JP6990396B2 - Biosensor with field effect transistor - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act (1) Publication 1 ▲ 1 ▼ Publication date: July 14, 2017 ▲ 2 ▼ Publication: Master's thesis presentation, Department of Materials Engineering, Graduate School of Engineering, University of Tokyo (Venue: Faculty of Engineering, University of Tokyo, Date: July 26, 2017) Abstracts ▲ 3 ▼ Publisher: Shoichi Nishitani ▲ 4 ▼ Contents of published invention: Shoichi Nishitani, Tokyo A part of the research on the biosensor equipped with the electric field effect transistor invented by Toshiya Sakata and Shoichi Nishitani was released in the abstract of the master's thesis presentation of the Department of Materials Engineering, Graduate School of Engineering, University of Tokyo. (2) Public 2 ▲ 1 ▼ Date: August 20, 2017 ▲ 2 ▼ Meeting name, venue: “254th ACS National Meeting & Expression”, August 20, 2017- Held on the 24th, Washington DC ▲ 3 ▼ Publishers: Shoichi Nishitani, Toshiya Sakata ▲ 4 ▼ Contents of the published invention: Shoichi Nishitani and Toshiya Sakata at the international conference "254th ACS National Meeting & Exhibition" Part of the research on biosensors equipped with field effect transistors invented by Shoichi Nishitani and Toshiya Sakata has been released. (3) Publication 3 ▲ 1 ▼ Publication date: August 25, 2017 ▲ 2 ▼ Publication: Proceedings of the 78th Japan Society of Applied Physics Autumn Academic Lecture ▲ 3 ▼ Publisher: Toshiya Sakata, Shoichi Nishitani ▲ 4 ▼ Contents of the published invention: Field-effect transistor invented by Toshiya Sakata and Shoichi Nishitani at the 78th JSAP Autumn Meeting Proceedings. Part of the research on biosensors equipped with is published.

The present invention relates to a biosensor including a field effect transistor, and more particularly to a modification of a gate electrode surface for suppressing noise from a non-detection target and a method thereof.

The development of next-generation medical care such as portable medical care and bioimplantable sensors requires biosensing technology to measure small molecule biomarkers, which are indicators of diseases and health conditions, quickly, accurately, and easily. A biosensor for such biosensing is a kind of chemical sensor that measures a chemical substance, and generally, a molecular identification element that recognizes only the target substance to be measured and an information that the recognition is made electrically. It is composed of a signal conversion element that converts a physical signal such as a simple signal.

Among various biosensors, a biosensor using a field effect transistor (FET) can detect minute changes in the potential (electricity density) at the sensor interface caused by the adsorption and bonding of molecules. Therefore, it is useful as a method for measuring low molecular weight target compounds with high sensitivity. Furthermore, the FET biosensor can (1) take no time and effort to measure, (2) can perform real-time measurement, (3) can perform unlabeled and non-invasive electrical measurement, (4). It also has features such as miniaturization and integration by semiconductor microfabrication technology.

As a conventional FET biosensor, an attempt has been made to improve selectivity by modifying the sensor surface with a site that can specifically interact with a target compound (for example, Patent Document 1 by the present inventors). .. However, even when such a highly selective configuration is adopted, the response noise derived from the contaminants is generated in the measurement targeting the biological sample containing a large amount of contaminants, especially in the measurement targeting small molecule compounds. The impact was great. Therefore, in order to selectively detect such low molecular weight compounds, more advanced device design, particularly design of the electrode interface, is required, but the device concept as a platform has not yet been established.

Japanese Patent No. 5447716

Therefore, the present invention provides a field-effect transistor biosensor capable of suppressing response noise due to impurities and detecting a target molecule such as a small molecule compound with high sensitivity and high selectivity, and a method for constructing a novel sensing interface for that purpose. The task is to do.

As a result of diligent studies to solve the above problems, the present inventor, unlike the conventional method aiming at specific bonding / adsorption of a target molecule on the gate electrode surface (interface) of a field effect transistor, is on the electrode surface. By providing a polymer layer having a certain thickness and capturing / eliminating (filtering) impurities by the polymer layer, only the target molecule is selectively permeated to the electrode surface, and low molecular weight compounds are detected with high sensitivity. We have found that we can provide a possible biosensor. Further, when modifying the surface of the electrode with the polymer layer, the first layer as a base is first immobilized with a diazonium compound salt, and then the polymer chain is extended by atom transfer radical polymerization to form a second filter region. It has been found that by forming a layer, it is possible to form a polymer layer in which the desired density and thickness can be controlled. The present invention has been completed based on these findings.

That is, the present invention, in one aspect,
<1> A biosensor equipped with a field effect transistor as a detection element.
The surface of the gate electrode of the field effect transistor is surface-modified with a polymer layer, and the polymer layer has a detection region in which the presence of a substance to be detected is detected as a potential change and a substance other than the substance to be detected. Consists of a filter area with a trap for specifically trapping contaminants
The capture portion is located at a position exceeding the Debye length from the surface of the gate electrode, the biosensor;
<2> The biosensor according to <1> above, wherein the polymer layer has a thickness of 10 to 100 nm.
<3> The biosensor according to <1> or <2> above, wherein the polymer layer is immobilized on the surface of the gate electrode by a covalent bond at the aryl group at the end of the polymer chain constituting the layer;
<4> The biosensor according to any one of <1> to <3> above, wherein the field effect transistor is an extension gate type field effect transistor;
<5> The biosensor according to any one of <1> to <4> above, wherein the gate electrode is a gold gate electrode;
<6> The biosensor according to any one of <1> to <5>, wherein the filter region is made of a polymer having a polymethacrylic acid ester skeleton; and <7> the capture portion is phenylboronic acid. A biosensor according to any one of <1> to <6> above is provided.

Moreover, in another aspect, the present invention is:
<8> A method for surface modification of a gate electrode of a field effect transistor for use in a biosensor, wherein a solution containing (a) a diazonium compound salt is brought into contact with the gate electrode, and a first layer is formed on the surface of the gate electrode. The step of forming the second layer on the first layer by extending the polymer chain from the end of the first layer by atom transfer radical polymerization, and (c) forming the second layer. The method comprising the step of introducing a trapping portion for specifically trapping contaminants other than the substance to be detected into the polymer chain;
<9> In the step (a), a first layer is formed by using a solution containing a compound having a thiol group at the terminal in addition to the diazonium compound salt, and then the compound and the gate having the thiol group are irradiated with ultraviolet rays. The method according to <9> above, which comprises a step of decomposing only the covalent bond formed on the surface of the electrode;
<10> The method according to <9> above, wherein the atom transfer radical polymerization in the step (b) is carried out in the presence of a photoactivation catalyst;
<11> The method according to any one of <8> to <10> above, wherein the field effect transistor is an extension gate type field effect transistor;
<12> The method according to any one of <8> to <11> above, wherein the gate electrode is a gold gate electrode;
<13> The method according to any one of <8> to <12> above, wherein the diazonium compound salt is an aryl diazonium salt;
<14> The method according to any one of <8> to <13>, wherein the polymer chain has a polymethacrylic acid ester skeleton; and <15>, the capture portion is phenylboronic acid, the above <8>. > To <14> according to any one of 1.

According to the biosensor of the present invention, the gate electrode surface of a field effect transistor (FET) is modified with a polymer layer, and a trapping portion for selectively capturing impurities is introduced into the polymer layer to eliminate the size. The two filtering effects of specific capture can significantly suppress unwanted contaminant-derived noise signals, resulting in highly sensitive and selective detection of targeted small molecule compounds on the order of nM. .. The design of such a sensing interface is completely different from the conventional method aiming at how to specifically bond the target molecule to the electrode interface.

Further, in the modification of the gate electrode surface, a method of forming a strong bond with the electrode surface using a diazonium compound salt and then forming a polymer layer by atom transfer radical polymerization above the bond is used to obtain a desired density. And it is possible to pattern a multilayer polymer layer whose thickness can be controlled.

FIG. 1 is a schematic view showing the overall configuration of the biosensor of the present invention. FIG. 2 is a schematic diagram showing a comparison between the detection mechanism (B) and the conventional method (A) in the present invention. FIG. 3 is a diagram showing the structure of the polymer layer in the present invention. FIG. 4 is a diagram showing the results of real-time FTE measurement by the biosensor of the present invention.

Hereinafter, embodiments of the present invention will be described. The scope of the present invention is not limited to these explanations, and other than the following examples, the present invention can be appropriately modified and implemented without impairing the gist of the present invention.

1. 1. Overall Configuration of Biosensor FIG. 1 shows the overall configuration of the biosensor of the present invention in a preferred embodiment. The biosensor 10 includes a sensing unit 11 for identifying the presence of a substance to be detected, and a field effect transistor (FET) 12 as a detection unit. The biosensor 10 identifies the substance to be detected contained in the sample in the sensing unit 11, and converts the identified information into an electrical signal in the FET 12 to detect the presence and concentration of the substance to be detected in the sample. do. Here, the sample can be a sample collected non-invasively, that is, a biological fluid other than blood, such as sweat, tears, and saliva.

In FIG. 1, as a typical example, a so-called extended-gate type FET having a configuration in which the gate electrode 21 is electrically connected to the metal electrode 32 via the metal wire 36 is used. Although the case will be described as an example, the biosensor according to the present invention is not limited to such an example. For example, as the detection element, a normal FET that immobilizes the measurement target on the insulating film may be used.

Low-molecular-weight biomarkers are suitable as target substances for detection by the biosensor of the present invention, and such biomarkers are typically amino acids such as cysteine and tyrosine, glucose which is closely related to diabetes, and the like. Examples include biological compounds such as dopamine and dopamine, which are neurotransmitters, and histamine, which is involved in allergic response. However, the present invention is not limited to these, and lactic acid, uric acid, sialic acid, sialyl lactose, fructose, paromomycin, kanamycin, L-epinephrine and the like can also be targeted.

The sensing unit 11 includes a gate electrode (extended gate electrode) 21 and a polymer layer 22 provided on the gate electrode 21. As will be described later, the polymer layer generates a noise signal when detecting the response of the target substance to be detected, and selectively captures impurities that affect the signal of the substance to be detected. Alternatively, it has a function of eliminating it, whereby only the substance to be detected is transmitted to the vicinity of the interface of the gate electrode 21.

In the embodiment shown in FIG. 1, in the sensing unit 11, a container 23 is formed by providing a cylindrical wall portion on one side surface of the gate electrode 21, and a sample containing an identification substance is added to the container 23. Will be done. The gate electrode 21 can be preferably formed of gold (Au), silver (Ag), or copper (Cu), but is more preferably gold. It means having a property. The capacity of the container 23 is not particularly limited, but it is preferable that a small amount of body fluid equal to or larger than the amount of the sample required for the measurement of the biosensor (for example, about 0.1 μL to 1 μL) can be contained.

The FET 12 is an element that can be electrically connected to the above-mentioned sensing unit 11 and detects a change resulting from the interaction between the above-mentioned target substance and the sensing unit 11. As shown in FIG. 1, the FET 12 includes a source electrode 34 and a drain electrode 35 formed on the surface of the semiconductor substrate 31, and a gate insulating film 33 formed on the source electrode 34 and the drain electrode 35. As the FET 12, an n-channel MOSFET (Metal Oxide Semiconductor Field Effect Transistor) (n-MOS) is suitable, but a p-channel MOSFET (p-MOS), an n-channel junction FET, or a p-channel junction FET is used. You can also do it. A metal electrode 32 is formed on the gate insulating film 33. The metal electrode 32 is electrically connected to the gate electrode 21 on the sensing portion side via the wiring 36. The metal electrode 32 can be formed of Au, Ag, Cu, or the like.

The material of the semiconductor substrate 31 is not particularly limited, but is Si, GaAs, a transparent oxide semiconductor (for example, ITO, IGZO, IZO), an organic semiconductor, and a carbon semiconductor (for example, carbon nanotube, graphene semiconductor, diamond). Known semiconductors such as semiconductors) can be appropriately selected and used. When a carbon semiconductor is used, it is preferable in that the measurement sensitivity of the sensor can be made higher than when Si is used.

The gate insulating film 33 is provided on the surface of a portion of the semiconductor substrate 31 sandwiched between the source electrode 34 and the drain electrode 35 (that is, the p-type semiconductor portion in the FET of FIG. 1), and has SiO ₂ and Si ₃ N. It can be formed of an oxide such as ₄ (SiN _x ), Ta ₂ O ₅ , Al ₂ O ₃ , or a nitride or the like.

The source electrode 34 and the drain electrode 35 are electrically connected to the power supply 37 and the ammeter 38, and are formed so as to measure the drain current flowing from the source electrode 34 to the drain electrode 35. When the charge density on the gate insulating film 33 changes, the magnitude of the drain current changes. That is, in order to keep the drain current constant, it is necessary to change the gate voltage as the charge density on the gate insulating film 33 changes. The FET 12 electrically measures the change in charge density on the gate insulating film 33 by measuring the change in the gate voltage.

At this time, if necessary, the reference electrode 24 may be provided as shown in FIG. The reference electrode 24 is electrically connected to the sensing unit 11, forms a closed circuit together with the source electrode 34 and the drain electrode 35, is an electrode that serves as a reference potential for voltage measurement in the FET, and may be grounded. Practically, it is necessary for voltage measurement in FET, but it is not always essential to provide the reference electrode 24 if the target substance can be measured by another method.

The general detection principle of the FET biosensor is described in, for example, Japanese Patent No. 5447716 (Patent Document 1), and the document can be referred to.

2. 2. Polymer layer on the surface of the gate electrode The biosensor of the present invention is characterized in that the surface of the gate electrode (extended gate electrode) 21 is surface-modified by the polymer layer. By providing the polymer layer, it has a function of selectively capturing or eliminating non-detection target substances, that is, contaminants, which generate a noise signal to the signal of the detection target substance which is originally intended. As a result, only the substance to be detected can be transmitted to the vicinity of the interface of the gate electrode 21, and only the signal derived from the target substance to be detected can be detected.

FIG. 2 shows a schematic diagram showing a comparison between the detection mechanism in the present invention and the conventional method. FIG. 2A is a conventional approach of specifically detecting a target molecule by modifying the surface of the gate electrode of a field effect transistor with a substance that can selectively bind to the target molecule to be detected. On the other hand, FIG. 2B shows a completely different approach in the present invention. That is, in the present invention, by providing a polymer layer (polymer filter) on the surface of the gate electrode, unwanted contaminants invade the electrode interface due to the two filtering effects of "size exclusion" and "specific capture". This is an approach in which the S / N ratio can be improved because only the target molecule can reach the electrode interface while preventing the detection of electrical signals derived from contaminants. Specifically, due to the presence of the polymer layer, large molecules such as proteins cannot reach the electrode interface due to the size exclusion effect, and for small contaminants, the detection of target molecules in the polymer layer. By introducing a trapping portion that selectively traps the contaminants that are an inhibitor, the contaminants are captured at the stage of the polymer layer to prevent further invasion into the electrode interface.

The structure of the polymer layer in the biosensor of the present invention will be described below. The polymer layer is a filter having an i) a detection region in which the presence of a substance to be detected is detected as a potential change, and ii) a capture unit for specifically capturing impurities other than the substance to be detected. It consists of areas. The structure of the polymer layer is shown in FIG. 3 as a preferred embodiment of the polymer layer. This embodiment assumes that the small molecule compound to be detected is L-cysteine, which is a kind of amino acid, and that dopamine, which is a small molecule compound of the same degree, is present as a contaminant. Preferably, the polymer layer has a thickness of 10-100 nm.

As shown in FIG. 3, the polymer layer has a detection region and a filter region formed by extending the polymer from the detection region. The detection region is covalently coupled to the gate electrode and its thickness is within a few nanometers, preferably within the Debye length, typically within 5 nm. Here, the Debye length is a region in the vicinity of the gate electrode where a potential change due to the presence of the compound can be observed. The Debye length depends on the concentration of the solution in the sensing unit 11 used for the measurement, and may be 1, 2, 3, or 4 nm depending on the case, or may be in the range of 5 to 10 nm. Sometimes. The layer of the filter region is also important in controlling the density of the entire final polymer layer.

Then, above the detection region, there is a filter region composed of a polymer chain having a predetermined length. In the filter region, the polymer chain is introduced with a trapping portion for specifically binding and trapping contaminants. The trapping portion exists at a position exceeding the Debye length from the surface of the gate electrode. In the example shown in FIG. 3, a boronic acid structure that can specifically bind to and capture dopamine, which is a contaminant, is introduced as a capture portion. Phenylboronic acid is known to be able to form stable esters with diol-containing molecules and have a very high binding affinity for catechol groups. Therefore, the dopamine present in the sample is trapped by phenylboronic acid in the filter region and cannot penetrate the detection region within the device length, so that a potential response is not generated. On the other hand, since L-cysteine, which is a target molecule, does not have an affinity for phenylboronic acid, it can pass through the filter region and selectively reach the detection region, so L-cysteine is selected. Can be detected.

The detection region is fixed to the surface of the gate electrode by a covalent bond at the end thereof, and is preferably fixed by a covalent bond between the carbon atom at the end of the detection region and a metal such as Au constituting the electrode. More preferably, the aryl group at the end of the detection region (that is, the aryl group at the end of the polymer chain) can be immobilized by a covalent bond with the surface of the gate electrode.

As the polymer constituting the polymer chain in the filter region, a hydrophilic polymer can be used, and typically a polymer having a polymethacrylic acid ester skeleton. For example, polymethyl methacrylate, ethyl polymethacrylate and the like can be mentioned. Further, a hydrophilic polymer known in the art such as polyethylene glycol can also be used. Further, the polymer chains constituting the filter region can be crosslinked with each other to form a stronger structure.

As the capture unit, for example, phenylboronic acid and its derivative can be used, but the present invention is not limited to this, and it is possible to selectively interact with the target contaminants to bind and capture them. Any functional group or the like can be used as long as it is. For example, vinylpyridine can be used for acidic target compounds, acrylamide or methacrylamide can be used for neutral target compounds, and it has a melamine structure when trapping uric acid (2-vinyl-4). , 6-diamino-1,3,5-triazine) can be used. Further, in some cases, by using a molecular template polymer for the filter region, it is possible to adopt an embodiment in which the target compound is captured by the template portion in the polymer.

The polymer layer in the present invention is preferably a self-assembled monolayer (SAMs: Self-Assembled Monolayers). In general, a self-assembled monolayer is a monolayer that binds to and accumulates on a solid surface and is spontaneously formed by an intermolecular force or the like that acts on each other.

3. 3. Surface modification method of gate electrode (formation of polymer layer)
In one aspect, the invention also relates to a method of surface modification of a gate electrode of a field effect transistor for use in a biosensor. Specifically, the method of the present invention is characterized by including the following steps (a) to (c).
(A) A step of bringing a solution containing a diazonium compound salt into contact with the gate electrode to form a first layer on the surface of the gate electrode.
(B) A step of extending a polymer chain from the end of the first layer by atom transfer radical polymerization to form a second layer on the first layer, and (c) the polymer chain forming the second layer. A step of introducing a trapping unit for specifically capturing contaminants other than the substance to be detected.

The step (a) is a step of forming the first layer corresponding to the detection region in the polymer layer. The first layer also serves as a base layer for forming a filter region described later. Therefore, it is preferable that the thickness of the first layer is such that the detection region is within the range of the Debye length. Typically, the thickness is preferably within the range of 5 nm from the surface of the gate electrode. The first layer may be formed as a self-assembled monolayer.

By performing a reaction to form such a first layer portion using a diazonium compound salt, a strong covalent bond with the surface of a gate electrode such as Au is formed by an —C— bond instead of the conventionally used —S— bond. Can be immobilized. Preferably, the diazonium compound salt is an aryl diazonium salt.

More specifically, the diazonium compound salt binds to the electrode surface via reactive carbon radicals. The generation of carbon radicals is carried out by electrochemical reduction and can be bonded to the electrode surface within a few cycles by applying a reduction potential in water or an organic solvent using a potential cycle (several seconds to minutes). Order).

The advantage of surface modification using such a diazonium compound salt, particularly aryldiazonium salt, is that it is a simple and rapid modification method; it can be applied to various substrate surfaces such as carbon, metal, semiconductor, etc .; p- of the diazonium salt. It can be mentioned that any functional group can be flexibly introduced by designing a substituent; and that it has a strong covalent bond between the aryl = electrode surface. From the viewpoint of the polymerization reaction of the polymer chain in the step (b) described later, the p-substituted group of the diazonium salt is preferably a hydroxyalkyl or nitro group.

As a modification of the step (a), a first layer is formed using a solution containing a compound having a thiol group at the terminal in addition to the diazonium compound salt, and then the thiol group (-SH) is formed by irradiation with ultraviolet rays. It is also possible to carry out a step of decomposing only the compound having and the covalent bond formed on the surface of the gate electrode. A schematic diagram of this process is shown below.

In this case, in addition to the layer formation derived from the diazonium compound salt as described above, a layer formed by an —S— bond derived from a thiol group is also formed, and the —S— bond is formed by irradiation with ultraviolet rays (UV). Since it is decomposed, the layer portion derived from the —S— bond can be removed, and only the layer portion derived from the diazonium compound salt can remain. Therefore, it is possible to control the density of the first layer by adjusting the ratio of the diazonium compound salt and the thiol group-containing compound and the like.

The step (b) is a step of forming a polymer chain above the end of the first layer formed in the above (a) to form a second layer corresponding to the filter region in the polymer layer.

Specifically, as shown in (1) in the following scheme, atom transfer radical polymerization (ATRP:
Atom Transfer Radical Preparation) is used to extend a polymer chain from the end of the first layer to form a second layer on top of the first layer. Atom transfer radical polymerization has the advantage that the polymer distribution and chain length can be controlled.

In order to carry out the atom transfer radical polymerization reaction, an alkyl halide group such as an alkyl bromide which is a reaction initiator is introduced at the end of the first layer. Then, the monomer polymerization reaction is carried out in the presence of a catalyst. Examples of the monomer used here include a methacrylate ester and the like, and methyl methacrylate (MAA) is preferable. More preferably, a combination of methyl methacrylate and 2-hydroxyethyl methacrylate can also be used for controlling hydrophilicity. In addition to these, as described above, various hydrophilic polymers can also be used as the polymer constituting the second layer.

As the catalyst in the atom transfer radical polymerization reaction, a catalyst known in the art can be used, but a metal ligand complex is preferable. More preferably, the catalyst is a catalyst that can be activated by light irradiation (photoactivation catalyst), and examples of such photoactivation catalysts include tris (2-phenylpyridinato) iridium (III) (fac-). Ir (ppy) ₃ ) can be mentioned. By using the polymerization reaction using such a photoactivation catalyst, the polymer elongation can be controlled only in the region irradiated with light, so that the polymer layer can be three-dimensionally patterned by lithography or the like.

The step (c) is a step of introducing a catching portion for specifically catching impurities other than the substance to be detected into the polymer chain formed in the above step (b). The introduction can be carried out by a method known in the art, depending on the type of capture unit desired. As a preferred example, as shown in (2) in the above scheme, when the polymer chain is a polymethacrylic acid ester skeleton, it is a capture part by an amidation reaction using a phenylboronic acid compound having an amino group. Phenylboronic acid can be introduced.

As described above, the polymer layers (first layer and second layer) obtained by the surface modification of the present invention preferably have a thickness of 10 to 100 nm as a whole. Further, since the thickness of the first layer is within the range corresponding to the Debye length, the capture portion introduced in the step (c) exists at a position where the distance from the gate electrode surface exceeds the Debye length. Will be. As a result, the contaminants captured by the capture unit cannot enter the region within the Debye length (that is, the detection region), so that the noise signal can be suppressed, and the noise signal can be suppressed, and the S / N ratio is high and the sensitivity is selective. It becomes possible to detect the target molecule.

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.

[reagent]
2- (4-Aminophenyl) Ethanol (APE), 2-bromoisobutyryl bromide (BiBB), 4-nitrobenzenediazonium tetrafluoroborate (NBD), 1,1-diphenyl-2-picrylhydrazyl (DPPH) , And ethyl 2-bromoisobrate (EBiB) were commercially available from Tokyo Chemical Industry. Tetrafluoroborate (HBF 4), sodium nitrite (NaNO ₂ ), triethylamine (TEA), methacrylic acid (MAA), 2-hydroxyethylmethacrylate (HEMA), N, N'-methylenebisacrylamide (MBAAm), Tris (2-Phenylpyridinato) Iridium (III) (fac-Ir (ppy) ₃ ), 4- (4,6-dimethoxy-1,3,5-triazine-2-yl) -4-methylmorpholinium Chloride (DMT-MM), m-aminophenylboronic acid (APBA), dopamine hydrochloride, 3- (3,4-dihydroxyphenyl) -L-alanine (L-DOPA), cysteine, N, N-dimethylformamide ( DMF), 1M hydrochloric acid, dichloromethane, solid NaCl, Na ₂ HPO ⁴ , NaH ₂ PO ⁴ , distilled water, ethanol, and methanol were commercially available products from Wako Pure Chemical Industries.

1. 1. Modification of Gate Electrode Surface The surface of the extended gate electrode (Au electrode) of the FET was modified by the following procedure to form a polymer layer. As a starting material, a method using a diazonium salt using an OH group terminal and a method using a diazonium salt having a nitro group terminal were carried out.

1-1 Modification using diazonium salt with OH end (a) Grafting of aryl diazonium with OH end in gold electrode
100 mL of 0.5 M HCl was stirred in ice-cold water in a 300 mL beaker. 28 mg (0.2 mmol) 2- (4-aminophenyl) ethanol (APE) was added and stirred for 5 minutes. Next, 1 mL of 200 mM NaNO ₂ 2 mL in water was added, and the mixture was stirred for 15 minutes. The washed Au electrode was immersed in the solution and a potential cycle from 0.4 V to -0.3 V was performed at 50 mV / s. After 5 cycles, the substrate was thoroughly washed with methanol and water.

⁽ B) Introduction of Polymer Initiator Next, the esterification reaction of the terminal hydroxyl group of the phenyl group with the alkyl bromide was carried out according to the following procedure13. The OH-Au electrode was immersed in 50 mL of dichloromethane in a capped beaker and stirred in ice-cold water. 712 μL (5 mmol) of triethylamine (TEA) was added. Next, 628 μL (5 mmol) of 2-bromoisobutyryl (BiBB) bromide was added dropwise to the mixture. The mixture was stirred below 4 ° C. for 2 hours and at room temperature. 24hours. The obtained Au electrode (Br-Au) was thoroughly washed with methanol and water.

(C) Polymer Polymerization Surface-initiated polymerization using photo-mediated atom transfer radical polymerization (ATRP) was carried out on the surface into which alkyl bromide was introduced by the following procedure. 3 mL (35 mmol) of methacrylic acid (MAA) and 1.9 mL (15 mmol) of 2-hydroxyethyl methacrylate (HEMA) were dissolved in 45 mL of methanol. The mixture was deoxygenated for 30 minutes by bubbling nitrogen gas. Next, the Br-Au substrate was immersed in the solution in the capped beaker using a stirrer. Next, 0.69 mg of fac-Ir (ppy) ₃ was added to the mixture. The mixture was irradiated with ultraviolet light for 24 hours. After the polymerization reaction, the substrate was immersed in methanol for 1 hour and then washed thoroughly with water.

(D) Introduction of phenylboronic acid into polymer Amidation of methacrylic acid and m-aminophenylboronic acid (APBA) was performed using DMT-MM as a water-soluble catalyst. 16.1 mg (0.1 mmol) APBA and 28.2 mg (0.1 mmol) DMT-MM were dissolved in 10 mL of methanol. The polymer coated Au electrode was immersed in the solution and stirred for 12 hours.

1-2 Modification using a diazonium salt having a nitro end (a) Grafting of an aryldiazonium having a nitro group end
2.4 mg (0.01 mmol) of 4-nitrobenzene diazonium tetrafluoroborate (NBD) and 98.8 mg (0.25 mmol) of nBu ₄ PF ₆ were dissolved in 10 mL of acetonitrile (1 mM and 25 mM, respectively). 8.1 mg (0.02 mmol) DPPH was added for monolayer modification. The washed Au electrode was immersed in the mixture and a potential cycle of 0.3 V to -0.7 V was performed at 50 mV / s. After a total of 5 cycles, the samples were sonicated in methanol for 2 minutes and then thoroughly washed with distilled water.

(B) Reduction of nitro group Nitrobenzene was reduced to aniline by the following procedure. The NB-Au substrate was immersed in 1/9 v% ethanol / water. The potential cycle was from 0.2 V to 1.2 V at 50 mV / s. After 10 cycles, the modified Au electrode was washed with methanol and water.

(C) Introduction of Polymer Initiator Alkyl bromide was introduced in the same procedure as in 1-1 (b) above.

(D) Polymer polymerization and introduction of phenylboronic acid Polymer polymerization and introduction of phenylboronic acid were carried out on the electrode surface in the same procedure as in 1-1 (c) and (d) above.

2. 2. Real-time FET measurement
A junction FET (K246-Y9A, Toshiba) was connected to the FET real-time monitoring system, and the gate was connected to the Au electrode. Prior to measurement, a 1 mL ring was encapsulated around the sensing surface using a commercially available superglue. The surface potential was measured by applying a constant IDS = 700 μA while automatically changing the V _S in the device, and was extracted as V _out . Here, the value of V _out is a measure of V _T or surface potential. For pH measurement, pH was adjusted using 100 mM phosphate buffer (5.6,6.0,6.4,6.8 and 7.2) prepared in the following procedure. First, 200 mM Na ₂ HPO ₄ and Na H ₂ PO ₄ in distilled water were prepared. Next, each solution was added to 20 mL of distilled water at the ratios shown in Table 1. The pH of the solution was measured using a pH / ion meter (F-53, HORIBA, Ltd.). All samples started at pH 7.2. After stabilization of the surface potential, the pH was changed from 7.2 to 5.6 and then the pH was returned to 7.2 by changing the solution twice.

In the measurements, instead of changing the solution to control the sample concentration, a syringe pump (Harvard) was used to continuously inject the target molecule at a constant rate (typically 5 μL / min). This causes the concentration to increase continuously and gradually. Time was converted to concentration based on the following assumptions: (1) Since the system is covered with parafilm, evaporation of the medium is negligible. (2) The concentration is sufficiently low so that the densities of the buffer solution and the target solution are equal. (3) kμL / min can be converted to k / 60μL / s. Then, assuming that the initial volume is V1 (μL) and the concentration of the injected target solution is M (mol / L), the concentration Ct (mol / L) at time t1 (s) can be expressed by the following relational expression. can.

2-1 Detection ability of FET biosensor of Au electrode First, the detection ability of low molecular weight biological compound was evaluated for the FET biosensor having no surface modification by the polymer layer. Au was used as the extension gate electrode. The results are shown in Table 2 below. In the table, "sensitivity" is the detection limit by the sensor. "Intensity" is the maximum change in surface potential. The "direction" is the direction of the signal shift. "Charge" is the charge that a biomolecule has at pH 7.4.

In the table, "sensitivity" is the detection limit by the sensor. "Intensity" is the maximum change in surface potential. The "direction" is the direction of the signal shift. "Charge" is the charge that a biomolecule has at pH 7.4.

As a result, it was found that the Au gate electrode can detect small molecule compounds other than fructose and tyrosine even in a state where surface modification such as a specific binding site for a target molecule is not performed. Here, since the detection response does not correlate with the charge of the molecule itself, it is considered that such response is not derived from the simple adsorption of the biomolecule on the Au surface. Although the theoretical elucidation has not always been made, it is possible that the FET response was observed due to the electrochemical reaction such as oxidation between these biomolecules and Au.

2-2 Sensing by FET biosensor with polymer layer introduced Next, selective detection of L-cysteine was performed using the FET biosensor having a polymer layer on the surface of the extended gate electrode adjusted in 1-1 above. Dopamine and L-DOPA were used as contaminants. The obtained results are shown in FIGS. 4A to 4D. In all the results, in addition to the one introduced with phenylboronic acid (PBA), the one using a modified electrode without PBA was measured as a comparative example.

FIG. 4A is a result showing the change in the surface potential when dopamine is continuously injected. After injection, the difference in response of the two different FETs with and without PBA was clearly observed. The surface potential was reduced in both cases, but the one coated with the PBA-free polymer showed a stronger response. This result indicates that dopamine was trapped in the polymer layer and did not reach the detection area.

Similar results were observed with the addition of L-DOPA (FIG. 4B). When 1 mM L-DOPA was added at a concentration of about 1600 nM, the surface potentials of both devices dropped sharply to the same value, but this was due to the limited number of PBA units in the polymer layer, so all at high concentrations. This is because L-DOPA cannot be captured.

Next, the result when L-cysteine which is a target molecule is added is shown in FIG. 4C. The presence or absence of PBA in the polymer had little effect on the response to cysteine. This is because cysteine passed through the polymer without binding to PBA and reached the detection surface. L-Cysteine could be detected on the order of nM.

Finally, a mixed solution of 0.03 mM L-DOPA and cysteine was injected (Fig. 4D). Sensors without PBA-containing polymers showed greater response. Therefore, in the case of the PBA-containing polymer, it is suggested that the contaminant L-DOPA was trapped in the polymer layer. This result demonstrates highly sensitive and selective detection of L-cysteine, a low molecular weight biomolecule.

10 Biosensor 11 Sensing unit 12 FET (detection unit)
21 Gate electrode (extended gate electrode)
22 Polymer layer 23 Container 24 Reference electrode 31 Semiconductor substrate 32 Metal electrode 33 Gate insulating film 34 Source electrode 35 Drain electrode 36 Metal wire 37 Power supply 38 Electrometer

Claims (13)

A biosensor equipped with a field effect transistor as a detection element.
The surface of the gate electrode of the field effect transistor is surface-modified with a polymer layer.
The polymer layer includes a first layer constituting a detection region in which the presence of a substance to be detected is detected as a potential change, and a trapping portion for specifically capturing impurities other than the substance to be detected. It consists of a second layer that constitutes the filter area to have.
The second layer is formed on the first layer by extending a polymer chain from the upper end of the first layer.
The polymer layer is covalently immobilized with the surface of the gate electrode at the aryl group at the lower end of the first layer.
The polymer layer has a thickness of 10-100 nm and has a thickness of 10-100 nm.
The trapping portion exists at a position exceeding the Debye length from the surface of the gate electrode.
The biosensor. The biosensor according to claim 1 , wherein the field effect transistor is an extension gate type field effect transistor. The biosensor according to claim 1 or 2 , wherein the gate electrode is a gold gate electrode. The biosensor according to any one of claims 1 to 3 , wherein the filter region is made of a polymer having a polymethacrylic acid ester skeleton. The biosensor according to any one of claims 1 to 4 , wherein the trapping portion is phenylboronic acid. A method for surface modification of the gate electrode of a field effect transistor for use in a biosensor.
(A) A step of bringing a solution containing a diazonium compound salt into contact with the gate electrode to form a first layer immobilized on the surface of the gate electrode by a covalent bond with the surface .
(B) A step of extending a polymer chain from the end of the first layer by atom transfer radical polymerization to form a second layer on the first layer, and (c) the polymer chain forming the second layer. A process of introducing a trapping unit for specifically capturing contaminants other than the substance to be detected,
Including
The polymer layer composed of the first layer and the second layer has a thickness of 10 to 100 nm.
The method. In the step (a), a first layer is formed by using a solution containing a compound having a thiol group at the terminal in addition to the diazonium compound salt, and then the surface of the compound having the thiol group and the gate electrode is irradiated with ultraviolet rays. 6. The method of claim 6 , comprising the step of decomposing only the covalent bonds formed in. The method according to claim 7 , wherein the atom transfer radical polymerization in the step (b) is carried out in the presence of a photoactivation catalyst. The method according to any one of claims 6 to 8 , wherein the field effect transistor is an extension gate type field effect transistor. The method according to any one of claims 6 to 9 , wherein the gate electrode is a gold gate electrode. The method according to any one of claims 6 to 10 , wherein the diazonium compound salt is an aryl diazonium salt. The method according to any one of claims 6 to 11 , wherein the polymer chain has a polymethacrylic acid ester skeleton. The method according to any one of claims 6 to 12 , wherein the trapping portion is phenylboronic acid.

Images