KR20100121969A - Fabrication of high-performance transducers for fet-type bioelectronic noses using polypyrrole nanotubes conjugated with human olfactory receptors - Google Patents

Abstract

PURPOSE: A manufacturing method of an olfactory sense nano biosensor which combines the human olfactory receptor protein and a conductivity polymer nanofiber is provided to attach the human olfactory receptor protein which applies to the receptor to the 1D conductive polymer nano material, thereby providing unlabelled FET olfactory nano bio sensor. CONSTITUTION: An electrode substrate is a surface modified to amine radical. The conductivity polymer nanomaterial having functional group uses condensation reaction and is fixed to the electrode substrate which is surface-modified. As the olfactory receptor protein uses the condensation reaction, the conductive polymer nano material having the functional group fixed to the electrode substrates is attached. An electric signal according to the chemical/electrical characteristic change between a first and a second transistor uses a FET(Field Effect Transistor) array utilizing the nano biosensor medium and is detected.

Description

Fabrication of Olfactory Nanobiosensors incorporating Human Olfactory Receptor Proteins and Conductive Polymer Nanofibers {Fabrication of High-Performance Transducers for FET-Type Bioelectronic Noses Using Polypyrrole Nanotubes conjugated with Human Olfactory Receptors}

The present invention relates to the fabrication of an unlabeled field effect transistor artificial olfactory sensor using a one-dimensional conductive polymer nanomaterial with olfactory receptor protein. A conductive polymer composite was used as the secondary transducer. By immobilizing a polymer nanomaterial covalently bonded to an olfactory receptor protein having a high affinity to specific target odor microparticles on an electrode substrate, an ultra-small nanobiosensor matrix having high sensitivity and selectivity will be developed.

The olfactory field is the least known of human senses due to its complexity. In 1991, Buck and Axel reported olfactory receptor genes from rat olfactory epithelial tissue, and a full-fledged study of vertebrate olfactory systems began (Buck and Axel, Cell, vol. 66, pp. 175-187). Industrial diversification (medical diagnostics, quality assessment, environmental monitoring) has led to an increasing number of industries. Therefore, the development of the base technology related to this situation is necessary, qualitative of the odor particulates associated with various odors. Development of devices for quantitative analysis is required. In particular, the development of small size, high reliability, high sensitivity next generation sensor is required internationally.

Currently, research on next-generation sensor materials is being actively conducted on carbon nanotube, metal 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 high manufacturing cost, chiral-dependent physical properties, and inert surface. In contrast, conductive polymers exhibit remarkable changes in conductivity, weight, volume, and color for specific analytes due to reversible oxidation / reduction reactions due to conjugated double bond structures. , Low weight, flexibility, etc. (Polymer Science and Technology, vol. 18, pp. 306-310, 2007). In addition, one-dimensional conductive polymer nanostructures, such as nanorods, nanofibers, and nanotubes, have a relatively high surface area, which can provide increased sensitivity and real-time response through increased interaction with analytes. Lett., Vol. 4, pp. 491-496).

In general, in order to induce a selective response to a target material using a biosensor, a variety of receptors are attached to a signal transducer through adsorption, capture, and covalent bonds. Among them, the method of immobilizing olfactory receptor proteins through covalent bonds has the advantage of providing very good chemical / physical stability. However, since most metals, inorganic semiconductors, and carbon nanotubes, including polymeric nanomaterials, have inert surfaces, additional surface treatment steps are required to introduce functional groups to these surfaces.

Detection and recognition of biomaterials typically proceeds in solution. Thus, inorganic semiconductor nanomaterials, including carbon nanotubes, were fixed directly on the electrode substrate through photolithography or electron beam lithography. However, conductive polymer nanomaterials have the disadvantage of being unsuitable for lithography processes due to 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 production of high-performance sensor manufacturing technology based on the production of one-dimensional conductive nanoparticles having a chemical functional group on the surface is strongly required for industrial applications of all technologies.

SUMMARY OF THE INVENTION An object of the present invention is to provide a one-dimensional conductive polymer nanomaterial having a controlled surface functional group through copolymerization of a conductive polymer monomer and a functional monomer in an inverse phase emulsion to solve such a conventional problem at once. Fabrication of non-labeled field effect transistor olfactory nanobiosensors capable of detecting odorous substances selectively binding to olfactory receptors by immobilizing nanomaterials on electrode substrates using functional groups and covalently attaching olfactory receptors to nanomaterial surfaces And it aims to provide a detection method of odor microparticles through this.

After numerous experiments and in-depth studies, the inventors have chemically bonded one-dimensional nanomaterials to an electrode substrate surface-modified using surface functional groups introduced through copolymerization, which is a completely different method. In addition, the olfactory receptor is also covalently attached to the surface of the nanomaterial to realize a stable field effect transistor array in solution. It has been found that the fabricated field effect transistor sensor can exhibit a selective and significantly improved sensitivity (in fM units) to the target odor particulates, thus leading to the present invention.

The present invention is to construct a field effect transistor channel region by using a one-dimensional conductive polymer nano-material attached to the olfactory receptor, and to detect the odor microparticles that selectively react with the olfactory receptor.

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

(A) surface modification of the electrode substrate with an amine group (-NH ₂ );

(B) fixing the conductive polymer nanomaterial having a functional group on the surface modified electrode substrate prepared above using a condensation reaction; And

(C) preparing an olfactory nanobiosensor by attaching an olfactory receptor to a conductive polymer nanomaterial having a functional group fixed on an electrode substrate using a condensation reaction; And

(D) providing a means for sensing an electrical signal according to a change in chemical and electrical properties between the primary and secondary transistors using a field effect transistor array utilizing the prepared nanobiosensor medium. have.

According to the present invention, covalent bonding of one-dimensional conductive polymer nanomaterials on an electrode substrate can cause increased interactions with target molecules due to the high surface area and anisotropic electrical properties, and furthermore attach olfactory receptors. This completely reproduces the role as an artificial nose (electronic nose), an olfactory nanobiosensor, and is a new polymer nanomaterial artificial electronic nose method that has not been reported at all.

Biosensor experiments are generally conducted in solution, which requires a device to maintain stable electrical contact, but does not require a separate device by covalently fixing the polymer nanomaterial and the electrode. In particular, the prepared unlabeled field effect transistor polymer olfactory nanobiosensor showed excellent selectivity for odor particles and excellent sensitivity in femtomol (fM) units beyond nanomolar (nM) units.

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.

The electrode substrate introduced in step (A) comprises an array of 100 finger pairs with a width of 10 μm, a length of 3500 μm, a thickness of 20 nm, and a gap of 10 μm. The electrode substrate was surface modified (-NH ₂ ) using 400 mL of 1 wt% 3-aminepropyltriethoxysilane aqueous solution to react with the functional group (-COOH) of the one-dimensional conductive polymer nanomaterial.

The kind of the silane coupling agent is not limited, and 3-aminepropyltriethoxysilane (3-aminoproyltrimethoxysilane) and 3-aminepropyltriethoxysilane (3-3-propyl) may introduce an amine group (-NH ₂ ). aminopropyltriethoxysilane).

The preparation of the conductive polymer nanomaterial having the functional group used in step (B) is preferably made with reference to the paper published in the laboratory, but is not particularly limited. (Reference: Chem Com. 2003, pp. 720 721) The form of the conductive polymer is preferably nanotubes (nanotube), nanorod (nanorod), nano ellipsoidal (nanoellipsoidal), but is not particularly limited. The diameter of the nanomaterial is preferably 10 to 1000 nanometers. In the nanomaterial, the functional group is preferably a carboxyl group (-COOH), and in order to perform polymerization together, the electrode surface is preferably surface-modified with an amine group (-NH ₂ ). When a coupling agent is used together, the condensation reaction can be easily induced between the carboxyl group and the amine group, and the conductive polymer nanomaterial having the functional group can be fixed to the electrode substrate.

The concentration ratio of the conductive polymer nanomaterial having the functional group is preferably in the range of 0.01 wt% to 10 wt%.

As the coupling agent used, 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium (DMT-MM) was used, and the concentration of DMT-MM was 0.1 wt. A range of% to 10 wt% is suitable, but may be higher or lower than this range.

It is preferable to leave at room temperature for 6 to 12 hours for the coupling reaction.

There are about 380 olfactory receptors that have a function in human nose olfactory epithelial cells, and the olfactory receptor protein introduced in the olfactory nanobiosensor manufacturing step (C) of the present invention is artificially expressed in Escherichia coli and is an olfactory receptor protein. In the case of the N terminal includes an amine group (-NH ₂ ). 3.31 μL of olfactory receptors were mixed with 40 μL of conductive polymer nanomaterial (0.1 wt%) with carboxylated (-COOH) functional groups, and then injected with 40 μL of coupling agent (1 wt%) at room temperature. The condensation reaction was induced by standing for 12 hours. Through this method, olfactory receptor nanobiosensor media chemically fixed to an electrode substrate can be easily prepared. The nanobiosensor medium is a source and a drain at each of the two ends of the electrode, and a conductive polymer nanomaterial to which the olfactory receptor protein is attached constitutes a channel region.

The content of the olfactory receptor protein is preferably 1:15 to 1:30 compared to the content of the polymer nanomaterial.

The agent used for the condensation reaction is not particularly limited, but 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium (DMT-MM) is preferably used. Do.

In step (D), target microparticles are detected using the prepared nanobiosensor medium. The matrix for sensor transducers, which convert chemical signals generated by chemical reactions between target molecules and olfactory receptors into electrical signals, consists of source and drain electrodes and channels that carry electrical signals. The liquid-ion gate is used, and the gate potential can be adjusted through the reference electrode and the counter electrode. Biochemical reactions between olfactory receptor proteins and odor particulates in field effect transistor arrays are used as primary transducers, and polymer nanomaterials are used as secondary transducers that convert signals from primary transducers to electrical signals. Through these primary and secondary transducers, current changes can be seen, and real-time monitoring of current flow between the source and drain is achieved through the accumulation or reduction of charge carriers in the polymer conductive nanoparticles.

In order to verify the sensitivity of the olfactory receptor nanobiosensor, amyl butyrate was used as the target odor microparticles, and 2 μL of the olfactory receptor nanobiosensor was injected at regular intervals. The change in current appearing at this time was measured. In addition, butyl butyrate, propyl butyrate, and hexyl bytyrate, which are non-target odor particulates, were used to verify the selectivity of the olfactory receptor nanobiosensor. Was carried out.

Control experiments were conducted to investigate the role of the olfactory receptor protein. It used a lipid membrane layer of Escherichia coli that does not contain olfactory receptor proteins, and there was no chemical / physical reaction with the target odor particulate.

As a result of measuring the current value according to the constant voltage of the nano biosensor prepared according to the content of the olfactory receptor protein, it was confirmed that the source-drain current change value decreases as the content increases.

About 380 human olfactory receptor proteins are known, each of which binds specifically to different types of target odor particles.

The constant voltage of the matrix is preferably in the range of 5 mV to 50 mV considering that the material is a polymer.

[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

In order to covalently bond the functional polymer nanotubes to the electrode substrate, the electrode substrate was first placed in a beaker containing 500 mL of 3-aminopropyltrimethoxysilane (5 wt%) solution and the stirring was performed at RPM 600. The electrode surface was modified with an amine functional group (-NH ₂ ) by standing for 6 hours at. As a result, an electrode substrate into which an amine group was introduced was obtained.

[Example 2]

The experiment was carried out in the same manner as in Example 1, but the surface modifier for modifying the amine functional group on the electrode substrate was carried out using 3-aminopropyltriethoxysilane. As a result, the same result as in Example 1 was obtained.

Example 3

The experiment was carried out in the same manner as in Example 1, but the surface modifier for modifying the amine functional group on the electrode substrate was carried out using vinyltrimethoxysilane. As a result, the same result as in Example 1 was obtained.

Example 4

In order to obtain the polymer nanomaterial with functional group, 0.15 parts by weight of the polymer monomer without functional group is added to the amount of the polymer monomer with functional group, and 100 nm in diameter and 60 S / conductivity through the melt polymerization method. A carboxylated nanomaterial, m, could be obtained.

Example 5

The experiment was carried out in the same manner as in Example 4, but the carboxylated nanomaterial having a diameter of 200 nanometers and a conductivity of 8 S / m was obtained through a reverse polymerization process by adding 0.3 weight ratio.

Example 6

40 μL of the polymer nanotubes prepared in Example 5 (0.01 wt%) and 1 wt% coupling agent (4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium ( DMT-MM)) 40 μL was dropped on the electrode substrate, and then condensation reaction was performed at room temperature for 6 hours. As a result, the polymer nanomaterial could be covalently fixed on the electrode substrate. (Fig. 1,2)

Example 7

The experiment was carried out in the same manner as in Example 6, but using the prepared 100 nanometer carboxylated polypyrrole nanorod (rod). As a result, it was confirmed that the polymer nanorad was covalently bonded on the electrode substrate.

Example 8

The experiment was carried out in the same manner as in Example 6, but the prepared 100 nanometer carboxylated polypyrrole nano ellipsoidal (ellipsoidal) was used. As a result, it was confirmed that the polymer nanoellipsoid was covalently bonded on the electrode substrate.

Example 9

3.31 μL of the olfactory receptor protein is dropped on the electrode substrate prepared in Example 4, and left at room temperature for 10 hours. As a result, it was confirmed that the olfactory receptor nanobiosensor medium was prepared. (Fig. 3)

Example 10

The experiment was carried out in the same manner as in Example 9, except that 6.61 μL of the olfactory receptor protein was used. As a result, it was confirmed that the olfactory receptor nanobiosensor medium was prepared. And it was confirmed that much improved than the example 9 in the sensitivity test.

Example 11

The experiment was carried out in the same manner as in Example 9, except that 13.24 μL of the olfactory receptor protein was used. As a result, it was confirmed that the olfactory receptor nanobiosensor medium was prepared. And it was confirmed that the sensitivity test was further improved than Examples 9 and 10.

Example 12

The experiment was performed in the same manner as in Example 10, but in order to perform a control experiment, a lipid membrane layer of Escherichia coli which did not express an olfactory receptor protein was separated and used. As a result, no chemical or physical reactions were seen in future sensitivity results.

Example 13

As a result of measuring the current value of the olfactory receptor nanobiosensor medium prepared in Examples 9, 10, and 11 according to the constant voltage (5 mV / s), the source-drain current change value decreased as the content of the olfactory receptor protein was increased. I could confirm that.

Example 14

In order to investigate the sensitivity of the target odor microparticles to the olfactory receptor nanobiosensors prepared in Examples 9, 10, and 11, 2 μL was sequentially injected from 1 pM to 100 μM of amyl butrate (AB). As a result, it was confirmed that the sensitivity, the highest sensitivity was confirmed that the olfactory receptor nanobiosensor prepared in Example 11.

Example 15

The experiment was carried out in the same manner as in Example 14, but butyl butyrate (BB) was used. As a result, it was confirmed that the sensitivity is significantly lower than the AB.

Example 16

The experiment was carried out in the same manner as in Example 14, but propyl butyrate (PB) was used. As a result, it was confirmed that the sensitivity is significantly lower than the AB.

Example 17

The experiment was carried out in the same manner as in Example 14, but using hexyl butyrate (HB). As a result, it was confirmed that the sensitivity is significantly lower than the HB.

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 schematic diagram of a source-drain gate field effect transistor biosensor fabricated in Example 6 of the present invention.

2 is a scanning electron micrograph of the carboxylated polypyrrole nanotube prepared in Example 6 of the present invention placed on an electrode substrate.

FIG. 3 is a scanning electron micrograph of a 200 nanometer polypyrrole nanotube covalently bonded to a carboxylated polypyrrole nanotube and an olfactory receptor prepared through Example 9 of the present invention.

Claims (10)

Surface-modifying the electrode substrate with an amine group (-NH ₂ ); Immobilizing a conductive polymer nanomaterial having a functional group on the surface modified electrode substrate prepared above using a condensation reaction; And Preparing an olfactory nanobiosensor by attaching an olfactory receptor protein to a conductive polymer nanomaterial having a functional group fixed on an electrode substrate using a condensation reaction; And Manufacture of olfactory nanobiosensors that sense electric signals according to chemical / electrical property changes between primary and secondary transistors using field effect transistor arrays using the prepared nanobiosensor media and can effectively detect specific odor particles Way. The method of claim 1, wherein during the surface modification of the electrode substrate, the material used is 3-aminopropyltrimethoxysilane, 3-aminepropyltriethoxysilane, vinyltrimethoxy Method for producing olfactory nanobiosensors using vinyltrimethoxysilanes. The method according to claim 1, wherein the conductivity of the conductive polymer nanomaterial having a functional group is 0.001 to 100 S / m. The method of manufacturing a olfactory nanobiosensor according to claim 1, wherein the conductive polymer nanomaterial having a functional group has a diameter of 10 to 1000 nanometers. According to claim 1, wherein the polymer nanomaterial having a functional group of the nanorod (nanorod), nanotubes (nanotube), nanoellipsoidal (nanoellisoidal) manufacturing method of the olfactory nanobiosensor. The method according to claim 1, wherein the ratio of the carboxyl (-COOH) functional groups generated in the conductive polymer is in the range of 0.05 to 0.5 parts by weight relative to the polymer. The method of manufacturing a olfactory nanobiosensor according to claim 1, wherein the condensation reaction time ranges from 1 hour to 24 hours when the polymer nanomaterial having the functional group is fixed to the surface-modified electrode substrate.        The method of claim 1, wherein the amine group of the olfactory receptor protein included in the lipid membrane enables selective immobilization of carboxyl groups on the surface of the polymer nanomaterial.        The method of claim 1, wherein the condensation reaction time of the olfactory receptor receptor is 1 hour to 24 hours.        According to claim 1, in order to verify the excellent sensitivity and selectivity of the manufactured olfactory receptor nanobiosensor odor particles are amyl butyrate, butyl butyrate, propyl butyrate, hexyl Method for detecting the olfactory nanobi sensor, characterized in that using butyl (hexyl butrate).

Images