KR20190103718A - Boronic acid-carbon dot complex having selective detection properties of dopamine, a method for producing the same, and a dopamine biosensor using the same - Google Patents

Abstract

The present invention relates to a boronic acid-carbon dot complex which can detect the presence of dopamine by changing the fluorescent color, and can easily detect dopamine with the naked eye using the fluorescent color that emits light by detecting an amount of dopamine through a change in photosensitivity of color; a preparation method thereof; and a dopamine biosensor using the same.

Description

Boronic acid-carbon dot complex having selective detection properties of dopamine, a method for producing the same, and a dopamine biosensor using the same}

The present invention relates to a boronic acid-carbon dot complex having a selective detection property of dopamine, a method for preparing the same, and a dopamine biosensor using the same. More specifically, the presence of dopamine can be detected by changing the fluorescence color, and The present invention relates to a boronic acid-carbondot complex which can easily detect dopamine with the naked eye through a fluorescent color that senses the amount of dopamine by changing the photosensitivity, and a preparation method thereof and a dopamine biosensor using the same.

Dopamine (Dopamine, DA) is a catecholamine family neurotransmitter, and is known to play an important role in the function of hormones, kidneys, myocardium and the central nervous system. Abnormal levels of dopamine in the central nervous system have been implicated as a major cause of several neurological disorders, including schizophrenia, epilepsy, Parkinson's disease, attention deficit disorder, and hyperactivity disorder.

Dopamine content in blood or urine is an important biomarker in medical diagnosis of neurological diseases, but the concentration of dopamine in vivo is very low. For example, the concentration of dopamine in extracellular fluid of the central nervous system is less than 100 nM. Moreover, the concentration of dopamine varies very widely from 1.0 × 10 ⁻⁷ M to 1.0 × 10 ⁻³ M over time and location.

In general, the techniques used for the detection of dopamine include immunoassay, spectrophotometry, chromatography, and electrochemical methods, but in the case of chromatography, spectral resolution is limited and very time consuming, and electrochemical methods are similar to redox. There was a limit in detecting very low concentrations of dopamine due to interference from the analyte with the potential.

On the other hand, the fluorescent material is a nano-sized fluorescent material such as quantum dot (quantum dot) and carbon nanoparticles (Carbon dot). Quantum dots have been studied intensively in biological applications because of their excellent optical properties, but there are problems of toxicity, environmental hazards, UV light excitation, photochromic or chemical instability.

On the other hand, carbon nanoparticles have excellent solubility in water, strong luminescence, high biocompatibility, ease of surface functionalization, long-term photo discoloration prevention, and low cost. However, carbon nanoparticle aqueous solution maintains stability for a long time. It is not easy to store, compared to solid systems, and has limitations that are not easy to use.

Accordingly, the inventors of the present invention have completed the present invention in an effort to provide a dopamine biosensor having excellent stability, dopamine selectivity and detection performance, long-term storage, and ease of use.

KR 10-1764005 B1 KR 10-1069310 B1

The present invention has been made to solve the above problems, it is an object of the present invention to provide a boronic acid-carbon dot complex having a selective detection properties of dopamine, a preparation method thereof and a dopamine biosensor using the same.

On the other hand, the object of the present invention is not limited to the above-mentioned object, other objects that are not mentioned will be clearly understood from the following description.

Boronic acid-carbon dot composite having a selective detection characteristic of dopamine according to the present invention for achieving the above object is a carbon dot surface modified with a diacrylate-based polymer and a monosaccharide; It is combined with the functional group of the carbon dot, aminophenylboronic acid represented by the following formula (1); includes.

[Formula 1]

In addition, according to the present invention, a method for preparing a boronic acid-carbon dot complex having selective detection properties of dopamine is prepared by (1) preparing a mixture comprising a diacrylate-based polymer and a monosaccharide, and subjecting the mixture to solvent-thermal synthesis. Reacting to prepare a surface-modified carbon dot; And (2) mixing and reacting the surface-modified carbon dot, aminophenylboronic acid represented by the following Formula 1, and a crosslinking agent to complete the boronic acid-carbon dot complex.

In addition, the dopamine biosensor using boronic acid-carbon dot complex having selective detection properties of dopamine according to the present invention has -COOH or -OH functional group, and carbon dot and -NH ₂ functional group in which acrylic functional group is introduced at the terminal. A boronic acid-carbondot complex introduced and chemically bonded to the —COOH or —OH functional group of the carbon dot, and including aminophenylboronic acid represented by the following Formula 1; And a base portion functionalized with a methacrylate-based polymer and having an acrylic functional group introduced to the surface thereof, wherein the boronic acid-carbon dot complex and the boron acid-based reaction are reacted between the acrylic functional groups of the base portion. It is characterized in that the carbon dot composite is immobilized.

By solving the above problems, the boronic acid-carbon dot complex of the present invention includes carbon dot having fluorescent properties and aminophenyl boronic acid which can be combined with dopamine, thereby detecting the presence and amount of dopamine and emitting fluorescent color. Through the naked eye there is an effect that can easily determine the presence and amount of dopamine.

In addition, the method of preparing the boronic acid-carbon dot composite of the present invention synthesizes the surface-modified carbon dot through a solvent thermal synthesis reaction, and the coupling reaction of the carbon dot and aminophenyl boronic acid, selective dopamine in a simple process There is an effect that can easily synthesize a complex having a detection characteristic.

On the other hand, the dopamine biosensor using the boronic acid-carbon dot complex of the present invention can be easily fixed to the base portion into which the acrylic functional group is introduced through the acrylic functional group of the boronic acid-carbon dot complex, and thus has excellent bonding strength and stability. It can be used for thin film-based biosensors such as glass and glass.

In addition, since the dopamine biosensor using the boronic acid-carbon dot complex of the present invention is in a solid state, it is easy to transport, and selectivity and detection performance are not degraded over time, so that it can be stored for a long time, and dopamine can be easily detected. It works.

1 is a view schematically showing a method for producing a BA-CD composite according to an embodiment of the present invention.
2 is a view schematically showing a manufacturing method of glass BA-CD which is a dopamine biosensor according to an embodiment of the present invention.
3 is a view for confirming the structure of BA-CD according to an embodiment of the present invention, (a) is a DLS spectrum of BA-CD aqueous solution (0.25mg / ml), (b) is a TEM image, (c) shows the FT-IR spectra of CD and BA-CD aqueous solutions.
4 shows the fluorescence and UV-Vis spectra of BA-CD aqueous solution (0.35 mg / ml) according to the excitation wavelength.
FIG. 5 schematically shows the formation of boronate ester complexes via boronate ester linkages between APBA and DA.
6 is a diagram showing the fluorescence characteristics of the APBA / DA mixed aqueous solution, (a) is the PL spectrum of the APBA / DA mixed aqueous solution (500uM / 500uM) according to pH (wavelength: 360nm), (b) at 500mA When the concentration of DA or APBA is fixed, the PL spectrum (excitation wavelength: 477 nm) of the APBA / DA mixed aqueous solution according to the concentration of APBA (black circle) or DA (red square) is shown.
Figure 7 shows the PL spectrum of the APBA / DA mixed aqueous solution, (a) is a PL spectrum according to the DA concentration when the APBA concentration (500uM) is fixed, (b) APBA when the DA concentration (500uM) is fixed It is PL spectrum according to concentration, and (c) and (d) show PL spectrum and PL intensity (excitation wavelength: 477 nm) of APBA / DA mixed aqueous solution mixed in the same concentration.
8 shows an increase in (a) PL spectrum (excitation wavelength: 360 nm) and (b) PL intensity of BA-CD / DA mixed aqueous solution according to the concentration of aqueous solution of BA-CD (excitation wavelength: 477 nm).
9 is a diagram for evaluating DA detection performance of BA-CD aqueous solution, (a) and (b) are PL intensity and PL spectral image according to the concentration of BA-CD aqueous solution, (c) is BA-CD / It is PL intensity | strength when DA mixed aqueous solution is excited at 477 nm, (d) shows the PL spectrum of APBA, DA, APBA / DA, and BA-CD / DA aqueous solution.
Figure 10 shows the PL spectrum (excitation wavelength: 360 nm) of the mixed aqueous solution containing (a) the aqueous solution containing biopolymers in the aqueous CD solution, and (b) the PL spectrum of the mixed aqueous solution mixed with the aqueous solution of BA-CD (excitation wavelength: 360 nm) (the biopolymers are glucose, lactose, ascorbic acid, urea and dopamine, and the final concentration of the aqueous biopolymer solution was adjusted to 0.25 mg / ml and 40 uM, respectively).
11 shows (FF ₀ ) / F ₀ (excitation wavelength: 477 nm) of the mixed solution of (a) the aqueous solution containing biopolymers and (b) the (FF) of the mixed aqueous solution mixed with the BA-CD aqueous solution. ₀ ) / F ₀ (excitation wavelength: 477 nm) (biopolymers are glucose, lactose, ascorbic acid, urea and dopamine, F and F ₀ are PL intensities before and after addition of an aqueous solution containing biopolymers). Final concentrations of analytes tested were adjusted to 0.25 mg / ml and 40 uM, respectively).
12 is a view for evaluating the dopamine detection performance of the glass BA-CD, a dopamine biosensor according to an embodiment of the present invention, (a) is a PL spectrum (excitation wavelength: 360 nm), (b) is an image according to different fluorescence quantum yields (Φs), and (c) shows PL intensity (excitation wavelength: 477 nm).
FIG. 13 shows (FF ₀ ) / F ₀ and images when the glass BA-CD excited at 360 nm was added to an aqueous solution of biopolymers and then excited at 477 nm (biopolymers were (i) glucose, (ii ) Lactose, (iii) ascorbic acid, (iv) urea, (v) uric acid and (vi) dopamine, F and F0 are PL strengths before and after glass BA-CD is injected, and the concentration of the aqueous biopolymer solution is 40 uM. to be).
14 is a view for evaluating the stability of the glass BA-CD, a dopamine biosensor according to an embodiment of the present invention, (a) is a glass BA that was added to the aqueous dopamine solution for 0 weeks, 2 weeks and 4 weeks, respectively -PL spectrum (excitation wavelength: 360 nm) and image of -CD, (b) shows PL intensity (excitation wavelength: 477 nm).
FIG. 15 is a PL spectrum (excitation wavelength: 360 nm) evaluating the detection performance of dopamine in human serum, (a) a BA-CD aqueous solution added with serum, and (b) a PL spectrum of glass BA-CD added to serum. It is shown.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the embodiments are to make the disclosure of the present invention complete, and those skilled in the art to which the present invention pertains. It is provided to inform the full scope of the invention. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In this specification, the singular forms also include the plural unless specifically stated otherwise in the phrases.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. On the other hand, illustrated and detailed description of the configuration and its operation and effects can be easily understood from those skilled in the art will be briefly or omitted and will be described in detail with respect to the parts related to the present invention.

According to an aspect of the present invention, a carbon dot surface-modified with a diacrylate-based polymer and a monosaccharide; And aminophenylboronic acid bonded to a functional group of the carbon dot; provides boronic acid-carbon dot complex having selective detection properties of dopamine.

Carbon dot is a fluorescent material, has excellent solubility in water, strong luminescence, high biocompatibility, ease of surface functionalization, long-term photo discoloration prevention and low cost.

The diacrylate-based polymer is added to introduce an acrylic functional group at the terminal of the carbon dot to be synthesized, and may be, for example, polyethylene glycol diacrylate, polypropylene glycol diacrylate or polybutylene glycol diacrylate, Preferably polyethylene glycol diacrylate (PEGDA) can be used.

Monosaccharide is added to introduce a -COOH or -OH functional group at the end of the carbon dot to be synthesized, for example, may be lactose, glucose, galactose or mannose, preferably lactose (Lactose) can be used.

Accordingly, the terminal of the surface-modified carbon dot includes a functional group -COOH or -OH, and the terminal of the aminophenyl boronic acid includes a -NH ₂ functional group, the functional group of the surface-modified carbon dot terminal and The functional group of the aminophenyl boronic acid terminal is characterized by chemical bonding.

The aminophenylboronic acid may be introduced to the surface of the carbon dot by covalently bonding to a functional group introduced at the terminal of the carbon dot. As described later, aminophenylboronic acid has a property capable of reversible binding with dopamine, thereby serving to detect dopamine.

Specifically, since B (OH) ₂ of aminophenylboronic acid reacts with cis-diol of dopamine, it is possible to selectively detect dopamine through aminophenylboronic acid.

In this case, the aminophenyl boronic acid may be a compound represented by Formula 1, specifically, the aminophenyl boronic acid is a group consisting of 2-aminophenylboronic acid, 3-aminophenylboronic acid, 4-aminophenylboronic acid, and mixtures thereof. It may be any one selected from. As a preferred embodiment of the present invention, aminophenylboronic acid may use 3-aminophenylboronic acid.

[Formula 1]

In an embodiment of the present invention, by using PEGDA as a diacrylate-based polymer and lactose as a monosaccharide, a carbon dot into which -COOH or -OH functional groups are introduced together with an acrylic functional group can be synthesized. The boronic acid-carbondot complex was formed by allowing a -COOH or -OH functional group of to form a chemical bond with the -NH ₂ functional group of the aminophenylboronic acid.

According to another aspect of the invention, (1) preparing a mixture comprising a diacrylate-based polymer and a monosaccharide, and preparing a surface-modified carbon dot by solvent thermal synthesis reaction of the mixture; And (2) mixing and reacting the surface-modified carbon dot, aminophenylboronic acid represented by Formula 1 below, and a crosslinking agent to complete a boronic acid-carbon dot complex; and boronic acid-carbon It provides a method for producing a dot composite.

In this case, a detailed description of the diacrylate-based polymer, carbon dot, monosaccharide and aminophenylboronic acid is the same as described above, and the part will be referred to.

Specifically, the terminal of the surface-modified carbon dot includes a -COOH or -OH phosphorus functional group, the terminal of the aminophenyl boronic acid includes a -NH ₂ functional group, the functional group of the surface-modified carbon dot terminal and The functional group of the aminophenyl boronic acid terminal is characterized by chemical bonding.

In addition, as a preferred embodiment of the present invention, the crosslinking agent may use EDC [1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride] and sulfo-NHS (sulfo-N-hydroxysuccinimide).

In one embodiment of the present invention, after preparing a mixture comprising a diacrylate-based polymer, monosaccharides, and solvent thermal synthesis to synthesize a carbon dot having an -COOH or -OH functional group with an acrylic functional group at the end, This was reacted with aminophenylboronic acid having a -NH ₂ functional group at the end, and coupling reaction with NHS and EDC.HCl to prepare a boronic acid-carbon dot complex in which aminophenylboronic acid and carbon dot formed a chemical bond.

According to another aspect of the present invention, the -COOH or -OH functional group, and the carbon dot and -NH ₂ functional group introduced at the terminal and the acrylic functional group is introduced at the terminal to chemically bond to the -COOH or -OH functional group of the carbon dot A boronic acid-carbondot complex comprising aminophenylboronic acid represented by Formula 1; And a base portion functionalized with a methacrylate-based polymer and having an acrylic functional group introduced to the surface thereof, wherein the boronic acid-carbon dot complex and the boron acid-based reaction are reacted between the acrylic functional groups of the base portion. It provides a dopamine biosensor using boronic acid-carbon dot complex characterized in that the carbon dot complex is immobilized.

The substrate portion is not particularly limited as long as it can be used as a substrate, and preferably a glass substrate can be used.

The methacrylate polymer is added to introduce an acrylic functional group to the surface of the substrate portion, for example, (trimethoxysilyl) propyl methacrylate, (triethoxysilyl) propyl methacrylate or (tributoxysilyl). Propyl methacrylate, preferably (trimethoxysilyl) propyl methacrylate (TMSPMA) can be used.

Specifically, dopamine is detected by changing the fluorescence color and fluorescence intensity due to the boronate ester complex which is the product of cis-diol reaction of B (OH) ₂ and dopamine of aminophenylboronic acid.

The dopamine biosensor according to the present invention can be easily fixed to the base portion into which the acrylic functional group is introduced through the acrylic functional group, and thus has excellent bonding strength and stability, which can be used for thin film-based biosensors such as film and glass, and is easy to transport since it is a solid state. Easy and long term storage

Hereinafter, the present invention will be described in more detail with reference to Examples, but the following Examples are not intended to limit the scope of the present invention, which will be construed as to help the understanding of the present invention.

<Example>

(1) material

Polyethyleneglycol diacrylate (PEGDA; weight average molecular weight (Mw) = 400 g / mole) was purchased from Polysciences, USA.

Polyethylene glycol (PEG; Mw = 380 ~ 420g / mole), Lactose, Ammonium (99.998%, trace metal basis), N-hydroxy-succinimide (NHS) ), 3-aminophenylboronic acid monohydrate (APBA; 98%), Phosphate buffer solution (PBS; 1M, pH = 7.4), D-(+)-glucose (D- (+)-Glucose, Uric acid, Urea, L-ascorbic acid, Dopamine hydrochloride, Human serum, 4- (dicyano Methylene) -2-methyl-6- (4dimethylaminostyryl) -4H-pyran [4- (dicyanomethylene) -2-methyl-6- (4dimethylaminostyryl) -4H-pyran, DCM, dye content 98%), benzoyl Treated tubing tubes (Dialysis tubing benzoylated, MWCO = 2,000 Da) and TMSPMA were purchased from Sigma-Aldrich.

1- (3- (Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC-HCl) was purchased from Tokyo Chemical Industries.

2-Ketoglutaric acid (KGA) was purchased from Acros Organic, Belgium.

H ₂ SO ₄ , pH buffer solutions were purchased from Deoksan, Korea, and deionized water (DI) was purified using a reverse osmosis system (PureFO, Romax).

(2) Synthesis of Surface Modified Carbon Dots (CD)

Referring to FIG. 1, 410 μl of 0.1M NH ₄ Cl solution, 100 mg of lactose powder, 590 μl of deionized water, 100 μl of H ₂ SO _4, and 3 ml of PEGDA were sequentially added to a vial. The mixture was uniformly stirred to a solution and then reacted at 160 ° C. for 20 minutes using a microwave reactor (Monowave 300, Anton paar, Austria).

Thereafter, the mixture was dispersed in 10 ml of deionized water and centrifuged (V1512-Vision, V3000i, Vision Scientific, Korea) for 2 minutes at 3700 rpm to remove small or aggregated particles.

The centrifuged solution was purified by a dialysis tube to remove excess samples remaining in the initial samples without participating in the reaction for 24 hours, thereby modifying the surface and introducing acrylic functional groups, -OH functional groups, or -COOH functional groups at the ends. Dots (hereinafter referred to as "CD") were synthesized.

On the other hand, it was carried out in the same manner as the synthesis method except for using PEG, not PEGDA to compare the surface modified carbon dot according to an embodiment of the present invention, the carbon dot (hereinafter referred to as "CD (PEG) Is free of acrylic functional groups at the ends.

(3) Preparation of boronic acid-carbon dot complex (BA-CD)

Referring to FIG. 1, the reactive CD solution was dispersed in 10 ml of 1 M PBS (pH 7.4) and centrifuged at 3700 rpm for 2 minutes to remove large or aggregated particles.

Then, 7.6 mg of EDC.HCl was added to the centrifuged CD solution, stirred for 10 minutes in the dark, and then 1.15 mg of NHS was further added under the same experimental conditions. After standing for 2 hours at room temperature, 15.45 mg of APBA was added to the mixed solution and incubated for 24 hours at room temperature.

Finally, the incubated mixed solution was purified by a dialysis tube to remove excess samples remaining in the initial samples without participating in the reaction for 24 hours, and then -50 using a freeze dryer (fd-1000, EYELA, Japan). By freeze drying at -60 ° C for 24 hours, boronic acid-carbondot complex (hereinafter referred to as "BA-CD") was prepared.

On the other hand, except that the CD (PEG) was used in order to compare with the BA-CD prepared according to an embodiment of the present invention was carried out in the same manner as described above, the composite thus prepared (hereinafter "BA-CD (PEG) ) ") Does not have an acrylic functional group at the end.

(4) Preparation of dopamine (DA) biosensor (glass BA-CD)

Referring to FIG. 2, after the glass substrate was washed with ethanol and dried, an oxygen plasma treatment was performed for 5 minutes using a plasma generator (Femto Science Inc, Korea). 200 µl of TMSPMA solution was spin-coated at 3000 rpm for 50 seconds using a spin coater (SPIN-1200D, Midas, Korea) on the cleaned glass substrate, and the glass substrate coated with TMSPMA for 10 minutes in an oven at 60 ° C. Dried. The glass substrate thus prepared includes acrylic functional groups on its surface.

A mixture of 0.3 mg BA-CD, 1 ml PEGDA and 100 μl KGA (concentration 0.04 M in water) was then spin coated at 200 rpm for 10 seconds to form 410 μm thick on a glass substrate.

While keeping the mixture at a distance of 5.5 cm from the spin coated glass substrate, use a UV curing machine (Innocure 100N, Litchtzen, Korea) to allow the reaction between TMSPMA's acrylic functional groups, BA-CD and PEGDA on the glass substrate for 1 minute. It was. After curing, the glass substrate (hereinafter referred to as "glass BA-CD") to which BA-CD was immobilized was washed with deionized water.

On the other hand, except that BA-CD (PEG) was used in order to compare with the glass BA-CD prepared according to an embodiment of the present invention was carried out in the same manner as the manufacturing method, the biosensor manufactured as "glass BA -CD (PEG) ".

(5) measurement

UV-Vis, Photoluminescence (PL) and Fourier-transform infrared spectra (FTIR spectra) are UV-2401 PC spectrophotometers (Shimadzu, Japan), RF-5301 PC (Shimadzu, Japan) And FT / IR-620 spectrum analyzer (Jasco, Japan).

The hydrodynamic diameter of the CD was measured using a dynamic light scattering (DLS) device (Scatteroscope I, K-One Nano, Korea).

The structure of the CD was imaged using a transmission electron microscope (TEM, Titan G2 ChemiSTEM CsProbe, FEI Company, Netherland, 200kv).

TEM samples were dip coated onto a 200-mesh carbon coated copper grid (G75, Ted Pella Inc., Redding, Calif.) And then the solvent was evaporated at 24 ° C. The carbon coated copper grid was prepared by coating a thin collodion film on the grid and then coating the carbon using a carbon coater (208 Carbon Coater, Cressington, Singapore).

Photographs of BA-CD, BA-CD / DA aqueous solution and glass BA-CD were taken with a smartphone camera (SM-N900K, SAMSUNG, Korea).

Experimental Example

(1) Confirmation of the structure of boronic acid-carbon dot complex (BA-CD)

In order to confirm the structure of the BA-CD prepared according to the present invention, dynamic light scattering (DLS) analysis, transmission electron microscope (TEM) analysis and FT-IR analysis were performed.

3 is a view for confirming the structure of BA-CD according to an embodiment of the present invention, (a) is a DLS spectrum of BA-CD aqueous solution (0.25mg / ml), (b) is a TEM image (C) shows FT-IR spectra of CD and BA-CD aqueous solutions.

4 shows fluorescence and UV-Vis spectra of BA-CD aqueous solution (0.35 mg / ml) according to the excitation wavelength.

Referring to (a) of FIG. 3, the size of the synthesized BA-CD shows a rather narrow distribution, and has a diameter of about 1.4 to 2.41 nm.

Referring to FIG. 3 (b), a CD synthesized according to the present invention has a diameter of ˜3.17 ± 14.3 nm, similar or consistent with previously reported carbon dots, and a grid of 2.05 A corresponding to 100 graphite planes. Since it is a clear lattice stripe seen in the gap, it can be confirmed that it is graphite (see the enlarged image inserted at the bottom right).

In addition, referring to (c) of Figure 3, looking at the results of the FT-IR analysis of CD (ATR mode), BA-CD (ATR mode) and glass BA-CD (KBr mode) prepared according to the present invention, C Stretching peaks for the ═O (3431 cm ⁻¹ ) functional group, COC (1725 cm ⁻¹ ) binding, and —OH (1352 cm ⁻¹ ) functional group can be seen, and these peaks show CD and BA-CD It can be seen that PEGDA, a carboxyl group and a hydroxyl group are present on the surface.

In addition, stretching peaks for the CH ₂ (1457 cm ^-1 ) bond and the CH (2871 cm ^-1 ) bond were observed, respectively, and these peaks showed PEGDA on the surface of the CD and BA-CD. Stretch vibration peaks for C = C (1637 cm ⁻¹ ) binding from functional groups can be identified.

In particular, in the FT-IR spectrum of BA-CD, a new amide II band was observed at 1575 cm ^-1 due to the amide bond formed by the EDC crosslinking agent, and the peak of boronic acid (1352 cm ^-1 ) was COC (1352 cm ^-1 ). Overlaid with the stretch band.

In addition, the fluorescence and UV-Vis analysis results of 0.35 mg / ml BA-CD aqueous solution at different excitation wavelengths with reference to FIG. 4 can confirm π-π ^* and np ^* transitions at 360 and 270 nm, respectively. It represents the typical transition of CD. The intensity of the fluorescence spectrum is highly dependent on the excitation wavelength, and the fluorescence maximum intensity was observed at 360 nm, close to the wavelength of the n-π ^* transition. The wavelength at maximum intensity increases in proportion to the excitation wavelength, and the change in the fluorescence wavelength when the excitation wavelength changes is commonly observed in carbon dots.

From the above results, it can be confirmed that BA-CD having an acrylic functional group and boronic acid functional group introduced therein was successfully synthesized.

(2) Evaluation of Dopamine Detection Performance of APBA

FIG. 5 schematically shows the formation of boronate ester complexes via boronate ester linkages between APBA and DA.

Referring to FIG. 5, APBA and DA combine with boronic acid esters to form boronate ester complexes (hereinafter referred to as "APBA / DA complexes").

The fluorescence characteristics of this aqueous solution of APBA / DA complex (hereinafter referred to as "APBA / DA mixed aqueous solution") were analyzed under different pH.

6 shows a fluorescence characteristic of the APBA / DA mixed aqueous solution, where (a) is the PL spectrum of the APBA / DA mixed aqueous solution (500 uM / 500 uM) according to pH (excitation wavelength: 360 nm), and (b) When the concentration of DA or APBA is fixed at 500 mA, the PL spectrum (excitation wavelength: 477 nm) of the APBA / DA mixed aqueous solution according to the concentration of APBA (black circle) or DA (red square) is shown.

Referring to (a) of FIG. 6, almost no fluorescence emission occurred at pH 9 or less, and it was confirmed that strong fluorescence emission appeared at pH 10 or higher. This is because the pKa value of APBA is 8.8 or more, so that the boronic acid ester bond is formed at a higher pH than the pKa of APBA by deprotonation.

That is, it can be seen that the fluorescence emission of the APBA / DA mixed aqueous solution is due to the formation of the APBA / DA complex.

All experiments for dopamine detection were then performed at pH 10 unless otherwise noted.

Figure 7 shows the PL spectrum of the APBA / DA mixed aqueous solution, (a) is a PL spectrum according to the DA concentration when the APBA concentration (500uM) is fixed, (b) APBA when the DA concentration (500uM) is fixed It is PL spectrum according to concentration, and (c) and (d) show PL spectrum and PL intensity (excitation wavelength: 477 nm) of APBA / DA mixed aqueous solution mixed in the same concentration.

Referring to (a) and (b) of FIG. 7, the highest PL strength was measured when 500uM of DA solution was fixed to 500uM of APBA solution, and 500uM of APBA solution was added to 500uM of DA solution. In one case the highest PL intensity was measured.

In addition, as a result of examining the PL strength of the solution mixed with the DA aqueous solution and the APBA aqueous solution at the same concentration, the PL strength of the solution mixed with 500 μM of both the DA aqueous solution and the APBA aqueous solution was measured to be the highest.

Accordingly, PL spectra were measured using a solution in which both the DA aqueous solution and the APBA aqueous solution were mixed at the same concentration of 500 uM.

Referring to FIGS. 6B and 7D, the fluorescence intensity and the PL intensity of the APBA / DA mixed aqueous solution were maximized at 477 nm, which is the cis-diol of B (OH) ₂ and DA of APBA. It means that the boronate ester complex is formed through the reaction.

However, referring back to (c) of FIG. 7, when the concentrations of both the DA aqueous solution and the APBA aqueous solution were 80 uM, the PL intensity dropped sharply, and when the concentrations were all 20 uM or less, the PL intensity was too low to show fluorescence emission. .

From the above results, it was confirmed that only a small amount of DA was difficult to detect with the APBA aqueous solution.

(3) Evaluation of Dopamine Detection Performance of BA-CD Aqueous Solution

In (c) of FIG. 7, BA-CD for the DA aqueous solution fixed at 20 uM was based on the result that the mixed aqueous solution in which both the DA aqueous solution and the APBA aqueous solution were mixed at 20 uM or less did not exhibit fluorescence emission due to very low PL intensity. To determine the dopamine detection performance of the aqueous solution.

In relation to Figure 8a is a PL spectrum (excitation wavelength: 360nm) of BA-CD / DA mixed aqueous solution according to the concentration of the BA-CD aqueous solution, Figure 8b is a BA-CD / DA mixed aqueous solution of the concentration of the BA-CD aqueous solution (b) shows an increase in PL intensity (excitation wavelength: 477 nm).

Referring to FIG. 8A, the PL strength was measured higher in the BA-CD / DA aqueous solution compared to the BA-CD aqueous solution, and was higher as the concentration of the BA-CD aqueous solution was increased.

Referring to FIG. 8B, the PL strength (excitation wavelength: 477 nm) of the BA-CD / DA aqueous solution according to the concentration of the BA-CD aqueous solution can be confirmed, and the PL strength of the BA-CD aqueous solution reaches 0.25 mg / ml. It increased until then, but then drastically decreased. That is, the maximum PL intensity was observed in a 0.25 mg / ml aqueous solution of BA-CD which could meet the same concentration conditions, so all experiments for dopamine detection thereafter were carried out using a 0.25 mg / ml aqueous solution of BA-CD unless otherwise stated. Was performed using.

9 is a diagram for evaluating DA detection performance of BA-CD aqueous solution, (a) and (b) are PL intensity and PL spectral image according to the concentration of BA-CD aqueous solution, (c) is BA-CD / It is PL intensity | strength when DA mixed aqueous solution is excited at 477 nm, (d) shows the PL spectrum of APBA, DA, APBA / DA, and BA-CD / DA aqueous solution.

Referring to (a) and (b) of FIG. 9, the pure BA-CD solution to which the DA aqueous solution was not added showed a tendency of increasing the PL strength with increasing concentration, and showed blue in the PL spectrum.

9 (c) shows the PL intensity at 477 nm as a function of Φ, where a new peak appeared up to 477 nm as Φ increased until Φ = 130 uM, and the PL intensity (430 of au). ) Then appeared to be saturated. In addition, it can be seen that the linear range and detection limit (LOD) are 0 to 40 uM and 0.32 uM, respectively.

9 (d), APBA (120uM), DA (120uM), APBA / DA (120 / 120uM) and BA-CD / DA (0.25mgml) to confirm the dopamine detection performance of BA-CD aqueous solution -1/120 uM) PL spectra were analyzed. The aqueous solution of DA and APBA showed almost no fluorescence emission, and the aqueous solution of BA-CD / DA showed about 4 times higher PL intensity than the aqueous solution of APBA / DA.

Thus, it could be reliably confirmed that CD increased the fluorescence emission of the boronate ester complex, which boronic acid combined with amide bonds, resulting in photoinduced electron transfer from electron-rich CD to DA / APBA complex, increasing fluorescence intensity Because

Meanwhile, an aqueous solution containing glucose, lactose, ascorbic acid, urea and dopamine was used as a biopolymer in order to evaluate the dopamine selectivity of the aqueous BA-CD solution and the aqueous CD solution.

10 shows the PL spectrum (excitation wavelength: 360 nm) of the mixed aqueous solution containing the aqueous solution containing the biopolymers in (a) the aqueous CD solution, and (b) the PL spectrum of the mixed aqueous solution mixed in the aqueous solution of BA-CD (excitation) Wavelength: 360 nm) (The biopolymers are glucose, lactose, ascorbic acid, urea and dopamine, and final concentrations of the analytes tested were adjusted to 0.25 mg / ml and 40 uM, respectively).

11 shows (FF ₀ ) / F ₀ (excitation wavelength: 477 nm) of a mixed solution obtained by mixing an aqueous solution containing biopolymers with (a) a CD aqueous solution, and (b) a mixed aqueous solution mixed with a BA-CD aqueous solution. (FF ₀ ) / F ₀ (excitation wavelength: 477 nm) (biopolymers are glucose, lactose, ascorbic acid, urea and dopamine, F and F ₀ are PL before and after adding an aqueous solution containing biopolymers). Intensity, and final concentrations of the analytes tested were adjusted to 0.25 mg / ml and 40 uM, respectively).

Referring to (a) and (b) of FIG. 10, in the case of the aqueous CD solution to which the biopolymer aqueous solution was added, the PL strength of all the biopolymers was measured very low, and in the case of the BA-CD aqueous solution to which the biopolymer aqueous solution was added. For all biopolymers except dopamine, PL strength was similar to that of aqueous CD solution, but the PL strength for dopamine was very high.

Referring to (a) and (b) of FIG. 11, in the case of the aqueous CD solution to which the aqueous biopolymer solution was added, the (FF ₀ ) / F ₀ values for all the biopolymers were hardly changed within 3%. in the case of the addition of BA-CD aqueous solution for all biopolymers, except for dopamine (FF ₀₎ / F ₀ value of I've found similarly to the case of CD aqueous solution, the (FF ₀₎ / F ₀ value for the dopamine It was measured very high.

From the above results, it can be seen that the new peak at 477 nm is due to the formation of the boronate ester complex, and that the BA-CD aqueous solution is very selective for dopamine.

(4) Confirmation of the structure of dopamine biosensor (glass BA-CD)

The structure of the dopamine biosensor including the BA-CD complex prepared according to the present invention was confirmed.

In relation to this, the FT-IR analysis results of the glass BA-CD on which the BA-CD is immobilized on the glass substrate will be described with reference to FIG. 3 (c).

Specifically, by calculating the ratio (r _{(C = C / C = O))} between the C = C stretching band of the stretching vibration and the peak of 1637cm ^-1 1725cm ^-1 C = O bond in the reaction of acrylic functional groups Confirmed. At this time, when the reaction between the acrylic functional groups occur a decrease C = C stretching band at 1637cm ^-1, C = O stretching vibration peak at 1725cm ^-1 is derived from the acrylic functional group and the CD (and BA-CD), peak Is considered a reference peak because it does not change by the reaction between the acrylic functional groups.

R _{(C = C / C = O)} for BA-CD peeled from BA-CD and glass BA-CD is 0.45 and 0.24, respectively, which is a double bond of acrylic functional group while BA-CD is fixed on the glass substrate Indicates this disappears.

From the above results, it can be confirmed that the dopamine sensor glass BA-CD was successfully prepared by immobilizing the BA-CD complex having an acrylic functional group on the glass substrate functionalized with TMSPMA and introducing the acrylic functional group.

(5) Evaluation of Dopamine Detection Performance of Dopamine Biosensor

In order to evaluate the dopamine sensitivity of the dopamine biosensor including the BA-CD complex prepared according to the present invention, a dopamine detection experiment was performed by immersion in an aqueous dopamine solution for each concentration.

12 is a view for evaluating the dopamine detection performance of the glass BA-CD, a dopamine biosensor according to an embodiment of the present invention, (a) is a PL spectrum (excitation wavelength: 360 nm), (b) is an image according to different fluorescence quantum yields (Φs), and (c) shows PL intensity (excitation wavelength: 477 nm).

Referring to (a) and (b) of FIG. 12, as the concentration of the DA aqueous solution is increased, the PL intensity of the glass BA-CD increases, and dopamine can be detected by PL spectroscopy, and the blue of the glass BA-CD without DA is present. The release turned to green release as the concentration of the aqueous DA solution increased.

12 (c) shows the PL intensity at 477 nm as a function of Φ. The linear range of the spectrum is 0 to 40 uM, and the detection limit (LOD) is 0.2 uM, similar to that of the BA-CD aqueous solution. It has similar sensitivity to BA-CD aqueous solution.

In this way, by using the glass BA-CD immobilized on the glass substrate as a dopamine sensor, the presence of dopamine can be easily seen through the color change, and the transport is easy, and the detection performance is improved. It has the advantage of long term storage while maintaining

(6) Evaluation of Dopamine Selectivity of Dopamine Biosensors

In order to evaluate the dopamine selectivity of the dopamine biosensor comprising the BA-CD complex prepared according to the present invention, a detection experiment was carried out using an aqueous biopolymer containing a substance other than dopamine. Specifically, the aqueous biopolymer solution is prepared using glucose, lactose, ascorbic acid, urea, uric acid, and dopamine, respectively, and after immersing glass BA-CD in 5 ml of the aqueous biopolymer solution for 4 hours and 30 minutes, glass The moisture on the BA-CD was removed to analyze the PL intensity and image.

Relevant to FIG. 13 shows (FF ₀ ) / F ₀ and images when the glass BA-CD excited at 360 nm was added to an aqueous solution of biopolymers, and then excited at 477 nm (biopolymers are (i) glucose, ( ii) lactose, (iii) ascorbic acid, (iv) urea, (v) uric acid and (vi) dopamine, F and F ₀ are PL strengths before and after glass BA-CD, and concentration of aqueous biopolymer solution Are 40 uM each).

Looking at the image inserted in Figure 13, in other biopolymer solution except dopamine, the blue color of the original color of the glass BA-CD appeared as it does not change, but (vi) a distinct green color was observed in the dopamine solution.

In addition, referring to (FF ₀ ) / F ₀ in FIG. 13, the value was nearly zero in the aqueous solution of biopolymer except for dopamine, but was measured at about 30 in the solution of (vi) dopamine.

From this, it can be seen that the dopamine biosensor according to the present invention has a high selectivity for dopamine, through which the presence of dopamine can be selectively distinguished from other biopolymers in the blood without interference.

(7) Evaluation of stability of dopamine biosensor

The stability of the dopamine biosensor comprising the BA-CD complex prepared according to the present invention was evaluated. Specifically, glass BA-CD not immersed in 40uM aqueous dopamine solution, glass BA-CD immersed for 2 weeks and glass BA-CD immersed for 4 weeks were prepared and analyzed for each PL spectrum, image and PL intensity. .

In relation to Figure 14 is a view for evaluating the stability of the glass BA-CD dopamine biosensor according to an embodiment of the present invention, (a) is a glass that was immersed in dopamine aqueous solution for 0 weeks, 2 weeks and 4 weeks, respectively PL spectrum (excitation wavelength: 360 nm) and image of BA-CD, and (b) shows PL intensity (excitation wavelength: 477 nm).

Referring to (a) and (b) of FIG. 14, the glass BA-CD which was immersed in the dopamine aqueous solution for 0, 2, and 4 weeks, respectively, showed the same PL spectrum and green emission. The PL strength did not drop, and the dopamine detection performance of glass BA-CD did not decrease for 4 weeks.

From this, the dopamine biosensor according to the present invention has not changed PL strength even after 4 weeks of preparation and immersion in the dopamine aqueous solution, so the stability of the biosensor is determined to be 4 weeks or more, and the solid state small glass type ( glass type) It can be used as a biosensor, so it is easy to carry and use when dopamine detection test is needed.

(8) Evaluation of Dopamine Detection Performance in Human Serum

To evaluate the dopamine sensitivity in human serum of the dopamine biosensor comprising the BA-CD complex and BA-CD complex prepared according to the present invention, a detection experiment was performed using dilute serum (× 10 with DI water).

15 is a PL spectrum (excitation wavelength: 360 nm) evaluating the detection performance of dopamine in human serum, (a) is an aqueous solution of BA-CD added with serum, and (b) is a glass BA-CD added to serum. PL spectrum is shown.

In addition, Table 1 below shows the amount of dopamine in human serum measured by BA-CD aqueous solution and glass BA-CD.

sample original DA in Human serum (uM) added DA
(uM) measured DA
(uM) recovery
(%) RDS
(n = 3,%) One
(BA-CD solution) 0 10 10.17 101 1.84 2
(BA-CD solution) 0 40 41.10 103 1.72 3
(glass BA-CD) 0 10 11.22 112 1.17 4
(glass BA-CD) 0 40 38.97 97 1.63

The dopamine originally present in human serum was almost 0 uM. Dopamine was added after centrifugation to remove large molecules. The concentration of the added dopamine aqueous solution was determined from the calibration curve (Fig. 9 (c) and Fig. 13 (c)) using a BA-CD aqueous solution and glass BA-CD.

Referring to (a) and (b) of FIG. 15, it can be seen that BA-CD and glass BA-CD increase in PL intensity in proportion to the concentration of aqueous dopamine solution, and show similar PL spectra, showing similar dopamine detection sensitivity. .

In addition, referring to Table 1, the amount of dopamine and the amount of dopamine detected in the serum can be seen. The recoveries (measured dopamine amount / added dopamine amount) for samples 1 and 2 with 10 uM and 40 uM dopamine, respectively, were 101% and 103%, respectively, and the recoveries for samples 3 and 4 were 112% and 97%, respectively. Appeared. Small deviations of 100% were observed to reflect interference by other biopolymers in the serum, but these deviations are relatively small.

From this, the dopamine biosensor prepared according to the present invention can confirm that dopamine can be detected in a sample containing dopamine of 10 μM or more.

Boronic acid-carbon dot complex (BA-CD) of the present invention was prepared simply by coupling PEGDA doping and BA to the surface of the carbon dot through a solvent thermal synthesis reaction, turn-on fluorescence for dopamine detection Based optical biosensor (glass BA-CD).

The aqueous solution of the boronic acid-carbon dot complex optimized at a concentration of 0.25 mg / ml at pH 10 showed a linear range of 0 to 40 uM and a detection limit (LOD) of 0.3 uM, and showed excellent dopamine detection performance with high selectivity to dopamine. Confirmed.

Immobilized on a glass substrate functionalized with TMSPMA through an acrylic functional group introduced at the end of BA-CD to prepare a glass substrate in the solid state (BA-CD) coated with BA-CD, the biosensor of the present invention. The glass BA-CD has excellent stability, selectivity, and detection performance, and has a linear range of 0 to 40 uM and a detection limit (LOD) of 0.2 uM, similar to the stability of BA-CD aqueous solution.

In addition, the glass BA-CD in the solid state is easy to transport, can be stored for a long time, selectivity and detection performance is not degraded over time, and it is convenient to use as it can confirm the detection of dopamine with the naked eye.

Boronic acid-carbondot complex and dopamine biosensor according to the present invention has a blue fluorescent color of the original, but can detect the presence of dopamine by changing the color to a new fluorescent color green for the sample containing dopamine, Changes in photosensitivity make it easy to visually check the amount of dopamine. The boronic acid-carbon dot complex can be applied to other solid state systems, and can be utilized for the development of thin film-based biosensors.

The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the claims that follow may be better understood. Those skilled in the art will appreciate that the present invention can be embodied in other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. The scope of the invention is indicated by the following claims rather than the above description, and all changes or modifications derived from the claims and their equivalents should be construed as being included in the scope of the invention.

Claims (8)

Carbon dots surface-modified with diacrylate-based polymers and monosaccharides; And
Boronic acid-carbon dot complex comprising a; and is bonded to the functional group of the carbon dot, aminophenylboronic acid (aminophenylboronic acid) represented by the following formula (1).
[Formula 1]

The method of claim 1,
At the end of the surface-modified carbon dot includes a functional group -COOH or -OH,
At the terminal of the aminophenyl boronic acid contains a functional group -NH ₂ ,
The boronic acid-carbon dot complex, characterized in that the functional group of the surface-modified carbon dot terminal and the functional group of the aminophenyl boronic acid terminal.
The method of claim 1,
The aminophenyl boronic acid is any one selected from the group consisting of 2-aminophenyl boronic acid, 3-aminophenyl boronic acid, 4-aminophenyl boronic acid and mixtures thereof.
(1) preparing a mixture comprising a diacrylate-based polymer and a monosaccharide, and preparing a surface-modified carbon dot by solvent thermal synthesis reaction of the mixture; And
(2) mixing and reacting the surface-modified carbon dot, aminophenylboronic acid represented by Formula 1 below, and a crosslinking agent to complete the boronic acid-carbon dot complex; and boronic acid-carbon dot Method for preparing a composite.
[Formula 1]

The method of claim 4, wherein
At the end of the surface-modified carbon dot includes a functional group -COOH or -OH,
At the terminal of the aminophenyl boronic acid contains a functional group -NH ₂ ,
The surface-modified carbon dot terminal functional group and aminophenyl boronic acid terminal functional group is characterized in that the chemical bonding of the functional group.
The method of claim 4, wherein
EDC [1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride] and NHS (N-hydroxy-succinimide) are used as the crosslinking agents.
Aminophenyl boron represented by the following general formula (1) by chemically bonding a -COOH or -OH functional group and an acrylic functional group introduced into the terminal and a carbon dot and -NH ₂ functional group introduced into the terminal and chemically bonding to the -COOH or -OH functional group of the carbon dot Boronic acid-carbondot complex comprising acid; And
And a substrate portion functionalized by a methacrylate-based polymer and having an acrylic functional group introduced to the surface thereof.
A dopamine biosensor using boronic acid-carbondot complex, characterized in that the boronic acid-carbondot complex is immobilized on the substrate portion through a reaction between the boronic acid-carbondot complex and an acrylic functional group of the substrate portion.
[Formula 1]

The method of claim 7, wherein
Boron acid-carbon dot complex characterized in that the dopamine is detected by the change in fluorescence color and fluorescence intensity due to the boronate ester complex which is the product of cis-diol reaction of B (OH) ₂ and dopamine of aminophenylboronic acid Dopamine biosensors used.

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