KR20110065232A - Method for detecting analytes using fluorescence quenching induced by gold nanoparticle enlargement - Google Patents

Abstract

PURPOSE: A method for detecting an analyte of high sensitivity using fluorescence reduction by gold nanoparticles is provided to apply to manufacturing various fluorescence-based optical biosensor. CONSTITUTION: A method for detecting an analyte by fluorescence reduction due to the growth of gold nanoparticles comprises: a step of adding gold nanoparticles and fluorescence dye to a gold nanoparticle growth solution to prepare a mixture; a step of adding a reducible analyte to the mixture to induce the growth of gold nanoparticles; and a step of measuring fluorescence intensity of the mixture to detect the reducible analyte. The gold nanopaticle growth solution is HAuCl_4, NaAuCl_4, AuCl_3, or K(AuCl_4). The fluorescence dye is fluorescein, rhodamine, eosin, elexa, or rose bengal.

Description

Method for Detecting Analytes Using Fluorescence by Gold Nanoparticle Growth {Method for Detecting Analytes Using Fluorescence Quenching Induced by Gold Nanoparticle Enlargement}

The present invention relates to a method for detecting a high sensitivity analyte using fluorescence reduction by growth of gold nanoparticles, and more particularly, to a high sensitivity using growth of gold nanoparticles induced by a reducing agent as an analyte and a decrease in fluorescence signal of a fluorescent dye. It relates to a high sensitivity analyte detection method using fluorescence reduction by the growth of gold nanoparticles, characterized by detecting the analyte.

Recent rapid developments in the field of nanotechnology have made it possible for metal nanoparticles to participate as major labels in biometric processes (Niemeyer CM., Angew Chem, 40: 4128-4158, 2001).

For example, using metal nanoparticle-nucleotide conjugates, methods such as surface plasmon resonance, surface-enhanced Raman scattering, quartz-crystal microbalance, etc. Research and development results have been published demonstrating that DNA hybridization can be detected (Cao et al., Science, 297: 1536-1540, 2002; He et al., J Am Chem Soc , 122: 9071-9077). , 2000).

In addition, various redox enzymes are measured by measuring the absorbance change of gold nanoparticles when gold nanoparticles grow through reduction of gold ions by a reducing agent such as H ₂ O ₂ generated during the enzymatic reaction in various redox reactions. It is reported that the activity of (Zayats, M. et al., Nano Lett. , 5: 21-25, 2005; Baron, R., Anal. Chem., 77: 1566-1571, 2005; Xiao , Y. et al., Angew.Chem ., Int. Ed. , 43: 4519-4522, 2004).

On the other hand, research into the development of fluorescence-based optical biosensors using the growth technology of gold nanoparticles has been continued (Bultzingslowen C. et al., Anal. Chem. Acta , 480: 275-283, 2003; Pickup, JC et al., Biosens. Bioelectron , 20: 2555-2565, 2005). Typically, gold nanoparticles have been introduced into fluorescence-based assays to detect biologically important analytes (Griffin, J. et al., Chem. Eur. J. , 15: 342-351, 2009; Lee, S. et al., Angew.Chem ., Int. Ed. , 47: 2804-2807, 2008). Most of the fluorescence-based assays incorporate FRET phenomena, and the design of the FRET-based detection probes requires modification of the fluorescent molecules to adjust the arrangement of the metal nanoparticles in a specific direction or distance. Manufacturing is not easy (Zhu, L. et al., Lab Chip , 6: 115-120, 2006).

Accordingly, the present inventors have made efforts to develop a simple and highly sensitive fluorescence-based sensing technology using gold nanoparticles. As a result, when the fluorescent signal disappearance of the fluorescent dye due to the gold nanoparticle growth reaction induced by the analyte is used, It was confirmed that the analyte can be detected and the present invention was completed.

An object of the present invention is to detect the analyte with high sensitivity by using the fluorescent signal degradation of the fluorescent dye caused by the growth of the gold nanoparticles induced by the reducing agent as an analyte, fluorescence decrease by the growth of gold nanoparticles To provide a high sensitivity analyte detection method using.

In order to achieve the above object, the present invention comprises the steps of: (a) preparing a mixture by adding gold nanoparticles and fluorescent dye to the growth solution of gold nanoparticles; (b) adding a reducible analyte to the mixture to induce gold nanoparticle growth; And (c) detecting a reducible analyte by measuring the fluorescence intensity of the mixture containing the grown gold nanoparticles, using a decrease in fluorescence caused by the growth of the gold nanoparticles. do.

The present invention also comprises the steps of (a) preparing a mixture of gold nanoparticle growth solution, gold nanoparticles, fluorescent dyes and enzymes; (b) adding an analyte to the mixture to induce a reaction of the analyte with an enzyme; And (c) measuring the fluorescence intensity of the mixture to which the analyte is added, and comparing the fluorescence intensity of the fluorescent dye to detect the analyte using the degree of fluorescence signal degradation. Provided is a method for detecting an analyte using a decrease in fluorescence signal.

The present invention also provides a biosensor for reducing analyte detection used for growth of gold nanoparticles, comprising a growth solution of gold nanoparticles, gold nanoparticles and a fluorescent dye.

The present invention also provides a biosensor for detecting a substrate of the enzyme, including a growth solution of gold nanoparticles, gold nanoparticles, a fluorescent dye, and an enzyme.

According to the present invention, the analyte can be detected with higher sensitivity than the method of measuring the absorbance change after the growth of gold nanoparticles, and can be applied to the manufacture of various fluorescence-based optical biosensors.

The present invention in one aspect, (a) adding a gold nanoparticles and a fluorescent dye to the growth solution of gold nanoparticles to prepare a mixture; (b) adding a reducible analyte to the mixture to induce gold nanoparticle growth; And (c) detecting a reducible analyte by measuring the fluorescence intensity of the mixture containing the grown gold nanoparticles, using a decrease in fluorescence caused by the growth of the gold nanoparticles. (FIG. 1A).

According to the present invention, when the grown gold nanoparticles and the fluorescent dye are present at the same time, the emission band of the fluorescent dye and the absorption band of the grown gold nanoparticles overlap, and thus the fluorescent dye of the fluorescent dye is deteriorated. It is based on the principle of detecting analytes, in particular reducing agents, which induce the growth of particles.

In the present invention, the reducible analyte is H ₂ O ₂ , hydroquinone (HQ), adrenaline, noradrenaline, dopamine, L-Dopa, mercaptosuccinic acid, 4-aminophenol, 3-aminophenol, 1,4-phenylenediamine, aniline, triethylamine, indole, 4-bromoaniline ,, 1-methylindole, 3-amino-1-propanol, pyridine, 3-indole propionic acid, glucine, DL-tryptophan, Sodium cirate, citric acid, sodium boro hydride, hydroxylamine, HCl, acetone , oxalic acid and b-diketone may be characterized in that the reducing agent selected from the group consisting of, but is not limited to a reducing agent capable of reducing gold ions.

In the present invention, the gold nanoparticle growth solution is a gold ion generating material selected from the group consisting of HAuCl ₄ , NaAuCl ₄ , AuCl ₃ and K (AuCl ₄ ) It may be characterized as an aqueous solution, but is not limited to a solution capable of producing gold ions in the aqueous solution.

In the present invention, the fluorescent dye is selected from the group consisting of fluorescein (fluorescein), rhodamines (rhodamine), eosin (Eosin), Alexa (rose) and rose bengal (rose bengal) However, if the material can exhibit fluorescence is not limited thereto.

In the present invention, gold nanoparticles 10 ^-4 to 10 ^-2 parts by weight and fluorescent dyes 10 ^-6 to 10 ^-2 parts by weight based on 100 parts by weight of the gold growth solution may be added. If the amount of the gold nanoparticles added is less than 10 ^-4 parts by weight, the effect of the gold nanoparticles on the fluorescent dye is weak, and if it exceeds 10 ^-2 parts by weight, the change of the gold nanoparticles is insignificant, which makes it impossible to make accurate measurements. . Further, when the amount of the fluorescent dye added is less than 10 ^-6 parts by weight, the stability of the fluorescence signal is lowered. When the amount of the fluorescent dye is more than 10 ^-2 parts by weight, the effect on the total fluorescence intensity due to the change of the gold nanoparticles is weakened. If less, there is a problem that can not be measured.

In the present invention, may be characterized in that the addition of the gold growth solution 100 parts by weight of the analyte 10 ^-18 to 10 ^-1 parts by weight, for, if the addition amount of analytes out of the above numerical range, the analysis accuracy of water detection There is a problem that is lowered.

In another aspect, the present invention, (a) preparing a mixture of gold nanoparticle growth solution, gold nanoparticles, fluorescent dyes and enzymes; (b) adding an analyte to the mixture to induce a reaction of the analyte with an enzyme; And (c) measuring the fluorescence intensity of the mixture to which the analyte is added, and comparing the fluorescence intensity of the fluorescent dye to detect the analyte using the degree of fluorescence signal degradation. It relates to a method for detecting an analyte using a decrease in fluorescence signal by

In the present invention, when the gold nanoparticles grown by the electrons generated during the enzyme reaction and the fluorescent dye are present at the same time, the emission band of the fluorescent dye and the absorption band of the grown gold nanoparticles overlap (overlap), the fluorescence of the fluorescent dye is reduced The phenomenon is based on the principle of detecting enzyme substrates that induce the growth of gold nanoparticles.

In the present invention, the enzyme is AChE (acetylthiocholinesterase), choline oxidase (choline oxidase), glucose oxidase (glucose oxidase), cytochrome oxidase (cytochrome oxidase), ascorbic xanthine oxidase (xanthine oxidase), polyphenols It is selected from the group consisting of oxidase (polyphenol oxidase), catechol oxidase (catechol oxidase), lysyl oxidase NADPH oxidase (NADPH oxidase), monoamine oxidase (monoamine oxidase) and laccase (laccase) The analyte may be acetylthiocholine, acetylcholine, hydroquinone, glucose, adrenaline, noradrenaline, dopamine, L-Dopa, mecaptosuccinic acid, 4-aminophenol, 3-aminophenol, 1,4-phenylenediamine, aniline, triethylamine, indole, 4-bromoaniline, 1-methylindole, 3-amino-1-propanol, pyridine, 3-indole propionic acid, glucine, DL-tryptophan, Sodium cirate, citric acid, sodium boro hydride, hydroxylamine, HCl, acetone, oxalic acid and b-diketone It can be an enzyme substrate selected from the group consisting of, but is not limited to a reducing agent capable of inducing the growth of gold nanoparticles by the reduction of gold ions after the reaction between the enzyme and the enzyme substrate, and an enzyme and enzyme substrate capable of releasing electrons. .

In the present invention, the gold nanoparticle growth solution may be characterized in that the aqueous solution of a gold ion generating material selected from the group consisting of HAuCl ₄ , NaAuCl ₄ , AuCl ₃ , K (AuCl ₄ ), gold in an aqueous solution Any solution capable of generating ions is not limited thereto.

In the present invention, the fluorescent dye may be selected from the group consisting of fluorescein, rhodamine, eosin, and rose bengal, but may exhibit fluorescence. Substance is not limited thereto.

In the present invention, the growth of gold was added to 100 parts by weight of the gold nano-particles 10 ^-4 to 10 ^-2 parts by weight of a fluorescent dye 10 ^-6 to 10 ^-2 parts by weight of the enzyme 10 ^-8 to 10 ^-2 parts by weight of It can be characterized by. If the amount of the gold nanoparticles added is less than 10 ^-4 parts by weight, the effect of the gold nanoparticles on the fluorescent dye is weak, and if it exceeds 10 ^-2 parts by weight, the change of the gold nanoparticles is insignificant, which makes it impossible to make accurate measurements. . Further, when the amount of the fluorescent dye added is less than 10 ^-6 parts by weight, the stability of the fluorescence signal is lowered. When the amount of the fluorescent dye is more than 10 ^-2 parts by weight, the effect on the total fluorescence intensity due to the change of the gold nanoparticles is weakened. If less, there is a problem that can not be measured. In addition, when the addition amount of the enzyme is 10 ^-8 to 10 ^-2 parts by weight, it is possible to derive an enzyme reaction that releases a sufficient amount of electrons to induce the gold nanoparticle growth reaction.

In yet another aspect, the present invention relates to a biosensor for reducing analyte used for growing gold nanoparticles, including a growth solution of gold nanoparticles, gold nanoparticles, and a fluorescent dye.

In the biosensor for reducing analyte detection used for the growth of gold nanoparticles, 10 ^-4 ~ 10 ^-2 parts by weight of gold nanoparticles and fluorescent dyes 10 ^-6 ~ 10 ^-2 to 100 parts by weight of the gold growth solution The weight part may be included.

In another aspect, the present invention relates to a biosensor for detecting a substrate of the enzyme, including a growth solution of gold nanoparticles, gold nanoparticles, a fluorescent dye, and an enzyme.

In the substrate detection of the enzyme biosensor for the growth of gold solution 100 parts by weight of the gold nano-particles 10 ^-4 to 10 ^-2 parts by weight of a fluorescent dye 10 ^-6 to 10 ^-2 parts by weight of an enzyme for 10 ^-8 ~ 10 ^-2 parts by weight may be included.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only to illustrate the invention, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1 Reducing Agent Detection Experiment Using Fluorescence Reduction by Gold Nanoparticle Growth Induced by Reducing Agent

1-1. In case of using fluorescein as fluorescent dye

A mixture was prepared by adding 75 µl of 0.22 µM aqueous gold nanoparticles solution and 10 µl of 1 mM fluorescein to 2.5 ml of 0.2 mM HAuCl ₄ aqueous solution.

_2.5 μl of 10 mM H ₂ O ₂ as a reducing agent was added to the mixture to induce the growth of gold nanoparticles.

The fluorescence intensity of the mixture to which the H ₂ O ₂ was added was measured by using a fluorescence spectrophotometer (RF-5301PC, Shimadzu, Japan) to conduct an H ₂ O ₂ detection experiment.

In addition, a digital camera was used to take a picture of the mixture to which the H ₂ O ₂ was added and observed.

As a result, as shown in Figure 1 (B), it can be seen that after the growth of the gold nanoparticles, the fluorescence intensity was remarkably decreased, and even in the photograph, the green fluorescence was significantly reduced after the growth of the gold nanoparticles. I could confirm it.

1-2. When rhodamine B is used as a fluorescent dye

A mixture was prepared by adding 75 µl of 0.22 µM gold nanoparticle aqueous solution and 5 µl of 1 mM rhodamine B to 2.5 ml of 0.2 mM HAuCl ₄ aqueous solution.

_2.5 μl of 10 mM H ₂ O ₂ as a reducing agent was added to the mixture to induce the growth of gold nanoparticles.

The fluorescence intensity of the mixture to which the H ₂ O ₂ was added was measured by using a fluorescence spectrophotometer (RF-5301PC, Shimadzu, Japan) to conduct an H ₂ O ₂ detection experiment.

In addition, a digital camera was used to take a picture of the mixture to which the H ₂ O ₂ was added and observed.

As a result, as shown in FIG. 1C, it can be seen that the fluorescence intensity was significantly decreased after the growth of the gold nanoparticles, and that the fluorescence of the orange was markedly lowered after the growth of the gold nanoparticles. I could confirm it.

Experimental Example 1 Correlation between Reducing Agent, Gold Nanoparticle Growth and Fluorescence Decay of Fluorescent Dye

In accordance with Example 1-1, a reducing agent detection experiment using fluorescence reduction by growth of gold nanoparticles induced by a reducing agent was conducted, but the concentration of the reducing agent added, that is, H ₂ O ₂ , was increased in the range of 0 to 200 μM. The reducing agent detection experiment was performed.

After inducing the growth of gold nanoparticles according to Example 1-1, using a transmission electron microscope (TEM) (HD-2300A, Hidachi, Japan) to take a picture of the gold nanoparticles according to the H ₂ O ₂ concentration, H The correlation graph between the normalized degree of quenching and the average size of Au nanoparticles (Avr. Size of Au NPs (nm)) of ₂ O ₂ concentration was derived.

As a result, as shown in (A) i ~ vi of Figure 2, it was found that the size of the gold nanoparticles increases as the concentration of H ₂ O ₂ increases. In addition, as shown in Figure 2 (B), it was found that as the concentration of H ₂ O ₂ increases, the standard grade of fluorescence decrease and the average size of the gold nanoparticles also increases. Here, increasing the standard grade of fluorescence reduction means that fluorescence reduction is intensified.

Experimental Example 2 Reducing Agent Detection Experiment Using Fluorescence Reduction by Gold Nanoparticle Growth Induced by Reducing Agent According to Reducing Agent Type

1-1. Experiment without gold nanoparticles and gold growth solution

5 μl of a fluorescent dye fluorescein was added to 2.5 ml of phosphate buffer to prepare a 5 μM aqueous solution of fluorescein (hereinafter referred to as Dye only).

_2.5 μl of 10 mM H ₂ O ₂ , 5 μl of 10 mM hydroquinone and 5 μl of 1 mM acetylthiocholine were added to the aqueous fluorescein solution to induce the growth of gold nanoparticles.

The fluorescence intensities of three mixtures of 40 μM of H ₂ O _2, 40 μM of hydroquinone, and 40 μM of acetylthiocholine and an aqueous fluorescein solution were measured using a fluorescence spectrophotometer (RF-5301PC, Shimadzu, Japan), and a reducing agent detection experiment was performed.

As a result, as shown in (A) of FIG. 3, fluorescence deterioration did not occur in the measurement of fluorescence intensity of three mixtures of 40 μM of H ₂ O _2, 40 μM of hydroquinone, and 40 μM of acetylthiocholine, respectively, and an aqueous solution of fluorescein.

From this, it was found that only when the gold nanoparticles and the gold nanoparticle growth solution were present, the growth of the gold nanoparticles was caused by the reducing agent and the fluorescence was lowered, thereby detecting the reducing agent. That is, when the reducing agent was added to the solution containing only the fluorescent dye, no fluorescence was found.

1-2. Experiment using gold nanoparticles and / or gold growth solution

5 μl of a fluorescent dye fluorescein was added to 2.5 ml of phosphate buffer to prepare a 5 μM aqueous solution of fluorescein (hereinafter referred to as Dye only).

In addition, a mixture (hereinafter referred to as + Au NPs) of 0.22 μM gold nanoparticles aqueous solution was added to the aqueous fluorescein solution.

In addition, a mixture (hereinafter referred to as + HAuCl ₄ ) of 5 μl of a 1 mM HAuCl ₄ aqueous solution of gold nanoparticle growth solution was prepared in the aqueous fluorescein solution.

In addition, a mixture (hereinafter, + Au NPs + HAuCl ₄ ) was prepared by adding 75 μl of 0.22 μM gold nanoparticle solution and 5 μl of 1 mM HAuCl ₄ solution to the fluorescein aqueous solution.

Thereafter, ₂₀₀ μM of H ₂ O ₂ , a reducing agent, was added to the aqueous fluorescein solution and three mixtures to induce the growth of gold nanoparticles.

Fluorescence intensities of the fluorescein aqueous solution and three mixtures of H ₂ O ₂ added thereto were measured using a fluorescence spectrophotometer (RF-5301PC, Shimadzu, Japan), and a reducing agent detection experiment was performed.

As a result, as shown in Figure 3 (B), in the case of the aqueous solution of fluorescein fluorescence did not occur, it was confirmed that the fluorescence decrease occurs in the mixture of gold nanoparticles and / or HAuCl ₄ added.

As a result, it was confirmed that fluorescent dyes, gold nanoparticles and / or gold nanoparticles growth solution and reducing agent should all be present in order to observe fluorescence deterioration.

Experimental Example 3: Sensitivity Change of Detection Experiment Using Fluorescence Reduction and Absorbance Change

A mixture was prepared by adding 75 µl of 0.22 µM gold nanoparticle aqueous solution and 5 µl of 1 mM fluorescein to 5 µl of 0.1 mM HAuCl ₄ aqueous solution.

10 μl of hydroquinone as a reducing agent was added to the mixture to induce the growth of gold nanoparticles.

The fluorescence intensity and the absorbance of the hydroquinone-added mixture were measured using a fluorescence spectrophotometer (RF-5301PC, Shimadzu, Japan) and an ultraviolet-vis spectrophotometer (UV-vis spectrophotometer Biospec-mini, Shimadzu, Japan), respectively. Detection experiment was performed.

As a result, as shown in Fig. 3A, as the hydroquinone concentration increased from 0 μM to 6 μM, the fluorescence intensity decreased, and the detection limit of the hydroquinone was 0.006 μM. In addition, as a specific example of the decrease in fluorescence, the degree of fluorescence reduction of the mixture to which 0.8 μM hydroquinone was added when the wavelength was 535 nm was about 13%.

In addition, as shown in (B) of FIG. 3, as the hydroquinone concentration increased from 0 µM to 6 µM, the absorbance increased, but it was found that the hydroquinone concentration was almost constant without changing the fluorescence intensity in the range up to 0.2 µM. . In addition, as a specific example of the increase in absorbance, the fluorescence deterioration degree of the mixture containing 0.8 μM hydroquinone at a wavelength of 535 nm was only about 3.5%.

As a result, hydroquinone can be detected using a decrease in fluorescence intensity and an increase in absorbance of the mixture to which hydroquinone is added, but the change in fluorescence intensity according to the hydroquinone change can be detected when the fluorescence intensity decrease is used. Since it appears large, it turned out that detection sensitivity is excellent.

Example 2 Enzyme Substrate Detection Method Using Degradation of Gold Nanoparticle Growth Fluorescence Signal Induced by Enzyme Reaction

A mixture of 5 μl of AChE (acetylcholinesterase), 5 μl of 0.1 M HAuCl ₄ aqueous solution, 0.22 μM gold nanoparticle aqueous solution and 5 μl fluorescein was prepared.

5 μl of acetylcholine, which is a substrate of AChE, was added to the mixture, and the experiments were performed with the concentrations of acetylcholine changed to 0 μM, 0.02 μM, 0.1 μM, 0.4 μM, 0.8 μM, and 1.6 μM.

The fluorescence intensity of the mixture to which acetylcholine was added was measured using a fluorescence spectrophotometer (RF-5301PC, Shimadzu, Japan), and acetylcholine detection experiments were performed.

As a result, as shown in (a) and (b) of Figure 5, it was found that as the concentration of acetylcholine increases, the fluorescence intensity of the mixture to which the acetylcholine is added decreases.

Experimental Example 4: Measurement of time required for fluorescence reduction

According to the method of Example 1-1, H ₂ O ₂ and hydroquinone detection experiment using the fluorescent signal degradation was performed. In addition, according to Example 2, an acetylthiocholine detection experiment using fluorescence reduction was performed.

As a result, as shown in Figure 6, when using the acetylthiocholine it was confirmed that the time required to reduce the fluorescence within 5 minutes. This result is due to the rapid reduction of HAuCl ₄ by the electrons produced in the reaction between AChE and acetylthiocholine.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 is a schematic diagram (A) of a fluorescence reduction reaction by growth of gold nanoparticles induced by reduction of gold ions in the presence of a reducing agent and AuCl ₄ ⁻ , before growth of gold nanoparticles based on H ₂ O ₂ as a reducing agent ( rhodamine before and after growth of gold nanoparticles (B) as a catalyst for the change in fluorescence intensity (B) of the fluoresein-containing solution and the reductant H ₂ O ₂ in solid and dotted lines Fluorescence intensity change (C) of the B-containing solution is shown.

FIG. 2 shows TEM images of gold nanoparticles after growth reaction when H ₂ O ₂ concentrations are (i) 0 μM, (ii) 2 μM, (iii) 10 μM, (iv) 40 μM and (v) 120 μM and (vi) 200 μM (A); Fluorescence decrease according to H ₂ O ₂ concentration (●) and H ₂ O ₂ This is a graph (B) showing the average size (○) change of gold nanoparticles.

3 is a graph showing a change in fluorescence intensity of the analyte according to the wavelength (A) and a graph showing a change in fluorescence intensity according to the presence or absence of gold nanoparticles and gold growth solution (B).

Figure 4 shows the fluorescence intensity (A) of the fluoroscein (5 μM) -containing gold nanoparticle growth solution according to the wavelength after the growth of gold nanoparticles in the presence of hydroquinone at a concentration of 0-6 μM; And it is a graph showing the UV-vis absorbance spectrum (B) according to the wavelength.

Figure 5 is a graph showing the change in fluorescence intensity according to the wavelength and the concentration of acetylthiocholine (a) and a graph showing the standard grade of fluorescence degradation according to the concentration of acetylthiocholine (b).

6 is a graph showing the change in fluorescence intensity over time according to the type of analyte.

Claims (17)

A method for detecting an analyte, using fluorescence reduction by growth of gold nanoparticles, comprising the following steps: (a) preparing a mixture by adding gold nanoparticles and a fluorescent dye to the growth solution of gold nanoparticles; (b) adding a reducible analyte to the mixture to induce gold nanoparticle growth; And (c) detecting a reducible analyte by measuring the fluorescence intensity of the mixture containing the grown gold nanoparticles. The method of claim 1, wherein the reducible analyte is H ₂ O ₂ , hydroquinone (HQ), adrenaline, noradrenaline, dopamine, L-Dopa, mercaptosuccinic acid, 4-aminophenol, 3-aminophenol, 1,4-phenylenediamine, aniline , triethylamine, indole, 4-bromoaniline ,, 1-methylindole, 3-amino-1-propanol, pyridine, 3-indole propionic acid, glucine, DL-tryptophan, Sodium cirate, citric acid, sodium boro hydride, hydroxylamine, HCl, acetone, oxalic acid and b-diketone. The gold nanoparticle growth solution of claim 1, wherein the gold nanoparticle growth solution is selected from the group consisting of HAuCl ₄ , NaAuCl ₄ , AuCl ₃ and K (AuCl ₄ ). It is an aqueous solution. The fluorescent dye of claim 1, wherein the fluorescent dye is selected from the group consisting of fluorescein, rhodamine, eosin, Alexa, and rose bengal. Way. The method of claim 1, wherein the growing gold solution 100 parts by weight of the gold nano-particles 10 of about ^-4 to 10 ^-2 parts by weight of a fluorescent dye 10 ^-6 to 10 ^-2 parts by weight of the enzyme 10 ^-8 to 10 ^-2 parts by weight Adding. The method of claim 1, wherein 10 parts by weight of analyte 10 ^-18 to 10 ^-1 is added to 100 parts by weight of the gold growth solution. A method for detecting an analyte, using a decrease in fluorescence signal by growth of gold nanoparticles, comprising the following steps: (a) preparing a mixture of gold nanoparticle growth solution, gold nanoparticles, fluorescent dyes and enzymes; (b) adding an analyte to the mixture to induce a reaction of the analyte with an enzyme; And (c) measuring the fluorescence intensity of the mixture to which the analyte is added, and comparing the fluorescence intensity of the fluorescent dye to detect the analyte using the degree of fluorescence signal degradation. The method of claim 7, wherein the enzyme is acetylthiocholinesterase (AChE), choline oxidase (glucose oxidase), glucose oxidase (cytochrome oxidase), ascorbate oxidase (ascorbic oxidase), Xanthine oxidase, polyphenol oxidase, catechol oxidase, lysyl oxidase NADPH oxidase, monoamine oxidase and lacamine (oxidase) laccase) selected from the group consisting of. The method of claim 7, wherein the analyte is acetylthiocholine, acetylcholine, hydroquinone, glucose, adrenaline, noradrenaline, dopamine, L-Dopa, mecaptosuccinic acid, 4-aminophenol, 3-aminophenol, 1,4-phenylenediamine, aniline, triethylamine, indole , 4-bromoaniline ,, 1-methylindole, 3-amino-1-propanol, pyridine, 3-indole propionic acid, glucine, DL-tryptophan, Sodium cirate, citric acid, sodium boro hydride, hydroxylamine, HCl, acetone, oxalic acid And b-diketone. The method of claim 7, wherein the gold nanoparticle growth solution is an aqueous solution of a gold ion generating material selected from the group consisting of HAuCl ₄ , NaAuCl ₄ , AuCl ₃ , K (AuCl ₄ ). The method of claim 7, wherein the fluorescent dye is selected from the group consisting of fluorescein, rhodamine, eosin, and rose bengal. The method of claim 7, wherein the gold growth solution to 10 with gold nanoparticles 10 ^-4 parts by weight based on 100 parts by weight of ^-2, a fluorescent dye 10 ^-6 to 10 ^-2 parts by weight of the enzyme 10 ^-8 to 10 ^-2 parts by weight Adding. The method according to claim 7, wherein 10 to ¹⁸ to 10 ^-1 parts by weight of the analyte is added to 100 parts by weight of the gold growth solution. A biosensor for the detection of reducible analytes used in the growth of gold nanoparticles, comprising growth solutions of gold nanoparticles, gold nanoparticles, and fluorescent dyes. The biosensor according to claim 14, wherein 10 ^-4 to 10 ^-2 parts by weight of gold nanoparticles and 10 ^-6 to 10 ^-2 parts by weight of fluorescent dyes are included with respect to 100 parts by weight of the gold growth solution. Biosensor for detecting the substrate of the enzyme, comprising a growth solution of gold nanoparticles, gold nanoparticles, fluorescent dyes and enzymes. According to claim 16, wherein the gold nano-gold for the growth solution 100 parts by weight of the particles 10 ^-4 to 10 ^-2 parts by weight of a fluorescent dye 10 ^-6 to 10 ^-2 parts by weight of the enzyme 10 ^-8 to 10 ^-2 parts by weight Biosensor included.

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