electrode of the MOSFET;
A control circuit comprising a counter electrode for controlling the potential of the sensor electrode to generate a chemical reaction of a double-stranded recognizer on the sensor electrode;
A switch for switching whether to cause the chemical reaction by short-circuiting the gate electrode to a ground point or a reference voltage source;
Measuring means for measuring a physical quantity indicating the electrical characteristics of the MOSFET before and after the chemical reaction;
A nucleic acid detection apparatus comprising: determination means for determining whether or not a difference between physical quantities indicating electrical characteristics of the MOSFET generated before and after the chemical reaction is greater than a predetermined value. The control circuit further includes a reference electrode for measuring a potential in the vicinity of the sensor electrode,
The nucleic acid detection device according to claim 1, wherein the potential of the counter electrode is controlled based on information from the reference electrode. The nucleic acid detector according to claim 1 or claim 2, characterized in that it comprises the sensor electrode into a plurality on the same substrate. The nucleic acid detection device according to any one of claims 1 to 3 , wherein a plurality of the control circuits are installed. In a nucleic acid detection method for detecting a target nucleic acid molecule having a specific sequence contained in a specimen based on the intensity of characteristic modulation of a MOSFET (metal-oxide semiconductor field-effect transistor),
Measuring a first physical quantity indicative of the electrical characteristics of the MOSFET;
A sample containing a target nucleic acid molecule is introduced onto a sensor electrode on which a probe nucleic acid molecule is immobilized in advance, and a hybridization reaction is caused between the target nucleic acid molecule and the probe nucleic acid molecule on the sensor electrode,
Introducing a reagent containing a double-stranded recognizer on the sensor electrode, causing a binding reaction between the double-stranded recognizer and the hybridized nucleic acid molecule,
Controlling the potential of the sensor electrode to cause a chemical reaction of the double-stranded recognizer,
Measuring a second physical quantity indicative of the electrical characteristics of the MOSFET;
Calculating a difference between the first physical quantity and the second physical quantity;
A method for detecting a nucleic acid, comprising determining whether or not the magnitude of the difference is larger than a predetermined value. In a nucleic acid detection method for detecting a target nucleic acid molecule having a specific sequence contained in a specimen based on the intensity of characteristic modulation of a MOSFET (metal-oxide semiconductor field-effect transistor),
A sample containing a target nucleic acid molecule is introduced onto a sensor electrode on which a probe nucleic acid molecule is immobilized in advance, and a hybridization reaction is caused between the target nucleic acid molecule and the probe nucleic acid molecule on the sensor electrode,
Measuring a first physical quantity indicative of the electrical characteristics of the MOSFET;
Introducing a reagent containing a double-stranded recognizer on the sensor electrode, causing a binding reaction between the double-stranded recognizer and the hybridized nucleic acid molecule,
Controlling the potential of the sensor electrode to cause a chemical reaction of the double-stranded recognizer,
Measuring a second physical quantity indicative of the electrical characteristics of the MOSFET;
Calculating a difference between the first physical quantity and the second physical quantity;
A method for detecting a nucleic acid, comprising determining whether or not the magnitude of the difference is larger than a predetermined value. In a nucleic acid detection method for detecting a target nucleic acid molecule having a specific sequence contained in a specimen based on the intensity of characteristic modulation of a MOSFET (metal-oxide semiconductor field-effect transistor),
A sample containing a target nucleic acid molecule is introduced onto a sensor electrode on which a probe nucleic acid molecule is immobilized in advance, and a hybridization reaction is caused between the target nucleic acid molecule and the probe nucleic acid molecule on the sensor electrode,
Introducing a reagent containing a double-stranded recognizer on the sensor electrode, causing a binding reaction between the double-stranded recognizer and the hybridized nucleic acid molecule,
Measuring a first physical quantity indicative of the electrical characteristics of the MOSFET;
Controlling the potential of the sensor electrode to cause a chemical reaction of the double-stranded recognizer,
Measuring a second physical quantity indicative of the electrical characteristics of the MOSFET;
Calculating a difference between the first physical quantity and the second physical quantity;
A method for detecting a nucleic acid, comprising determining whether or not the magnitude of the difference is larger than a predetermined value. In a nucleic acid detection method for detecting a target nucleic acid molecule having a specific sequence contained in a specimen based on the intensity of characteristic modulation of a MOSFET (metal-oxide semiconductor field-effect transistor),
A sample containing a target nucleic acid molecule is introduced onto a sensor electrode on which a probe nucleic acid molecule is immobilized in advance, and a hybridization reaction is caused between the target nucleic acid molecule and the probe nucleic acid molecule on the sensor electrode,
Introducing a reagent containing a double-stranded recognizer on the sensor electrode, causing a binding reaction between the double-stranded recognizer and the hybridized nucleic acid molecule,
Controlling the potential of the sensor electrode to cause a chemical reaction of the double-stranded recognizer,
Measuring a first physical quantity indicative of the electrical characteristics of the MOSFET;
Control the potential of the sensor electrode again to cause a chemical reaction of the double-stranded recognizer,
Measuring a second physical quantity indicative of the electrical characteristics of the MOSFET;
Calculating a difference between the first physical quantity and the second physical quantity;
A method for detecting a nucleic acid, comprising determining whether or not the magnitude of the difference is larger than a predetermined value.

Images