KR101110170B1 - Method for selectively detecting cysteine via water-soluble fluorescent hyperbranched conjugated polymer-metal ions complexation - Google Patents

Abstract

The present invention is a metal ion-soluble superbranched conjugated polymer composite having a property of dissolving in water by the introduction of a sulfonate or ammonium salt ionic group capable of reacting with a metal ion, and a method for preparing the same, and the metal ion-soluble superbranched copolymer The present invention relates to a sensor including a liquefied polymer composite, and more particularly, to a metal ion-soluble superbranched conjugated polymer composite having a large number of receptors combined with a metal ion, which can be used as a chemical sensor and a biosensor, and a method of manufacturing the same. And it relates to a sensor comprising the same.
The metal ion-water soluble hyperbranched conjugated polymer composite according to the present invention introduces a receptor capable of reacting with a specific biomaterial, especially metal ions such as cobalt, in the side chain, and thus cobalt-bonded water soluble hyperbranched conjugated polymer composite As a biosensor material capable of reacting with amino acids such as cysteine, it is possible to quantitatively and qualitatively analyze cysteine, an amino acid, so that diagnostics, medical research, clinical experiments, and chemical analysis can be widely used.

Description

Method for selectively detecting cysteine via water-soluble fluorescent hyperbranched conjugated polymer-metal ions complexation

The present invention relates to the application of a chemical sensor and a biosensor using a water-soluble hyperbranched conjugated polymer compound having a property of dissolving in water by the introduction of sulfonic acid or sulfonic acid ionic groups, and more specifically, in the side chain The present invention relates to a metal ion-soluble superbranched conjugated polymer composite having a large number of ionic groups capable of reacting and which can be used as a chemical sensor and a biosensor based on metal ions combined with ionic groups. The present invention also relates to metal ion-soluble superbranched conjugated polymer complexes for chemical sensors and biosensors and to selective detection of amino acids, in particular cysteines, using the same.

The polymers that are closely related to our lives go beyond conventional polymers that have been recognized as plastics, and now organic light emitting diodes, organic thin film transistors, and organic memory devices. It is being transformed into specific materials such as electronic materials such as chemical, biosensor, and analytical chemistry (J. H, Burroughes, DDC Bradley, AR Brown, RN Marks, K. Mackay, RH Friend, PL Burns). , AB Holmes, Nature, 347, 539, 1990; CD Dimitrakopolous, P. Malenfant, Adv. Mater., 14, 99, 2002; F. Garnier, Acc. Chem. Res., 32, 209, 1999; P. Peumans , A. Yakimov, SR Forrest, J. Appl. Phys., 93, 3693, 2003).

The use of organic conjugated polymers in analytical chemistry has evolved both qualitatively and quantitatively. This is because the conductivity, oxidation-reduction potential difference, absorption or emission spectra are sensitive to changes in the environment, which are basic characteristics of conjugated polymers. In particular, if the conjugated polymer having a molecule or ion recognition function recognizes the target material and brings the above characteristics change, it can be applied as a sensor. Among various applications as sensors, researches using color shift derivative compounds for sensor and patterning are being conducted. Recently, a lot of attention has been paid to molecules capable of recognizing ions through color shifts, electrochemical or optical responses for applications in the chemical and bio fields. Although the response of the sensing material shows an excellent response, additional recognition devices are needed to recognize the fluorescence response signal to the outside, and recently, many color-shifting compounds that can be identified by the human eye according to the purpose of use have been studied. (AP De silva, HQN Gunaratne, T. Gunnlaugsson, AJM Huxley, CP McCoy, JT Rademacher, TE Rice, Chem. Rev., 97, 1515, 1997; D. Esteban-Gomez, L. Fabbrizzi, M. Licchelli, J. Org.Chem., 70, 5717, 2005).

Hyperbranched polymers are spotlighted as ideal materials for a wide range of applications due to their unique physical and chemical properties. The most characteristic property is that it has a lower viscosity than similar linear polymers in solution or in the molten state. This phenomenon can be explained by the characteristics of the branching properties of the hyperbranched polymer. The hyperbranched polymer does not take the form of a random coil of the linear polymer in a solution state but takes a globular shape. In the case of the linear polymer, the viscosity increases linearly in a certain molecular weight range, but if the limit is exceeded, the viscosity rapidly increases. This is explained by the entanglement of polymer chains, which does not occur in hyperbranched polymers. Thus, hyperbranched polymers have a lower viscosity than linear polymers. The branching properties of these hyperbranched polymers also affect the relative solubility in various solvents. Hyperbranched polymers have higher chemical reactivity and higher solubility than similar linear polymers. Linear polymers have a limited number of side chains, whereas hyperbranched polymers have branching properties, which increase the number of terminal groups depending on the degree of branching. You will have greater responsiveness. Superbranched polymers having these characteristics can be utilized as sensor materials through conjugation (J. Feng, Y. Li, M. Yang, J. Polym. Sci., Part A: Polym. Chem. 46, 222, 2008; W.-Y. Lai, R. Xia, Q.-Y. He, PA Levemore, W. Huang, DDC Bradley, Adv. Mater. 20, 1, 2008; R. Kikkeri, I. Garcia-Rubio, PH Seeberger, Chem. Commun. 235, 2009).

Cysteine is a sulfur-containing amino acid that exists naturally in small amounts in many proteins. Cysteine contains only thiol groups among the 20 basic amino acids. The thiol group undergoes a redox reaction when the cysteine is oxidized to form cystine. Because of this ability to be involved in redox, cysteine has antioxidant capacity. Cysteine is a major source of sulfur in human metabolism. Thus, cysteine belongs to the class of non-essential amino acids, but is essential for children, the elderly, and people with certain metabolic or sulfur absorption disorders. And there are many advantages such as: Cysteine is present in α-keratin, a major component of nails, skin, and hair, a component of collagen, and maintains skin elasticity. It is effective in treating rheumatoid arthritis and has chelating ability to remove metal ions in the body, which is involved in lipolysis and muscle production. Conversely, homocysteine, which has one more alkyl group than cysteine, causes blood coagulation at higher concentrations, causing oxidation of bad-cholesterol and causing it to precipitate in arteries. It also causes dementia, neural tube defects, complications during pregnancy, inflammatory bowel disease, and osteoporosis. Detection with fluorine turn-on using coumarins to detect homocysteine or cysteine with this particularity has been reported (RPM Steegerstheunissen, GHJ Boers, Trijbels, FJM Trijbels, TKABN Eskes, N. Engl. J. Med., 324, 199, 1991; PM Ueland, SE Vollset, Clin. Chem., 50, 1293, 2004; K.-S. Lee, T.-K. Kim, JH Lee, H.-J. Kim, JI Hong, Chem. Commun., 6173, 2008).

On the basis of the binding properties of the metal ions and amino acids as described above, efforts have been made to detect amino acids by color sensors due to color change by low molecular weight and high molecular materials. The pigment compound having a receptor that can react with the metal ion in solution state changes color or fluorescent color by reacting with the metal ion, and as the amino acid is added again, It is known to induce the change of the color and fluorescent color of a pigment compound. The detection of these amino acids is not carried out mostly in water, but in an organic solvent, and therefore the applicability is limited.

In addition, the biosensor using a linear polymer compound has a low solubility in water, a limited reactivity to metal ions, and a low efficiency, which makes it difficult to use a biosensor with high sensitivity.

The present invention is to solve the problems of the prior art as described above, by increasing the number of sulfonic acid, or sulfonate which is a side chain of a water-soluble conjugated polymer capable of ionic bonding with cobalt ions to use as a chemical sensor and a biosensor It is an object of the present invention to provide a water-soluble hyperbranched conjugated polymer compound having increased solubility and sensitivity, and a method for detecting cysteine through fluorescence change of a polymer due to interaction of cobalt ions and cysteine bound thereto.

It is another object of the present invention to provide a water-soluble hyperbranched conjugated polymer compound capable of being applied to chemical sensors and biosensors that selectively recognize only amino acids, in particular cysteine, to show a detection signal as a change in color and fluorescence intensity.

The present invention relates to a metal-soluble superbranched conjugated polymer compound composite for a chemical sensor and a biosensor having a property of dissolving in water by the introduction of a sulfonic acid and a sulfonate ionic group, and more particularly, to a side chain. Metal-soluble super-branched conjugated polymer complex, chemical sensor comprising the same, which has a water-soluble solubility and has a large number of ionic groups capable of reacting with metal ions in the side chain, which can be used as a chemical sensor and a biosensor. It relates to a selective detection method of cysteine using the same.

Hereinafter, the present invention will be described in detail.

The water-soluble hyperbranched conjugated polymer compound used to form the metal ion-water soluble hyperbranched conjugated polymer composite of the present invention may be introduced into a conjugated polymer side chain with sulfonic acid or sulfonate capable of ionic bonding with metal ions. In order to implement a hyperbranched structure, sulfonic acid or sulfonate introduced into the side chain of the water-soluble conjugated polymer is present as a functional group for imparting solubility to water and reactivity to metal ions. It is indicated by 1.

[Formula 1]

[In Formula 1, Ar is (C6-C20) arylene or (C2-C12) heteroarylene including 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 sulfonic acid or sulfonate at the terminal; m is an integer from 1 to 30.]

[Formula 2]

[In Formula 2, R ₁ , R ₂ , Ar, and R ₃ are the same as defined in Formula 1, and n is an integer of 5 to 199.]

Terminals of the alkyl groups of R ₁ and R ₂ may be independently substituted with sulfonic acid or sulfonate, and the alkyl groups of R ₁ and R ₂ may be independently methyl, ethyl, i-propyl, n-propyl, or i-butyl. , n-butyl, t-butyl, n-pentyl, i-pentyl or n-hexyl, wherein Ar is selected from phenylene or benzothiadiazole or bisthienylbenzothiadiazole .

Although the molecular weight of the water-soluble hyperbranched conjugated polymer compound of Chemical Formula 1 according to the present invention is not limited in principle, the number average molecular weight (Mn) is preferably 3,000 to 100,000, and the range is appropriately adjusted according to the characteristics required for the purpose. Can be used.

 The water-soluble hyperbranched conjugated polymer used in the present invention is a polymer filed by the present inventor (Korean Patent Application No. 10-2010-0003084), and a sulfonic acid or sulfonic acid salt is introduced into the side chain, thereby being able to bind with metal ions. Have it.

The water-soluble hyperbranched conjugated polymer compound of Chemical Formula 1 having arylene or heteroarylene according to the present invention in the main chain has sulfonic acid or sulfonic acid salt introduced into the side chain, so that it can be combined with metal ions. It is useful for forming liquefied polymer complexes and for detecting amino acids using the interaction of metal ions with amino acids in the complex. Utilizing the properties of the amine group, the carboxylic acid group or the thiol group of the amino acid, it is expected to inhibit the stability of the superbranched conjugated polymer and the metal ion complex in which the amine group, the carboxylic acid group or the thiol group are already formed in the aqueous solution. Due to the interaction of the metal ion-superbranched polymer complex is decomposed, the amino acid can be detected by the change in color and fluorescence of the polymer complex. In particular, as can be seen in the embodiment, it can be used as a purpose for selectively detecting cysteine.

On the other hand, the linear water-soluble conjugated polymer compound of formula (2) also reacts with the sulfonic acid or sulfonic acid salt of the side chain to form a metal ion-polymer complex, and detects amino acids using the reaction between the metal ion and the amino acid. It may be useful, but because of the limited number of sulfonic acids or sulfonic acid salts present in the side chain of the linear polymer, the number of ionic bonds with the metal ions is small, so that it can be seen that the solubility is low and the detection efficiency and selectivity are low as shown in the examples below. have.

As described above, the metal ion-water soluble hyperbranched conjugated polymer complex according to the present invention, that is, the metal ion-water soluble hyperbranched conjugated polymer complex introduced with a large number of receptors (sulfonic acid) that can easily bind with amino acids (cysteine) is a specific bio It can be used as a cognitive substance for materials, and especially when combined with metal ions (cobalt ions), it has high selectivity for amino acids (cysteine), which can be used in a variety of chemical sensors and biosensors. It can be widely used for chemical analysis.

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.

Preparation Example Preparation of Water-Soluble Conjugated Polymer Compound

Preparation Example 1 Water-Soluble Hyperbranched Conjugated Polymer Compound A (Formula 1, R ₁ = R ₂ = -CH ₂ CH ₂ CH ₂ CH ₂ S (= O) ₂ OH, Ar = 1,4-phenylene, R Preparation of ₃ = -B (OH) ₂ )

0.5 g (0.925 mmol) of 1,4-dibromobenzene-2,5-bis-4-butoxysulfonic acid, 0.204 g (1.232 mmol) of 1,4-benzenediboronic acid and tris (4-bromophenyl) amine 0.074 g (0.308 mmol), and 0.067 g (0.06 mmol) of a tetrakis (triphenylphosphine) palladium catalyst were dissolved in a dehumidified 15 mL of DMF and 8 ml of 2M Na ₂ CO ₃ , followed by 90 It was refluxed for 40 hours at ℃. After the reaction, the mixture was cooled to room temperature, poured into acetone to precipitate crystals, and the precipitate was filtered. After dissolving the solid obtained by filtration in tertiary distilled water, the water-soluble hyperbranched conjugated polymer compound A which has a molecular weight of 12,400 or more was obtained by filtration using an osmosis membrane.

¹ H NMR (300 MHz, D ₂ O) δ = 7.7 to 6.6 (8H, aromatic), 4.2 to 3.7 (6H, alkyl), 3.2 to 2.8 (6H, alkyl), 2.1 to 1.6 (12H, alkyl) ppm

Preparation Example 2 Water-Soluble Hyperbranched Conjugated Polymer Compound B (Formula 1, R ₁ = R ₂ = -CH ₂ CH ₂ CH ₂ CH ₂ S (= O) ₂ OH, Ar = 4,7-benzothiadia Preparation of the _{sol, R 3 = -B (OH)} 2)

0.5 g (0.925 mmol) of 1,4-dibromobenzene-2,5-bis-4-butoxysulfonic acid, 0.233 g (1.406 mmol) of 1,4-benzenediboronic acid and tris (4-bromophenyl) amine 0.037 g (0.154 mmol and 0.027 g (0.0925 mmol) of 4,7-dibromo-2,1,3-benzothiadiazole and 0.081 g (0.07 mmol) of tetrakis (triphenylphosphine) palladium catalyst Was dissolved in a mixed solution of 18 mL of THF and 8 mL of 2M Na ₂ CO ₃ and refluxed for 40 hours at 90 ° C. After the reaction, the mixture was cooled to room temperature, poured into acetone to precipitate crystals, and the precipitate was filtered. After dissolving the solid obtained by filtration in tertiary distilled water, the water-soluble hyperbranched conjugated polymer compound B which has a molecular weight of 12,400 or more was obtained by filtration using an osmosis membrane.

¹ H NMR (300 MHz, D ₂ O) δ = 8.1 to 6.8 (8H, aromatic), 4.1 to 3.8 (6H, alkyl group), 3.3 to 2.8 (6H, alkyl group), 2.3 to 1.8 (12H, alkyl group) ppm.

[Preparation Example 3] The water-soluble second branch Conjugated polymer compound C (Formula 1, R ₁ = R ₂ = -CH ₂ CH ₂ CH ₂ CH ₂ S (= O) ₂ OH, Ar = 4,7-bisthienylbenzothiadiazole, R ₃ Preparation of = -B (OH) ₂ )

0.3 g (0.555 mmol) of 1,4-dibromobenzene-2,5-bis-4-butoxysulfonic acid, 0.122 g (0.74 mmol) of 1,4-benzenediboronic acid and tris (4-bromophenyl) amine 0.044 g (0.185 mmol), 4,7-bis (5-bromothiophen-2-yl) benzo-2,1,3-thiadiazole 0.021 g (0.056 mmol), and tetrakis (triphenylforce) 0.067 g (0.06 mmol) of the pin) palladium catalyst were dissolved in a mixed solution of 15 mL of DMF and 8 ml of 2M Na ₂ CO ₃ which had been dehumidified, and refluxed at 90 DEG C for 40 hours. After the reaction, the mixture was cooled to room temperature, poured into acetone to precipitate crystals, and the precipitate was filtered. After dissolving the solid obtained by filtration in tertiary distilled water, the water-soluble hyperbranched conjugated polymer compound C which has a molecular weight of 12,400 or more was obtained by filtration using an osmosis membrane.

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.

[Comparative Manufacturing Example 1] The water-soluble linear ball liquefied polymer compound D (formula _{2, R 1 = R 2 =} -CH 2 CH 2 CH 2 CH 2 S (= O) 2 OH, Ar = 1,4- phenylene, R Preparation of ₃ = -B (OH) ₂ )

0.5 g (0.825 mmol) of 1,4-dibromobenzene-2,5-bis-4-butoxysulfonic acid, 0.183 g (1.11 mmol) of 1,4-benzenediboronic acid, and tetrakis (triphenylphosphine) 0.065 g (0.06 mmol) of the palladium catalyst were dissolved in a mixed solution of 30 mL of THF and 18 mL of 2M Na ₂ CO ₃ , which had been dehumidified, and refluxed at 90 ° C. for 48 hours. After the reaction, the mixture was cooled to room temperature, poured into acetone to precipitate crystals, and the precipitate was filtered. After dissolving the solid obtained by filtration in tertiary distilled water, the water-soluble linear conjugated polymer compound D which has a molecular weight of 10,000 or more was obtained by the filtration using an osmosis membrane.

¹ H NMR (300 MHz, D ₂ O) δ = 7.7 to 6.8 (8H, aromatic), 4.2 to 3.7 (6H, alkyl group), 3.2 to 2.8 (6H, alkyl group), 2.1 to 1.6 (12H, alkyl group) ppm.

[Comparative Preparation Example 2] Water-soluble linear ball liquefied polymer compound E (formula _{2, R 1 = R 2 =} -CH 2 CH 2 CH 2 CH 2 S (= O) 2 OH, Ar = 4,7- benzothiazol Oh diamond Preparation of the _{sol, R 3 = -B (OH)} 2)

0.5 g (0.825 mmol) of 1,4-dibromobenzene-2,5-bis-4-butoxysulfonic acid and 0.202 g (1.22 mmol) of 1,4-benzenediboronic acid and 4,7-dibromo-2, 0.027 g (0.0925 mmol) of 1,3-benzothiadiazole, and 0.07 g (0.061 mmol) of tetrakis (triphenylphosphine) palladium catalyst were dehumidified with 18 mL of THF and 8 mL of 2M Na ₂ CO. ₃ was dissolved in a mixed solution and refluxed at 90 ° C. for 40 hours. After the reaction, the mixture was cooled to room temperature, poured into acetone to precipitate crystals, and the precipitate was filtered. The solid obtained by filtration was melt | dissolved in tertiary distilled water, and the filtration using the osmosis membrane obtained the water-soluble super branched conjugated polymer compound E which has a molecular weight of 12,400 or more.

¹ H NMR (300 MHz, D ₂ O) δ = 8.0-6.8 (8H, aromatic), 4.2-3.6 (6H, alkyl group), 3.4-2.7 (6H, alkyl group), 2.3-1.8 (12H, alkyl group) ppm.

[Comparative Manufacturing Example 3] The water-soluble linear ball liquefied polymer compound F (formula _{2, R 1 = R 2 =} -CH 2 CH 2 CH 2 CH 2 S (= O) 2 OH, Ar = 4,7- bis Im in Neal Preparation of Benzothiadiazole, R ₃ = -B (OH) ₂ )

0.3 g (0.555 mmol) of 1,4-dibromobenzene-2,5-bis-4-butoxysulfonic acid and 0.131 g (0.79 mmol) of 1,4-benzenediboronic acid and 4,7-bis (5-bro) 0.021 g (0.056 mmol) of thiophen-2-yl) benzo-2,1,3-thiadiazole, and 0.045 g (0.04 mmol) of tetrakis (triphenylphosphine) palladium catalyst were dehumidified. It was dissolved in a mixture of mL of DMF and 8 ml of 2M Na ₂ CO ₃ and refluxed at 90 ℃ for 40 hours. After the reaction, the mixture was cooled to room temperature, poured into acetone to precipitate crystals, and the precipitate was filtered. After dissolving the solid obtained by filtration in tertiary distilled water, the water-soluble hyperbranched conjugated polymer compound F which has a molecular weight of 12,400 or more was obtained by the filtration using an osmosis membrane.

¹ H NMR (300 MHz, D ₂ O) δ = 8.1 to 7.3 (3.1H, aromatic), 7.2 to 6.7 (2H, aromatic), 4.1 (2.8H, alkyl group), 3.2 (3H, alkyl group), 2.2 to 1.6 (5H, alkyl group) ppm.

[ Example Performance Evaluation with Cysteine Sensors

[ Example  One] Anionic  Performance Evaluation of Polymers into Cysteine Sensors I

To compare the biochemical detection ability of the anionic polymer compounds prepared in Preparation Example 1 and Comparative Preparation Example 1 with respect to cysteine, the polymer compounds were dissolved in 0.006 molar concentration sodium phosphate buffer solution (pH 7.4), respectively, 2.0 × 10. ^{Adjusted to -5} molarity. Add the 2.5 × 10 ^-4 molar concentration of cobalt ions to the color and fluorescent color change of the solution and the 8.75 × 10 ^-4 molar concentration of amino acid (cysteine) to change the color and fluorescence color of the solution. It observed using. As a result, it was observed that the anionic hyperbranched conjugated polymer compound of Formula 1 and the anionic linear conjugated polymer compound of Formula 2 exhibited different fluorescence changes as cobalt ions and cysteine were added.

In the anionic hyperbranched conjugated polymer of Chemical Formula 1 according to Preparation Example 1, as the cobalt ions are added, the fluorescence of the polymer is reduced before addition. As a result of measuring fluorescence by a fluorescence photometer, in the case of the anionic hyperbranched conjugated polymer compound of Chemical Formula 1, the blue fluorescence at 416 nm was reduced by 29.6% by the addition of cobalt ions. On the basis of this, it was confirmed that the anionic hyperbranched conjugated polymer compound and the cobalt ion were bonded.

As the amino acid was added to the anionic hyperbranched conjugated polymer composite bound to the cobalt ions, it was observed that the color change and the fluorescent color changed, respectively. In particular, in the case of cysteine, the color and fluorescence intensity were different before and after cysteine addition, as the absorption and fluorescence of visible light of the polymer-high-balt ion composite before cysteine increased.

As a result of measuring the color change by the Uv-vis spectrometer, lysine, alanine, tyrosine, glycine, leucine, tryptophan, asparagine, valine, aspartic acid, arginine, The absorption at 352 nm was unchanged by the addition of serine, isoleucine, threonine, methionine, glutamine, phenylalanine, glutamic acid and histidine. In the case of cysteine, unlike the case of the previous 18 amino acids, it was found to increase significantly than the absorption of the polymer-cobalt ion complex before cysteine addition. The addition of cysteine increased the absorption at 352 nm by 362.9%.

As a result of measuring fluorescence by a fluorescence photometer, lysine, alanine, tyrosine, glycine, leucine, tryptophan, asparagine, valine, aspartic acid, arginine, serine, isoleucine in the case of anionic hyperbranched conjugated polymer compound There was no change in blue fluorescence at 416 nm by addition of threonine, methionine, glutamine, phenylalanine, glutamic acid and histidine. In the case of cysteine, unlike the previous 18 cases, the fluorescence of the polymer-cobalt ion complex before cysteine addition was significantly decreased. When cysteine was added, the blue fluorescence at 416 nm decreased by 75.8%.

On the other hand, in the anionic linear conjugated polymer of Chemical Formula 2 according to Comparative Preparation Example 1, as the cobalt ions are added, the fluorescence of the polymer before addition decreases. As a result of measuring fluorescence by a fluorescence photometer, in the case of the anionic hyperbranched conjugated polymer compound of Chemical Formula 2, the blue fluorescence at 416 nm was reduced by 22.9% by the addition of cobalt ions. Based on this, it was confirmed that the anionic linear conjugated polymer compound and the cobalt ion were bound. Similarly, the addition of amino acids to the anionic linear conjugated polymer compound bound to the cobalt ion showed different color and fluorescent color changes, respectively.

In the case of the anionic linear conjugated polymer compound-cobalt ion complex of Chemical Formula 2, the color change was measured by Uv-vis spectroscopy. As a result, lysine, alanine, tyrosine, glycine, leucine, tryptophan, asparagine, valine, aspartic acid, The absorption at 352 nm was unchanged by the addition of arginine, serine, isoleucine, threonine, methionine, glutamine, phenylalanine, glutamic acid and histidine. In the case of cysteine, unlike in the case of the previous 18 amino acids, it was found to slightly increase than the absorption of the polymer-cobalt ion complex before cysteine addition. The addition of cysteine increased the absorption at 352 nm by 162.9%.

As a result of measuring fluorescence by a fluorescence photometer, lysine, alanine, tyrosine, glycine, leucine, tryptophan, asparagine, valine, aspartic acid, arginine, serine, There was no change in blue fluorescence at 416 nm by addition of isoleucine, threonine, methionine, glutamine, phenylalanine, glutamic acid and histidine. In the case of cysteine, unlike the previous 18 cases, it was slightly reduced than the fluorescence of the polymer before cysteine addition. When cysteine was added, the blue fluorescence at 416 nm decreased by 45.2%.

[ Example  2] Anionic  Performance Evaluation of Polymers into Cysteine Sensors II

To compare and confirm the biochemical detection capability of the anionic polymer compounds prepared in Preparation Example 2 and Comparative Preparation Example 2 for cysteine, the polymer compounds were dissolved in 0.006 molar concentration sodium phosphate buffer solution (pH 7.4), respectively, 2.0 × 10. ^{Adjusted to -5} molarity. Add the 2.5 × 10 ^-4 molar concentration of cobalt ions to the color and fluorescent color change of the solution and the 8.75 × 10 ^-4 molar concentration of amino acid (cysteine) to change the color and fluorescence color of the solution. It observed using. As a result, it was observed that the anionic hyperbranched conjugated polymer compound of Formula 1 and the anionic linear conjugated polymer compound of Formula 2 exhibited different fluorescence changes as cobalt ions and cysteine were added.

In the case of the anionic hyperbranched conjugated polymer of Formula 1 according to Preparation Example 2, the fluorescence of the polymer before addition decreases as cobalt ions are added. As a result of measuring fluorescence by a fluorescence photometer, in the case of the anionic hyperbranched conjugated polymer compound of Chemical Formula 1, the blue fluorescence at 416 nm was reduced by 28.4% by the addition of cobalt ions. On the basis of this, it was confirmed that the anionic hyperbranched conjugated polymer compound and the cobalt ion were bonded.

As the amino acid was added to the anionic hyperbranched conjugated polymer composite bound to the cobalt ions, it was observed that the color change and the fluorescent color changed, respectively. In particular, in the case of cysteine, the color and fluorescence intensity were different before and after cysteine addition, as the absorption and fluorescence of visible light of the polymer-high-balt ion composite before cysteine increased.

As a result of measuring the color change by the Uv-vis spectrometer, lysine, alanine, tyrosine, glycine, leucine, tryptophan, asparagine, valine, aspartic acid, arginine, The absorption at 352 nm was unchanged by the addition of serine, isoleucine, threonine, methionine, glutamine, phenylalanine, glutamic acid and histidine. In the case of cysteine, unlike the case of the previous 18 amino acids, it was found to increase significantly than the absorption of the polymer-cobalt ion complex before cysteine addition. The addition of cysteine increased the absorption at 352 nm by 364.1%.

As a result of measuring fluorescence by a fluorescence photometer, lysine, alanine, tyrosine, glycine, leucine, tryptophan, asparagine, valine, aspartic acid, arginine, serine, isoleucine in the case of anionic hyperbranched conjugated polymer compound There was no change in blue fluorescence at 416 nm by addition of threonine, methionine, glutamine, phenylalanine, glutamic acid and histidine. In the case of cysteine, unlike the previous 18 cases, the fluorescence of the polymer-cobalt ion complex before cysteine addition was significantly decreased. When cysteine was added, the blue fluorescence at 416 nm decreased by 73.8%.

On the other hand, in the anionic linear conjugated polymer of Formula 2 according to Comparative Preparation Example 2, as the cobalt ions are added, the fluorescence of the polymer before addition decreases. As a result of measuring fluorescence by a fluorescence photometer, in the case of the anionic hyperbranched conjugated polymer compound of Chemical Formula 2, the blue fluorescence at 416 nm was reduced by 22.9% by the addition of cobalt ions. Based on this, it was confirmed that the anionic linear conjugated polymer compound and the cobalt ion were bound. Similarly, the addition of amino acids to the anionic linear conjugated polymer compound bound to the cobalt ion showed different color and fluorescent color changes, respectively.

In the case of the anionic linear conjugated polymer compound-cobalt ion complex of Chemical Formula 2, the color change was measured by Uv-vis spectroscopy. As a result, lysine, alanine, tyrosine, glycine, leucine, tryptophan, asparagine, valine, aspartic acid, The absorption at 352 nm was unchanged by the addition of arginine, serine, isoleucine, threonine, methionine, glutamine, phenylalanine, glutamic acid and histidine. In the case of cysteine, unlike in the case of the previous 18 amino acids, it was found to slightly increase than the absorption of the polymer-cobalt ion complex before cysteine addition. The addition of cysteine increased the absorption at 352 nm by 161.6%.

As a result of measuring fluorescence by a fluorescence photometer, lysine, alanine, tyrosine, glycine, leucine, tryptophan, asparagine, valine, aspartic acid, arginine, serine, There was no change in blue fluorescence at 418 nm by addition of isoleucine, threonine, methionine, glutamine, phenylalanine, glutamic acid and histidine. In the case of cysteine, unlike the previous 18 cases, it was slightly reduced than the fluorescence of the polymer before cysteine addition. When cysteine was added, the blue fluorescence at 418 nm was reduced by 44.2%.

[ Example  3] Anionic  Performance Evaluation of Polymers into Cysteine Sensors III

To compare and confirm the biochemical detection ability of the anionic polymer compounds prepared by Preparation Example 3 and Comparative Preparation Example 3 for cysteine, the polymer compounds were dissolved in 0.006 molar concentration sodium phosphate buffer solution (pH 7.4), respectively, and 2.0 × 10. ^{Adjusted to -5} molarity. Add the 2.5 × 10 ^-4 molar concentration of cobalt ions to the color and fluorescent color change of the solution and the 8.75 × 10 ^-4 molar concentration of amino acid (cysteine) to change the color and fluorescence color of the solution. It observed using. As a result, it was observed that the anionic hyperbranched conjugated polymer compound of Formula 1 and the anionic linear conjugated polymer compound of Formula 2 exhibited different fluorescence changes as cobalt ions and cysteine were added.

In the case of the anionic hyperbranched conjugated polymer of Chemical Formula 1 according to Preparation Example 3, as the cobalt ions are added, the fluorescence of the polymer is reduced before addition. As a result of measuring fluorescence by a fluorescence photometer, in the case of the anionic hyperbranched conjugated polymer compound of Chemical Formula 1, the blue fluorescence at 416 nm was reduced by 27.6% by the addition of cobalt ions. On the basis of this, it was confirmed that the anionic hyperbranched conjugated polymer compound and the cobalt ion were bonded.

As the amino acid was added to the anionic hyperbranched conjugated polymer composite bound to the cobalt ions, it was observed that the color change and the fluorescent color changed, respectively. In particular, in the case of cysteine, the color and fluorescence intensity were different before and after cysteine addition, as the absorption and fluorescence of visible light of the polymer-high-balt ion composite before cysteine increased.

As a result of measuring the color change by the Uv-vis spectrometer, lysine, alanine, tyrosine, glycine, leucine, tryptophan, asparagine, valine, aspartic acid, arginine, The absorption at 353 nm was unchanged by the addition of serine, isoleucine, threonine, methionine, glutamine, phenylalanine, glutamic acid and histidine. In the case of cysteine, unlike the case of the previous 18 amino acids, it was found to increase significantly than the absorption of the polymer-cobalt ion complex before cysteine addition. The addition of cysteine increased the absorption of 353 nm by 359.9%.

As a result of measuring fluorescence by a fluorescence photometer, lysine, alanine, tyrosine, glycine, leucine, tryptophan, asparagine, valine, aspartic acid, arginine, serine, isoleucine in the case of anionic hyperbranched conjugated polymer compound There was no change in blue fluorescence at 416 nm by addition of threonine, methionine, glutamine, phenylalanine, glutamic acid and histidine. In the case of cysteine, unlike the previous 18 cases, the fluorescence of the polymer-cobalt ion complex before cysteine addition was significantly decreased. When cysteine was added, the blue fluorescence at 418 nm decreased by 71.1%.

On the other hand, in the anionic linear conjugated polymer of Formula 2 according to Comparative Preparation Example 3, as the cobalt ions are added, the fluorescence of the polymer before addition decreases. As a result of measuring fluorescence by a fluorescence photometer, in the case of the anionic hyperbranched conjugated polymer compound of Chemical Formula 2, the blue fluorescence at 418 nm was reduced by 21.3% by the addition of cobalt ions. Based on this, it was confirmed that the anionic linear conjugated polymer compound and the cobalt ion were bound. Similarly, the addition of amino acids to the anionic linear conjugated polymer compound bound to the cobalt ion showed different color and fluorescent color changes, respectively.

In the case of the anionic linear conjugated polymer compound-cobalt ion complex of Chemical Formula 2, the color change was measured by Uv-vis spectroscopy. As a result, lysine, alanine, tyrosine, glycine, leucine, tryptophan, asparagine, valine, aspartic acid, There was no change in absorption at 353 nm by the addition of arginine, serine, isoleucine, threonine, methionine, glutamine, phenylalanine, glutamic acid and histidine. In the case of cysteine, unlike in the case of the previous 18 amino acids, it was found to slightly increase than the absorption of the polymer-cobalt ion complex before cysteine addition. The addition of cysteine increased the absorption of 353 nm by 164.1%.

As a result of measuring fluorescence by a fluorescence photometer, lysine, alanine, tyrosine, glycine, leucine, tryptophan, asparagine, valine, aspartic acid, arginine, serine, There was no change in blue fluorescence at 418 nm by addition of isoleucine, threonine, methionine, glutamine, phenylalanine, glutamic acid and histidine. In the case of cysteine, unlike the previous 18 cases, it was slightly reduced than the fluorescence of the polymer before cysteine addition. When cysteine was added, the blue fluorescence at 418 nm decreased by 41.1%.

As a result of Examples 1, 2, and 3, the prepared polymer compound exhibits a decrease in fluorescence according to the addition of cobalt ions, and an optional biosensor in which absorption and fluorescence change vary according to the type of amino acid added. Although it can be confirmed that the linear polymer (Comparative Preparation Examples 1, 2 and 3) having low solubility and having a limited number of sulfonic acids in the side chain, the cysteine was selectively detected, but the fluorescence intensity was decreased relatively. Low sensitivity results were obtained. On the other hand, in the case of hyperbranched polymers (manufacture examples 1, 2, 3) containing a large number of sulfonic acids in the side chain, cysteine was selectively and efficiently detected, thereby greatly increasing absorption and greatly reducing fluorescence.

Based on this, hyperbranched conjugated polymer having a high content of ionic groups in the side chain selectively detects an amino acid (cysteine) as shown in Formula 1, whereas linear conjugated polymer such as Formula 2 is relatively insoluble. As the number of side chain sulfonic acid or sulfonic acid salt is limited, it can be confirmed that the efficiency is low.

Claims (13)

A metal ion-soluble superbranched conjugated polymer composite having a metal ion bonded to an anion group of a sulfonic acid or a sulfonic acid salt of a side chain of a water-soluble hyperbranched conjugated polymer compound represented by Formula 1 below.
[Formula 1]

[In Formula 1, Ar is (C6-C20) arylene or (C2-C12) heteroarylene including 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 sulfonic acid or sulfonate at the terminal; m is an integer from 1 to 30.] The method of claim 1,
The alkyl groups of R ₁ and R ₂ are each independently selected from methyl, ethyl, i-propyl, n-propyl, i-butyl, n-butyl, t-butyl, n-pentyl, i-pentyl or n-hexyl Metal ion-soluble superbranched conjugated polymer composite, characterized in that one. The method of claim 1,
Ar is a metal ion-soluble super-branched conjugated polymer composite, characterized in that at least one selected from phenylene, benzothiadiazole and bisthienylbenzothiadiazole. The method of claim 1,
The water-soluble hyperbranched conjugated polymer compound of Chemical Formula 1 has a number average molecular weight of 3,000 to 100,000, the metal ion-soluble superbranched conjugated polymer composite. The method of claim 1,
Metal ion-water-soluble hyperbranched conjugated polymer composite, characterized in that the metal ion is a transition metal ion. The method of claim 1,
Metal ion-soluble superbranched conjugated polymer composite, characterized in that the metal ion is cobalt ion. A sensor comprising a metal ion-soluble superbranched conjugated polymer composite according to any one of claims 1 to 6. The method of claim 7, wherein
The sensor is characterized in that for selective detection of amino acids using a fluorescence photometer. The method of claim 8,
And said sensor is for selective detection of cysteine. A method for detecting cysteine, comprising using the sensor according to claim 7. A method for producing the metal ion-water soluble hyperbranched conjugated polymer composite according to claim 1 by dissolving a water-soluble superbranched conjugated polymer compound represented by Chemical Formula 1 in an aqueous solution and administering metal ions thereto.
[Formula 1]

[In Formula 1, Ar is (C6-C20) arylene or (C2-C12) heteroarylene including 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 sulfonic acid or sulfonate at the terminal; m is an integer from 1 to 30.] 12. The method of claim 11,
The alkyl groups of R ₁ and R ₂ are each independently selected from methyl, ethyl, i-propyl, n-propyl, i-butyl, n-butyl, t-butyl, n-pentyl, i-pentyl or n-hexyl Method for producing a metal ion-soluble hyperbranched conjugated polymer composite, characterized in that one. 12. The method of claim 11,
Ar is a method for producing a metal ion-soluble superbranched conjugated polymer composite, characterized in that at least one selected from phenylene, benzothiadiazole and bisthienylbenzothiadiazole.