KR100944940B1 - Fabrication method of label-free field-effect transistor biosensors based on 1D conducting polymer nanomaterials - Google Patents

Abstract

The present invention relates to the fabrication of a non-labeled field effect transistor biosensor device using a one-dimensional conductive polymer nanomaterial with an aptamer receptor, wherein an aptamer having a high affinity for a specific target material is shared. A method of detecting biomaterials such as proteins by immobilizing bonded one-dimensional conductive polymer nanomaterials on an electrode substrate and monitoring their current changes in a liquid-ion gate field effect transistor array is provided.

According to the present invention, there is an advantage that one-dimensional conductive polymer nanomaterial having controlled surface functional groups can be easily manufactured by using a simple and effective reversed phase emulsion copolymerization method. Furthermore, the prepared conductive polymer nanomaterial is fixed by chemical bonding on the electrode substrate to maintain a good electrical contact with the metal electrode, and the aptamer receptor is chemically / physically stable fixed by covalent bonding. Introduced to the material surface. Aptamer receptors induce charge accumulation and reduction in conductive polymers through reaction with specific target materials. One-dimensional nanomaterials have the advantage of providing enhanced sensitivity and real-time response because of their increased surface area and unidirectional electrical properties, which enhance interaction with the analytes.

 One-dimensional nanomaterials, conductive polymers, aptamers, field effect transistors, unlabeled biosensors

Description

Fabrication method of label-free field-effect transistor biosensor using one-dimensional conducting polymer nanomaterial with aptamer receptor {Fabrication method of label-free field-effect transistor biosensors based on 1D conducting polymer nanomaterials}

The present invention relates to a method of manufacturing a one-dimensional conductive polymer nanomaterial with an aptamer receptor and to fabricating a non-labeled field effect transistor biosensor device using the conductive nanoparticles, and having a high affinity for a specific target material. A method of detecting biomaterials such as proteins by immobilizing covalently bonded one-dimensional conductive polymer nanomaterials on an electrode substrate and monitoring their current changes in a field effect transistor array is provided. .

With the rapid development of the information industry, the development of plastic electronics materials is required internationally. In particular, interest in the development of small size, high reliability, and high sensitivity next generation sensors is continuously increasing. Currently, research on next-generation sensor materials is being actively conducted on carbon nanotubes, metals, and inorganic semiconductor nanomaterials. Carbon nanotubes are under continuous research through the development of various synthetic methods, but are limited in practical use due to the disadvantages of expensive manufacturing costs, chirality-dependent electrical properties, and inactive surfaces. In contrast, conductive polymers have various advantages such as diversity in molecular design, ease of processing, low weight, and flexibility (polymer science and technology, vol. 18, pp. 306-310, 2007).

The oxidation level of the conductive polymer can be easily controlled by chemical or electrochemical doping / dedoping. And this leads to sensitive and rapid reactions (changes in electrical conductivity or color) to certain particular chemical / biological species (Chem. Rev., vol. 100, pp. 2537-2574, 2000). This feature enables the application of conductive polymers to various sensor activities. In fact, conductive polymers have been known to be more sensitive to external environmental fluctuations than other sensing materials due to their inherent transfer properties such as electrical conductivity and energy transfer (see Acc. Chem. Res., Vol. 31, pp. 201-). 207, 1998). In addition, one-dimensional conductive polymer nanostructures, such as nanorods, nanofibers and nanotubes, have a relatively high surface area, providing increased sensitivity and real-time response through increased interaction with analytes. Nano Lett., Vol. 4, pp. 491-496, 2004; Nano Lett., Vol 4, pp. 671-675, 2004). Despite these advantages, the absence of reproducible and reliable methods for producing nanoparticles has been a significant obstacle to the development of sensors using conductive polymer nanostructures. In particular, the method of manufacturing one-dimensional conductive polymer nanomaterials has been limited to the use of expensive templates such as porous aluminum oxide membranes or polycarbonate membranes. There is a fatal disadvantage that only a very small amount of product can be produced by the step (Chem. Mater., Vol. 8, pp. 2382-2390; Science, vol. 296, pp. 1997).

In general, a biosensor introduces various kinds of receptors into a signal transducer for a selective response to a specific target material. The receptor is attached to the signal sensing surface mainly through adsorption, entrapment, and covalent bonds. Among them, the method of attaching the receptor through covalent bonding has an advantage of providing very good chemical / physical stability compared to other methods. However, covalent bonding of the receptor requires chemical functional groups available on the surface of the signal detector. Most metal, inorganic semiconductor, and polymeric nanomaterials, including carbon nanotubes, have inert surfaces and therefore require additional surface treatment steps to introduce surface functional groups.

Detection and recognition of biomaterials typically proceeds in solution. For this purpose, inorganic semiconductor nanomaterials, including carbon nanotubes, were fixed directly on the electrode substrate via photolithography or electron beam lithography. However, conductive polymer nanomaterials have disadvantages that are not suitable for lithography processes because of possible chemical and physical damage. Most conductive polymers also exhibit low adhesion to electrode substrates made of silicon, glass, metal, and the like. Due to these problems, the development of biosensors using conductive polymer nanostructures has been considerably limited.

Therefore, the development of simple and efficient manufacturing technology that can easily manufacture one-dimensional conductive nanoparticles having surface chemical functional groups and high-performance sensor manufacturing technology based thereon is strongly required for industrial application of all technologies.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for preparing a one-dimensional conductive polymer nanomaterial having a controlled surface functional group through copolymerization of a conductive polymer monomer and a functional monomer on an inverse phase emulsion, in order to solve this conventional problem at once. By using a functional group to fix the nanomaterial to the electrode substrate and covalently attaching the aptamer receptor to the surface of the nanomaterial, a non-labeled field effect capable of detecting a target substance selectively binding to the aptamer An object of the present invention is to provide a transistor biosensor and a method for detecting a target substance using the same.

After numerous experiments and in-depth studies, the present inventors have carried out methods of chemically bonding one-dimensional nanomaterials to surface-modified electrode substrates using surface functional groups introduced through copolymerization, which are completely different from the known methods. By fixing and aptamer receptors also covalently attached to the surface of the nanomaterial, it was confirmed that a stable field effect transistor array could be realized in solution. It has been found that the fabricated field effect transistor sensor can exhibit selective and significantly improved sensitivity to the target material and has led to the present invention.

The present invention is to construct a field effect transistor channel region using a one-dimensional conductive polymer nano-material with an aptamer receptor, and to detect a target material using the same.

The field effect transistor biosensor manufacturing step according to the present invention,

(A) adding a surfactant to the nonpolar solvent at 1-80 ° C .;

(B) adding and stirring a metal salt and an aqueous oxidizing agent to form a cylindrical micelle; And

(C) preparing a one-dimensional conductive polymer nanomaterial having controlled surface functional groups by dropwise addition of a functional monomer copolymerized with the conductive polymer monomer; And

(D) immobilizing the nanomaterials on the surface modified electrode substrate through a chemical reaction; And

(E) obtaining a sensor medium by chemically attaching an aptamer receptor to the surface of the nanomaterial; And

(F) providing a detection means for detecting a change in electrical properties of the sensor medium using a field effect transistor arrangement using a liquid-ion gate.

According to the present invention, a method of implementing a non-labeled field effect transistor biosensor by covalently fixing a one-dimensional conductive polymer nanomaterial on an electrode substrate and attaching an aptamer receptor is an entirely new method that has not been reported so far. In addition, the introduction of functional monomers during the polymerization process eliminates the need for a separate complex surface treatment process. In addition, the conductive polymer nanomaterials are covalently attached onto the substrate to maintain stable electrical contact between the polymer nanomaterials and the electrodes even in solution. In particular, the manufactured unlabeled field effect transistor biosensor showed excellent selectivity and sensitivity to the target material.

Unless specifically stated in the specification, the numerical range of temperature, content, size, etc. means a range capable of optimizing the manufacturing method of the present invention.

In step (A) the surfactant is dissolved in a nonpolar solvent at a temperature of 1-80 ° C. to form spherical micelles.

The type of surfactant that can be used in the preparation method of the present invention is not particularly limited, as the inventors mentioned in Patent Application No. 10-2006-0019432, most of the anionic surfactants that can be applied to the emulsion polymerization Can be used. Among them, surfactants such as AOT (dioctyl sulfosuccinate, sodium salt), SDS (sodium dodecylsulfate) and SDBS (sodium dodecylbenzenesulfonate) are preferred.

Non-limiting examples of nonpolar solvents include hexane, heptane, octane, benzene, toluene, xylene and the like.

In step (B) the spherical micelles are transformed into cylindrical micelles through interaction with metal salts or oxidants. As the inventors mentioned in patent application No. 10-2006-0019432, the metal salt and the oxidizing agent aqueous solution can be appropriately selected according to the type of the surfactant, mainly iron (III) chloride (FeCl ₃ ), iron (III) chloride hydrate (FeCl ₃ (H ₂ O) ₆ ), iron (III) sulfate (Fe ₂ (SO ₄ ) ₃ ) and the like that can be used simultaneously in the metal salt and oxidizing agent that can be used by dissolving in water.

In step (C), the chemical copolymerization of the conductive polymer monomer and the functional monomer on the cylindrical micelle surface forms a one-dimensional conductive polymer nanomaterial. The type and degree of introduction of the surface functional group can be easily controlled by adjusting the type and the amount of the injection of the functional monomer in the polymerization process.

The monomer used for the polymerization is preferably pyrrole, aniline, thiophene and derivatives thereof, which are conductive polymer monomers.

The functional monomer used for the polymerization may be appropriately selected depending on the type of the monomer, and non-limiting examples include pyrrole-2-carboxylic acid, pyrrole-3-carboxylic acid (pyrrole-3). -carboxylic acid, pyrrole-2-sulphonic acid, 2-aminobenzoic acid, 3-aminobenzoic acid, aniline-2-sulphonic acid various functional monomers such as (aniline-2-sulfonic acid), thiophene-2-carboxylic acid, and thiophene-3-carboxylic acid. Can be used.

In step (D), the one-dimensional conductive polymer nanomaterials having the surface functional groups are covalently fixed to the surface-modified microelectrode array substrate surface. The electrode substrate of the silicon or glass component introduces a functional group capable of reacting with the functional group of the conductive polymer nanomaterial to the surface through a silane coupling agent treatment.

The type of the silane coupling agent is not limited, and a silane coupling agent having a terminal group suitable for covalent bonding may be appropriately selected according to the functional group type of the conductive polymer nanomaterial, and a non-limiting example is 3-aminepropyltrimethoxysilane. Various kinds of silane coupling agents such as (3-aminopropyltrimethoxysilane), 3-aminopropyltriethoxysilane, vinyltrimethoxysilane, and carboxyethylsilanetriol may be used. .

The concentration of the silane coupling agent may be used in the range of 0.01% to 10% by weight relative to the total solution, but is not limited to these ranges and may be less or more than the above range.

In step (E), the aptamer receptor includes end groups covalently bonded to the surface functional groups of the one-dimensional conductive polymer nanomaterial. The introduction amount of the aptamer receptor may be controlled by the amount of surface functional groups of the one-dimensional conductive polymer nanomaterial.

The type of aptamer receptor is not limited, and may be appropriately selected according to the type of target material to be detected. Examples of the aptamer receptor include various aptamers such as DNA aptamer, RNA aptamer, and peptide aptamer. Can be.

In step (F), two microelectrode bands are used as source and drain electrodes, respectively, and the one-dimensional conductive polymer nanomaterial constitutes the channel region. Liquid-ion gates are used, and the gate potential can be adjusted through the reference and counter electrodes. In the field effect transistor array, the current flow between the source and the drain can be changed through the accumulation and reduction of charge carriers in the one-dimensional conductive nanoparticle through the specific reaction of the aptamer receptor and the target material. The change in conductivity generated by the signal detection unit is quantified in real time using an electrical conversion device.

One-dimensional conductive polymer nanomaterials prepared by the method of the present invention can easily introduce a controlled amount of functional groups on the surface without a complicated surface process. The surface functional group is used to covalently fix the one-dimensional conductive polymer nanomaterials to the electrode substrate, and the aptamer receptor can also be stably attached to the surface of the nanomaterial through covalent bonding. The fabricated sensor device is capable of increased interaction with the target material due to the high surface area and anisotropic electrical properties peculiar to the one-dimensional conductive nanoparticles, resulting in a very fast and reproducible reaction. The non-labeled field effect transistor sensor device according to the present invention is not limited to a specific analyte, but may be applied as a detection means for various analytes expected in the future, and their use does not depart from the scope of the present invention.

      EXAMPLE

Although specific examples of the present invention will be described with reference to the following Examples, the scope of the present invention is not limited thereto.

      Example 1

A reactor containing 20 mL of hexane was added to a reactor adjusted to 15 ° C. using a thermostat, and 7.9 mmol of AOT was added thereto, followed by stirring to form a micelle. After introducing 0.5 mL of iron trichloride solution (7 M) into the reaction, a mixture of 6 mmol of pyrrole and 0.4 mmol of pyrrole-3-carboxylic acid was slowly added dropwise using a pipette. After the polymerization proceeded with stirring at 15 ° C. for about 2 hours, excess ethanol was added to the reactor. The reaction solution was transferred to a separatory funnel and the precipitated carboxylated polypyrrole nanoparticle layer was recovered. As a result of carboxylated polypyrrole nanoparticles prepared using an electron microscope, particles of the form of nanotubes having a diameter of about 200 nm and a length of more than 5 μm were observed (FIGS. 1,2).

Example 2

A reactor containing 20 mL of hexane was added to a reactor adjusted to 15 ° C. using a thermostat, and 7.9 mmol of AOT was added thereto, followed by stirring to form a micelle. After introducing 0.5 mL of an aqueous solution of iron trichloride (7 M) into the reaction, a mixture of 6 mmol of pyrrole and 0.2 mmol of pyrrole-3-carboxylic acid was slowly added dropwise using a pipette. After the polymerization proceeded with stirring at 15 ° C. for about 2 hours, excess ethanol was added to the reactor. The reaction solution was transferred to a separatory funnel and the precipitated carboxylated polypyrrole nanoparticle layer was recovered. As a result of carboxylated polypyrrole nanoparticles prepared using an electron microscope, particles of the form of nanotubes having a diameter of about 200 nm and a length of 5 μm or more were observed (FIGS. 3 and 4).

Example 3

As a result of analyzing the carboxylated polypyrrole nanotubes prepared in Examples 1 and 2 by X-ray photoelectron spectroscopy, the characteristic peak related to the carboxyl group was confirmed at 289 eV, and the quantitative element ratio The _polypyrrole nanoparticles ( A _{C _289} / A _{C _ total} = 0.054) prepared in Example 1 having a higher injection amount of the functional monomer pyrrole-3-carboxylic acid through analysis were _compared to the nanoparticles prepared in Example 2 ( _{Compared with} A _{C _289} / A _{C _ total} = 0.032), the amount of carboxylic acid introduced was higher (FIG. 5).

Example 4

A photolithography technique is used to pattern the interdigitated microelectrode array on the glass substrate. The electrode structure comprises an array of 80 finger pairs with a width of 10 μm, a length of 4000 μm, a thickness of 40 nm, and an interspacing of 10 μm. The electrode substrate was surface modified using 5 wt% 3-aminepropyltrimethoxysilane aqueous solution to react with the functional groups of the one-dimensional conductive polymer nanomaterial. 10 μL of an ethanol solution containing 0.1 wt% of the carboxylated polypyrrole nanotubes obtained in Examples 1 and 2 was added with 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium ( DMT-MM) was mixed with an ethanol solution containing 10 wt% and then exposed to a surface modified electrode substrate. After the reaction, the remaining reactants are removed using distilled water and water is removed using nitrogen gas. For aptamer receptor attachment, 10 μL of a 10 wt% DMT-MM aqueous solution and 2 μL of 1 μM thrombin aptamer (5′-GGTTGGTGTGGTTGG-3 ′) are reacted with the nanotubes. The 3 'end of the thrombin aptamer was linked with an amine linker to form a covalent bond through shrinkage reaction with the carboxyl group on the surface of the nanotube. After the reaction was completed, residual reactants were removed using distilled water and water was removed using nitrogen gas.

As illustrated in the accompanying drawings of FIG. 6, a field effect transistor array using a liquid-ion gate G was implemented. The channel region is made of polypyrrole nanotubes between the source (S) and drain (D) electrodes, and the source-drain voltage is applied and the current change (ㅿ I ) using a potentiostat connected to a computer. / I ₀ = ( I - I ₀ ) / I ₀ , I and I ₀ represent real-time measured current and initial current value, respectively.

To investigate the selectivity for thrombin protein, bovine serum albumin (BSA) was used as the non-target protein. As shown in FIG. 7, when sequentially injecting 90 nM of thrombin and bovine serum albumin, a decrease in source-drain current was observed only for thrombin. Therefore, the fabricated biosensors were found to exhibit excellent selectivity for specific target materials.

Example 5

In order to investigate the role of the aptamer receptor in the biosensor manufactured in Example 4, polypyrrole nanotubes without aptamer receptor were applied to the channel region. As shown in FIG. 8, no change in source-drain current was observed when injecting thrombin, indicating that the selective reaction between the aptamer receptor and thrombin results in a change in source-drain current.

Example 6

In the same manner as in Example 4, thrombin was injected in the concentration range of 50 nM to 500 nM. The source-drain current change was investigated and the calibration curve for that concentration range was recorded as shown in FIG. 9.

Example 7

In the same manner as in Example 4, but the sensor response to the thrombin in the serum of human blood was investigated. As shown in Figure 10, when the injection of 166 nM thrombin was able to confirm the source-drain current change.

Those skilled in the art will be able to make various applications and modifications within the scope of the present invention based on the above contents.

1 is a scanning electron micrograph of the carboxylated polypyrrole nanotube prepared in Example 1 of the present invention.

2 is a transmission electron micrograph of the carboxylated polypyrrole nanotube prepared in Example 1 of the present invention.

3 is a scanning electron micrograph of the carboxylated polypyrrole nanotube prepared in Example 2 of the present invention.

4 is a transmission electron micrograph of the carboxylated polypyrrole nanotube prepared in Example 2 of the present invention.

5 is an X-ray photoelectron spectroscopy spectrum of carboxylated polypyrrole nanotubes prepared in Examples 1 and 2 of the present invention. CPNT-1 and CPNT-2 refer to carboxylated polypyrrole nanotubes prepared in Examples 1 and 2, respectively. Arrows point to peaks associated with carboxyl groups (source, drain, liquid-ion gate and gate potential are denoted S, D, G, and Eg, respectively).

Figure 6 is a schematic diagram showing a liquid-ion gate field effect transistor biosensor applying the one-dimensional conductive polymer nanomaterial with aptamer attached to the channel region according to the present invention.

7 is a graph showing a source-drain current change curve when thrombin is injected into a sensor manufactured in Example 4 of the present invention. T and B refer to thrombin and bovine serum albumin, respectively. And CPNT-1 and CPNT-2 means carboxylated polypyrrole nanotubes prepared in Examples 1 and 2, respectively.

8 is a graph showing a current change curve for thrombin when a carboxylated polypyrrole nanotube to which a aptamer receptor is not attached is applied to a sensor fabricated in Example 4 of the present invention in a channel region. T and B refer to thrombin and bovine serum albumin, respectively.

9 is a graph showing a current change calibration curve for the concentration of 50-500 nM thrombin of the sensor manufactured in Example 4 of the present invention. CPNT-1 and CPNT-2 refer to carboxylated polypyrrole nanotubes prepared in Examples 1 and 2, respectively.

10 is a graph showing the current change curve for 166 nM thrombin in serum of human blood. CPNT-1 and CPNT-2 refer to carboxylated polypyrrole nanotubes prepared in Examples 1 and 2, respectively. T refers to thrombin.

Claims (7)

Adding a surfactant to the nonpolar solvent at 1-80 ° C .; Adding and stirring a metal salt and an aqueous oxidizing agent to form a cylindrical micelle; And Preparing a one-dimensional conductive polymer nanomaterial having a controlled surface functional group by dropwise addition of a functional monomer copolymerized with the conductive polymer monomer; And Immobilizing the nanomaterials on the surface-modified electrode substrate through a chemical reaction; And Obtaining a sensor medium by chemically attaching an aptamer receptor to the surface of the nanomaterial; And Providing a detection means for detecting a change in electrical properties of the sensor medium using a field effect transistor array using a liquid-ion gate. The method of claim 1, wherein the conductive polymer monomer is pyrrole-2-carboxylic acid, pyrrole-3-carboxylic acid, which is pyrrole, aniline, thiophene and derivatives thereof. ), Pyrrole-2-sulphonic acid, 2-aminobenzoic acid, 3-aminobenzoic acid, aniline-2-sulphonic acid (aniline- Biosensor characterized by selecting one of 2-sulfonic acid), thiophene-2-carboxylic acid, and thiophene-3-carboxylic acid Manufacturing method. The method of claim 1, wherein the functional monomer has a functional group that can be connected to the aptamer receptor through a chemical bond, regardless of the type. The method of claim 1, wherein the channel region is configured by fixing one-dimensional nanomaterials having functional groups through covalent bonds on the surface-modified electrode substrate. The method of manufacturing a biosensor according to claim 1, wherein one-dimensional conductive polymer network form or mesh form composed of a plurality of nanoparticles is applied to the channel region. The method of claim 1, wherein each of the two microelectrode bands having an interdigitated shape is used as a source and a drain electrode to form a field effect transistor array. The method of claim 1, wherein the aptamer receptor is covalently bonded to the surface functional group of the one-dimensional conductive polymer nanomaterial.

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