WO2022080543A1 - Nanoparticules comprenant des nanoagrégats d'or à base d'enzymes, et leur procédé de fabrication - Google Patents

Nanoparticules comprenant des nanoagrégats d'or à base d'enzymes, et leur procédé de fabrication Download PDF

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WO2022080543A1
WO2022080543A1 PCT/KR2020/014347 KR2020014347W WO2022080543A1 WO 2022080543 A1 WO2022080543 A1 WO 2022080543A1 KR 2020014347 W KR2020014347 W KR 2020014347W WO 2022080543 A1 WO2022080543 A1 WO 2022080543A1
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gold nanoclusters
gold
nanoparticles
protein
glucose
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PCT/KR2020/014347
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English (en)
Korean (ko)
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오병근
이명준
신정협
송지애
김태환
이상남
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서강대학교 산학협력단
(주)유니언스진
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Publication of WO2022080543A1 publication Critical patent/WO2022080543A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors

Definitions

  • the present invention relates to a nanoparticle composed of an enzyme-based gold nanocluster and a method for manufacturing the same. It relates to particles and a method for manufacturing the same.
  • Hydrogen peroxide and glucose are representative physiologically active substances to be measured using a sensor.
  • the hydrogen peroxide plays an important role in physiological processes such as cell growth and apoptosis in the human body, but hydrogen peroxide is present in high concentrations in the human body.
  • blood sugar is high or low, it can cause cellular damage and cancer, and although glucose in the blood plays an important role in our body, it can affect the body and cause diseases such as diabetes.
  • a sensor for measuring hydrogen peroxide and a sensor for measuring glucose as described in the following patent documents are being developed.
  • Patent No. 10-1823114 registered on January 23, 2018
  • Patent No. 10-1450385 (Registered on Oct. 06, 2014) "Biosensor for measuring glucose concentration and manufacturing method therefor"
  • the present invention has been devised to solve the above problems,
  • An object of the present invention is to provide nanoparticles for measuring hydrogen peroxide, in which a protein-gold nanoclusters and a peroxidase-gold nanoclusters are aggregated, and having high sensitivity and improved stability, and a method for preparing the same.
  • the present invention provides nanoparticles for measuring glucose having high sensitivity and improved stability in which protein-gold nanoclusters, peroxidase-gold nanoclusters and glucose oxidase-gold nanoclusters are aggregated, and a method for manufacturing the same. There is a purpose.
  • the present invention is implemented by an embodiment having the following configuration in order to achieve the above object.
  • the nanoparticles according to the present invention include a protein-gold nanoclusters and an enzyme-gold nanoclusters bound to the protein-gold nanoclusters.
  • the enzyme-gold nanoclusters include peroxidase-gold nanoclusters
  • the protein-gold nanoclusters include gold nanoclusters and the gold nanoclusters.
  • a protein surrounding the peroxidase-gold nanoclusters is characterized in that it comprises a gold nanoclusters and a peroxidase surrounding the gold nanoclusters.
  • the nanoparticles according to the present invention are formed by aggregation of the protein-gold nanoclusters and the peroxidase-gold nanoclusters, have a spherical shape, and have a fluorescence intensity when reacted with hydrogen peroxide characterized by change.
  • albumin is used as the protein.
  • the enzyme-gold nanoclusters include peroxidase-gold nanoclusters and glucose oxidase-gold nanoclusters
  • the protein-gold nanoclusters include A gold nanoclusters and a protein surrounding the gold nanoclusters
  • the peroxidase-gold nanoclusters include a gold nanoclusters and a peroxidase surrounding the gold nanoclusters
  • the glucose oxidase-gold nanoclusters include It is characterized in that it comprises a gold nano-clusters and a glucose oxidase surrounding the gold nano-clusters.
  • the nanoparticles according to the present invention are formed by agglomeration of the protein-gold nanoclusters, the peroxidase-gold nanoclusters, and the glucose oxidase-gold nanoclusters, and have a spherical shape. It is characterized in that it is used for glucose measurement.
  • the color of the mixed solution changes according to the presence and amount of glucose.
  • the senor for measuring glucose according to the present invention includes a metal electrode and nanoparticles for measuring glucose bound to the metal electrode by a linker, wherein the nanoparticles for measuring glucose are a fifth It is characterized in that the nanoparticles are used.
  • the method for producing nanoparticles according to the present invention includes a mixed solution preparation step of preparing a mixed solution by mixing protein-gold nanoclusters and enzyme-gold nanoclusters, and protein through desolvation -
  • protein-gold nanoclusters and peroxidase-gold nanoclusters are mixed.
  • protein-gold nanoclusters, peroxidase-gold nanoclusters, and glucose oxidase-gold nanoclusters are characterized by mixing.
  • the present invention can obtain the following effects by the present embodiment above.
  • nanoparticles for measuring hydrogen peroxide are formed by aggregation of protein-gold nanoclusters and peroxidase-gold nanoclusters, and thus have the effect of sensing hydrogen peroxide with high sensitivity while improving stability.
  • the present invention provides the effect that the nanoparticles for glucose measurement are formed by aggregation of protein-gold nanoclusters, peroxidase-gold nanoclusters and glucose oxidase-gold nanoclusters, thereby improving stability and enabling the sensing of glucose with high sensitivity there is
  • 1 is a reference diagram for explaining the manufacturing process of nanoparticles for measuring hydrogen peroxide according to an embodiment of the present invention.
  • FIG. 2 is a reference diagram for explaining a change in fluorescence of nanoparticles for measuring hydrogen peroxide according to an embodiment of the present invention.
  • Figure 3 is a reference diagram for explaining the manufacturing process of the nanoparticles for glucose measurement according to another embodiment of the present invention.
  • FIG. 4 is a reference diagram for explaining a sensor and an electrochemical test method using the same according to another embodiment of the present invention.
  • FIG. 5 is a TEM image of nanoparticles for measuring hydrogen peroxide according to an embodiment of the present invention.
  • FIG. 6 is a graph showing the results of DLS analysis of nanoparticles for measuring hydrogen peroxide according to an embodiment of the present invention.
  • FIG. 7 is an image showing the results of photographing the nanoparticles for measuring hydrogen peroxide according to an embodiment of the present invention in sunlight and ultraviolet rays.
  • FIG. 8 is a graph showing an absorption wavelength and an emission wavelength of nanoparticles for measuring hydrogen peroxide according to an embodiment of the present invention.
  • FIG. 9 is a TEM image of nanoparticles for measuring glucose according to another embodiment of the present invention.
  • FIG. 10 is a graph showing the results of DLS analysis of nanoparticles for glucose measurement according to another embodiment of the present invention.
  • FIG 11 is an image showing the results of taking the nanoparticles for glucose measurement in ultraviolet light according to another embodiment of the present invention.
  • FIG. 12 is a graph for confirming that the fluorescence intensity of the nanoparticles for measuring hydrogen peroxide according to an embodiment of the present invention is changed by reacting with hydrogen peroxide.
  • FIG 13 is an image showing the results of photographing the nanoparticles for measuring hydrogen peroxide in ultraviolet light to confirm that the fluorescence intensity changes by reacting the nanoparticles for measuring hydrogen peroxide according to an embodiment of the present invention.
  • 14 and 15 are graphs showing the decrease in fluorescence intensity according to the concentration of hydrogen peroxide to confirm that the fluorescence intensity is changed by reacting the nanoparticles for measuring hydrogen peroxide with hydrogen peroxide according to an embodiment of the present invention.
  • 16 is a photographed image showing a result of selective hydrogen peroxide detection of nanoparticles for measuring hydrogen peroxide according to an embodiment of the present invention.
  • 17 and 18 are images showing the results of photographing the mixed solution for confirming that glucose can be measured using the nanoparticles for glucose measurement and the TMB solution according to another embodiment of the present invention.
  • 19 is an SEM image of a sensor according to another embodiment of the present invention.
  • 21 to 22 are graphs showing CV measurement results of a sensor according to another embodiment of the present invention.
  • 23 is a graph showing the linearization result of the reduction peak current value according to the glucose concentration of the sensor according to another embodiment of the present invention.
  • 24 and 25 are graphs showing amperometric i-t measurement results of a sensor according to another embodiment of the present invention.
  • 26 is a graph showing a result of selective glucose detection by a sensor according to another embodiment of the present invention.
  • 27 is a graph showing a glucose detection result in an actual sample of a sensor according to another embodiment of the present invention.
  • An embodiment of the present invention relates to a nanoparticle composed of an enzyme-based gold nanocluster, wherein the nanoparticle includes a protein-gold nanocluster and an enzyme-gold nanocluster coupled to the protein-gold nanocluster.
  • a conventional gold nanocluster is a material in which dozens of gold atoms are aggregated to have a size of about 2 nm or less, and has the same properties as molecules having discontinuous energy levels and thus emits fluorescence.
  • protein-gold nanoclusters and enzyme-gold nanoclusters were prepared using proteins (including enzymes) to stabilize the gold nanoclusters, and using them, the nanocluster to produce particles.
  • the enzyme-gold nanoclusters stabilized using an enzyme-Gold nanoclusters have all of the enzyme properties, fluorescence properties and conductivity. Nanoclusters also have the same catalytic properties as enzymes and have fluorescent properties and conductivity.
  • the protein-gold nanoclusters include gold nanoclusters and proteins surrounding the gold nanoclusters.
  • a general protein other than an enzyme may be used, for example, a protein capable of forming a self-conjugate having cohesiveness, a protein containing residues such as cysteine and tyrosine, etc. may be used, specifically human serum albumin, bovine serum Albumin, albumin such as ovalbumin, lysozyme, etc. may be used.
  • the protein-gold nanoclusters may be prepared by a conventional method, for example, a protein solution and a solution containing gold ions are mixed, and an aqueous alkali solution is added to reduce the gold ions (Au 3+ ) penetrating into the protein.
  • a plurality of gold ions are located in the protein by the bonding of gold ions to residues present in the protein, Au 3+ is reduced to Au 1+ , and some Au 1+ is converted to Au 0 by an aqueous alkali solution. It is reduced to form a protein-gold nanocluster in which the protein surrounds the gold nanocluster.
  • the enzyme-gold nanoclusters include gold nanoclusters and an enzyme surrounding the gold nanoclusters.
  • Various enzymes can be used for the enzyme, and since the enzyme is also a protein, the enzyme-gold nanoclusters are prepared by the same conditions as the protein-gold nanoclusters production method except that an enzyme solution is used instead of a normal protein solution.
  • the enzyme-gold nanoclusters one type or another type of a plurality of enzyme-gold nanoclusters may be used. As shown in FIGS. 1 and 2, when peroxidase-gold nanoclusters are used, the , , peroxidase-gold nanoclusters agglomerated to form hydrogen peroxide measurement nanoparticles have a spherical shape.
  • Nanoparticles for glucose measurement in which clusters are agglomerated with each other have a spherical shape, and when the nanoparticles are coupled to an electrode to constitute an electrochemical sensor, an electrical signal that changes depending on the presence and amount of glucose can be obtained, It can be used in electrochemical sensors to measure.
  • the nanoparticles for glucose measurement, glucose and TMB solution are mixed, the nanoparticles for glucose measurement convert glucose into hydrogen peroxide and gluconic acid, and the hydrogen peroxide is converted into water by the nanoparticles for glucose measurement It is reduced and at the same time TMB is oxidized, so it changes color to blue. That is, the higher the concentration of glucose, the bluer the color of the mixed solution of the nanoparticles for glucose measurement, glucose and TMB.
  • Another embodiment of the present invention relates to a sensor for measuring glucose comprising a metal electrode and nanoparticles for measuring glucose bound to the metal electrode by a linker as shown in FIG. 4 .
  • Another embodiment of the present invention relates to a method for producing nanoparticles composed of an enzyme-based gold nano-cluster, wherein the method for producing nanoparticles is mixed with a protein-gold nano-cluster used as a template and an enzyme-gold nano-cluster
  • the mixed solution preparation step is a step of preparing a mixed solution by mixing the protein-gold nanoclusters used as a template and the enzyme-gold nanoclusters, wherein the enzyme-gold nanoclusters are one or different types of a plurality of enzyme-gold Nanoclusters may be used.
  • the enzyme-gold nanoclusters are one or different types of a plurality of enzyme-gold Nanoclusters may be used.
  • FIG. 1 when peroxidase-gold nanoclusters are used, nanoparticles for measuring hydrogen peroxide may be prepared through the above preparation method, and as shown in FIG. 3 , peroxidation When using the enzyme-gold nanoclusters and the glucose oxidase-gold nanoclusters, nanoparticles for measuring glucose can be prepared.
  • the desolvation step is a step of reacting by adding a desolvation solution containing ethanol to the mixed solution so that the protein-gold nanoclusters and the enzyme-gold nanoclusters aggregate to form particles through desolvation.
  • Desolvation containing ethanol The solution is added to the mixed solution so that the protein and enzyme are aggregated into a certain shape and size through desolvation, so that the protein-gold nanoclusters and the enzyme-gold nanoclusters are agglomerated to form particles.
  • the desolvation solution may further contain acetonitrile in addition to ethanol, and it is preferable that the desolvation solution is added dropwise rather than being mixed into the mixed solution at once.
  • the crosslinking step is a step of adding an additional crosslinking agent after the desolvation step so that the particles formed in the desolvation step are crosslinked to have a solid shape. and amine groups between the enzymes so that the particles formed in the desolvation step have a solid shape.
  • the crosslinking agent may be, for example, glutaraldehyde.
  • the contents subjected to the crosslinking step are centrifuged to remove impurities to obtain nanoparticles.
  • BSA solution (100 mg/mL) was prepared by dissolving bovine serum albumin in tertiary distilled water, and 0.5 mL of a HAuCl 4 solution (10 mM) dissolved in tertiary distilled water was mixed with 0.5 mL of the BSA solution, and then inside the BSA 10ul of sodium hydroxide (1M) was additionally added to reduce the gold ions made of Au 3+ to Au 0 , and reacted while mixing at 900 RPM at 37 ° C. for 24 hours.
  • the gold nano-clusters and the protein (peroxidase) surrounding the gold nano-clusters were the same as in Example 1 except that HRP was used instead of BSA.
  • a gold nanocluster (HRP-AuNC) was prepared.
  • Glucose oxidation comprising gold nanoclusters and a protein (glucose oxidase) surrounding the gold nanoclusters, except that glucose oxidase (GOx) was used instead of BSA, and the other conditions were the same as in Example 1.
  • Enzyme-gold nanoclusters (GOx-AuNC) were prepared.
  • nanoparticles for sensing were prepared using BSA-AuNC, HRP-AuNC, and GOx-AuNC prepared in Example 1.
  • the nanoparticles for measuring hydrogen peroxide prepared in 2 of Example 2 were confirmed by TEM, and the results are shown in FIG. 5, and DLS analysis was performed on the nanoparticles for measuring hydrogen peroxide, and the results are shown in FIG. ,
  • the nanoparticles for measurement of hydrogen peroxide were photographed in daylight and ultraviolet light, and the results are shown in FIG. , the absorption wavelength and emission wavelength of the nanoparticles for measurement of hydrogen peroxide were confirmed, and the results are shown in FIG. 8 .
  • the nanoparticles for glucose measurement prepared in 3 of Example 2 were confirmed by TEM, and the results are shown in FIG. 9, and DLS analysis was performed on the nanoparticles for glucose measurement, and the results are shown in FIG. , The glucose measurement nanoparticles were photographed in ultraviolet light, and the results are shown in FIG. 11 .
  • the conjugated enzyme particle (CENP) was formed in the same manner as in Example 2 3, except that BSA, HRP and GOx were used instead of BSA-AuNC, HRP-AuNC and GOx-AuNC.
  • the nanoparticles for measuring hydrogen peroxide have a spherical shape and have a nano size.
  • the nanoparticles for measuring hydrogen peroxide have fluorescence properties. It can be seen that the nanoparticles have an absorption wavelength peak of 248 nm and an emission wavelength peak of 720 nm.
  • the nanoparticles for glucose measurement have a spherical shape and have a nano size.
  • the nanoparticles for glucose measurement have fluorescence properties, but include BSA, HRP and GOx. It can be seen that the composite enzyme particles do not have fluorescence properties.
  • FIGS. 14 and 15 are an HPNP photograph, and the photograph on the right is an HPNP mixed with 100 mM hydrogen peroxide), BSA-AuNC, HRP-AuNC and By varying the concentration of hydrogen peroxide to be mixed for each HPNP, the fluorescence intensity was measured with an absorption wavelength of 248 nm and an emission wavelength of 720 nm, and the fluorescence intensity reduction rate was calculated, and the results are shown in FIGS. 14 and 15 .
  • the peroxidase particles (ENP) were formed in the same manner as in Example 2 3 except that BSA and HRP were used instead of BSA-AuNC, HRP-AuNC and GOx-AuNC.
  • the fluorescence intensity of the peroxidase particles is close to 0, so that the fluorescence intensity is not a particle having a fluorescence property, and the fluorescence intensity of HPNP is about 8000.
  • the intensity of fluorescence is 3000.
  • the lowering 14 and 15 it can be seen that the detection limit of BSA-AuNC is 10 ⁇ M and that of HRP-AuNC is 1 ⁇ M, whereas the detection limit of HPNP is 10 nM, and that HPNP is BSA-AuNC and HRP-AuNC It can be seen that the decrease rate is large at a low concentration of hydrogen peroxide compared to .
  • Blank means a case in which the same amount of tertiary distilled water is mixed in HPNP to have the same concentration as other solutions.
  • FIGS. 17 and 18 After mixing the nanoparticles for glucose measurement, PBS, and TMB solution, glucose of different concentrations was added to react, and the result was photographed with a digital camera, and the results are shown in FIGS. 17 and 18 .
  • Fig. 17 (a) is an image when glucose is not mixed
  • Fig. 17 (b) is an image when 2 mM glucose is mixed
  • Fig. 18 (a) is an image when glucose is not mixed.
  • (b) of FIG. 18 is an image when 100 ⁇ M of glucose is mixed.
  • a washing solution was prepared by mixing sulfuric acid and hydrogen peroxide in a ratio of 7:3, and the gold plate (1 ⁇ 1.5 cm) was immersed in the washing solution to remove impurities, washed with 70% ethanol and tertiary water, and dried.
  • FIG. 19 is a scanning electron microscope (SEM) image of the gold substrate before (a) and after (b) GNP fixation
  • FIG. 20 is an atomic force microscope (AFM) of the gold substrate before (a) and after (b) GNP fixation.
  • SEM scanning electron microscope
  • AFM atomic force microscope
  • sensor 3 in which GOx-AuNC was immobilized was manufactured in the same manner as in Example 7 2, except that GOx-AuNC was used.
  • Example 7 2 Except that CENP was used instead of GNP, the other conditions were the same as those of Example 7 2 to prepare a sensor 4 to which CENP was fixed.
  • Example 7 The electrochemical characteristics of the sensors 1 to 4 prepared in Example 7 were analyzed through cyclic voltammetry, and the results are shown in FIGS. 21 to 23 .
  • a CHI-660e device was used, a three-electrode system using PBS as an electrolyte was used, and each of sensors 1 to 4 was As a working electrode, an Ag/AgCl electrode was used as a reference electrode, and a platinum electrode was used as a counter electrode, respectively.
  • 21 shows CV graphs of sensors 1 to 3 when glucose 1uM is added
  • FIG. 22 shows CV graphs of sensors 1 and 4 when glucose 1uM is added
  • FIG. 23 is a reduction peak current according to glucose concentration at -0.55V. It is a graph showing the result of linearization of values.
  • the It Amperometry method was performed to confirm the real-time reaction by concentration of glucose.
  • a three-electrode system using PBS as an electrolyte was used, and sensor 1 was As the working electrode, Ag/AgCl electrode was used as a reference electrode and platinum electrode as a counter electrode, respectively, and it was performed with a potential of -0.55V, a sampling interval of 0.1 sec, and a sensitivity of 5 ⁇ 10 6 (A/V).
  • 10 ⁇ l of the prepared glucose solution was added so that the final concentration of glucose was 5, 10, 20, 40, 80, 160, 320 nM, and amperometric it curve data were obtained, and the results are shown in FIGS. 24 and 25 .

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Abstract

La présente invention concerne des nanoparticules comprenant des nanoagrégats d'or à base d'enzymes et leur procédé de fabrication, les nanoparticules étant formées par l'agglomération de nanoagrégats de protéine-or et de nanoagrégats d'enzyme-or, et peuvent ainsi améliorer la stabilité et la sensibilité de détection.
PCT/KR2020/014347 2020-10-16 2020-10-20 Nanoparticules comprenant des nanoagrégats d'or à base d'enzymes, et leur procédé de fabrication WO2022080543A1 (fr)

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