JP2006138846A - Nucleic acid detecting sensor, nucleic acid detecting chip, and nucleic acid detecting device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To significantly enhance sensitivity of a nucleic acid detecting sensor using an FET (field-effect transistor). <P>SOLUTION: In the nucleic acid detecting sensor for detecting a target nucleus acid molecule 109 with a special alignment included in specimen on the basis of the intensity of characteristics modulation of FET, by fixing at least a probe nucleus acid molecule 102 hybridizable with the target nucleus acid molecule to a gate 101 of the aforementioned FET, a gate width of the FET is obtained in the order of (ε<SB>0</SB>ε<SB>r</SB>k<SB>B</SB>T/e<SP>2</SP>n)<SP>1/2</SP>, where ε<SB>0</SB>is the dielectric constant of vacuum, ε<SB>r</SB>the relative permittivity of channel domain, k<SB>B</SB>the Boltzmann constant, T the absolute temperature of the channel domain of FET, e the elementary charge, and n the carrier density. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a nucleic acid detection sensor, a nucleic acid detection chip, and a nucleic acid detection device that detect a target nucleic acid molecule contained in a specimen using a field-effect transistor (FET).

Conventionally, there are nucleic acid detection sensors for detecting nucleic acid molecules that detect whether or not a target nucleic acid molecule is contained in a specimen using an FET (see, for example, Non-Patent Document 1, Patent Documents 1 and 2). ).
Toshiya Sakata et al., “Detection of DNA hybridization using gene transistors”, Proc. 1179 (2003) JP 2003-322633 A Japanese National Patent Publication No. 2001-511246

  However, in the prior art, a method for efficiently detecting a signal at the level of one nucleic acid molecule using an FET and a method for quantitative analysis in a wide concentration range are not clearly described.

  In view of the conventional problems, an object of the present invention is to provide a nucleic acid detection sensor, a nucleic acid detection chip, and a nucleic acid detection device that dramatically improve sensitivity in a nucleic acid detection sensor using an FET.

According to the nucleic acid detection sensor of the present invention, in the nucleic acid detection sensor for detecting a target nucleic acid molecule having a specific sequence contained in a specimen based on the intensity of characteristic modulation of an FET (field-effect transistor), At least one probe nucleic acid molecule capable of hybridizing with the target nucleic acid molecule is immobilized on the gate of the FET, ε ₀ is the dielectric constant of vacuum, ε _r is the relative dielectric constant of the channel region, and k _B is the Boltzmann constant , T is the absolute temperature of the channel region of the FET, e is the elementary charge, and n is the carrier density, the gate width of the FET is
_{(Ε 0 ε r k B T} / e 2 n) 1/2
It is characterized by becoming an order.

According to the nucleic acid detection sensor of the present invention, in the nucleic acid detection sensor for detecting a target nucleic acid molecule having a specific sequence contained in a specimen based on the intensity of characteristic modulation of an FET (field-effect transistor), At least one probe nucleic acid molecule capable of hybridizing with the target nucleic acid molecule is immobilized on the gate of the FET, ε ₀ is the dielectric constant of vacuum, ε _r is the relative dielectric constant of the channel region, and k _B is the Boltzmann constant , T is the absolute temperature of the channel region of the FET, e is the elementary charge, and n is the carrier density, the gate length of the FET is
_{(Ε 0 ε r k B T} / e 2 n) 1/2
It is characterized by becoming an order.

According to the nucleic acid detection chip of the present invention, in the nucleic acid detection sensor for detecting a target nucleic acid molecule having a specific sequence contained in a specimen based on the intensity of characteristic modulation of an FET (field-effect transistor), At least one probe nucleic acid molecule capable of hybridizing with the target nucleic acid molecule is immobilized on the gate of the FET, ε ₀ is the dielectric constant of vacuum, ε _r is the relative dielectric constant of the channel region, and k _B is the Boltzmann constant , T is the absolute temperature of the channel region of the FET, e is the elementary charge, and n is the carrier density, the gate width of the FET is
_{(Ε 0 ε r k B T} / e 2 n) 1/2
In a nucleic acid detection chip having a plurality of nucleic acid detection sensors of the order of t, a detection time is t and a diffusion constant of a nucleic acid molecule is D = 1.6 × 10 ⁻⁶ cm ² / s, the unit on the nucleic acid detection chip The number of the nucleic acid detection sensors per area is on the order of (Dt) ^-1 .

According to the nucleic acid detection apparatus of the present invention, in the nucleic acid detection sensor for detecting a target nucleic acid molecule having a specific sequence contained in a specimen based on the intensity of characteristic modulation of an FET (field-effect transistor), At least one probe nucleic acid molecule capable of hybridizing with the target nucleic acid molecule is immobilized on the gate of the FET, ε ₀ is the dielectric constant of vacuum, ε _r is the relative dielectric constant of the channel region, and k _B is the Boltzmann constant , T is the absolute temperature of the channel region of the FET, e is the elementary charge, and n is the carrier density, the gate width of the FET is
_{(Ε 0 ε r k B T} / e 2 n) 1/2
The nucleic acid detection sensor of the order of the nucleic acid and the nucleic acid detection sensor are different in the nucleic acid probe molecule, and the nucleic acid probe molecule having a base sequence that is not complementary to the nucleic acid molecule contained in the sample is fixed to the gate. Zero level detection sensors, two capacitive elements connected to the drain terminals of the sensors, and charges of the capacitive elements charged at a predetermined voltage value via FETs included in the sensors. A sense amplifier that discharges and amplifies a difference in discharge efficiency from each FET, and a determination unit that determines presence or absence of nucleic acid detection based on the difference in discharge efficiency are provided.

According to the nucleic acid detection apparatus of the present invention, in the nucleic acid detection sensor for detecting a target nucleic acid molecule having a specific sequence contained in a specimen based on the intensity of characteristic modulation of an FET (field-effect transistor), At least one probe nucleic acid molecule capable of hybridizing with the target nucleic acid molecule is immobilized on the gate of the FET, ε ₀ is the dielectric constant of vacuum, ε _r is the relative dielectric constant of the channel region, and k _B is the Boltzmann constant , T is the absolute temperature of the channel region of the FET, e is the elementary charge, and n is the carrier density, the gate width of the FET is
_{(Ε 0 ε r k B T} / e 2 n) 1/2
The nucleic acid detection sensor of the order of the nucleic acid and the nucleic acid detection sensor are different in the nucleic acid probe molecule, and the nucleic acid probe molecule having a base sequence that is not complementary to the nucleic acid molecule contained in the sample is fixed to the gate. Zero level detection sensors, differential pairs using FETs included in the sensors as input transistors, and output voltages of the differential pairs generated by applying a common reference voltage to the differential pairs And determining means for determining the presence or absence of nucleic acid detection.

  According to the nucleic acid detection sensor, nucleic acid detection chip, and nucleic acid detection device of the present invention, the sensitivity can be dramatically improved.

Hereinafter, a nucleic acid detection sensor, a nucleic acid detection chip, and a nucleic acid detection device according to embodiments of the present invention will be described in detail with reference to the drawings.
The nucleic acid detection sensor 100 included in the nucleic acid detection device of the present embodiment of the present invention includes a MOSFET (metal-oxide semiconductor field-effect transistor) and a substrate, and the MOSFET usually includes a plurality of nucleic acid probes (Probe DNA) 102. It is attached. The MOSFET includes a gate 101, a source 103, and a drain 104, and the nucleic acid probe molecule 102 is attached on the gate 101. As shown in FIG. 1, the source 103 and the drain 104 are connected via a body 106, and a gate 101 is stacked on the body 106 via a gate oxide film 105. The source 103, drain 104, and body 106 are formed on a BOX (Buried Oxide) 107 that is a buried oxide film. As shown in FIG. 1, the sensor 100 can be manufactured using a wafer having an SOI (Silicon On Insulator) structure, but it is obvious that a person skilled in the art can manufacture an equivalent using a bulk Si substrate. Will be understood.

  In the nucleic acid detection device of this embodiment, it is determined whether or not the target nucleic acid molecule has been detected based on the intensity of modulation of the electrical characteristics of the MOSFET. In this embodiment, the shape of the gate 101 is narrowed in the direction connecting the source 103 and the drain 104 (that is, the gate width W of the gate 101 is narrowed). In this case, even a small change in the number of charges generated on the gate greatly modulates the electrical characteristics of the MOSFET, so that a small number of target nucleic acid molecules can be detected.

  Furthermore, in this embodiment, the channel length of the MOSFET (that is, the gate length L in FIG. 1) is made equal to or longer than the gate width W. In this case, since many nucleic acid probe molecules 102 are immobilized in the gate length direction (that is, the direction connecting the source 103 and the drain 104), the nucleic acid probe molecule 102 and the target nucleic acid molecule 109 are located at any position in the gate length direction. Even if this coupling occurs, the modulation of the MOSFET can be surely induced. That is, an operation equivalent to taking a logical sum of signals from a large number of nucleic acid probe molecules 102 is performed. In addition, the presence of a small number in the sample solution increases the probability of binding to any one of a large number of probes due to the dense arrangement within the surface of the chip that contacts the droplets of the sample to be analyzed. Even nucleic acid molecules can be detected rapidly.

More specifically, how much the gate length and gate width of the gate 101 are set will be described. When binding of the target nucleic acid molecule 109 occurs in a certain probe nucleic acid molecule 102, the change in the number of charges generated on the gate 101 side electrostatically induces a change in the charge state in the channel via the gate oxide film 106. Now, the Debye length of the carrier in the region of the body 106 where the channel is formed is
(Ε ₀ ε _{rk B} T / e ² n) ^1/2 (Formula 1)
Given in. Here, ε ₀ is the dielectric constant of vacuum, ε _r is the relative dielectric constant of the channel region, k _B is the Boltzmann constant, T is the absolute temperature of the channel region, e is the elementary charge, and n is the carrier density. When the monovalent charge changes on the gate side, the charge state is expected to change greatly in the region within the radius given by (Equation 1) in the channel.

  Therefore, if the gate width and gate length of the gate 101 equivalent to the diffusion distance calculated by (Equation 1) are determined, it is considered that large FET characteristic modulation can be obtained with a small number of target nucleic acid molecules. That is, the gate width is on the order of the length calculated by (Expression 1), and the gate length is also the length calculated by (Expression 1). That is, the gate width and gate length are set to the same number of digits as the length calculated by (Equation 1) (that is, at most about 10 times or 1/10). More preferably, the gate width is on the order of the length calculated by (Equation 1), and the gate length is longer than the length of the gate width.

  In this embodiment, assuming a material having a carrier density equivalent to that of a normal MOSFET, the length calculated by (Equation 1) is about 50 nm. Therefore, using this length, for example, the gate width Is 50 nm. There is no problem even if the gate width is about 100 nm, but it is more preferably about 50 nm or less. On the other hand, since the gate length is set to be equal to or longer than the gate width, it is about 50 nm or longer.

  Further, since the diameter of the nucleic acid molecule is about 2 nm, when the nucleic acid probe molecules 102 are densely attached on the gate 101 having a gate width of 50 nm, about 25 nucleic acid probe molecules 102 are arranged in a direction perpendicular to the channel. Will be. When a target nucleic acid molecule 109 having the same length as a nucleic acid probe molecule 102 having a length of about 20 base pairs binds to one of the nucleic acid probe molecules 102, a charge change equivalent to the number of base pairs occurs. It is expected that a change in physical characteristics (for example, a change in the threshold voltage of the MOSFET) will appear greatly due to this change in charge.

  A plurality of nucleic acid detection sensors 100 are arranged on the chip. The accuracy of detecting the target nucleic acid molecule varies depending on how the nucleic acid detection sensor 100 is arranged on the chip. For example, since the nucleic acid detection sensor 100 is densely arranged in the surface of the chip that comes into contact with the droplet of the analyte to be analyzed, the probability of binding to any one of a large number of probes is increased. Even a small number of nucleic acid molecules can be rapidly detected. More preferably, the integration density of the sensor array is determined so that the sensors are arranged at an interval shorter than the diffusion distance of the nucleic acid molecules. Furthermore, by counting the number of sensors that have detected the target nucleic acid molecule, it is possible to estimate the concentration of the target nucleic acid molecule or the number of molecules of the target nucleic acid molecule. Details of this arrangement will be described later with reference to FIGS.

  Next, a nucleic acid detection apparatus for detecting the modulation of the electrical characteristics of the MOSFET induced by the binding between the target nucleic acid molecule 109 and the nucleic acid probe molecule 102 using the above-described nucleic acid detection sensor 100 will be described. Since this modulation appears as, for example, a variation in threshold voltage value, the nucleic acid detection device detects this variation in threshold voltage value. In this embodiment, as shown below, two types of nucleic acid detection devices for detecting this physical phenomenon are provided. One is a device (FIGS. 2 and 4) that directly converts a digital signal to output whether or not the target nucleic acid molecule 109 has been detected, and the other is an analog voltage value based on fluctuations in the threshold voltage value. This is an output type device (FIGS. 7, 8, and 9). A feature of these apparatuses is that the determination is performed by comparing the nucleic acid detection sensor 100 with the zero level detection sensor to which a nucleic acid probe molecule having no base sequence complementary to the target nucleic acid molecule 109 is fixed. With this feature, the detection accuracy of the target nucleic acid molecule 109 can be improved.

  Next, an example of a nucleic acid detection apparatus that detects nucleic acids using the nucleic acid detection sensor 100 shown in FIG. 1 will be described with reference to FIG. FIG. 2 is an example of a nucleic acid detection apparatus using a cross-coupled inverter.

  2 includes a reference electrode 201, a reference voltage power supply 202, a charging voltage source input terminal 203, charging switches 204 and 205, a control pulse input terminal 206, a power supply voltage 207, a reference potential 208, and a sense amplifier control switch. 209, storage capacitors 210 and 211, output signal amplifiers 212 and 213, sense amplifier 214, MOSFET 215 included in nucleic acid detection sensor 100, 216 included in nucleic acid detection sensor 200, nucleic acid probe molecule 217, and nucleic acid detection sensor 200 Become.

  The apparatus shown in FIG. 2 includes a circuit that determines whether or not a change in the threshold voltage of the MOSFET included in the nucleic acid detection sensor 100 to which the nucleic acid probe molecule 102 is attached has occurred. This circuit is equivalent to that used in a read circuit of a flash memory, and a nucleic acid detection MOSFET is used in a portion corresponding to a MOSFET having a floating gate used in the flash memory. The circuit also includes a reference electrode for controlling the surface potential of the MOSFET. In this nucleic acid detection apparatus, the nucleic acid detection sensor 100 whose surface potential is controlled by the reference electrode 201 is a MOSFET for nucleic acid detection capable of binding the target nucleic acid molecule 109, and a nucleic acid detection sensor paired with this sensor. Reference numeral 200 denotes a zero-level detection sensor to which a nucleic acid probe molecule 217 that cannot bind the target nucleic acid molecule 109 is attached. The zero level detection sensor 200 is the same as the nucleic acid detection sensor 100 except that a nucleic acid probe molecule 217 is attached instead of the nucleic acid probe molecule 102.

In the circuit of FIG. 2, the discharge time of the capacitor 210 in which the sense amplifier 214 determines the amount of saturation current that varies as a result of variation in the threshold voltage of the MOSFET that varies depending on the presence or absence of nucleic acid binding on the nucleic acid detection sensor 100. Is compared with the discharge time of the capacitor 210 determined by the threshold voltage value of the sensor 200 for detecting the zero level. The sense amplifier 214 senses which one of the nucleic acid detection sensor 100 and the zero level detection sensor 200 lowers the voltage first, and outputs the voltage value of each sensor relative to the counterpart. That is, the sense amplifier 214 outputs a digital value of 1 or 0 corresponding to each sensor. The output signal is amplified by the amplifiers 212 and 213 to a size that can be easily determined.
In order to make the presence or absence of nucleic acid detection in the nucleic acid detection sensor 100 correspond to a digital value of 0 or 1, the ratio between the storage capacitor 210 and the storage capacitor 211 is the discharge time of the nucleic acid detection sensor 200 and the nucleic acid detection sensor 100. Is set in advance to be exactly half of both discharge times when the target nucleic acid molecule 109 is bound and not. In addition, since the discharge time depends on the reference electrode potential, the reference voltage power source 202 must also be set in advance. That is, to operate the circuit of FIG. 2, it is necessary to determine the following parameters in advance.
(1) Ratio of capacitance of storage capacitor 210 and storage capacitor 211
(2) Voltage value of the reference voltage power source 202 that determines the potential of the reference electrode 201 with respect to the reference potential 208
Describing in more detail (1), the time constant of the discharge time of the storage capacitor 210 when hybridization (double strand formation) is detected is τ ₁ ′, and the storage capacitor 210 when hybridization is not detected Is set to τ ₁ , and the discharge time constant of the storage capacitor 211 is set to τ ₂ .
τ ₁ ′ <τ ₂ <τ ₁ (Formula 2)
Should be established. However, this (Equation 2) assumes a case where the MOSFET of the nucleic acid detection sensor 100 is n-type and the threshold voltage value of the MOSFET decreases due to the effect of a positively charged intercalating agent due to hybridization. When no intercalating agent is used, the threshold value of the n-type MOSFET is raised, so that the inequality sign in (Equation 2) is reversed. More preferably, τ ₂ is an intermediate value between τ ₁ and τ ₁ ′,
τ ₂ = (τ ₁ + τ ₁ ′) / 2 (Formula 3)
It is desirable to set so that

These (Equation 2) and (Equation 3) are converted into a capacitance ratio. Assuming that the MOSFETs of the nucleic acid detection sensor 100 and the zero level detection sensor 200 are operating in the saturation region, the current flowing through the nucleic acid detection sensor 100 is:
i = μCW (V _GS −V _th ) ² / L (Formula 4)
It is expressed. Here, C is the capacitance of the MOSFET oxide film, μ is the surface channel mobility, W is the gate width, L is the gate length, V _GS is the gate-source voltage, that is, the voltage between the reference electrode 3 and the source 103 in this case. Show. _Vth indicates the threshold voltage value of the MOSFET, and this threshold voltage value varies depending on whether or not hybridization occurs. If the threshold voltage value when hybridization is detected is V _th ′, the threshold voltage value when it is not detected is V _th, and the currents corresponding to the respective voltage values are i ′ and i, τ ′ ₁ , τ ₁ , Τ ₂ are approximately,
τ ₁ '= C ₁₀ V _pre / i'
τ ₁ = C ₁₀ V _pre / i (Formula 5)
τ ₂ = C ₁₁ V _pre / i
It is expressed. Here, C ₁₀ and C ₁₁ represent capacitances of the storage capacitor 210 and the storage capacitor 211, respectively, and V _Pre represents a voltage value input from the charging voltage source input terminal 203. By substituting (Equation 4) and (Equation 5) into (Equation 2), the conditions to be satisfied by C ₁₀ and C ₁₁ are determined as follows. That is,
1 <C ₁₀ / C ₁₁ <(V _GS −V _th ′) ² / (V _GS −V _th ) ² (Formula 6)
It is. Further, the more desirable condition represented by (Expression 3) is that (Expression 4) and (Expression 5) are used.
C ₁₀ / (2C ₁₁ −C ₁₀ ) = (V _GS −V _th ′) ² / (V _GS −V _th ) ² (Expression 7)
It is determined as follows.

Next, a procedure for detecting a nucleic acid using the circuit shown in FIG. 2 will be described with reference to FIG.
First, the control unit (not shown) turns off the charging switches 204 and 205 for switching whether or not the storage capacitors 210 and 211 are charged (step S301). Further, the sense amplifier control switch 209 that controls the sense amplifier 214 is turned off (step S301). Further, the reference voltage power source 202 is adjusted as an initial setting so that the voltage between the reference electrode 201 and the source 103 of the nucleic acid detection sensor 100 satisfies the above (Expression 6) or (Expression 7) (Step S301). ).

  Next, the charging switches 204 and 205 are turned on, and the charging voltage is applied to the storage capacitors 210 and 211 via the charging voltage source input terminal 203 (step S302). Since the same voltage value is applied to the storage capacitors 210 and 211, the same amount of charge is stored in the storage capacitors 210 and 211. Thereafter, the sense amplifier control switch 209 is turned on to operate the sense amplifier 214 (step S303).

  Then, the charging switches 204 and 205 are turned off (step S304), and the presence or absence of nucleic acid detection is determined from a digital value of 0 or 1 detected by the sense amplifier 214 after a predetermined time has elapsed. Even if the order of step S303 and step S304 is reversed, the operation is correct.

Next, an example in which the circuit of FIG. 2 is modified will be described with reference to FIG. The same parts as those shown in FIG.
The modification of FIG. 4 is a case where a sense amplifier 401 in which the sense amplifier 214 is formed only by an nMOS is used. The operation principle is basically the same as in FIG. 2, but a differential amplifier 402 needs to be added. In this circuit, each node to which the storage capacitors 210 and 211 are connected converges to a certain potential between V _pre that is a voltage value input from the charging voltage source input terminal 203 and the reference potential 208. The difference between the nodes is amplified by the differential amplifier 402 and further amplified by the output amplifier 403, and a digital value of 0 or 1 having a finally detectable magnitude can be obtained from the output signal terminal 405.

Further, by arranging the nucleic acid detection sensor 100 densely on the chip substrate, the concentration can be analyzed. When t is given as the detection time, the surface density of the nucleic acid detection sensor 100 is set to (Dt) ⁻¹ or more when D is the diffusion constant of the nucleic acid molecule (1.6 × 10 ⁻⁶ cm ² / s). For example, the nucleic acid molecule can be detected within the detection time t. In other words, the surface density of the nucleic acid detection sensor 100 is such that the sample droplets are introduced at a higher density than at least one MOSFET nucleic acid detection sensor 100 in a circle whose radius is the diffusion distance of nucleic acid molecules. An array of nucleic acid detection sensors 100 is formed at an integrated density such that it exists in the region to be formed.

For example, when the area density of the nucleic acid detection sensor 100 is 10 ⁶ / cm ² on a chip substrate, the distance between adjacent sensors is about 10 μm. At this time, as a scale l representing the diffusion distance of the nucleic acid molecule,
l = (Dt) ^1/2 (Formula 8)
Since t is calculated as several seconds, detection in at least several minutes is possible. That is, in a few minutes, the nucleic acid molecule is considered to bind to any of a large number of existing sensors. Of course, if the surface density of the number of sensors is further increased, an increase in detection speed can be expected.

  It is also possible to perform high-speed quantitative analysis using a chip in which the nucleic acid detection sensors 100 are densely arranged. As a method proposed so far, there is a method as described in JP-A-2004-309462. According to this method, as shown in the upper part of FIG. 5, a large number of nucleic acid probe molecules exist in a large sensor area, and a signal obtained by binding only a part of the nucleic acid probe molecule to the target nucleic acid molecule is buried in the background signal. In order to prevent this, it has been proposed to use an electrode with a small area as shown in the middle part of FIG. Although this method can also detect nucleic acids with high sensitivity, there is a drawback that the reaction time must be lengthened to increase sensitivity.

  On the other hand, in the embodiment of the present invention, unlike this method, as shown in the lower part of FIG. 5, it is only necessary to bind a nucleic acid to any one of a large number of sensors. In the case of quantitative analysis, the number of sensors determined to have detected nucleic acids by digital values is counted. The higher the concentration of nucleic acid, the greater the number of sensors that have detected the nucleic acid. Based on the ratio of the number of nucleic acid detection sensors that have detected nucleic acids to the total number of nucleic acid detection sensors, the nucleic acid concentration of the target nucleic acid molecule contained in the sample is estimated.

  Furthermore, in the case of quantitative analysis of many types of nucleic acids, as shown in FIG. 6, an array of sensors to which a nucleic acid probe having a certain base sequence is fixed is arranged on the surface of a plurality of chips, and a sample is introduced here. do it.

  Of course, it is not necessary to arrange the nucleic acid probe molecules 102 of the same type together at one place. Even if they are arranged regularly or randomly, quantitative analysis of nucleic acids is possible if the surface density is constant.

(Modification of this embodiment)
Unlike FIGS. 2 and 4, a nucleic acid detection apparatus using a differential pair will be described with reference to FIGS.
The apparatus shown in FIGS. 7 and 8 includes a circuit that uses a differential pair to determine whether or not a change in the threshold voltage of the MOSFET included in the nucleic acid detection sensor 100 to which the nucleic acid probe molecule 102 is attached has occurred. It is a waste. Whether or not a nucleic acid is detected in the nucleic acid detection sensor 100 provided in the present embodiment can also be determined using a circuit using this differential pair. That is, the MOSFET used as the nucleic acid detection sensor 100 and the MOSFET used as the zero level detection sensor 200 are arranged in the differential pair.

  For example, as shown in FIG. 8, the output voltage itself generated by setting the potential of the reference electrode 201 to a predetermined value is measured, or, as shown in FIG. 7, two more transistors are inserted than in FIG. Then, the nucleic acid may be detected as a change in the offset voltage of the voltage follower circuit formed thereby.

  Further, for example, as shown in FIG. 9, even if a double-gate MOS structure element capable of controlling the potential of the back gate is used, the MOSFET included in the nucleic acid detection sensor 100 to which the nucleic acid probe molecule 102 is attached is used. It can be determined whether or not a threshold voltage change has occurred. That is, the potential of the nucleic acid detection sensor 100 to which the nucleic acid probe molecule 102 is immobilized and the zero level detection sensor 200 are controlled via the reference electrode 201, and the fluctuation of the offset voltage of the voltage follower circuit is controlled as in FIG. Can be measured.

  According to the above-described embodiment, the sensitivity is improved by setting the gate width of the FET of the detection sensor to about the Debye length of the electron in the channel region and setting the gate length to about the Debye length of the electron in the channel region. It can be improved dramatically. Furthermore, it is possible to detect one nucleic acid molecule at a very high speed. In addition, by arranging a plurality of detection sensors densely on the detection chip, it is possible to simultaneously provide a quantitative analysis technique in a very wide concentration range. In addition, high-precision measurement can be performed in a short time without performing labeling of the target nucleic acid or amplification of nucleic acid such as PCR (polymerase chain reaction).

  Note that the present invention is not limited to the above-described embodiment as it is, and can be embodied by modifying the constituent elements 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.

The figure which shows one of the sensors for nucleic acid detection arrange | positioned in multiple numbers by the nucleic acid detection chip concerning this embodiment of this invention. The figure which shows an example of the nucleic acid detection apparatus which detects a nucleic acid using the nucleic acid detection sensor of FIG. The flowchart which shows operation | movement of the nucleic acid detection apparatus of FIG. The figure which shows the nucleic acid detection apparatus which is a modification of FIG. The figure which shows the principle of quantitative analysis. The figure which shows the case where the quantitative analysis of the nucleic acid of another kind is carried out. It is a modification of FIG. 2, Comprising: The figure which shows an example of the nucleic acid detection apparatus which uses a differential pair. FIG. 9 is a diagram showing another example of the nucleic acid detection apparatus in FIG. 7 which is a modification of FIG. 2 and uses a differential pair. FIG. 5 is a diagram showing an example of a nucleic acid detection apparatus using a double gate MOS, which is a modification of FIG. 2.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 ... Sensor for nucleic acid detection, 101 ... Gate, 102, 217 ... Nucleic acid probe molecule, 103 ... Source, 104 ... Drain, 105 ... Gate oxide film, 106 ... SOI 107 ... buried oxide film (BOX), 108 ... silicon substrate, 109 ... target nucleic acid molecule, 200 ... sensor for zero level detection, 201 ... reference electrode, 202 ... reference voltage Power source, 203 ... charging voltage source input terminal, 204, 205 ... charging switch, 206 ... control pulse input terminal, 207 ... power supply voltage, 208 ... reference potential, 209 ... Sense amplifier control switch, 210, 211 ... Storage capacitor, 212, 213 ... Amplifier, 214, 401 ... Sense amplifier, 215, 216 ... MOSFET, 02 ... differential amplifier, 403 ... output amplifier, 405 ... output signal terminal.

Claims (8)

In a nucleic acid detection sensor for detecting a target nucleic acid molecule having a specific sequence contained in a specimen based on the intensity of characteristic modulation of an FET (field-effect transistor),
Immobilizing at least one probe nucleic acid molecule capable of hybridizing with the target nucleic acid molecule to the gate of the FET;
ε ₀ is the vacuum dielectric constant, ε _r is the relative dielectric constant of the channel region, k _B is the Boltzmann constant, T is the absolute temperature of the channel region of the FET, e is the elementary charge, and n is the carrier density. width _{(ε 0 ε r k B T} / e 2 n) 1/2
Nucleic acid detection sensor, characterized in that   The nucleic acid detection sensor according to claim 1, wherein the gate length of the FET is on the same order as the gate width of the FET and is larger than the gate width. In a nucleic acid detection sensor for detecting a target nucleic acid molecule having a specific sequence contained in a specimen based on the intensity of characteristic modulation of an FET (field-effect transistor),
Immobilizing at least one probe nucleic acid molecule capable of hybridizing with the target nucleic acid molecule to the gate of the FET;
ε ₀ is the vacuum dielectric constant, ε _r is the relative dielectric constant of the channel region, k _B is the Boltzmann constant, T is the absolute temperature of the channel region of the FET, e is the elementary charge, and n is the carrier density. long, _{(ε 0 ε r k B T} / e 2 n) 1/2
Nucleic acid detection sensor, characterized in that In a nucleic acid detection chip comprising a plurality of nucleic acid detection sensors according to any one of claims 1 to 3,
When the detection time is t and the diffusion constant of the nucleic acid molecule is D = 1.6 × 10 ⁻⁶ cm ² / s, the number of the nucleic acid detection sensors per unit area on the nucleic acid detection chip is
1 / Dt
A nucleic acid detection chip characterized by being on the order of or higher.   5. The nucleic acid detection chip according to claim 4, wherein the nucleic acid concentration of a target nucleic acid molecule contained in the sample is estimated based on a ratio of the number of nucleic acid detection sensors that detect nucleic acids to the total number of nucleic acid detection sensors. . A nucleic acid detection sensor according to any one of claims 1 to 3,
A nucleic acid probe molecule is different from the nucleic acid probe molecule, and a zero level detection sensor in which a nucleic acid probe molecule having a base sequence that is not complementary to a nucleic acid molecule contained in a specimen is fixed to a gate;
Two capacitive elements respectively connected to the drain terminals of the sensors;
A sense amplifier that discharges the charge of each capacitive element charged at a predetermined voltage value through an FET included in each sensor, and amplifies a difference in discharge efficiency from each FET at that time,
A nucleic acid detection apparatus comprising: determination means for determining presence or absence of nucleic acid detection based on the difference in discharge efficiency. A nucleic acid detection sensor according to any one of claims 1 to 3,
A nucleic acid probe molecule is different from the nucleic acid probe molecule, and a zero level detection sensor in which a nucleic acid probe molecule having a base sequence that is not complementary to a nucleic acid molecule contained in a specimen is fixed to a gate;
A differential pair using an FET included in each sensor as an input transistor;
A nucleic acid detection apparatus comprising: a determination unit that determines whether or not nucleic acid is detected based on a magnitude of an output voltage of a differential pair generated by applying a common reference voltage to the differential pair. In a nucleic acid detection chip comprising a plurality of nucleic acid detection sensors according to any one of claims 1 to 3,
When the detection time is t and the diffusion constant of the nucleic acid molecule is D, the number of the nucleic acid detection sensors per unit area on the nucleic acid detection chip is
1 / Dt
A nucleic acid detection chip characterized by being on the order of or higher.

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