KR100989290B1 - Water-soluble conjugated polymers for chemical sensors and biosensors, method for producing the same and sensors containing the same - Google Patents

Abstract

The present invention relates to a water-soluble conjugated polymer compound containing benzothiadiazole or bisthienylbenzothiadiazole, and more particularly, to a water-soluble conjugated (conjugated) polymer that can be used as a chemical sensor and a biosensor. It relates to a compound and a method for preparing the same.
The polymer compound according to the present invention can be used as a cognitive substance for the protein, and in particular, since the selectivity for lysozyme, which is one of the representative proteins, is high, it is possible to quantitatively and qualitatively analyze the protein lysozyme as a biosensor. Medical research, clinical trials, and chemical analysis can be widely used.

Description

Water-soluble conjugated polymers for chemical sensors and biosensors, method for producing the same and sensors containing the same}

 The present invention relates to a water-soluble conjugated polymer compound containing benzothiadiazole or bisthienylbenzothiadiazole, and more particularly, can selectively recognize a specific protein and be used as a chemical sensor and a biosensor. The present invention relates to a water-soluble conjugated polymer compound and a preparation method thereof. The present invention also relates to a water-soluble conjugated polymer compound for a biosensor and a selective detection method for a protein using the same.

 In many cases, such as diagnostics, medical research, clinical trials, or chemical analysis, it is necessary to quickly and accurately analyze the concentrations of various proteins in test solutions. In this analysis, various types of biosensor materials using materials that are selective for a specific protein are used. The general sensing signal of the sensor uses electrical properties such as electricity, resistance, and potential difference, or optical properties such as color and fluorescence. Among them, the change of color and fluorescent color can be easily distinguished by the naked eye, so it is one of the easy methods to measure even without special equipment.

In general, low molecular weight materials are widely used as optical sensors for changing the color and fluorescent colors, and the measurement target material can detect various chemical species such as molecules as well as ions.

Conjugated polymers are capable of converting chemical signals into measurable electrical or optical signals, and in particular, they have increased sensitivity (amplification) when expressing signals in response to interaction with the object under test. And sensor materials such as anion detection and explosive (aromatic nitro compound) detection (DT McQuade, AE Pullen, TM Swager, Chem. Rev. 100, 2537, 2000). In addition, the water-soluble conjugated polymer is widely used as a sensor material for low molecular weight biomaterials, proteins, DNA, etc. by using the advantages of increasing the sensitivity of the conjugated polymer and dissolving in water (C. Li, M. Numata, M. Yu, S. Wang, S. Shinkai, Angew. Chem. Int. Ed. 42, 4803, 2003; I.-B. Kim, JN Wilson, UHF Bunz, Chem. Commun. 10, 1273, 2005; BS Gaylord, AJ Heeger, GC Bazan, Proc. Natl. Acad. Sic. USA 99, 10954, 2002).

Among them, the development of a sensor material for detecting a protein is currently attracting much attention, and in particular, the detection of a protein by a change in color or fluorescent color of the sensor material is widely used due to the sensitivity of the detection signal. Among proteins, the detection of lysozyme has become a physiologically important factor. Lysozyme is an antimicrobial enzyme found in animal tissues, saliva, and tears that prevent bacterial infection by hydrolyzing mucopolysaccharides on the cell walls of bacteria. Therefore, the detection of lysozyme is necessary to maintain homeostasis through the immune system in the human body.

Amphoteric electrolyte materials, such as proteins, have inherent isoelectric points, resulting in negative or positive charges depending on pH. If the protein becomes negatively charged by adjusting the pH in an aqueous solution, when a pigment compound containing a cation is added, the protein and the pigment compound may be aggregated to change color or fluorescent color, and conversely, if the protein becomes positively charged, A pigment compound having an anion can be added to change the color or fluorescent color by association.

Efforts to detect proteins with color sensors due to color change due to the characteristics of the proteins have been attempted by small molecules and polymer materials. They are known to induce a change in color or fluorescence color by association when a positive or negatively charged ionic property is approached in an aqueous solution and a protein of opposite charge approaches (SW Thomas III, GD Joly, TM Swager, Chem. Rev. 107). , 1339, 2007).

The biosensor using the compound as described above was measured in the form of simply quenching (fluorescence quenching) the change in fluorescence color. That is, when proteins and compounds are associated, they are measured in the form of the same increase and decrease of fluorescence, not change of color or fluorescence. In this case, it is difficult to detect a protein having a similar isoelectric point. Lysozyme and cytochrome C have isoelectric points of 11.0 and 10.0, respectively. When the cationic protein and the anionic pigment compound were measured in neutral (pH 7) state, the fluorescence was reduced in the aqueous solution containing both proteins. In addition, BSA (bovine serum albumin) has an isoelectric point of 4.9, which shows a negative charge in the neutral state, but shows a decrease in fluorescence through hydrophobic groups and hydrophobic bonds of the anion pigment compound, which affects the detection of proteins and affects the sensitivity of sensor molecules. The selectivity can be reduced.

The conjugated polymer as described above has a significant drop in solubility in water due to hydrophobic groups in the polymer backbone, making it difficult to use as a biosensor as a water-soluble polymer sensor material.

 The present invention is to solve the problems of the prior art as described above, the object of the present invention is to provide a water-soluble conjugated polymer compound and a method for producing the same can be applied to chemical sensors and biosensors by increasing the solubility of the conjugated polymer in water It is done.

 It is another object of the present invention to provide a water-soluble conjugated polymer compound and a method for preparing the same, which can be applied to chemical sensors and biosensors that selectively recognize only specific proteins, especially lysozyme, and thus display a detection signal as a change in fluorescence color.

 In addition, the present invention is prepared by adjusting the repeating unit of the polymer compound causing the fluorescence change to benzothiadiazole or bisthienyl benzothiadiazole to produce a multi-fluorescence color chemical sensor and biosensor that changes the fluorescence color differently To provide a way.

The present invention relates to a water-soluble conjugated polymer compound, a method for producing the same, and a sensor including the same.

More specifically, the present invention is a water-soluble conjugated polymer having benzothiadiazole or bisthienylbenzothiadiazole in the polymer main chain and containing an ionic group in the side chain to impart water solubility to the polymer. The present invention relates to a compound, a method for preparing the same, and a sensor for selectively recognizing a specific protein including the same.

The present invention relates to a water-soluble conjugated polymer compound containing benzothiadiazole or bisthienylbenzothiadiazole, and more particularly, can selectively recognize a specific protein and be used as a chemical sensor and a biosensor. The present invention relates to a water-soluble conjugated polymer compound and a preparation method thereof. The present invention also relates to a water-soluble conjugated polymer compound for a biosensor and a selective detection method for a protein using the same.

Hereinafter, the present invention will be described in detail.

The water-soluble conjugated polymer compound according to the present invention has a polymerization unit selected from the following Chemical Formulas 1 to 4.

[Formula 1]

[Formula 2]

(3)

[Formula 4]

[In Formulas 1 to 4, R ₁ and R ₂ are each independently a linear or branched C1 to C6 alkyl group substituted with sulfonate or tri (C1-C7) alkylammonium salt at the terminal;

Ar is (C6-C20) arylene or (C3-C12) heteroarylene containing at least one hetero atom selected from N, O and S;

a, b, c and d are mole fractions, a and c are each independently a real number of 0 to 1, b is 1-a and d is 1-c.]

Terminals of the alkyl groups of R ₁ and R ₂ may be independently substituted with a sulfonate, trimethylammonium salt, ethyldimethylammonium salt, diethylmethylammonium salt, triethylammonium salt, and the alkyl groups of R ₁ and R ₂ are independent of each other. Is selected from methyl, ethyl, i-propyl, n-propyl, i-butyl, n-butyl, t-butyl, n-pentyl, i-pentyl or n-hexyl, wherein Ar is divalent phenylene or Selected from thienylene.

The molecular weight of the water-soluble conjugated polymer according to the present invention is not limited in principle, but is preferably 3,000 to 100,000 as the number average molecular weight (Mn), and the range can be appropriately adjusted according to the characteristics required for the use thereof.

Hereinafter, each reaction is explained in full detail, taking the manufacturing method of the water-soluble conjugated polymer compound by Suzuki coupling as an example.

(A) water soluble Conjugate Benzothiadiazole type  Preparation of Polymer Compound

The water-soluble conjugated polymer compound including the polymerized unit represented by the formula (1) according to the present invention is a benzothiazole derivative (I), a water-soluble monomer (III) and an aromatic monomer (IV) as described in Scheme 1 below. It is prepared by Suzuki coupling in the presence of a tetrakis (triphenylphosphine) palladium catalyst.

Scheme 1

[In Scheme 1,

Ar is (C6-C20) arylene or (C3-C12) heteroarylene containing at least one hetero atom selected from N, O and S;

R ₁ and R ₂ are each independently a linear or branched C 1 to C 6 alkyl group substituted with sulfonate or tri (C 1 -C 7) alkylammonium salt at the end;

,

또는

이고;Y is X when Cl, Br or I

,

or

ego;

,

또는

인 경우 Y는 Cl, Br 또는 I이고;X

,

or

When Y is Cl, Br or I;

a and b are mole fractions where a is a real number from 0 to 1 and b is 1-a.]

In addition, the mole fraction of a and the mole fraction of b of the water-soluble conjugated polymer compound having a polymerized unit represented by Formula 1 can be controlled by the equivalent ratio of the benzothiadiazole derivative (I) and the water-soluble monomer (III). have.

In the embodiment of the present invention as the aromatic monomer (IV) was prepared a water-soluble conjugated polymer compound using benzene-1,4-diboronic acid bis (pinacol) ester, but is not limited to this by Suzuki coupling Any monomer may be used as long as it can produce the above-mentioned conjugated benzothiazole-based high molecular compound.

Suzuki in the presence of a benzothiadiazole derivative (I) and a water-soluble monomer (III) in the presence of an equivalent amount of an aromatic monomer (IV) and a tetrakis (triphenylphosphine) palladium catalyst equal to the sum of the moles of (I) and (III) The polymer is formed by a coupling reaction (N. Miyaura, A. Suzuki, Chem. Rev. 95, 2457, 1995). The conjugated benzothiazole-based high molecular compound can be prepared by two methods, and more specifically, the first method is a benzothiazole derivative (I) having a halogen at both terminals, that is, Cl, Br, or I, and a water-soluble compound. The monomer (III) and aromatic monomer (IV) having functional groups such as borane or boronic acid or boron ester at both ends are polymerized by the Suzuki coupling reaction, and another method is in contrast to borane or boron at both ends. It is produced by polymerizing a benzothiazole derivative (I) having a functional group such as an acid or boron ester, and a water-soluble monomer (III) and a dihalo aromatic monomer (IV) by Suzuki coupling. Depending on which of the above two methods is used, an appropriate benzothiadiazole derivative and aromatic monomer (IV) can be selected and used.

 As another compound according to the present invention, the benzothiazole-based high molecular compound represented by Chemical Formula 2 is water-soluble with a benzothiazole derivative (I) as shown in the following Scheme 2 under reaction conditions similar to those of Chemical Formula 1 The monomer (III) is prepared by Suzuki coupling reaction in the presence of a tetrakis (triphenylphosphine) palladium catalyst, wherein the compound of formula 2 consists of a homopolymer, not a copolymer.

Scheme 2

[In Scheme 2,

R ₁ and R ₂ are each independently a linear or branched C 1 to C 6 alkyl group substituted with sulfonate or tri (C 1 -C 7) alkylammonium salt at the end;

,

또는

이고;Y is X when Cl, Br or I

,

or

ego;

,

또는

인 경우 Y는 Cl, Br 또는 I 이다.]
X

,

or

Where Y is Cl, Br or I.]

(B) water soluble Conjugate Biscyclinylbenzothiadiazole series  Preparation of Polymer Compound

The water-soluble conjugated polymer compound including the polymerized unit represented by the formula (3) according to the present invention, as described in Scheme 3 below, the bis cyclinyl benzothiazole derivative (II) and the water-soluble monomer (III) (II) It is prepared by Suzuki coupling in the presence of the same number of moles of aromatic monomer (IV) and tetrakis (triphenylphosphine) palladium catalyst.

Scheme 3

[In Scheme 3,

Ar is (C6-C20) arylene or (C3-C12) heteroarylene comprising at least one hetero atom selected from N, O and S;

R ₁ and R ₂ are each independently a linear or branched C 1 to C 6 alkyl group substituted with sulfonate or tri (C 1 -C 7) alkylammonium salt at the end;

,

또는

이고;Y is X when Cl, Br or I

,

or

ego;

,

또는

인 경우 Y는 Cl, Br 또는 I이고;X

,

or

When Y is Cl, Br or I;

c and d are mole fractions, c is a real number from 0 to 1 and d is 1-c.]

In the embodiment of the present invention as the aromatic monomer (IV) was prepared a water-soluble conjugated polymer compound using benzene-1,4-diboronic acid bis (pinacol) ester, but is not limited to this by Suzuki coupling Any monomer may be used as long as it can produce the above-mentioned conjugated bisthienylbenzothiazole-based polymer compound.

In addition, the molar fraction of c and the molar fraction of d of the water-soluble conjugated polymer compound having a polymerized unit represented by the formula (3) are equivalent ratios of the biscyclinylbenzothiadiazole derivative (II) to the water-soluble monomer (III). Can be adjusted by

 As another compound according to the present invention, the biscyclinylbenzothiadiazole-based high molecular compound represented by Formula 4 is represented by the following Scheme 4 under reaction conditions similar to that of Formula 2, wherein The diazole derivative (II) and the water-soluble monomer (III) are prepared by Suzuki coupling reaction in the presence of a tetrakis (triphenylphosphine) palladium catalyst, wherein the compound of Formula 4 consists of a homopolymer, not a copolymer.

Scheme 4

[In Scheme 4,

R ₁ and R ₂ are each independently a linear or branched C 1 to C 6 alkyl group substituted with sulfonate or tri (C 1 -C 7) alkylammonium salt at the end;

,

또는

이고;Y is X when Cl, Br or I

,

or

ego;

,

또는

인 경우 Y는 Cl, Br 또는 I 이다.]
X

,

or

Where Y is Cl, Br or I.]

Polymerization of formula (3) or (4) with a water-soluble conjugated polymer compound or bis-thienylbenzothiadiazole containing a polymer unit of formula (1) or (2) having a benzothiazole in the main chain according to the present invention A water-soluble conjugated polymer compound containing a unit or a sensor comprising the same is useful for detecting a protein. The sensor includes a chemical sensor and a biosensor. By taking advantage of the amphoteric electrolyte characteristics of the protein, it predicts the charge of the protein according to pH and adds a compound having the opposite charge to detect the protein by the change of color and fluorescence by association. In particular, as can be seen in the embodiment can be used as a purpose for selectively sensing in lysozyme.

As described above, the polymer compound according to the present invention can be used as a protein for a university cognitive substance, and in particular, since the selectivity for lysozyme is high, it is possible to quantitatively and qualitatively analyze protein lysozyme as a biosensor for diagnosis, medical research, and clinical experiment. Chemical analysis can be widely used. In addition, since the fluorescence characteristics of the benzothiazole-based polymer and the bis-thienyl benzothiazole-based polymer are different from each other, they can be widely used as multi-fluorescence color biosensors.

The polymer material of the present invention described so far can be applied to various technical fields such as chemical sensor materials and biosensor materials, and in particular, has physical properties corresponding to sensor functions such as responsiveness and selectivity by control of guest molecules. It can be applied to molecular recognition sensor materials.

The present invention will be described in detail through the following examples. However, these examples are for illustrative purposes only and the present invention is not limited thereto.

[ Production Example  One] Benzothiadiazole type  Preparation of Monomer

A) 4,7- Dibromo -2,1,3- Of benzothiadiazole (compound I-a, X = Br)  Produce

10 g (73.4 mmol) of 2,1,3-benzothiadiazole were added to 50 mL of bromic acid and then heated to 110 ° C. 11.3 mL (220 mmol) of bromine was added dropwise to the solution, followed by additional 20 mL of bromic acid for smooth stirring. After stirring for 2 hours the reaction was filtered, the filtrate was cooled to room temperature and filtered again. Finally, the solid obtained by filtration was washed three times with water, washed again with methanol and dried to obtain 16.37 g (75.8%) of 4,7-dibromo-2,1,3-benzothiadiazole.

¹ H NMR (300 MHz, CDCl ₃ ) δ = 7.75 (2H, aromatic) ppm.

B) 4,7-bis (4,4,5,5,- Tetramethyl -1,3,2- Dioxaborolane -2-yl) benzo-2,1,3-thiadiazole (compound I-b, X = 4,4,5,5- tetramethyl-1,3,2-dioxaborolane)  Produce

1 g (3.41 mmol) of 4,7-dibromo-2,1,3-benzothiadiazole and 1.96 g (20 mmol) of potassium acetate were added to 10 mL of 1,4-dioxane and heated to 80 ° C. 363 mg (1.43 mmol) of bis (pinacolato) diboron and 89 mg (0.71 mmol) of [1,1′-bis (diphenylphosphino) ferrocane] dichloropalladium (II) Put it. After stirring for 12 hours, the mixture was cooled to room temperature, and 30 mL of distilled water and 90 mL of ethyl acetate were added to the reaction solution to extract the product, and the organic layer was recovered. Magnesium sulfate was added to the collected organic layer, followed by stirring, followed by filtration to obtain a filtrate. After evaporation of the filtrate, the obtained solid was separated by column chromatography using a 97: 3 mixed solution of hexane and ethyl acetate, and then purified by 4,7-bis (4,4,5,5, -tetramethyl-1,3, 600 mg (46%) of 2-dioxaborolane-2-yl) benzo-2,1,3-thiadiazole were obtained.

¹ H NMR (300 MHz, CDCl ₃ ): δ = 8.04 (2H, aromatic), 1.41 (24H, alkyl group) ppm.

[ Production Example  2] Biscyclinylbenzothiadiazole series  Preparation of Monomer

A) 4,7- Bis (5-bromothiophen-2-yl) benzo -2,1,3-thiadiazole (compound II -a, X = Br Manufacturing

2 g (6.67 mmol) of 4,7-di (thiophen-2-yl) -2,1,3-benzothiadiazole were dissolved in 52 mL of chloroform and 52 mL of acetic acid, followed by 2.5 g ( 14.01 mmol) of N-bromosuccinimide (NBS) is added slowly. After stirring for 24 hours at room temperature, the reaction was filtered, and the obtained solid was recrystallized in DMF and dried to give 1 g of 4,7-bis- (5bromothiophen-2-yl) benzo-2,1,3 -Obtain thiadiazole (33%).

¹ H NMR (300 MHz, DMSO) δ = 8.16 (2H, aromatic), 7.98 (2H, aromatic), 7.41 (2H, aromatic) ppm.

B) 4,7-bis [5- (4,4,5,5,- Tetramethyl -1,3,2- Dioxaborolane -2 days) Thiophene -2 days] Benzo -2,1,3- Thiadiazol (compound II -b, X = 4,4,5,5- tetramethyl -1,3,2-dioxaborolane)

300 mg (0.65 mmol) of 4,7-bis (5-bromothiophen-2-yl) benzo-2,1,3-thiadiazole and 370 mg (3.77 mmol) of potassium acetate were mixed with 1,4-dioxine. It was added to 5 mL of saine and heated to 100 ℃. Add 363 mg (1.43 mmol) of bis (pinacolato) diboron and 89 mg (0.71 mmol) of [1,1′-bis (diphenylphosphino) ferrosene] dichloropalladium (II). After stirring for 12 hours, the mixture was cooled to room temperature, 20 mL of tertiary distilled water and 60 mL of ethyl acetate were added to the reaction solution to extract the product, and the organic layer was recovered. Magnesium sulfate was added to the collected organic layer, followed by stirring, followed by filtration to obtain a filtrate. After evaporating the obtained filtrate, the obtained solid was separated by column chromatography using 97: 3 mixed solution of hexane and ethyl acetate using 104 mg (29%) of 4,7-bis [5- (4,4,5, 5, -tetramethyl-1,3,2-dioxaborolane-2-yl) thiophen-2-yl] benzo-2,1,3-thiadiazole was obtained.

¹ H NMR (300 MHz, DMSO): delta = 8.18 (2H, aromatic), 8.01 (2H, aromatic), 1.45 (24H, alkyl group) ppm.

[ Production Example  3] Preparation of Monomer for Manufacturing Water-Soluble Polymer

A) 1,4- Dibromo -2,5- Bis (4-sulfonatobutoxy) benzenesodium salt (compound III -a, X = Br , R _One = R ₂ =- CH ₂ CH ₂ CH ₂ CH ₂ S (= O) (= O) ONa)

4 g (14.01 mmol) of 2,5-dibromo-1,4-hydroquinone and 1.8 g (44.8 mmol) of sodium hydroxide were added to 35 mL of ethanol and heated to 80 ° C. To the solution was added 3.44 g (44.8 mmol) of 1,4-butane sultone. After stirring for 12 hours, the mixture was cooled to room temperature and the reaction was filtered. The obtained solid was washed with ethanol and dried to obtain 7.85 g (96%) of 1,4-dibromo-2,5-bis (4-sulfonatobutoxy) benzenesodium salt.

¹ H NMR (300 MHz, D ₂ O): delta = 7.41 (2H, aromatic), 4.16 (4H, alkyl group), 3.08 (4H, alkyl group), 2.00 (4H, alkyl group), 1.96 (4H, alkyl group) ppm.

B) 1,4- Vis (3- Bromopropoxy ) -2,5-Dibromobenzene (Compound III -b, X = Br , R _One = R ₂ =- CH ₂ CH ₂ CH ₂ Br )

3.52 g (10.00 mmol) of 1,4-bis (3-bromopropoxy) benzene was dissolved in 80 mL of methylene chloride, and 0.01 g (0.18 mmol) of iron was added thereto, followed by stirring at room temperature. 3.43 g (21.4 mmol) of bromine are diluted in 40 mL of methylene chloride and slowly added to the solution. After stirring for 12 hours the reaction was filtered. 50 mL of tertiary distilled water was added to the obtained filtrate and the organic layer was recovered. Magnesium sulfate was added to the collected organic layer, followed by stirring, followed by filtration to obtain a filtrate. After evaporating the filtrate, the obtained solid was separated by column chromatography using a 4: 1 mixed solution of hexane and ethyl acetate using 4.03 g (79%) of 41,4-bis (3-bromopropoxy) -2. , 5-dibromobenzene was obtained.

¹ H NMR (300 MHz, CDCl ₃ ): δ = 7.14 (2H, aromatic), 4.13 (4H, alkyl group), 3.69 (4H, alkyl group), 2.35 (4H, alkyl group) ppm.

[ Example  1-3] Benzothiadiazole type  Preparation of Polymer Compound

[ Example  1] a polymer of formula (1) R _One = R ₂ = 4- Sulfonytobutyl , Ar = 1,4- Phenylene Manufacturing

26.5 mg (0.090 mmol) of 4,7-dibromo-2,1,3-benzothiadiazole ( compound Ia ) and 1,4-dibromo-2,5-bis (4-sulfonatobutoxy) benzene sodium salt (compound III -a) 474.4 mg (0.812 mmol ) and 1,4-dimethyl benzene-boronic acid bis (pinacol) ester 297.7 mg (0.902 mmol), and tetrakis (triphenylphosphine) palladium catalyst 3.5 mg ( 0.003 mmol) was removed from the dehumidified 6 mL of DMF and 9 mL of 2M Na ₂ CO ₃ It was dissolved in the mixed solution and refluxed at 90 ° C. for 48 hours. After the reaction, the mixture was cooled to room temperature, poured into a methanol / acetone / ether mixed solution having a volume ratio of 500 mL of 10:40:50 to precipitate crystals, and the precipitate was filtered. The solid obtained by filtration was dissolved in tertiary distilled water and then filtered through an osmosis membrane to obtain a polymer of Formula 1 having a molecular weight of 12,400 or more. The elemental analysis showed that x occupies a mole fraction of 10%.

¹ H NMR (300 MHz, D ₂ O) δ = 8.1 to 7.3 (2H, aromatic), 7.2 to 6.7 (2H, aromatic), 3.9 (4H, alkyl group), 2.8 (4H, alkyl group), 1.7 to 1.3 (7.8) H, alkyl group) ppm.

[ Example  2] a polymer of formula 1 ( R _One = R ₂ = 3- N, N, N - Trimethylammoniumpropyl , Ar = 1,4-phenylene)

26.5 mg (0.090 mmol) of 4,7-dibromo-2,1,3-benzothiadiazole ( compound Ia ) and 1,4-bis (3-bromopropoxy) -2,5-dibromobenzene ( Compound III- b ) 414 mg (0.812 mmol) and 297.7 mg (0.902 mmol) of 1,4-benzenediboronic acid bis (pinacol) ester, and 3.5 mg (0.003 mmol) of tetrakis (triphenylphosphine) palladium catalyst ) Dehumidified 9 mL toluene and 4.5 mL 2M Na ₂ CO ₃ It was dissolved in the mixed solution and refluxed at 90 ° C. for 48 hours. After the reaction, the mixture was cooled to room temperature and poured into 200 mL of methanol to precipitate crystals. The precipitate was filtered, and the obtained solid was washed with 200 mL of acetone and filtered. The solid obtained by filtration was dissolved in 10 mL of THF, cooled to -78 ° C, and slowly added 4 mL of trimethylamine. After raising the temperature of the reaction solution to room temperature, the mixture was stirred for 6 hours and poured into 200 mL of acetone to precipitate. The precipitated solid was dissolved in tertiary distilled water and filtered through an osmosis membrane to obtain a polymer of Formula 1 having a molecular weight of 12,400 or more. The elemental analysis showed that x occupies a mole fraction of 10%.

¹ H NMR (300 MHz, D ₂ O) δ = 8.1-7.3 (2H, aromatic), 7.2-6.7 (2H, aromatic), 4.1 (4H, alkyl group), 3.8-3.2 (6H, alkyl group), 2.9 (4H , Alkyl group), 1.7 (4H, alkyl group) ppm.

[ Example  3] a polymer of formula (2) R _One = R ₂ = 4- Sulfonytobutyl Manufacturing

4,7-bis (4,4,5,5, -tetramethyl-1,3,2-dioxaborolane-2-yl) benzo-2,1,3-thiadiazole ( Compound Ib ) 100 mg (0.258 mmol) and 1,4-dibromo-2,5-bis (4-sulfo Nei Tobu) benzene sodium salt (compound III -a) 151 mg (0.258 mmol ), and tetrakis (triphenylphosphine) 2.3 mg (0.002 mmol) of palladium catalyst were added with 6 mL of dehumidified DMF and 9 mL of 2M Na ₂ CO _3. It was dissolved in the mixed solution and refluxed at 90 ° C. for 48 hours. After the reaction, the mixture was cooled to room temperature, poured into a methanol / acetone / ether mixed solution having a volume ratio of 500 mL of 10:40:50 to precipitate crystals, and the precipitate was filtered. The solid obtained by filtration was dissolved in tertiary distilled water, and then filtered through an osmosis membrane to obtain a polymer of Formula 2 having a molecular weight of 12,400 or more.

H NMR (300 MHz, D ₂ O) δ = 8.3-7.6 (3H, aromatic), 7.4-7.0 (2H, aromatic), 4.1 (2H, alkyl group), 3.0 (2.5H, alkyl group), 1.9-1.6 (4.7) H, alkyl group) ppm.

[ Example  4-6] Biscyclinylbenzothiadiazole series  Preparation of Polymer Compound

[ Example  4] a polymer of formula (3) R _One = R ₂ = 4- Sulfonytobutyl , Ar = 1,4- Phenylene Manufacturing

4,7-bis (5-bromo-thiophen-2-yl) benzo -2,1,3- Im ah oxadiazole (Compound II -a) 21.6 mg (0.057 mmol ) and 1,4-dibromo -2 , 5-bis (4-sulfo Nei Tobu) benzene sodium salt (compound III -a) 300.0 mg (0.514 mmol ) and 1,4-dimethyl benzene-boronic acid bis (pinacol) ester 188.0 mg (0.571 mmol), and 3.5 mg (0.003 mmol) of tetrakis (triphenylphosphine) palladium catalyst were added to 8 mL of DMF with dehumidification and 12 mL of 2M Na ₂ CO _3. It was dissolved in the mixed solution and refluxed at 100 ° C. for 48 hours. After the reaction, the mixture was cooled to room temperature, poured into a methanol / acetone / ether mixed solution having a volume ratio of 500 ml of 10:40:50 to precipitate crystals, and the precipitate was filtered. The solid obtained by filtration was dissolved in tertiary distilled water and then filtered through an osmosis membrane to obtain a polymer of Formula 3 having a molecular weight of 12,400 or more. The elemental analysis showed that x occupies 8% mole fraction.

¹ H NMR (300 MHz, D ₂ O) δ = 8.1 to 7.3 (3.1H, aromatic), 7.2 to 6.7 (2H, aromatic), 4.0 (2.8H, alkyl group), 3.0 (3H, alkyl group), 2.0 to 1.5 (5H, alkyl group) ppm.

[ Example  5] a polymer of formula (3) R _One = R ₂ = 3- N, N, N - Trimethylammoniumpropyl , Ar = 1,4-phenylene)

4,7-bis [5- (4,4,5,5, -tetramethyl-1,3,2-dioxaborolane-2-yl) thiophen-2-yl] benzo-2,1,3- 41.2 mg (0.090 mmol) of thiadiazole ( compound II- b ) and 414 mg (0.812 mmol) of 1,4-bis (3-bromopropoxy) -2,5-dibromobenzene ( compound III- b ) And 297.7 mg (0.902 mmol) of 1,4-benzenediboronic acid bis (pinacol) ester, and 3.5 mg (0.003 mmol) of tetrakis (triphenylphosphine) palladium catalyst with 4.5 mL of dehumidified toluene and 4.5 mL 2M Na ₂ CO ₃ It was dissolved in the mixed solution and refluxed at 100 ° C. for 48 hours. After the reaction, the mixture was cooled to room temperature and poured into 200 mL of methanol to precipitate crystals. The precipitate was filtered, and the obtained solid was washed with 200 mL of acetone and filtered. The solid obtained by filtration was dissolved in 10 mL of THF, cooled to -78 ° C, and slowly added 4 mL of trimethylamine. After raising the temperature of the reaction solution to room temperature, the mixture was stirred for 6 hours and poured into 200 mL of acetone to precipitate. The precipitated solid was dissolved in tertiary distilled water and filtered through an osmosis membrane to obtain a polymer of Formula 1 having a molecular weight of 12,400 or more. The elemental analysis showed that x occupies a mole fraction of 10%.

¹ H NMR (300 MHz, D ₂ O) δ = 8.1 to 7.3 (3.1H, aromatic), 7.2 to 6.7 (2H, aromatic), 4.0 (2.8H, alkyl group), 3.7 to 3.2 (4.7H, alkyl group), 3.0 (3H, alkyl group), 2.0 (3.1H, alkyl group) ppm.

[ Example  6] a polymer of formula 4 ( R _One = R2 = 4- Sulfonytobutyl Manufacturing

4,7-bis [5- (4,4,5,5, -tetramethyl-1,3,2-dioxaborolane-2-yl) thiophen-2-yl] benzo-2,1,3- Sy O oxadiazole (compound II -b) 143 mg (0.258 mmol ) and 1,4-dibromo-2,5-bis (4-sulfo Nei Tobu) benzene sodium salt (compound III -a) 151 mg (0.258 mmol), and 2.3 mg (0.002 mmol) of tetrakis (triphenylphosphine) palladium catalyst were removed with 6 mL of dehumidified DMF and 9 mL of 2M Na ₂ CO _3. It was dissolved in the mixed solution and refluxed at 100 ° C. for 48 hours. After the reaction, the mixture was cooled to room temperature, poured into a methanol / acetone / ether mixed solution having a volume ratio of 500 mL of 10:40:50 to precipitate crystals, and the precipitate was filtered. The solid obtained by filtration was dissolved in tertiary distilled water, and then filtered through an osmosis membrane to obtain a polymer of Formula 4 having a molecular weight of 12,400 or more.

¹ H NMR (300 MHz, D ₂ O) δ = 8.2-7.7 (2H, aromatic), 7.6-7.1 (2H, aromatic), 4.0 (4H, alkyl group), 3.0 (4H, alkyl group), 2.0-1.5 (8H , Alkyl group) ppm.

[ Example  7] Manufactured Anionic  Performance Evaluation of Polymers as Protein Sensors

In order to confirm the biochemical detection ability of the polymers prepared by Examples 1, 3, 4 and 6, the polymer compounds were dissolved in 0.1 molar concentration of sodium phosphate buffer (pH 9.55). Each was set at 1.72 × 10 ⁻⁵ molarity. In addition, the fluorescence spectrophotometer changes the solution's fluorescence to 8.26 × 10 ^-6 molarity by adding lysozyme and cytochrome C, which are positively charged proteins at pH 9.55, and BSA having low isoelectric point but hydrophobic bond with the polymer main chain It was observed using.

As a result, it was possible to observe different fluorescence changes depending on the protein of the compound of Example 1 or the compound of Example 4, which is a polymer compound according to the invention. Particularly, in the case of lysozyme, the fluorescence of only the polymer before protein addition was decreased and a new fluorescence was shown, showing a completely different fluorescence color before and after lysozyme addition. As a result of measuring the fluorescence by the fluorescence photometer, in the polymer compound of Example 1, blue fluorescence at 430 nm was reduced by 93% by the addition of cytochrome C, and blue fluorescence at 430 nm and 535 nm when BSA was added. Green fluorescence increased by 115% and 193%, respectively. In the case of lysozyme, the fluorescence of the polymer before lysozyme addition decreased and new fluorescence appeared, unlike the previous two cases. When lysozyme was added, blue fluorescence at 430 nm decreased by 60% and green fluorescence at 535 nm increased by 116%.

In addition, in the high molecular compound of Example 4 according to the present invention, blue fluorescence at 430 nm was reduced by 94% in cytochrome C. When BSA was added, blue fluorescence at 430 nm and red fluorescence at 650 nm were 129%, respectively. And increased by 937%. In the case of lysozyme, the blue fluorescence of the polymer before the addition of lysozyme was decreased and a new red fluorescence appeared. When lysozyme was added, blue fluorescence at 417 nm decreased by 67% and red fluorescence at 650 nm increased by 557%.

As a result, it was confirmed that the prepared polymer compound can be used as a selective biosensor according to the protein. In addition, when benzothiadiazole is included in the polymer backbone, the lysozyme is detected to indicate green fluorescence, and when bisthienyl benzothiazole is included in the polymer backbone, the fluorescence is detected. It can be used as a sensor.

[ Example  8] Manufactured Cationic  Performance Evaluation of Polymers as Protein Sensors

In order to confirm the biochemical detection capability of the polymers of the polymer compounds prepared in Examples 2 and 5, the polymer compounds were dissolved in 0.1 molar concentration of sodium phosphate buffer (pH 2.5), respectively, at 1.72 × 10 ⁻⁵ molarity. Fit. A small amount of pepsin, a protein having a negative charge at pH 3.5, with a low isoelectric point was added thereto, and the change in fluorescence of the solution was observed using a fluorophotometer to a concentration of 8.26 × 10 ⁻⁶ .

As a result, different fluorescence changes were observed depending on the protein of Example 2 or Example 5, which is a polymer compound according to the invention. When pepsin was added to the compound of Example 2, blue fluorescence at 430 nm was reduced by 58% and green fluorescence at 535 nm was increased by 124%. In addition, the compound of Example 5 was reduced by 74% blue fluorescence of 417 nm, red fluorescence of 650 nm was increased by 638%.

As described above, pepsin is an enzyme acting in gastric acid, which is strongly acidic, has an isoelectric charge of 1.0 or less, resulting in a negative charge at pH 3.5, thereby changing the fluorescent color due to the association of the cationic polymers of Examples 2 and 5 with electrostatic attraction It could be confirmed that it can be used as a protein sensor.

Claims (7)

A water-soluble conjugated polymer compound having a polymerization unit of the formula (4).
[Chemical Formula 4]

[In Formula 4, R ₁ and R ₂ are each independently a linear or branched C1 to C6 alkyl group substituted with sulfonate or tri (C1-C7) alkylammonium salt at each end.] The method of claim 1,
The terminal of the alkyl group of R ₁ and R ₂ is independently water-soluble conjugated polymer compound, characterized in that substituted with sulfonate, trimethylammonium salt, ethyldimethylammonium salt, diethylmethylammonium salt, triethylammonium salt. The method of claim 1,
The water-soluble conjugated polymer compound is a water-soluble conjugated polymer compound, characterized in that the number average molecular weight (Mn) is 3,000 to 100,000. A water-soluble conjugated polymer compound of Chemical Formula 4 according to Scheme 4 was prepared by suzuki coupling bisthienylbenzothiazole derivative (II) and water-soluble monomer (III) in the presence of a tetrakis (triphenylphosphine) palladium catalyst. How to make.
Scheme 4

[In Scheme 4, R ₁ and R ₂ are each independently a linear or branched C 1 to C 6 alkyl group substituted with sulfonate or tri (C 1 -C 7) alkylammonium salt at each end;
Y is X when Cl, Br or I

,

or

ego;
X

,

or

Where Y is Cl, Br or I.] A sensor comprising the water-soluble conjugated polymer compound according to any one of claims 1 to 2. 6. The method of claim 5,
The sensor is characterized in that for detecting the protein. The method of claim 6,
The protein is a sensor, characterized in that the lysozyme.