JP5294339B2 - Method for detecting a substance to be detected in a sample - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detection method of a substance to be detected in a sample capable of reducing a noise, and having far superior sensitivity than ever before. <P>SOLUTION: In this method for detecting a substance to be detected in a sample by a sensor including a substrate 1, and a channel having a source electrode 3 and a drain electrode 4 provided oppositely at a prescribed interval on an insulating thin film 2 formed on the upper surface of the substrate 1, the channel is constituted of ultrafine fibers, and after dropping sample solution onto the channel, a solvent in the sample solution is vaporized. <P>COPYRIGHT: (C)2011,JPO&amp;INPIT

Description

  The present invention relates to a method for detecting a substance to be detected in a sample using a sensor, and in particular, a structure of a field effect transistor (hereinafter abbreviated as FET) or a single electron transistor (hereinafter abbreviated as SET). The present invention relates to a method for detecting a substance to be detected in a sample by using a sensor such as a biosensor having the above.

  The conventionally proposed biosensor forms a thin film on the electrode with a reactive group that selectively reacts with a specific molecule, and measures the change in potential when the thin film adsorbs the specific molecule. ing. Specifically, this is a method of detecting the amount of glucose by forming a thin film having glucose oxidase on an electrode and measuring a change in current value accompanying an oxidation reaction with glucose.

  Examples of this type of biosensor include the following Patent Documents 1 to 5 and Non-Patent Documents 1 and 2.

JP-A-10-260156 International Publication No. 02/48701 International Publication No. 01/44796 JP 2002-118248 A Japanese Patent Publication No.58-019984 Aizawa, chemical communication. 945 pages (1989) Alexander Star, Jean-Christophe P, Gabriel. Keith Bradley, and George Gruner, Vol. 3, No. 4, 459-463 (2003)

  However, since the conventional biosensor is a method that directly detects the current value associated with the chemical reaction as described above, the detection sensitivity is low and it is difficult to detect low concentrations of glucose. There was a drawback that the feature of selectivity could not be fully exhibited.

  An object of the present invention is to provide a method for detecting a substance to be detected in a sample, which can eliminate such drawbacks in the prior art, can reduce noise, and has a sensitivity far superior to that of the prior art.

In order to achieve the above object, a first means of the present invention includes: a substrate; and a channel having a source electrode and a drain electrode provided on the insulating thin film formed on the upper surface of the substrate so as to face each other at a predetermined interval. In a method of detecting a substance to be detected in a sample solution with a sensor having at least
The channel is composed of ultrafine fibers;
The sample solution is dropped on the channel, and then the solvent of the sample solution is evaporated.

In order to achieve the above object, the second means of the present invention comprises a substrate and a source electrode and a drain electrode provided on the first insulating thin film formed on the upper surface of the substrate so as to face each other at a predetermined interval. In a method for detecting a substance to be detected in a sample solution with a sensor having at least a channel having
The channel is composed of ultrafine fibers;
A second insulating thin film is formed on the surface of the substrate opposite to the channel, a back gate electrode is provided outside the second insulating thin film, and the second insulating thin film interacts with the substance to be detected. Modified with certain substances to
The sample solution is dropped between the modified portion and the back gate electrode, and then the solvent of the sample solution is evaporated.

According to a third means of the present invention, in the first or second means, the ultrafine fiber is a carbon nanotube.

According to a fourth means of the present invention, in the third means, a defect is introduced into the carbon nanotube.

According to a fifth means of the present invention, in the second means, the substance to be detected and the specific substance are interacting biopolymers.

According to a sixth means of the present invention, in the fifth means, the substance to be detected is an antigen or an antibody, and the specific substance is an antibody or an antigen.

  The present invention is configured as described above, and can provide a method for detecting a substance to be detected in a sample that can reduce noise and has a sensitivity far superior to that of the prior art.

It is a perspective view of a sensor concerning an embodiment of the present invention. It is a schematic block diagram of the sensor. It is the schematic which shows a mode that a to-be-detected substance is detected using the sensor. It is a schematic perspective view which shows the other detection mode of the to-be-detected substance of the sensor which concerns on embodiment of this invention. It is an enlarged schematic diagram between the insulating substrate and the gate electrode of the sensor. It is a schematic block diagram which shows a mode that a carbon nanotube is grown and formed in embodiment of this invention. It is a figure which shows the room temperature Coulomb diamond characteristic by a carbon nanotube single electron type transistor. It is a schematic perspective view which shows a mode that a carbon nanotube is grown and formed by the conventional method. It is a schematic perspective view which shows a mode that a carbon nanotube is grown and formed by the method of this invention. It is a schematic perspective view which shows the example of an arrangement | sequence of the catalyst by the method of this invention. It is an expansion perspective view of the catalyst. It is the top view (a) and sectional drawing (b) of the sensor which do not give a 2nd method. It is the top view (a) and sectional drawing (b) of a sensor which show the state after dripping a solution on the sensor. It is the top view (a) and sectional drawing (b) of the sensor of this invention. It is the top view (a) and sectional drawing (b) of a sensor which show the state after dripping a solution on the sensor. It is sectional drawing which shows the state which modified the back gate electrode with the sensor of this invention. It is sectional drawing which shows the state which carried out the molecular modification of the carbon nanotube directly with the sensor of this invention. It is sectional drawing which shows the state which modified the carbon nanotube with the indirect molecule | numerator with the sensor of this invention. It is a schematic block diagram which shows the other structure of the sensor of this invention. It is a schematic block diagram which shows other structure of the sensor of this invention. It is an IV characteristic curve figure at the time of FITC detection by the sensor of this invention. It is an IV characteristic curve figure at the time of Ni ion detection by the sensor of this invention. It is an IV characteristic curve figure at the time of hemagglutinin detection by the antigen antibody reaction of the sensor of this invention. It is an IV characteristic curve figure at the time of hemagglutinin detection by the antigen antibody reaction of the sensor of this invention. It is an IV characteristic curve figure at the time of hemagglutinin detection by the antigen antibody reaction of the sensor of this invention. It is an IV characteristic curve figure at the time of hemagglutinin detection by the antigen antibody reaction of the sensor of this invention. It is an IV characteristic curve figure at the time of hemagglutinin detection by the antigen antibody reaction of the sensor of this invention. It is an IV characteristic curve figure at the time of hemagglutinin detection by the antigen antibody reaction of the sensor of this invention. It is an IV characteristic curve figure at the time of hemagglutinin detection by the antigen antibody reaction by the sol gel method of the sensor of this invention. It is an IV characteristic curve figure at the time of hemagglutinin detection by the antigen antibody reaction by the sol gel method of the sensor of this invention. It is an IV characteristic curve figure at the time of hemagglutinin detection by the antigen antibody reaction by the sol gel method of the sensor of this invention. It is an IV characteristic curve figure at the time of hemagglutinin detection by the antigen antibody reaction by the sol gel method of the sensor of this invention. It is an IV characteristic curve figure at the time of hemagglutinin detection by the antigen antibody reaction by the sol gel method of the sensor of this invention. It is an IV characteristic curve figure at the time of hemagglutinin detection by the antigen antibody reaction by the sol gel method of the sensor of this invention. It is an IV characteristic curve figure at the time of calmodulin detection by the antigen antibody reaction of the sensor of this invention. It is an IV characteristic curve figure at the time of calmodulin detection by the antigen antibody reaction of the sensor of this invention.

  Next, embodiments of the present invention will be described with reference to the drawings. FIG. 1 is a perspective view of a SET biosensor according to an embodiment of the present invention, and FIG. 2 is a schematic configuration diagram of the SET biosensor.

In these drawings, 1 is a chip-like insulating substrate, 2 is an insulating thin film coated on the insulating substrate 1 and having a functional group such as a hydroxyl group, an amino group, or a carboxylic acid group on its surface (in this embodiment, a hydroxyl group). insulating thin film made of SiO _2), 3 and 4 at the source electrode and the drain electrode are formed at predetermined intervals on the thin film 2, prongs 5 and 6 on opposite portions of both electrodes 3 and 4 formed with (See FIG. 1).

  A carbon nanotube (hereinafter abbreviated as CNT) 7 into which defects are introduced is grown and formed between the tip portions 5 and 6 of both electrodes 3 and 4. A gate electrode 8 is formed on the surface of the substrate 1 opposite to the thin film 2.

  For the insulating substrate 1, for example, an inorganic compound such as silicon oxide, silicon nitride, aluminum oxide, or titanium oxide, or an organic compound such as acrylic resin or polyimide is used. For the electrodes 3, 4, and 8, for example, a metal such as gold, platinum, or titanium is used. The electrical connection relationship between the electrodes 3, 4 and 8 is as shown in FIG.

  In this embodiment, CNT is used as the nanotube-like structure. By using this nanotube-like structure, a very fine channel can be formed, and a highly sensitive sensor can be obtained.

  Note that a gap G is formed under the CNT 7 as shown in FIG. In this way, a sensor having a SET structure is configured. The basic configuration of SET and FET is the same, but in the channel used as a current path, the SET channel has a quantum dot structure, and the FET channel does not have a quantum dot structure. ing.

  In this transistor (SET and FET), the current value between the source electrode 3 and the drain electrode 4 changes sensitively to changes in the charge (more strictly, the spin electron state) on the gate electrode 8 and the CNT 7. Generally, SET is more sensitive than FET. In addition, it is rare that SET characteristics are observed without modification after CNT generation. However, by setting the CNT on the FET to the CNT generation temperature (high temperature of about 900 ° C.), the CNT is partially Breaks to form an island and exhibits SET current characteristics. The same result can be obtained by flowing a current (about − + several mA) larger than the operating current (about several μA).

  In the present invention, when a molecule adheres (interacts) to the gate electrode 8 or the CNT 7 of these transistors, the electronic state on the CNT changes indirectly or directly. From the change in the current between the four, molecules attached (interacted) can be detected. It is also possible to detect a molecule from a change in current when the gate electrode 8 or the CNT 7 itself is molecularly modified, or to detect a reaction between the modified molecule and another molecule.

  In particular, when the gate electrode 8 or the CNT 7 is modified (immobilized) with an antibody (or antigen), a specific antigen (or antibody) can be detected using an antibody-antigen reaction. By this technique, microorganisms such as pathogenic viruses and bacteria of infectious diseases can be detected with high sensitivity and at high speed. This method can be effectively used for prevention by early detection of infectious diseases and research of microorganisms, and since the element (sensor) itself is remarkably small, it is taken to the field and used for detection of infectious disease viruses and for these studies. be able to.

  FIG. 3 is a schematic view showing a state in which a substance to be detected is detected using the sensor. As shown in the figure, the sensor has a molecule detection portion 18 and a signal conversion portion 19, which are closely related.

In the figure, 12 is a protective film made of SiO ₂ , 13 is a specific substance (for example, an antibody) that selectively reacts or adsorbs (interacts with) the substance to be detected, and 14 selectively reacts with the specific substance 13. Alternatively, a substance to be detected (for example, an antigen) adsorbed (interacted), 15 is a sample solution containing the substance 14 to be detected.

  FIG. 4 and FIG. 5 are schematic views showing another state of detecting a substance to be detected using the sensor of the present invention, and FIG. 5 is an enlarged schematic view between the insulating substrate 1 and the gate electrode 8 of the sensor. is there. In this example, the sample solution 15 containing the substance to be detected 14 is interposed between the insulating substrate 1 and the gate electrode 8 to detect the substance 14 to be detected. In FIG. 5, reference numeral 20 is a molecule that maintains the orientation of a specific substance (for example, an antibody), and 21 is a substance other than the substance to be detected that exists in the sample solution 15. FIG. 5 shows a state in which a target substance (for example, antigen) 14 selectively interacts with a specific substance (for example, antibody) 13 such as reaction or adsorption.

Next, control of basic conductivity characteristics of CNT will be described.
(1) The type of catalyst used when applying CNT growth, applying an electric or magnetic field to arbitrarily design the growth position, direction, number, chirality, characteristics, etc. of the CNTs that are the basic elements of a biosensor device Optimize the shape and shape.

  FIG. 6 is a schematic configuration diagram showing an example of a method of patterning a catalyst and controlling the position and direction of CNTs while applying an electric field.

In the figure, 1 is an insulating substrate, 2 is a thin film made of SiO ₂ coated on the insulating substrate 1, 9a and 9b are catalyst layers made of iron or the like patterned on the SiO ₂ thin film 2, and 7 is With the CNT formed between the catalyst layers 9a and 9b by applying an electric field, the growth position, direction, number, chirality, characteristics, and the like are arbitrarily controlled. 10 is a reaction vessel, 11 is a hydrocarbon gas such as methane gas which is a raw material of CNT. The grown CNT has a length of about several μm (for example, about 3 μm), a diameter of about several nm, and is an ultrafine fibrous aggregate.

(2) Using the CNTs with controlled growth positions, directions, characteristics, etc. as non-invasive electrodes, a shape in the 4-probe method is produced.

  In the four-probe method, four needle-shaped electrodes (for example, electrode A, electrode B, electrode C, and electrode D) are placed on a sample in a straight line, and the outer two probes (for example, electrode A and electrode D) are arranged. A constant current is passed between them, the potential difference generated between the two inner probes (for example, electrode B and electrode C) is measured to obtain a resistance value, and the obtained resistance value is multiplied by the thickness of the sample and the correction coefficient RCF to obtain a sample. This is a method of calculating the volume resistance value.

(3) The portion where the electrode and the channel (CNT) overlap is localized using a high electric field electron beam or STM (Scanning Tunneling Microscopy) / AFM (Atomic Force Microscope). Welding is performed by an applied electric field to integrate the electrode and the channel (CNT).

(4) Next, the transport characteristics of the CNT are evaluated. The electrical conductivity characteristics to be evaluated include ballistic conductivity characteristics, whether spin injection is possible, and whether spin transport is possible.

(5) It has already been confirmed in the preliminary experiments of the present inventors that the introduction of defects into CNTs significantly changes the electrical characteristics of CNTs (by introducing defects into CNTs, Preliminary experiments confirm that a SET having a high Coulomb energy of 5000 K and operating at room temperature can be formed).

  Therefore, by introducing defects to the CNTs arbitrarily by STM / AFM processing or electron beams, CNTs whose conductivity characteristics can be controlled can be obtained.

  As a specific example of this CNT defect introduction method, for example, there is a method in which annealing is performed at substantially the same temperature (for example, about 800 ° C.) as when CNT is produced, and then naturally cooled. Here, the defect of CNT refers to a change in the shape of CNT, such as a part of carbon atoms jumping out by heat and barely connected in a state where CNT is cut off, The structure is not clear at present.

(6) The correlation between the defects in the CNT and the electrical characteristics of the CNT is examined. For example, the density, distribution, and size (size, energy barrier, etc.) of defects are evaluated by the scanning probe method (Kelvin probe method, Maxwell probe method, etc.), and the correlation between the defects in the CNT and the electrical characteristics of the CNT To clarify. Thus, by grasping the correlation between the defects in the CNTs and the electrical characteristics, it is possible to manufacture a SET with good reproducibility and uniformity of characteristics.

(7) According to the above (6), the electrical characteristics of the carbon nanotube can be controlled by controlling the introduction of defects.

  In the present invention, a SET that operates at room temperature can be manufactured using CNTs into which defects are introduced. Although the case where CNTs with defects are used is described here, CNTs without introducing defects can also be used.

  In order to avoid malfunctions due to floating charges and mobile charges, which were problems in conventional SETs, in the present invention, when two CNT-based SETs are made close to each other and a single charge is detected, both SETs are detected. The AND of the output characteristics (room temperature) is taken. As a result, both SETs operate only when there is a true charge, so that malfunctions due to floating charges and mobile charges can be avoided.

  In order to further increase the measurement speed, a system that uses a resonance circuit to operate with an alternating current is employed instead of the conventional direct current method using the above-described method. As described above, a single charge distribution can be measured at room temperature and at high speed without malfunction.

  FIG. 7 is a diagram showing room temperature coulomb diamond characteristics by SET using CNT. From this room temperature Coulomb diamond characteristic, it can be proved that the SET using the CNT of the present invention can operate at room temperature.

The SET of CNT used as well as formed on the substrate 1 as shown in FIG. 1, and coated with a protective film 12 made of a chip of SiO ₂ in order to operate in solution, as shown in FIG. 3, the SiO ₂ protective film A specific substance 13 such as an antibody is immobilized on 12. In this example, the protective film 12 is provided, but the protective film 12 may not be provided in some cases.

  A biosensor according to the present embodiment is installed in a sample solution 15 in which a substance to be detected 14 such as DNA is dissolved, and is operated with an alternating current using a resonance circuit. By measuring the interaction, the detection target substance 14 can be detected (evaluation of surface charge distribution characteristics).

  Next, the production of the signal conversion part of the sensor using CNT will be described in detail. Here, an FET-type or SET-type transistor is manufactured using the semiconductor properties of CNT. The manufacturing method includes a process of vapor deposition of a catalyst by a general lithographic method, CNT growth by thermal CVD, and an electrode manufacturing process.

  However, this has the following problems. First, control of CNT growth is not easy. Several methods of CNT growth have been proposed, but in the case of producing an element in which the electrodes of the signal conversion part are connected by a single CNT, the yield and structural stability of CNT cross-linking between the electrodes in the end is important. For this reason, it is important to determine the conditions (temperature, type of gas, flow rate, electric field, magnetic field, etc.) of the catalyst (mutual position, structure, size, etc.) and thermal CVD method.

  Furthermore, an electrode is prepared after CNT growth on the catalyst. At that time, the electrode may be peeled off from the substrate or a crack may be generated in the electrode, and the contact potential with the CNT may change the characteristics of the device. There is a possibility of affecting the strength, and in order to obtain stable current characteristics, it is necessary to study electrode materials.

  Therefore, in the embodiment, a novel technique (a first technique described later) is used particularly for various elements of the catalyst. In addition, when the CNT is directly modified with molecules, the solvent containing the molecules may cover the electrode, and the influence of the solvent covering the electrode surface on the connection between the electrode and a measuring instrument such as a probe will cause this. A prevention method (second method described later) was used.

  Further, even when a back gate type or side gate type element is used, a sample or vapor containing the sample may affect the gate electrode or the side gate electrode. This could be avoided by protecting the CNT (third method described later). In fact, in the detection results of various reactions using the back gate electrode and the side gate electrode, the detected substance vaporized and adhered to the CNT surface as well as the gate electrode and the side gate electrode. There is a possible example.

Specifically, in the first method, in order to form CNT growth nuclei, a catalyst is deposited on the SiO ₂ film using an electron beam lithography method. In this embodiment, both surfaces of the Si substrate having a thickness of 380 μm are covered with SiO ₂ films having a thickness of about 300 nm. The present invention relates to a technique in which a transition metal such as iron, nickel, cobalt, molybdenum, tungsten, or a catalyst containing these transition metal fine particles is used on the SiO ₂ film, and the catalyst is used as a CNT growth nucleus.

FIG. 8 is a diagram for explaining a conventional method. In FIG. 8, 1 is an Si insulating substrate having SiO ₂ films formed on both surfaces, 7 is CNT, 9a and 9b are catalysts, and 22a and 22b are electrodes later. Is the position where is formed. In the conventional method, as shown in the figure, the catalysts 9a and 9b are formed by vapor deposition one by one at a predetermined interval, and the CNT 7 grown from one catalyst 9a reaches the paired catalyst 9b. Connection between the catalysts 9a and 9b by the CNT 7 can be made.

  FIG. 9 is a diagram for explaining an embodiment (first method) of the present invention. As shown in FIG. 9, a plurality of dot-shaped catalysts 9a-1, 9a-1, 9a-n are arranged side by side, and a plurality of dot-shaped catalysts 9b-1, 9b-2, ... 9b-n are also formed at the position 22b where the other electrode is formed. It is formed so as to face the catalysts 9a-1, 9a-2, ... 9a-n. Thus, by increasing the number of installed catalysts 9, that is, the growth nuclei of CNTs and arranging them closely, it is possible to remarkably increase the probability that CNTs that originally tend to grow randomly from the catalyst 9 reach the paired catalysts 9. By this method, the yield can be increased by 10 times or more as compared with the prior art.

  FIG. 10 is a diagram illustrating an arrangement example of the catalyst 9 according to the present embodiment (first method). Each of the catalyst rows 9a-1, 9a-2,... 9a-n and the other catalyst row 9b-1 are arranged densely so that the distance L1 between the adjacent catalysts is 2 μm. , 9b-2,... 9b-n, the interval L2 is 4 μm. Note that the number of installed catalysts 9, the interval L1, and the interval L2 can be arbitrarily set.

  FIG. 11 is an enlarged perspective view of the catalyst 9. As shown in the figure, the catalyst 9 includes a support layer 25 made of Si or the like having a thickness of 50 nm, an intermediate layer 26 made of a transition metal such as Mo, Ta, or W having a thickness of 10 nm formed on the support layer 25. The formed top layer 27 is made of a transition metal such as Fe, Ni, and Co having a thickness of 3 nm. Therefore, the total height H of the catalyst 9 is 63 nm and the diameter D is 2 μm. The catalyst 9 having a multilayer structure is patterned by a thin film forming technique such as vapor deposition, sputtering, or ion plating.

  As shown in FIG. 6, the insulating substrate 1 on which the catalyst 9 is formed is placed in a reaction vessel 10 of a thermal CVD apparatus, and then a hydrocarbon gas 11 such as methane or ethane is injected to grow CNT 7 on the catalyst 9. Let

  In this embodiment, the growth of CNT 7 was performed according to the following procedure. The insulating substrate 1 on which the catalyst 9 was formed was heated from room temperature to 900 ° C. for 15 minutes. At this time, Ar was allowed to flow into the reaction vessel 10 at a flow rate of 1,000 sccm (a gas flow rate for 1 minute). While maintaining this temperature, methane and hydrogen are introduced at a flow rate of 1,000 sccm and 500 sccm, respectively, for 10 minutes, and then the reaction vessel 10 is cooled to room temperature over 120 minutes. At this time, Ar gas was allowed to flow into the reaction vessel 10 at 1,000 sccm.

  After producing CNTs 7 in this manner, electrodes (source electrode 3 and drain electrode 4) were deposited on the insulating substrate 1. For the electrode, Au is vapor-deposited or Ti is vapor-deposited, and then the surface is covered with Au. In particular, the latter has a feature that the peeling from the substrate and the generation of cracks in the electrode are small. The width of the electrode covering the catalyst is about 10 μm.

  Next, the second method described above will be described. A large number of electrodes (about 50 to 400) are formed simultaneously. When the CNT is directly modified, a solution containing the decorative molecule may be dropped on the CNT, and depending on the amount of the solution, the entire electrode may be covered. Once the surface of the electrode is covered with a solution, when measuring the current between the electrodes connected by CNT, a film is formed between the probe of the measuring device such as a prober and the electrode, and an accurate current value is obtained. There is no possibility.

  12 and 13 are diagrams for explaining a sensor not subjected to the second method. FIG. 12 shows a state before the solution is dropped, and FIG. 13 shows a state after the solution is dropped. In both figures, (a) is a plan view and (b) is a cross-sectional view. In the conventional sensor, when the electrodes 3 and 4 are small in size, as shown in FIG. 13, the entire electrodes 3 and 4 are often covered with a coating 28 formed by dropping the solution. Since the value of the current flowing between the electrodes 3 and 4 is as small as about 1 μA, if the film 28 exists between the probe of the measuring apparatus and the electrodes 3 and 4, accurate current measurement cannot be performed.

  Therefore, in the present invention, as shown in FIGS. 14 and 15, the length L3 of the electrodes 3 and 4 [see FIG. 14 (a)] is about 1.5 to 3 times longer than that of FIG. In this way, by increasing the length L3 of the electrodes 3 and 4, even if the molecular coating 28 that modifies the CNT 7 is formed, the end 29 of the electrodes 3 and 4 is not covered with the coating 28 (FIG. 15). See). By applying a probe of a measuring device such as a prober to the portion 29 not covered with the coating 28 using an optical microscope, the current flowing between the electrodes 3 and 4 can be accurately measured.

  In the present embodiment, in FIG. 14A, the width w1 of the tip portion of the electrodes 3 and 4 is 10 μm, the width W2 of the portion to which the probe is applied is 150 μm, and the length L3 is 500 μm. As shown in FIG. 14B, the CNT 7 is slightly curved between the electrodes 3 and 4, and a gap G is provided between the CNT 7 and the surface on the substrate 1 side.

  Next, the above-described third method will be described. CNT easily interacts with various molecules including water, its electronic state changes, and this change appears as a change in current value. This can be positively used as a gas sensor, and at the same time becomes a noise source when a back gate electrode, a side gate electrode or the like is used as a sensor.

  Therefore, in this embodiment, the CNT and a part of the electrode are covered with an insulating protective film to reduce noise. An insulating adhesive can be used to form the insulating protective film, but a passivation film widely used for spin coating can also be used. In particular, the increase in current observed when water was applied to the back gate electrode was not observed due to the formation of the insulating protective film. In addition, the formation of this insulating protective film makes it possible to ultrasonically clean the entire device and to clean the back gate electrode and the like with a stronger cleaning agent than ever before.

  The gate electrode of the sensor can be formed at various positions, and can have various structures depending on the use of the sensor and the ease of manufacture. Next, each structure of the sensor will be described.

(A) Structure in which the gate electrode is molecularly modified When a molecule adheres to the SiO ₂ film formed on the substrate, the value of the current flowing between the source electrode and the drain electrode changes. For example, the current value changes by applying a fluorescent molecule FITC (Fluorescein isothiocyanate) to the gate electrode. As an example of the antibody-antigen reaction, the SiO ₂ film is molecularly modified with an antibody (or antigen) and reacted with the corresponding antigen (or antibody) to detect a change in electrical signal. Since the molecule can be modified in a larger region than CNT, it is suitable for detection of many molecules. In addition, since the CNTs are not directly modified, damage to the CNTs due to cleaning after use can be avoided.

FIG. 16 is a diagram showing this structure. As shown in the figure, the SiO ₂ film opposite to the CNT 7 on the insulating substrate 1 is molecularly modified with a specific substance (for example, antibody) 13, and a substance to be detected (for example, an antigen) is interposed between the insulating substrate 1 and the gate electrode 8. ) Including a sample solution 15 is included.

(B) Structure obtained by direct molecular modification of CNT FIG. 17 is a diagram showing a structure obtained by direct molecular modification of CNT7. By directly molecularly modifying CNT7, the change of the electronic state on CNT7 by the modified molecule is larger than that when the backgate electrode 8 is molecularly modified, and this structure has high sensitivity.

(C) Structure obtained by indirectly molecularly modifying CNT FIG. 18 shows a structure obtained by indirectly molecularly modifying CNT7. In order to indirectly modify the CNT7, the CNT7 is covered with an insulating thin film 30 made of an organic compound such as an adhesive as shown in FIG. The modification of the electronic state of the CNT 7 is caused by the change of the electronic state caused in the insulating thin film 30 by the modifying molecule or the molecule attached to the surface, resulting in the change of the current.

(D) Structure using a side gate In this structure, an island is formed in the vicinity of the CNT on the substrate, and this is used as a gate. This structure has the advantage that the CNT7 itself is not damaged due to the modification of the CNT7 directly without taking the effort of molecular modification of the back surface of the substrate (back gate electrode). ing. It is a structure suitable for SET.

  In the case of the structure in which the aforementioned (A) back gate electrode is molecularly modified, it is preferable to stabilize current characteristics by covering a part of the CNT and the electrode with an insulating protective film. In the case of the structure in which (B) CNT is directly molecularly modified and the structure in which (C) CNT is indirectly molecularly modified, the electrodes 3 and 4 are covered with a film as described with reference to FIG. It is preferable to form a portion 29 that is not broken.

  FIG. 19 is a schematic configuration diagram for explaining still another structure. In this structure, the insulating substrate 1 itself is used as a channel (back channel), and the electrodes 3 and 4 are provided on the substrate 1 with the CNTs 7 therebetween. A recess 16 serving as a channel is formed on the back surface of the substrate 1, and the recess 16 can be detected on the back surface of the insulating substrate 1 by wetting it with a liquid containing a detection target substance.

  FIG. 20 is a schematic configuration diagram for explaining still another structure. Even in this structure, the insulating substrate 1 itself is used as a channel (back channel), but a probe 17 made of CNT or the like is provided in the channel of the substrate 1. The integrated back channel and probe 17 can be used, for example, as a probe of a scanning probe microscope.

Next, specific examples of the present invention will be described. As a preliminary experiment, a solution containing FITC, which is a fluorescent molecule, was dropped between the SiO ₂ film and the back gate electrode, and changes in current characteristics were observed. FIG. 21 shows IV characteristics when the gate voltage is −20 V and the FITC concentration is 0.64 nM.

  In the figure, the vertical axis represents the value of current flowing between the source electrode and the drain electrode (source-drain current) (A), and the horizontal axis represents the voltage value between the source electrode and the drain electrode (voltage between the source and drain) (V ). Also, the dotted line in the figure is the IV characteristic curve before FITC is attached, and the solid line is the IV characteristic curve after FITC is attached.

  As is apparent from this figure, it can be seen that the IV characteristics greatly change before and after the deposition of FITC.

(Specific example 1)
Next, detection of divalent ions using ion reaction will be described. The CNT of the CNT biosensor was directly modified with pyrene, and N- [5- (3′-maleimidopropylamino) -1-carboxypentyl] iminodiacetic acid {N- [5- (3′-Maleimidopropylamimo) -1- Carboxypentyl] iminodiacetic acid (hereinafter abbreviated as NTA) was bonded to the back gate electrode, a solution containing Ni ions was dropped, and the conduction characteristics were examined by IV characteristics in each case.

  FIG. 22 shows IV characteristics when no electric field is applied to the gate electrode. The vertical axis represents the current value (A) flowing between the source electrode and the drain electrode, and the horizontal axis represents the voltage value (V) between the source electrode and the drain electrode. In the figure, di is an IV characteristic curve after cleaning the back gate electrode, nta is an IV characteristic curve after NTA bonding, and ni is an IV characteristic curve after dropping a solution containing Ni ions. .

  As is apparent from this figure, the current increases by increasing the voltage between the source electrode and the drain electrode, but dv (the voltage between the source and the drain) in all systems (di, nta, and ni systems) = In the vicinity of 0 V, the current hardly increases and a semiconductor property is observed.

  Compared to the IV characteristic curve (di) after cleaning, the IV characteristic curve (nta) after NTA is bonded to the back gate electrode shows a significant decrease in current. In contrast, when Ni ions are added to the system, the current increases as shown by the IV characteristic curve (ni).

  Since NTA reacts not only with Ni ions but also with other divalent positive ions, other divalent positive ions can also be detected.

(Specific example 2)
Next, detection of anti-hemagglutinin (HA) antibody using an antigen-antibody reaction will be described.

The C-terminal of HA was cleaved at various levels (220, 250, 290, 320) and expression was attempted. Genes were introduced into 293T cells, and the expression of HA protein in the cells was confirmed using monoclonal antibody E2 / 3 and polyclonal antibodies. It was confirmed that the HA protein was secreted into the supernatant by Western blotting. HA _1-290 was expressed in large quantities and purified from the supernatant with a Ni ²⁺ column. The fraction containing the target HA protein was confirmed by ELISA (enzyme-linked immunosorbent assay) and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). It was. Although expression was also _observed for the shorter HA _1-220 , it was not used because it did not react with the monoclonal antibody.

As a control experiment, after binding NTA on the back gate electrode formed through the SiO ₂ film of the CNT biosensor, Ni ions were added to the system, and the dilution ratio of the stock solution was 10 ⁻¹⁰ to 10 ⁻⁵ . An IV characteristic curve was obtained by giving an HA antibody solution. At this time, since the back gate electrode has no HA, the HA antibody does not bind to the back gate electrode with orientation.

Next, it was immobilized on NTA on the back gate electrode formed through a SiO ₂ film by means of a His tag to which HA was previously attached, and an HA antibody was given in the same manner to obtain an IV characteristic curve. These IV characteristic curves are shown in FIGS. The gate voltage was −20V.

FIG. 23 is an IV characteristic curve when a solution containing Ni ions is given after NTA binding, and FIG. 24 is an IV characteristic curve when an HA antibody having a dilution ratio of 10 ⁻¹⁰ is given. 25 is an IV characteristic curve diagram when HA antibody having a dilution ratio of 10 ⁻⁸ is given, and FIG. 26 is an IV characteristic curve diagram when HA antibody having a dilution ratio of 10 ⁻⁷ is given. 27 shows an IV characteristic curve when HA antibody having a dilution ratio of 10 ⁻⁶ was given, and FIG. 28 shows an IV characteristic when HA antibody having a dilution ratio of 10 ⁻⁵ was given. FIG.

  In these figures, the dotted line in the figure indicates that the former back gate electrode has no HA, and the solid line indicates that the latter HA is fixed in advance to the NTA on the back gate electrode by a hysteresis tag.

  As is clear from these figures, when the voltage between the source electrode and the drain electrode is changed from 0 V to 1 V, there is almost no difference in the current value between the source electrode and the drain electrode (solid line and dotted line). When the voltage is increased to 1 V or higher, the current value rapidly increases in a system (solid line) in which HA is assumed to be fixed.

  From this, it is possible to detect HA antibody even in a highly diluted region as compared with conventional methods such as ELISA (e (nzyme-) l (inked) i (mmuno) s (orbent) a (say))). It turns out that it is.

(Specific example 3)
The detection of hemagglutinin (HA) using these antigen-antibody reactions gave similar results even when using the sol-gel method. Their IV characteristics are shown in FIGS. In all systems before the experiment of antigen-antibody reaction, a solution containing Ni ions is given after NTA binding. The gate voltage was −20V.

FIG. 29 is an IV characteristic curve diagram when HA antibody at a dilution rate of 10 ⁻⁷ was given without adding HA antigen, and FIG. 30 was when HA antibody at a dilution rate of 10 ⁻⁶ was given without adding HA antigen. the I-V characteristic curve diagram of FIG. 31 the I-V characteristic curve view when given HA antibody dilution ^10-5 without the HA antigen, Figure 32 dilution 10 after attaching the HA antigen ^- the I-V characteristic curve view when given HA antibody in ^6, FIG. 33 is the I-V characteristic curve view when given HA antibody dilution ^{10 -5} after attaching the HA antigen, FIG. 34 HA antigen It is an IV characteristic curve figure when the HA antibody of the dilution rate ^10-4 was given after attaching ^| subjecting. In these figures, ni is an IV characteristic curve when a solution containing Ni ions is given after NTA binding, and HA is immobilized on NTA on the SiO ₂ film back gate electrode by a His tag pre-attached with HA. Is.

As is clear from these figures, a large change appeared in the current value between the source electrode and the drain electrode particularly when the dilution ratios of the stock solution were 10 ⁻⁵ and 10 ⁻⁴ . Moreover, the detection sensitivity was about ELISA.

(Specific example 4)
Next, detection of anti-calmodulin (CaM) using an antigen-antibody reaction will be described. A DNA fragment containing the CaM gene cDNA was inserted into the SacI-XbaI site of the expression vector pBAD / gIII (manufactured by Invitrogen) to construct a CaM expression vector (pBAD / gIII / calmodulin).

  The vector was introduced into Escherichia coli LMG194 strain to obtain an rCaM expression clone. This claw was inoculated into 2 ml of LB / Ampicillin medium and cultured overnight.

After inoculating 5 ml of this culture solution into LB / Ampicillin medium and culturing with shaking at 37 ° C. until OD ₆₀₀ reaches 0.5, L-arabinose is added to a final concentration of 0.02%. The culture was shaken at 37 ° C for an hour. The cells were collected by centrifugation, suspended in NatI-Ve Binding Buffer (manufactured by Invitrogen), ultrasonically pulverized, partially purified using Probond ^™ Purification System (manufactured by Invitrogen), and then manufactured by HiLoad26 / 60 Superdex 75 pgm (manufactured by BioLoad 26/60 Superdex 75 pgm). ) Was used to purify uniformly by SDS / polyacrylamide electrophoresis to obtain rCaM.

After binding NTA to the SiO ₂ film of the CNT biosensor, rCaM is immobilized on the NTA on the SiO ₂ film with a His tag (His tag) attached in advance, and the dilution ratio of the stock solution is from 10 ⁻⁸ to 10 ⁻² An IV characteristic curve was obtained by giving a CaM antibody at a concentration of 1 to 5%. The result is shown in FIG. The gate voltage was −20V.

Curve (A) in the figure is an IV characteristic curve when washed after binding to NTA, and curve (B) is an IV characteristic curve and curve (C) when rCaM is bound to NTA by a pre-histage tag. Is an IV characteristic curve when an anti-CaM antibody having a dilution ratio of 10 ⁻⁸ is applied and reacted, and curve (2) is an anti-CaM antibody having a dilution ratio of 10 ⁻⁷ and reacted. The IV characteristic curve and curve (e) are the IV characteristic curve and curve (f) when the anti-CaM antibody having a dilution ratio of 10 ⁻⁶ is reacted and the stock solution has a dilution ratio of 10 the I-V characteristic curve when reacted giving anti CaM antibody ^-4, curve (g) is the I-V characteristic curve when the dilution ratio of the stock solution were reacted to give the anti-CaM antibody of ^10-2 It is.

  As is clear from these figures, when the voltage between the source electrode and the drain electrode is changed from 0 V to 0.5 V, the current value changes according to each concentration. From this, it can be seen that the anti-CaM antibody can be detected even in a region where the dilution of the stock solution is very high, like the HA antibody.

  The detection results of anti-CaM antibody and anti-HA antibody using ELISA are shown in the following table. In this measurement procedure, the primary antibody was diluted at the following dilution ratio and allowed to stand for 1 hour, the secondary antibody (anti-mouse HRPO standard antibody) was diluted 5000 times and allowed to stand again for 1 hour, and then TMB. A substrate having an absorption wavelength of 450 nm was produced with a color former and the absorbance was measured.

table

(Anti-CaM antibody) (Anti-HA antibody)
PBS Neg. Con 0.034 0.030
2.5 × 10 ⁻² 2.000 1.722
6.3 × 10 ⁻³ 2.439 2.725
1.6 × 10 ⁻³ 2.899 3.378
3.9 × 10 ⁻⁴ 2.300 3.132
0.98 × 10 ⁻⁴ 0.650 2.839
2.4 × 10 ⁻⁵ 0.177 1.413
6.1 × 10 ⁻⁶ 0.051 0.290

Table 1 shows that it is difficult to detect by ELISA at a dilution of 6.1 × 10 ⁻⁶ . On the other hand, in the specific examples 3 and 4, the sol-gel method shows sensitivity about ELISA, and the others are detected at a dilution of about 10 ⁻⁸ .

  CNTs were grown on the Si substrate, electrodes were formed at both ends thereof, and the back surface of the Si substrate opposite to the surface on which the CNTs were grown was activated with acid (sulfuric acid). -Mercaptopropyltriethoxysilane) is reacted at 180 ° C. to immobilize NTA. Next, Ni ions were added, antigens (CaM, HA) into which histidine was introduced were immobilized, reacted with diluted antibodies, washed, and a negative bias was applied to the back surface of the substrate to measure IV characteristics. .

FIG. 36 is a characteristic diagram showing changes in current value when 1.5 V is applied between the CNT electrodes after reacting the diluted CaM antibody with CaM immobilized as described above. As is clear from this figure, when the antigen was not immobilized, the current value hardly changed, but when immobilized, the current value increased as the antibody concentration increased. It was also found that antibody detection was possible in a dilution range of about 10 ⁻¹⁰ to 10 ⁻⁸ of the antibody stock solution.

When the detection limit of the antibody was examined using FLISA, it was found that the detection limit was obtained by diluting the antibody stock solution by about 10 ⁻⁶ . It was also found that the detection limit differs between CaM and HA and depends on the antigen and antibody.

  In each of the above specific examples, the case where the gate voltage is −20 V has been described. However, it has been found that detection is possible even with a gate voltage of about 0 V or a positive gate voltage with little change in the current value.

  When a CNT biosensor is applied to a solution, noise may be observed, causing a problem in data reliability. Therefore, noise can be remarkably reduced by evaporating the solvent (water) with a blower, a heater, a thermoelectric conversion element (Peltier element) or the like after dropping the sample solution (test solution) onto the sensor. The noise countermeasure is applied to a specific example in which the above solution is applied. In addition, the sample solution (test solution) can be cooled by a thermoelectric conversion element (Peltier element), liquid nitrogen, or the like, and the influence of a solvent such as water can be reduced. In particular, by freezing water to insulate, noise can be greatly reduced.

Conventional methods include ELISA and Western blotting, but their sensitivity is limited to a stock solution dilution ratio of about 10 ⁻⁵ . Sensitivity of the sensor of the present invention to which was subjected to a HA antibody detection, 10 ³ about the ELISA.

  In addition, since an electric signal is used, there are not many chemical reaction processes, and therefore the time required for detection is extremely short. Although the current characteristic was examined with the IV curve, it is possible to acquire the IV curve within a few seconds with a parameter analyzer.

  Conventionally known PCRs require temperature control because they involve temperature changes, but the sensor of the present invention can be used in a constant temperature environment, so temperature control is not required, and the configuration is simplified and miniaturized. Is possible. Examples that can be used in a constant temperature environment include the RT-PCR method, the ICAN method, and the LAMP method, but all of them have a drawback of requiring a long detection time.

  The sensor of the present invention can perform not only a single type of detection but also various types of sensing simultaneously on a single sample, and a plurality of types of detection at the same time. In addition, multiple samples can be detected in parallel using a plurality of sensors.

  The sensor using the nanotube-like structure in the channel of the present invention is strong and can be used repeatedly. However, since it is inexpensive, it can be disposable for detection of dangerous viruses and the like.

  Although the case where CNT is used has been described in the embodiment, it is also possible to use ultrafine fibers that are not in a tube shape.

In the above embodiment, one type of biosensor in which a DNA probe is formed has been described. For example, three CNT biosensors with a SiO ₂ film are provided on a substrate, and a DNA probe, a protein probe, and a sugar are provided on each SiO ₂ film. It is also possible to form lipid probes individually and measure different biopolymers (DNA, protein, glycolipid) simultaneously.

  In the above embodiment, the case where the surface charge distribution characteristics in DNA are evaluated has been described. However, the present invention can also be applied to detection of other biopolymers such as sugar chains, RNA, amino acids, sugars and viruses. It is also possible to detect changes in the electronic state in the process in which a protein such as rhodopsin emits protons in response to light, or changes in the electronic state due to structural changes in the dye.

  In the above-described embodiment, an example in which a nanotube-like structure is connected to the SET channel portion has been described. However, a nanotube-like structure can also be used for the FET channel portion.

  When microorganisms such as viruses enter the human body or other living organisms, antibodies against them start to interact. Therefore, viruses with antibodies can be detected from body fluids using the sensor of the present invention. For example, since the HA shown as the specific example is a protein called spike protein that covers the surface of the influenza virus, it can be detected by the sensor of the present invention, and infectious diseases such as influenza, SARS, and BSE can be detected with high sensitivity and high speed. it can.

  Since the sensor of the present invention has a small detection portion and uses an electric signal, the detection circuit can be formed into a chip, and can be used as a portable and inexpensive detector. This enables testing in the field and can be provided to any medical institution. This not only helps prevent defense against early detection of infectious diseases, but also helps combat bioterrorism.

  Also in the basic science field, it is possible to detect the binding strength of intermolecular interactions at the single molecule level by the sensor of the present invention and classify viruses and proteins by current characteristics. Therefore, it is possible to conduct basic experiments for drug discovery by searching for and designing molecules similar to antibodies. One molecule can also be detected over time. Furthermore, it can also be used as a basic circuit of a spectroscopic antigen-antibody reaction detector.

  By directly modifying the gate electrode and CNT of the sensor of the present invention with DNA, complementary DNA can be detected electrically with extremely high sensitivity. In addition, identification is possible through ultrasensitive and high-speed measurement of microorganisms such as infectious disease viruses and bacteria caused by DNA.

  Furthermore, environmental hormones, toxins, and inorganic substances can be detected by the sensor of the present invention. In addition, since the influence of the vapor of the sample can be detected, the target is not limited to liquid but can be applied to gas, and the concentration of specific substances such as harmful substances in the atmosphere and other gases can be measured.

1: Insulating substrate,
2: Insulating thin film
3: Source electrode,
4: Drain electrode,
7: Carbon nanotube (CNT),
8: Gate electrode,
13: Specific substance,
14: Substance to be detected
15: Sample solution.

Claims (6)

Detects a substance to be detected in a sample solution with a sensor having at least a substrate and a channel having a source electrode and a drain electrode provided on the insulating thin film formed on the upper surface of the substrate so as to face each other at a predetermined interval. In the way to
The channel is composed of ultrafine fibers;
A method for detecting a substance to be detected in a sample, comprising: dropping the sample solution onto the channel; and evaporating a solvent of the sample solution. A sensor to be detected in a sample solution by a sensor having at least a substrate and a channel having a source electrode and a drain electrode provided facing each other at a predetermined interval on a first insulating thin film formed on the upper surface of the substrate. In a method for detecting a substance,
The channel is composed of ultrafine fibers;
A second insulating thin film is formed on the surface of the substrate opposite to the channel, a back gate electrode is provided outside the second insulating thin film, and the second insulating thin film interacts with the substance to be detected. Modified with certain substances to
A method for detecting a substance to be detected in a sample , wherein the sample solution is dropped between the modified portion and the back gate electrode, and then the solvent of the sample solution is evaporated . In the detection method of the to-be-detected substance in the sample of Claim 1 or 2,
The method for detecting a substance to be detected in a sample, wherein the ultrafine fibers are carbon nanotubes . In the detection method of the to-be-detected substance in the sample of Claim 3,
A method for detecting a substance to be detected in a sample, wherein a defect is introduced into the carbon nanotube . In the detection method of the to-be-detected substance in the sample of Claim 2 ,
A method for detecting a substance to be detected in a sample, wherein the substance to be detected and the specific substance are interacting biopolymers . In the detection method of the to-be-detected substance in the sample of Claim 5,
A method for detecting a substance to be detected in a sample , wherein the substance to be detected is an antigen or an antibody, and the specific substance is an antibody or an antigen .

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