WO2014200164A1 - Composition having ultra-high sensitivity and high selectivity for detecting copper ion and fluorescent chemical sensor - Google Patents

Composition having ultra-high sensitivity and high selectivity for detecting copper ion and fluorescent chemical sensor Download PDF

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Publication number
WO2014200164A1
WO2014200164A1 PCT/KR2013/010418 KR2013010418W WO2014200164A1 WO 2014200164 A1 WO2014200164 A1 WO 2014200164A1 KR 2013010418 W KR2013010418 W KR 2013010418W WO 2014200164 A1 WO2014200164 A1 WO 2014200164A1
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phosphor
quencher
copper ion
copper ions
copper
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PCT/KR2013/010418
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French (fr)
Korean (ko)
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박경순
이애주
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한국기초과학지원연구원
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Publication of WO2014200164A1 publication Critical patent/WO2014200164A1/en

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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • C09K11/07—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials having chemically interreactive components, e.g. reactive chemiluminescent compositions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1003—Carbocyclic compounds
    • C09K2211/1007—Non-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1003—Carbocyclic compounds
    • C09K2211/1014—Carbocyclic compounds bridged by heteroatoms, e.g. N, P, Si or B
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1022—Heterocyclic compounds bridged by heteroatoms, e.g. N, P, Si or B
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6434—Optrodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7786—Fluorescence

Definitions

  • the present invention provides a composition for detecting copper ions having ultra-high sensitivity and high selectivity, a fluorescence chemical sensor comprising the same, a manufacturing method thereof, a selective detection and determination method of copper ions, a screening method of a copper ion chelating agent and a copper ion chelating agent It relates to a method of confirming the chelate effect of.
  • Copper is the third most abundant metal in essential heavy metals present in the human body, and is known to play an important role in various physiological processes.
  • copper ions convert oxygen molecules into reactive oxygen species, causing damage to proteins, nucleic acids and lipids. Indeed, excessive amounts of copper are toxic and cause various diseases due to excessive oxidative stress.
  • Diseases caused by abnormal intake of copper ions include cancer, cardiovascular disorders, Menques' disease, Wilson's disease, Alzheimer's disease, gastrointestinal disorders, hypoglycemia, dyslexia, and infant's liver disease. It is known to occur due to the accumulation of copper ions. Therefore, it is very important to detect residual copper ions in biological systems and environmental fields.
  • spectroscopy and voltage-current methods for detecting copper ions require complex pretreatment and labor before analysis.
  • an object of the present invention is to provide a composition for detecting copper ions.
  • Another object of the present invention is to provide a fluorescent chemical sensor for detecting copper ions.
  • Another object of the present invention to provide a method for confirming the chelate effect of a copper ion chelating agent.
  • the present invention is a phosphor; And a quencher that absorbs the luminous effect of the phosphor and exhibits a quencher effect, wherein the phosphor and the quencher include an azide group or an alkyne group, respectively, wherein the phosphor is an azide group or an alkyne group.
  • the quencher provides a composition for detecting copper ions, characterized in that it comprises an alkyne group or an azide group.
  • the present invention provides a fluorescent ion sensor for detecting copper ions comprising the composition for detecting copper ions.
  • the present inventors made an effort to develop a sensor capable of detecting copper ions quickly and easily with high sensitivity and selectivity.
  • a copper ion sensor using click chemistry and fluorescence resonance energy transfer (FRET) of azide / alkyne compounds. It was confirmed that copper ions can be selectively detected at high sensitivity (femtomol level) in mixed samples.
  • FRET fluorescence resonance energy transfer
  • a feature of the present invention is that the phosphor-quencher quenching system using click chemistry and fluorescence resonance energy transfer of azide / alkyne compounds is highly effective It selectively detects copper ions with sensitivity.
  • copper ions can be selectively detected (see FIG. 2), and the detection of such copper ions can be achieved in a short time (see FIG. 3). Even in the presence of copper ions can be detected specifically (see Figure 4), it is possible to detect copper ions with a high sensitivity of femtomol level (see Figure 5).
  • it is possible to evaluate the effect of the copper silver chelating agent see FIG. 6), to detect the copper silver present in the cell (see FIG. 7) and to quantify the copper ions present in the serum (FIG. 8).
  • the phosphor includes an azide group or an alkyne group, and the azide or alkyne group undergoes a cycloaddition reaction with an alkyne or azide compound bound to the quencher in the presence of copper ions (Cu 1+ ).
  • Phosphors that can be used in the present invention include, for example, Tavi's "FluoroTable” ' Common fluoiDphores (Source: Zeiss Corporation website and http://info.med.yale.edu/genetics/ward/tavi/FISHdyes2 itml). There is a disclosed phosphor.
  • the phosphor is cyanine, fluorescein, fluoresce (B0DIPY), tetramethylrodamine
  • the phosphor emits red or near infrared fluorescence.
  • Phosphors emitting red or near-infrared fluorescence include cyanine and alexa, and these phosphors emit and absorb light of near-infrared light, thereby minimizing interference or hop number with cells, blood, and biological tissues.
  • the quencher functions to absorb and quench the wavelength of light emitted from the phosphor.
  • An appropriate quencher may be selected and used according to the range of the emission wavelength of the phosphor, and it is preferable to use a quencher having the same or similar wavelength band as the emission wavelength of the phosphor.
  • the quencher is a black hole quencher (BHQ), a blackberry quencher (blackberry quencher, BBQ) or derivatives thereof.
  • BHQ black hole quencher
  • BBQ blackberry quencher
  • Exemplary phosphor and quencher pairs usable in the present invention are shown in Tables 1 and 2 below.
  • the composition for detecting copper ions further includes a reducing agent.
  • the reducing agent functions to reduce copper ions (II) to copper ions (I).
  • any reducing agent having such a function can be used without limitation, and examples of the reducing agent that can be used include sodium ascorbate.
  • the form of the fluorescent chemical sensor for detecting copper ions in the present invention is not particularly limited, and any form containing the composition of the present invention may be used without limitation.
  • Specific examples of the fluorescence chemical sensor may include a form in which the phosphor and the quencher (additional reducing agent) are contained in a container together with a solvent.
  • the present invention provides a composition for detecting copper ions or a method for producing a fluorescent chemical sensor comprising the following steps:
  • step (c) the phosphor to which the azide group obtained in step (a) is introduced and the quencher to which the alkyne group obtained in step (b) is introduced, or the phosphor to which the alkyne group obtained in step (a) is introduced and in step (b) Mixing the quencher into which the obtained azide group was introduced.
  • a phosphor and a quencher in which an azide or alkyne group is introduced are obtained.
  • Such phosphors and quencher may be produced directly or obtained by a method such as purchase.
  • a method of introducing an azide group into the phosphor and the quencher can be carried out according to the following formula 1 or 2.
  • A represents a phosphor or a quencher.
  • Scheme 1 introduces an azide group to a phosphor (or quencher) through reaction of a phosphor (or quencher) activated with hydroxysuccinimide and a compound containing both an amine group and an azide group.
  • Banungsik 2 shows a compound containing both a carboxyl group (or a quencher), an amine (N3 ⁇ 4) group and an azide glove, and a crosslinking agent, di-cylohexyldiimid (DCC), We show how to introduce an azide group into a phosphor (or quencher) via hydroxysuccinimide.
  • DCC di-cylohexyldiimid
  • reaction formula 1 as a compound that can be used to introduce an azide group into the hydroxysuccinimide-activated phosphor (or quencher), 4-azidoaniline hydrochloride, 11- Azidodo3,6,9-trioxoundecan-1-amine (11-az ido-3, 6, 9-1 ri oxaundecan-l-am i ne) Azidodo PEG 6 -amine (azido-PEG 6 - amine), azido -PEG 8 - amine (azid coming PEG -amine 8) and azido PEG 10 - amine (azido-PEG 10 -amine), etc., and includes all the other azide group with an amine group Any compound can be used without limitation.
  • a compound that can be used to introduce an azide group using a crosslinking agent DCC and hydroxysuccinimide to a phosphor having a carboxyl group (or a quencher) is 4-azidoaniline hydrochloride, 11 Azido-3, 6, 9-trioxoundecan-1-amine, azido-PEG 6 -amine, azido-PEG S -amine and azido-PEG 10 -amine; Any compound containing both a group and an amine group can be used without limitation.
  • the method of introducing an alkyne group to the phosphor and the quencher may be carried out according to the following formula 3 or 4.
  • A represents a phosphor or a quencher.
  • a compound which can be used to introduce an alkyne group using a crosslinking agent DCC and a catalyst 4-dimethylaminopyridine (DMAP) to a quencher (or phosphor) having a carboxyl group is 2-butyne- 1- (2-butyn-l-ol), 3-butyn-1- (3-butyn-l-ol), 3-butyn-2-ol (3—butyn-2-ol), 4-pentin 2-ol (4-pentyn-2-ol), 5-nuxen-2-yn-1- (5-611-2 ⁇ ]-1-00, 2-nucleine-1- (2-hexyn- 1-ol), 3-nucleine-1— (3-hexyn ⁇ l-ol), 3-nucleine-2 -ol (3-hexyn— 2-ol), 4-nucleine-3-ol (4-hexyn- 3-ol), 5-nucleine 3-ol (5-hexyn-3-ol), 5-n
  • step (c) the phosphor to which the azide group (or alkyne group) obtained in step (a) is introduced and the quencher (additionally reducing system) into which the alkyne group (or azide group) obtained in step (b) is introduced
  • suitable solvents include aqueous solutions, organic solvents and buffer solutions (eg PBS).
  • PBS buffer solutions
  • the complex itself obtained here may be used as a sensor for detecting copper ions, and may be implemented as a sensor device by additionally providing the complex to a sensor substrate.
  • the invention provides a method for the selective detection of copper ions in a sample comprising the following steps:
  • the term “detection” includes not only identifying the presence of copper ions in the sample (quantitative analysis), but also determining the amount of copper ions (quantitative analysis).
  • the amount of copper ions present in the sample can be found by substituting the degree of quenching (fluorescence intensity) of the phosphor identified in step (b) into a copper silver standard curve (see Test Example 7 below).
  • the selective detection method of copper ions according to the present invention by substituting the fluorescence intensity (fluorescence intensity) of the phosphor identified in step (b) after step (b) to the copper ion standard curve
  • the method may further include a step (step c) of determining the concentration of copper ions.
  • the detection method of the present invention it is also possible to detect copper ions present at very low concentrations (eg, femtomol level) in the sample.
  • the concentration of copper ions present in the sample is 1 femtomol (fM) or more.
  • the detection method of the present invention can be applied to the detection of copper ions present in biological samples and environmental samples (eg, water of rivers, rivers, seas, etc.).
  • the sample is selected from the group consisting of aqueous solution, organic solution, cells, cell lysate, blood, plasma, serum, lymph, saliva, urine, semen and ascites.
  • the sample is a cell, thereby enabling qualitative and quantitative analysis of intracellular copper ions by the present invention.
  • the sample is serum, and according to the present invention, qualitative and quantitative analysis of copper silver in serum is possible.
  • copper ion (I) can be detected.
  • the monovalent copper ions are used to form a cyclone between the azide group (or alkyne group) of the phosphor and the alkyne group (or azide group) of the quencher.
  • the reducing agent may not be included in the copper silver detection composition or the fluorescent chemical sensor.
  • the presence of copper ions can be determined by additionally treating the reducing agent with a mixture of the phosphor, quencher and sample, and then confirming the quenching of the phosphor. Specifically, if quenching of the phosphor does not occur by treatment with 0 reductant, there are no ions in the sample, and (ii) if quenching of phosphor occurs by the reducing agent treatment, copper ions (II) are present in the sample. Therefore, according to the detection method of this invention, copper silver (II) can also be detected.
  • FIG. 1 exemplarily shows the detection principle of the copper ion (II) detection method of the present invention.
  • II copper ion
  • FIG. 1 exemplarily shows the detection principle of the copper ion (II) detection method of the present invention.
  • a sample containing copper ions (Cu 2+ ) is added in the presence of an azide-functionalized f luorophore containing an azide group, an alkyne-functionalized quencher containing an alkyne group and a reducing agent, Cu 2+ is reduced to Cu 1+ by the reducing agent, and a cycloedition reaction occurs between the azide group of the phosphor and the alkyne group of the quencher through the catalytic reaction oligomer of the reduced Cu 1+ .
  • the fluorescent intensity of the phosphor is drastically reduced by the phosphor-quencher quenching mechanism, and copper ions can be detected by checking the degree of quenching.
  • Azide and alkyne groups are selectively reacted by Cu (I) ions, resulting in the mixing of other metal ions Even if present, only copper and silver can be detected selectively, and qualitative and quantitative analysis of copper ions is possible.
  • the invention provides a method for screening a copper ion chelating agent comprising the following steps:
  • the present invention provides a method for confirming the chelate effect of a copper ion chelating agent comprising the following steps:
  • the test material if the test material does not exhibit a copper ion chelating effect, the quenching of the phosphor occurs. Therefore, when quenching of the phosphor occurs, the test material can be determined as a non-copper silver chelating agent. In contrast, if the test material exhibits a copper ion chelating effect, there is no change in the fluorescence intensity of the phosphor or a substantial decrease. Therefore, if there is no change in the fluorescence intensity of the phosphor or does not substantially decrease, the test substance may be determined as a copper ion chelating agent.
  • the term "does not substantially reduce” means that the fluorescence intensity of the phosphor is reduced by the test substance, but the degree of reduction is small compared to the degree of fluorescence reduction by the non-copper chelating agent. .
  • the present invention can detect copper silver easily and quickly with high sensitivity based on click chemistry selectively binding in the presence of copper ions (I), and a fluorophore dye-quehcer quenching system system) the sensitivity and signal-to "noise ratio (signal-to-noise ratio) is excellent, using a.
  • copper ions can be detected in all solvents because they are dissolved in both aqueous solutions and organic solvents, and copper silver is also detected in biological samples such as plasma, serum, and urine, including water in rivers, lakes, and seas. You can use it.
  • the present invention is applicable to high throughput screening for evaluating the efficacy of existing copper ion chelating agents and developing new copper ion chelating agents.
  • the present invention can qualitatively and quantitatively detect copper ions present in intracellular or intracellular serum and can be applied as a diagnostic kit for diseases.
  • Red bars a mixture of Li +, Ca 2+ , Ni + and Hg 2+ ; Green bar: ' Mg 2+ , K + , a mixture of Pd 2+ and Co 2+ ; Blue bars: a mixture of Mn 2+ , Cd 2+ , Fe 2+ and Ag + .
  • Figure 6 shows that the sensor of the present invention can evaluate the efficacy of the copper chelating agent.
  • FIG. 7 shows that the sensor of the present invention can detect intracellular copper ions.
  • Figure 8 shows that the sensor of the present invention can quantify the amount of copper ions in serum.
  • azido-PEG 1 ( ⁇ Cy5.5 (25 ⁇ ), alkyne -BHQ3 (25 ⁇ ), sodium ascorbate (1 ⁇ ) Li +, Ca 2+ , Ni + , ⁇ ⁇ +, Mg 2+ , K + , Na + , Pd 2+ , Co 2+ , Mn 2+ , Cd 2+ 'Fe 2+ in PBS pH 7.0 solution
  • the fluorescence was measured, at which the copper ion concentration was 50 nM and the other metal silver concentration was 0.5 mM.
  • azido-PEG 10 -Cy5.5 25 ⁇ M
  • alkyne-BHQ3 25 ⁇ M
  • sodium ascorbic 1 ⁇
  • PBS pH 7.0
  • copper ion chelating agent that have no copper ion chelating effect.
  • the cultured ⁇ 293 ⁇ was treated with 100 ⁇ of CuS0 4 , followed by incubation for 12 hours, which is a time when Cu 2+ ions are reduced to Cu 1+ .
  • Cells were washed with culture medium and treated with azido-PEG 10 -Cy5.5 (25 ⁇ ) and alkyne—BHQ3 (25 ⁇ ) for 20 minutes, and then washed with cell culture and incubated for 4 hours. Intracellular copper ion detection was observed by confocal scanning laser microscope.
  • Blood was collected. The collected blood was left for 1 hour at the actual silver, centrifuged at 13,000 rpm for 10 minutes to obtain a serum.
  • 50-fold diluted serum and azido-PEG 10 -Cy5, 5 (25 ⁇ ) and alkyne-BHQ3C25 ⁇ ) were reacted for 30 minutes and then fluorescence intensity was measured.
  • the copper silver concentration in the serum was quantified from the copper ion standard curve obtained in the range of 10 ⁇ to 60 ⁇ .

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Abstract

The present invention relates to a composition having ultra-high sensitivity and high selectivity for detecting a copper ion; a fluorescent chemical sensor comprising the same; a method for manufacturing the same; a method for selectively detecting a copper ion; a method for screening a copper ion chelating agent; and a method for confirming a chelating effect of a copper ion chelating agent. The present invention is applicable in detecting a copper ion in water from a river, lake, ocean, or the like, and bio-samples, such as plasma, serum, and urine, and is applicable in high-rate mass-screening for evaluating the effect of the existing copper ion chelating agent and developing novel copper ion chelating agents. Further, the copper ion can be qualitatively and quantitatively detected in the serum in cells and in the body, and the present invention is applicable as a disease diagnosing kit.

Description

【명세서】  【Specification】
【발명의 명칭】  [Name of invention]
초고감도 및 높은 선택성을 갖는 구리이온 검출용 조성물 및 형광화학센서  Ultra High Sensitivity and High Selectivity Copper Ion Detection Composition and Fluorescent Chemical Sensor
【기술 분야】 [Technical field]
본 발명은 초고감도 및 높은 선택성을 갖는 구리이온 검출용 조성물, 이를 포함하는 형광화학센서， 이들의 제조방법， 구리이온의 선택적 검출방법 및 정량방법， 구리이온 킬레이트제의 스크리닝 방법 및 구리이온 킬레이트제의 킬레이트 효과 확인 방법에 관한 것이다.  The present invention provides a composition for detecting copper ions having ultra-high sensitivity and high selectivity, a fluorescence chemical sensor comprising the same, a manufacturing method thereof, a selective detection and determination method of copper ions, a screening method of a copper ion chelating agent and a copper ion chelating agent It relates to a method of confirming the chelate effect of.
【배경 기술】 [Background technology]
구리는 인체에 존재하는 필수 중금속 중쎄서 세 번째로 풍부한 금속으로서, 다양한 생리학적 과정에서 중요한 역할을 수행하는 것으로 알려져 있다. 그러나, 살아있는 세포에서 구리이온은 산소분자를 활성산소종으로 변환시켜 단백질， 핵산 및 지질의 손상을 유발시킨다. 실제로, 구리의 과도한 양은 독성을 나타내며， 과도한 산화성 스트레스로 인하여 다양한 질환을 유발시킨다. 비정상적인 구리이온의 섭취로 유발되는 질병으로는 암， 심혈관 장애， 멘케스병， 월슨병， 알츠하이머， 위장질환， 저혈당증， 난독증 및 유아 간질환 등이 있으며， 이러한 질환들은 특히 여러 기관들 중에서도 주로 간과 신장에서의 구리이온의 축적에 의해 발생하는 것으로 알려져 있다. 따라서, 생체 시스템 및 환경 분야에서 잔류 구리이온을 검출하는 것은 매우 중요하다.  Copper is the third most abundant metal in essential heavy metals present in the human body, and is known to play an important role in various physiological processes. However, in living cells, copper ions convert oxygen molecules into reactive oxygen species, causing damage to proteins, nucleic acids and lipids. Indeed, excessive amounts of copper are toxic and cause various diseases due to excessive oxidative stress. Diseases caused by abnormal intake of copper ions include cancer, cardiovascular disorders, Menques' disease, Wilson's disease, Alzheimer's disease, gastrointestinal disorders, hypoglycemia, dyslexia, and infant's liver disease. It is known to occur due to the accumulation of copper ions. Therefore, it is very important to detect residual copper ions in biological systems and environmental fields.
지금까지 구리이온을 검출하기 위해 다양한 화학센서， 금속 나노클러스터， 양자점 (quantum dot), 나노로드 (nanorod) , 비색법 (color imetric method) , 분광법 (spectroscopy) 및 전압- 전류법 (voltammetry) 등이 개발되어져 왔다. 그러나， 유기형광체， 화학센서 및 나노클러스터는 다른 금속 이온에 대한 교차활성도 (cross-activity), 수용액에서의 낮은 용해도 및 느린 반웅성 때문에 실제 웅용에는 한계가 있다.  To date, various chemical sensors, metal nanoclusters, quantum dots, nanorods, color imetric methods, spectroscopy and voltammetry have been developed to detect copper ions. It has been. However, organic phosphors, chemical sensors and nanoclusters have limited practical use because of their cross-activity to other metal ions, low solubility in aqueous solutions and slow reaction properties.
또한, 구리이온 검출을 위한 대부분의 형광화학센서는 가시광선 영역대의 빛올 내기 때문에 세포， 생체분자 및 조직에 의한 간섭 또는 자가 형광으로 인해 민감도가 떨어진다. 나아가, 그동안 개발되었던 구리이온을 검출하기 위한 형광화학센서 대부분은 소수성을 띄기 때문에 유기용매의 사용이 필요하며， 감도도 상대적으로 좋지 않아 실제 웅용에는 적합하지 않았다. In addition, most fluorescent chemical sensors for detecting copper ions are visible Because of the glow of the area, the sensitivity is reduced due to interference or autofluorescence by cells, biomolecules and tissues. Furthermore, most of the fluorescence chemical sensors developed for detecting copper ions have hydrophobic properties and require the use of organic solvents.
또한， 구리미온을 검출하기 위한 분광밥과 전압-전류법은 분석하기 전에 복잡한 전처리 과정과 노동력을 필요로 한다.  In addition, spectroscopy and voltage-current methods for detecting copper ions require complex pretreatment and labor before analysis.
따라서, 생체 시스템 및 환경 분야의 실제 적용을 위해서는, 높은 민감도와 선택성으로 신속 및 간편하게 구리이온을 검출할 수 있는 구리이온 검출법의 개발이 요구된다. 본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야와 수준 및 본 발명의 내용이 보다 명확하게 설명된다.  Therefore, for practical application in biological systems and environmental fields, development of a copper ion detection method capable of detecting copper ions quickly and simply with high sensitivity and selectivity is required. Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, so that the technical field and level to which the present invention belongs and the contents of the present invention are more clearly described.
【발명의 내용】 [Content of invention]
【해결하려는 과제]  [Action to be solved]
본 발명자들은 높은 민감도와 선택성으로 신속 간편하게 구리이온을 검출할 수 있는 센서를 개발하기 위하여 연구 노력하였다. 그 결과, 아자이드 /알킨화합물의 클릭 케미스트리 (click chemistry)와 형광 공명 에너지 전이 (Fluorescence resonance energy transfer , FRET)를 이용하여 구리이온 검출 센서를 개발하였으며, 이 센서를 사용하여 여러 종류의 금속이온이 흔재된 시료에서 구리이온을 높은 민감도 (펨토몰 수준)로 선택적으로 검출할 수 있음을 확인함으로써, 본 발명을 완성하게 되었다. 따라서, 본 발명의 목적은 구리이온 검출용 조성물을 제공하는 데 있다.  The present inventors have tried to develop a sensor that can detect copper ions quickly and easily with high sensitivity and selectivity. As a result, a copper ion detection sensor was developed using click chemistry and fluorescence resonance energy transfer (FRET) of azide / alkyne compounds. The present invention was completed by confirming that copper ions can be selectively detected at high sensitivity (femtomol level) in a common sample. Accordingly, an object of the present invention is to provide a composition for detecting copper ions.
본 발명의 다른 목적은 구리이온 검출용 형광화학센서를 제공하는 데 있다.  Another object of the present invention is to provide a fluorescent chemical sensor for detecting copper ions.
본 발명의 또 다른 목적은 구리이온 검출용 조성물 또는 형광화학센서의 제조방법을 제공하는 데 있다. 본 발명의 또 다른 목적은 시료 내 구리이온의 선택적 검출방법을 제공하는 데 있다. It is another object of the present invention to provide a composition for detecting copper ions or a method of manufacturing a fluorescence chemical sensor. Another object of the present invention is to provide a method for the selective detection of copper ions in a sample.
본 발명의 또 다른 목적은 세포내 또는 혈청에서의 구리이온 검출 및 정량분석 방법을 제공하는 데 있다.  It is another object of the present invention to provide a method for detecting and quantifying copper ions in cells or in serum.
본 발명의 또 다른 목적은 구리이온 킬레이트제의 스크리닝 방법을 제공하는 데 있다.  It is another object of the present invention to provide a method for screening a copper ion chelating agent.
본 발명의 또 다른 목적은 구리이온 킬레이트제의 킬레이트 효과 확인 방법을 제공하는 데 있다. ' 본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다. Another object of the present invention to provide a method for confirming the chelate effect of a copper ion chelating agent. "Detailed description of the invention below is a further object and advantage of the present invention become apparent from the claims and the drawings.
【과제의 해결 수단】 [Measures of problem]
본 발명의 일 양태에 따르면， 본 발명은 형광체; 및 상기 형광체의 발광효과를 흡수하여 소광효과를 나타내는 소광체를 포함하고, 상기 형광체 및 소광체는 각각 아자이드 (azide) 그룹 또는 알킨 (alkyne) 그룹을 포함하되， 형광체가 아자이드 그룹 또는 알킨그룹을 포함하는 경우 소광체는 알킨그룹 또는 아자이드 그룹을 포함하는 것을 특징으로 하는 구리이온 검출용 조성물을 제공한다.  According to one aspect of the invention, the present invention is a phosphor; And a quencher that absorbs the luminous effect of the phosphor and exhibits a quencher effect, wherein the phosphor and the quencher include an azide group or an alkyne group, respectively, wherein the phosphor is an azide group or an alkyne group. When included, the quencher provides a composition for detecting copper ions, characterized in that it comprises an alkyne group or an azide group.
또한， 본 발명의 다른 양태에 따르면, 본 발명은 상기 구리이온 검출용 조성물을 포함하는 구리이온 검출용 형광화학센서를 제공한다. 본 발명자들은 높은 민감도와 선택성으로 ·신속 간편하게 구리이온을 검출할 수 있는 센서를 개발하기 위하여 연구 노력하였다. 그 결과, 아자이드 /알킨화합물의 클릭 케미스트리 (click chemistry)와 형광 공명 에너지 전이 (Fluorescence resonance energy transfer, FRET)를 이용하여 구리이온 검출 센서를 개발하였으몌 이 센서를 사용하여 여러 종류의 금속이온이 혼재된 시료에서 구리이온을 높은 민감도 (펨토몰 수준)로 선택적으로 검출할 수 있음을 확인하였다.  Further, according to another aspect of the present invention, the present invention provides a fluorescent ion sensor for detecting copper ions comprising the composition for detecting copper ions. The present inventors made an effort to develop a sensor capable of detecting copper ions quickly and easily with high sensitivity and selectivity. As a result, we developed a copper ion sensor using click chemistry and fluorescence resonance energy transfer (FRET) of azide / alkyne compounds. It was confirmed that copper ions can be selectively detected at high sensitivity (femtomol level) in mixed samples.
본 발명의 특징은 아자이드 /알킨 화합물의 클릭 케미스트리와 형광 공명 에너지 전이를 이용한 형광체-소광체 소광 시스템을 훨：용하여 높은 민감도로 구리이온을 선택적으로 검출한다는 점이다. 하기 실시예에서 확인한 바와 같이, 본 발명에 의하면 구리이온을 선택적으로 검출할 수 있으며 (도 2 참조), 이러한 구리이온의 검출을 빠른 시간 안에 달성할 수 있으며 (도 3 참조), 다양한 금속이온의 존재 하에서도 구리이온을 특이적으로 검출할 수 있으며 (도 4 참조)， 펨토몰 수준의 높은 민감도로 구리이온을 검출할 수 있다 (도 5 참조). 나아가， 본 발명에 의하면 구리이은 킬레이트제의 효과를 평가할 수 있으며 (도 6 참조), 세포 내 존재하는 구리이은의 검출 (도 7 참조)과 '혈청에 존재하는 구리이온의 정량이 가능하다 (도 8 참조). A feature of the present invention is that the phosphor-quencher quenching system using click chemistry and fluorescence resonance energy transfer of azide / alkyne compounds is highly effective It selectively detects copper ions with sensitivity. As confirmed in the following examples, according to the present invention, copper ions can be selectively detected (see FIG. 2), and the detection of such copper ions can be achieved in a short time (see FIG. 3). Even in the presence of copper ions can be detected specifically (see Figure 4), it is possible to detect copper ions with a high sensitivity of femtomol level (see Figure 5). Furthermore, according to the present invention, it is possible to evaluate the effect of the copper silver chelating agent (see FIG. 6), to detect the copper silver present in the cell (see FIG. 7) and to quantify the copper ions present in the serum (FIG. 8).
상기 형광체는 아자이드 그룹 또는 알킨 그룹을 포함하며, 상기 아자이드 또는 알킨 그룹은 구리이온 (Cu1+)의 존재 시 소광체에 결합되어 있는 알킨 또는 아자이드 화합물과 사이클로에디션 (cycloaddition) 반응을 한다. 본 발명에서 사용할 수 있는 형광체에는， 예를 들어 Tavi's "FluoroTable" '· Common fluoiDphores (출처: Zeiss Corporation 웹사이트 및 http://info.med.yale.edu/ genetics/ward/tavi/FISHdyes2 itml)에 개시된 형광체가 있다. The phosphor includes an azide group or an alkyne group, and the azide or alkyne group undergoes a cycloaddition reaction with an alkyne or azide compound bound to the quencher in the presence of copper ions (Cu 1+ ). . Phosphors that can be used in the present invention include, for example, Tavi's "FluoroTable"' Common fluoiDphores (Source: Zeiss Corporation website and http://info.med.yale.edu/genetics/ward/tavi/FISHdyes2 itml). There is a disclosed phosphor.
본 발명의 일구현예에 따르면， 상기 형광체는 시아닌 (Cyanine), 플루오레신 (fluorescein), 보디피 (B0DIPY), 테트라메틸로드아민 According to one embodiment of the invention, the phosphor is cyanine, fluorescein, fluoresce (B0DIPY), tetramethylrodamine
(Trtramethylrhodamine) , 알렉사 (Alexa), 알로피코시아닌 (al iopicocyanine) 및 이들의 유도체로 구성된 군으로부터 선택된다. (Trtramethylrhodamine), Alexa, allopicocyanine and derivatives thereof.
본 발명의 일구현예에 따르면， 상기 형광체는 적색 또는 근적외선의 형광을 발광한다. 적색 또는 근적외선의 형광을 발광하는 형광체로는 시아닌과 알렉사가 있으며， 이들 형광체는 근적외선의 빛을 방출 및 흡수하므로 세포, 혈액 및 생체 조직 등과의 간섭 또는 홉수를 최소화할 수 있다.  According to one embodiment of the present invention, the phosphor emits red or near infrared fluorescence. Phosphors emitting red or near-infrared fluorescence include cyanine and alexa, and these phosphors emit and absorb light of near-infrared light, thereby minimizing interference or hop number with cells, blood, and biological tissues.
본 발명에서 상기 소광체는 형광체에서 나오는 빛의 파장을 흡수하여 소광시키는 기능을 한다. 형광체의 발광 파장의 범위에 따라 적절한 소광체를 선택하여 사용할 수 있으며, 형광체의 발광 파장과 동일하거나 유사한 파장대의 소광체를 사용함이 바람직하다.  In the present invention, the quencher functions to absorb and quench the wavelength of light emitted from the phosphor. An appropriate quencher may be selected and used according to the range of the emission wavelength of the phosphor, and it is preferable to use a quencher having the same or similar wavelength band as the emission wavelength of the phosphor.
본 발명의 일구현예에 따르면， 상기 소광체는 블랙홀 소광체 (blackhole quencher , BHQ), 블랙베리 소광체 (blackberry quencher, BBQ) 또는 이들의 유도체이다. According to one embodiment of the invention, the quencher is a black hole quencher (BHQ), a blackberry quencher (blackberry quencher, BBQ) or derivatives thereof.
본 발명에서 사용가능한 예시적인 형광체와소광체의 짝을 하기 표 1 및 2에 나타내었다.  Exemplary phosphor and quencher pairs usable in the present invention are shown in Tables 1 and 2 below.
【표 1】  Table 1
Figure imgf000006_0001
【표 2】
Figure imgf000006_0001
Table 2
Figure imgf000007_0001
본 발명의 일구현예에 따르면, 상기 구리이온 검출용 조성물은 환원제를 추가적으로 포함한다. 상기 환원제는 구리이온 (II)을 구리이온 (I)으로 환원시키는 기능을 한다. 본 발명에서는 이러한 기능을 하는 환원제라면 제한 없이 사용할 수 있으며, 사용가능한 환원제의 예로는 소듐 아스코빅 (sodium ascorbate)을 들 수 있다.
Figure imgf000007_0001
According to one embodiment of the present invention, the composition for detecting copper ions further includes a reducing agent. The reducing agent functions to reduce copper ions (II) to copper ions (I). In the present invention, any reducing agent having such a function can be used without limitation, and examples of the reducing agent that can be used include sodium ascorbate.
본 발명에서 구리이온 검출용 형광화학센서의 형태는 특별히 한정되지 않고, 본 발명의 조성물을 포함하는 형태라면 제한 없이 가능하다. 상기 형광화학센서의 구체적인 예로는， 상기 형광체 및 소광체 (추가적으로 환원제)가 용매와 함께 용기에 담겨있는 형태를 들 수 있다. 본 발명의 또 다른 양태에 따르면， 본 발명은 다음의 단계를 포함하는 구리이온 검출용 조성물 또는 형광화학센서의 제조방법을 제공한다:  The form of the fluorescent chemical sensor for detecting copper ions in the present invention is not particularly limited, and any form containing the composition of the present invention may be used without limitation. Specific examples of the fluorescence chemical sensor may include a form in which the phosphor and the quencher (additional reducing agent) are contained in a container together with a solvent. According to another aspect of the invention, the present invention provides a composition for detecting copper ions or a method for producing a fluorescent chemical sensor comprising the following steps:
(a) 아자이드 그룹 또는 알킨 그룹이 도입된 형광체를 얻는 단계; (b) 알킨 그룹 또는 아자이드 그룹이 도입된 소광체를 얻는 단계; 및 (a) obtaining a phosphor into which an azide group or an alkyne group is introduced; (b) obtaining a quencher in which an alkyne group or an azide group is introduced; And
(c) 단계 (a)에서 얻은 아자이드 그룹이 도입된 형광체와 단계 (b)에서 얻은 알킨 그룹이 도입된 소광체， 또는 단계 (a)에서 얻은 알킨 그룹이 도입된 형광체와 단계 (b)에서 얻은 아자이드 그룹이 도입된 소광체를 흔합하는 단계. (c) the phosphor to which the azide group obtained in step (a) is introduced and the quencher to which the alkyne group obtained in step (b) is introduced, or the phosphor to which the alkyne group obtained in step (a) is introduced and in step (b) Mixing the quencher into which the obtained azide group was introduced.
상기 단계 (a) 및 (b)에서는 아자이드 또는 알킨 그룹이 도입된 형광체와 소광체를 얻는다. 이러한 형광체와 소광체는 직접 제조하거나, 구매 등의 방법으로 수득할수도 있다.  In the above steps (a) and (b), a phosphor and a quencher in which an azide or alkyne group is introduced are obtained. Such phosphors and quencher may be produced directly or obtained by a method such as purchase.
본 발명의 일구현예에 따르면， 상기 형광체 및 소광체에 아자이드 그룹을 도입하는 방법은 하기 반웅식 1 또는 2에 따라 실시할 수 있다. 반웅식에서 A는 형광체 또는 소광체를 나타낸다.  According to one embodiment of the present invention, a method of introducing an azide group into the phosphor and the quencher can be carried out according to the following formula 1 or 2. In the reaction, A represents a phosphor or a quencher.
반웅식 1
Figure imgf000008_0001
Figure imgf000009_0001
Banungsik 1
Figure imgf000008_0001
Figure imgf000009_0001
상기 반응식 1은 하이드록시숙신이미드 (Hydroxysuccinimide)로 활성화된 형광체 (또는 소광체)와 아민 그룹과 아자이드 그룹을 모두 포함하는 화합물과의 반웅을 통하여 형광체 (또는 소광체)에 아자이드 그룹을 도입하는 방법을 보여주며， 반웅식 2는 카르복시 그룹을 갖는 형광체 (또는 소광체)와 아민 (N¾)그룹과 아자이드 그톱을 모두 포함하는 화합물을 가교제인 디-사이€로헥실디이미드 (DCC)와 하이드록시숙신이미드 (N-hydroxysuccinimide)를 통하여 형광체 (또는 소광체)에 아자이드 그룹을 도입하는 방법을 보여준다.  Scheme 1 introduces an azide group to a phosphor (or quencher) through reaction of a phosphor (or quencher) activated with hydroxysuccinimide and a compound containing both an amine group and an azide group. Banungsik 2 shows a compound containing both a carboxyl group (or a quencher), an amine (N¾) group and an azide glove, and a crosslinking agent, di-cylohexyldiimid (DCC), We show how to introduce an azide group into a phosphor (or quencher) via hydroxysuccinimide.
상기 반웅식 1에서, 하이드록시숙신이미드로 활성화된 형광체 (또는 소광체)에 아자이드 그룹을 도입하기 위해 사용할 수 있는 화합물로는, 4- 아지도아닐린 염산염 (4-azidoaniline hydrochloride), 11-아지도ᅳ3，6,9- 트리옥사운데칸 -1-아민 ( 11-az ido-3 , 6， 9- 1 r i oxaundecan-l-am i ne ) 아지도ᅳ PEG6-아민 (azido-PEG6— amine), 아지도 -PEG8-아민 (azid으 PEG8-amine) 및 아지도 PEG10-아민 (azido-PEG10-amine) 등이 있으며， 이 외 아자이드 그룹과 아민 그룹을 모두 포함하는 화합물이면 제한 없이 사용할 수 있다. In the reaction formula 1, as a compound that can be used to introduce an azide group into the hydroxysuccinimide-activated phosphor (or quencher), 4-azidoaniline hydrochloride, 11- Azidodo3,6,9-trioxoundecan-1-amine (11-az ido-3, 6, 9-1 ri oxaundecan-l-am i ne) Azidodo PEG 6 -amine (azido-PEG 6 - amine), azido -PEG 8 - amine (azid coming PEG -amine 8) and azido PEG 10 - amine (azido-PEG 10 -amine), etc., and includes all the other azide group with an amine group Any compound can be used without limitation.
상기 반응식 2에서, 카르복시 그룹을 갖는 형광체 (또는 소광체)에 가교제인 DCC와 하이드록시숙신이미드를 이용하여 아자이드 그룹을 도입하기 위해 사용할 수 있는 화합물로는, 4-아지도아닐린 염산염, 11- 아지도 -3 , 6 , 9-트리옥사운데칸 -1-아민， 아지도 -PEG6-아민， 아지도 -PEGS-아민 및 아지도 -PEG10-아민 등이 있으며， 이 외 아자이드 그룹과 아민 그룹을 모두 포함하는 화합물이면 제한 없이 사용할 수 있다. In Scheme 2, a compound that can be used to introduce an azide group using a crosslinking agent DCC and hydroxysuccinimide to a phosphor having a carboxyl group (or a quencher) is 4-azidoaniline hydrochloride, 11 Azido-3, 6, 9-trioxoundecan-1-amine, azido-PEG 6 -amine, azido-PEG S -amine and azido-PEG 10 -amine; Any compound containing both a group and an amine group can be used without limitation.
또한， 상기 형광체 및 소광체에 알킨 그룹을 도입하는 방법은 하기 반웅식 3 또는 4에 따라 실시할 수 있다. 반웅식에서 A는 형광체 또는 소광체를 나타낸다. In addition, the method of introducing an alkyne group to the phosphor and the quencher may be carried out according to the following formula 3 or 4. In the reaction, A represents a phosphor or a quencher.
Figure imgf000010_0001
Figure imgf000010_0001
상기 반응식 3에서， 하이드록시숙신이미드로 활성화된 소광체 (또는 형광체)에 알킨 그룹을 도입하기 위해 사용할 수 있는 화합물로는， 프로파질아민 (propargylamine), 2-메틸 -3-부틴 -2-아민 (2-methy卜 3-butyn-2- amine), 4-펜틴 -1ᅳ아민 (4-pentyn-l-amine)， 3-메틸 -1-펜틴 -3ᅳ아민 염산염 (3-methyl-l-pentyn-3-amine hydrochloride), 1-아민 -4—클로로 -2- 부틴 염산염 (l—amino—4—chloro—2— butyne hydrochloride) , 3一 에티닐아닐린 (3-ethynylani line) 및 4-에티닐아닐린 (4— ethynylani 1 ine) 등이 있으며， 이 외 알¾ 그룹과 아민 그룹올 모두 포함하는 화합물이면 제한 없이 사용할 수 있다.  In Scheme 3, a compound that can be used to introduce an alkyne group to a quencher (or phosphor) activated with hydroxysuccinimide, propargylamine, 2-methyl-3-butyn-2- Amine (2-methy 卜 3-butyn-2-amine), 4-pentyn-l-amine, 3-methyl-1-pentin-3 ᅳ amine hydrochloride (3-methyl-l) -pentyn-3-amine hydrochloride), 1-amine-4-chloro-2-butyne hydrochloride (l-amino—4—chloro-2, butyne hydrochloride), 3 ethynylaniline (3-ethynylani line) and 4- Ethynylaniline (4—ethynylani 1 ine), and the like, and any compound containing both an al¾ group and an amine group can be used without limitation.
상기 반응식 4에서， 카르복시 그룹을 갖는 소광체 (또는 형광체)에 가교제인 DCC와 촉매인 4-디메틸아미노피리딘 (DMAP)을 이용하여 알킨 그룹을 도입하기 위해 사용할 수 있는 화합물로는， 2-부틴 -1ᅳ을 (2-butyn-l- ol), 3-부틴 -1-을 (3-butyn-l-ol), 3-부틴 -2-올 (3— butyn-2-ol ) , 4-펜틴 -2- 올 (4-pentyn-2-ol) , 5-핵센-2-인-1-을(5- 611-2^]-1-00， 2-핵신 -1-을 (2- hexyn-1-ol), 3-핵신 -1—을 (3-hexynᅳ l-ol)， 3-핵신 -2ᅳ올 (3-hexyn— 2-ol)， 4- 핵신 -3-올 (4-hexyn-3-ol), 5-핵신 3-올 (5-hexyn-3-ol )， 9-운데신 -1-올 (9- undecyn-1-ol) 및 10-운데신 -1-을 (lO-undecyn-1-ol ) 등이 있으며， 이 외 알킨 그룹과 하이드톡시 그룹을 모두 포함하는 화합물이면 제한 없이 사용할 수 있다.  In the above Scheme 4, a compound which can be used to introduce an alkyne group using a crosslinking agent DCC and a catalyst 4-dimethylaminopyridine (DMAP) to a quencher (or phosphor) having a carboxyl group is 2-butyne- 1- (2-butyn-l-ol), 3-butyn-1- (3-butyn-l-ol), 3-butyn-2-ol (3—butyn-2-ol), 4-pentin 2-ol (4-pentyn-2-ol), 5-nuxen-2-yn-1- (5-611-2 ^]-1-00, 2-nucleine-1- (2-hexyn- 1-ol), 3-nucleine-1— (3-hexyn ᅳ l-ol), 3-nucleine-2 -ol (3-hexyn— 2-ol), 4-nucleine-3-ol (4-hexyn- 3-ol), 5-nucleine 3-ol (5-hexyn-3-ol), 9-undecine-1-ol and 9-undecyn-1-ol and 10-undecine-1- (lO- undecyn-1-ol) and the like, and any compound containing both an alkyne group and a hydroxy group can be used without limitation.
상기 단계 (c)에서는 단계 (a)에서 얻은 아자이드 그룹 (또는 알킨 그룹)이 도입된 형광체 및 단계 (b)에서 얻은 알킨 그룹 (또는 아자이드 그룹)이 도입된 소광체 (추가적으로 환원계)를 적절한 용매에서 흔합한다. 사용가능한 용매로는 수용액, 유기용매 및 완충용액 (예컨대， PBS) 등이 있다. 여기서 얻어진 흔합물 자체를 구리이온 검출용 센서로 사용할 수 있으며， 추가적으로 상기 흔합물을 센서 기판에 적절히 구비시킴으로써 센서장치로 구현할 수도 있다. 본 발명의 또 다른 양태에 따르면, 본 발명은 다음의 단계를 포함하는 시료 내 구리이온의 선택적 검출방법을 제공한다: In step (c), the phosphor to which the azide group (or alkyne group) obtained in step (a) is introduced and the quencher (additionally reducing system) into which the alkyne group (or azide group) obtained in step (b) is introduced In suitable solvents. Solvents that can be used include aqueous solutions, organic solvents and buffer solutions (eg PBS). have. The complex itself obtained here may be used as a sensor for detecting copper ions, and may be implemented as a sensor device by additionally providing the complex to a sensor substrate. According to another aspect of the invention, the invention provides a method for the selective detection of copper ions in a sample comprising the following steps:
(a) 상기 구리이온 검출용 조성물 또는 형광화학센서에 시료를 접촉시키는 단계; 및  (a) contacting a sample with the composition for detecting copper ions or a fluorescent chemical sensor; And
(b) 형광체의 소광을 확인하는 단계.  (b) confirming quenching of the phosphor.
본 명세서에서 사용된, 용어 "검출 "은 시료 내에 구리이온이 존재하는지 여부를 확인하는 것뿐만 아니라 (정성적 분석), 구리이온의 양을 알아내는 것 (정량적 분석)도 포함한다. 시료에 존재하는 구리이온의 양은， 단계 (b)에서 확인한 형광체의 소광 정도 (형광 세기)를 구리이은 표준곡선에 대입해봄으로써 알아낼 수 있다 (하기 시험예 7 참조).  As used herein, the term “detection” includes not only identifying the presence of copper ions in the sample (quantitative analysis), but also determining the amount of copper ions (quantitative analysis). The amount of copper ions present in the sample can be found by substituting the degree of quenching (fluorescence intensity) of the phosphor identified in step (b) into a copper silver standard curve (see Test Example 7 below).
따라서， 본 발명의 일구현예에 따르면， 본 발명에 의한 구리이온의 선택적 검출방법은， 단계 (b) 이후에 단계 (b)에서 확인한 형광체의 소광 (형광 세기)을 구리이온 표준곡선에 대입하여 구리이온의 농도를 알아내는 단계 (단계 c)를 더 포함할 수 있다.  Therefore, according to one embodiment of the present invention, the selective detection method of copper ions according to the present invention, by substituting the fluorescence intensity (fluorescence intensity) of the phosphor identified in step (b) after step (b) to the copper ion standard curve The method may further include a step (step c) of determining the concentration of copper ions.
본 발명의 검출방법에 의하면 시료 내에 매우 낮은 농도 (예컨대， 펨토몰 수준)로 존재하는 구리이온의 검출도 가능하다.  According to the detection method of the present invention, it is also possible to detect copper ions present at very low concentrations (eg, femtomol level) in the sample.
본 발명의 일구현예에 따르면, 상기 시료에 존재하는 구리이온의 농도는 1 펨토몰 (femtomol, fM) 이상이다.  According to one embodiment of the present invention, the concentration of copper ions present in the sample is 1 femtomol (fM) or more.
본 발명의 검출방법은 생체 시료 및 환경 시료 (예컨대， 하천， 강， 바다 등의 물)에 존재하는 구리이온의 검출에 적용될 수 있다.  The detection method of the present invention can be applied to the detection of copper ions present in biological samples and environmental samples (eg, water of rivers, rivers, seas, etc.).
본 발명의 일구현예에 따르면， 상기 시료는 수용액， 유기용액， 세포， 세포 용해물 (cell lysate), 혈액， 혈장, 혈청， 림프, 타액， 뇨， 정액 및 복수로 구성된 군으로부터 선택된다. 하나의 특정예에 따르면， 상기 시료는 세포이며， 이에 따라 본 발명에 의하여 세포내 구리이온의 정성 및 정량분석이 가능하다. 다른 특정예에 따르면, 상기 시료는 혈청이며， 이에 따라 본 발명에 의하여 혈청 내 구리이은의 정성 및 정량분석이 가능하다. 본 발명의 검출방법에 의하면, 구리이온 (I)의 검출이 가능하다. 즉， 시료에 1가의 구리이은 또는 1가와 2가의 구리이온이 존재하면， 1가의 구리이온에 의하여 형광체의 아자이드 그룹 (또는 알킨 그룹)과 소광체의 알킨 그룹 (또는 아자이드 그룹) 사이에 사이클로에디션 반웅이 일어나고， 이에 의하여 형광체와 소광체의 거리가 매우 가까워짐에 따라 형광체- 소광체 소광 메커니즘에 의해 형광체의 형광세기가 급격히 감소하게 되고， 이러한 소광 정도를 확인함으로써 구리이온 (I)을 검출할 수 있는 것이다. 이 경우, 상기 구리이은 검출용 조성물 또는 형광화학센서에는 환원제가 포함되지 않을 수 있다. According to one embodiment of the invention, the sample is selected from the group consisting of aqueous solution, organic solution, cells, cell lysate, blood, plasma, serum, lymph, saliva, urine, semen and ascites. According to one specific example, the sample is a cell, thereby enabling qualitative and quantitative analysis of intracellular copper ions by the present invention. According to another specific example, the sample is serum, and according to the present invention, qualitative and quantitative analysis of copper silver in serum is possible. According to the detection method of this invention, copper ion (I) can be detected. That is, if monovalent copper silver or monovalent and divalent copper ions are present in the sample, the monovalent copper ions are used to form a cyclone between the azide group (or alkyne group) of the phosphor and the alkyne group (or azide group) of the quencher. As the edition reaction occurs and the distance between the phosphor and the quencher is very close, the fluorescence intensity of the phosphor is drastically reduced by the phosphor-quencher quenching mechanism, and copper ions (I) can be detected by checking the degree of quenching. It can be. In this case, the reducing agent may not be included in the copper silver detection composition or the fluorescent chemical sensor.
한편， 본 발명의 검출방법을 실시한 결과， 형광체의 소광이 일어나지 않는 경우는 다음의 두 가지 경우가 있올 수 있다: (i) 구리이온이 존재하지 않거나, (Π) 구리이온 (II)이 존재하는 경우. 이러한 경우에는， 형광체， 소광체 및 시료의 흔합물에 환원제를 추가적으로 처리하여 준 후 형광체의 소광을 확인함으로써 구리이온의 존재를 알아낼 수 있다. 구체적으로, (0 환원제 처리에 의하여 형광체의 소광이 일어나지 않는다면 시료에 구이이온이 존재하지 않는 것이고， (ii) 반대로 환원제 처리에 의하여 형광체의 소광이 일어난다면 시료에 구리이온 (II)이 존재하는 것으로 판단할 수 있다. 따라서， 본 발명의 검출방법에 의하면 구리이은 (II)의 검출도 가능하다.  On the other hand, as a result of carrying out the detection method of the present invention, there are two cases in which quenching of the phosphor does not occur: (i) copper ions do not exist or (Π) copper ions (II) Occation. In this case, the presence of copper ions can be determined by additionally treating the reducing agent with a mixture of the phosphor, quencher and sample, and then confirming the quenching of the phosphor. Specifically, if quenching of the phosphor does not occur by treatment with 0 reductant, there are no ions in the sample, and (ii) if quenching of phosphor occurs by the reducing agent treatment, copper ions (II) are present in the sample. Therefore, according to the detection method of this invention, copper silver (II) can also be detected.
도 1은 본 발명의 구리이온 (II) 검출방법의 검출원리를 예시적으로 보여준다. 도 1을 참조하여 본 발명의 구리이온 검출방법을 구체적으로 설명한다. 아자이드 그룹을 포함하는 형광체 (azide-functionalized f luorophore), 알킨 그룹을 포함하는 소광체 (alkyne-functionalized quencher) 및 환원제가 존재하는 조건에서 구리이온 (Cu2+)을 함유하는 시료가 첨가되면, 환원제에 의하여 Cu2+가 Cu1+로 환원되고， 환원된 Cu1+에 의한 촉매반웅올 통하여 형광체의 아자이드 그룹과 소광체의 알킨 그룹 사이에 사이클로에디션 반웅이 일어난다. 이에 의하여 형광체와 소광체의 거리가 매우 가까워짐에 따라 형광체ᅳ소광체 소광 메커니즘에 의해 형광체의 형광세기가 급격히 감소하게 되고， 이러한 소광 정도를 확인함으로써 구리이온을 검출하게 된다. 아자이드 그룹과 알킨 그룹은 Cu(I)이온에 의해 선택적으로 반웅되므로, 다른 금속이온이 흔합되어 있더라도 구리이은만을 선택적으로 검출할 수 있으며, 구리이온의 정성 및 정량적 분석이 가능하다. 1 exemplarily shows the detection principle of the copper ion (II) detection method of the present invention. Referring to Figure 1 will be described in detail the copper ion detection method of the present invention. When a sample containing copper ions (Cu 2+ ) is added in the presence of an azide-functionalized f luorophore containing an azide group, an alkyne-functionalized quencher containing an alkyne group and a reducing agent, Cu 2+ is reduced to Cu 1+ by the reducing agent, and a cycloedition reaction occurs between the azide group of the phosphor and the alkyne group of the quencher through the catalytic reaction oligomer of the reduced Cu 1+ . Accordingly, as the distance between the phosphor and the quencher is very close, the fluorescent intensity of the phosphor is drastically reduced by the phosphor-quencher quenching mechanism, and copper ions can be detected by checking the degree of quenching. Azide and alkyne groups are selectively reacted by Cu (I) ions, resulting in the mixing of other metal ions Even if present, only copper and silver can be detected selectively, and qualitative and quantitative analysis of copper ions is possible.
. 본 발명의 또 다른 양태에 따르면， 본 발명은 다음의 단계를 포함하는 구리이온 킬레이트제의 스크리닝 방법을 제공한다: . According to another aspect of the invention, the invention provides a method for screening a copper ion chelating agent comprising the following steps:
(a) 환원제를 포함하는 상기 구리이온 검출용 조성물 또는 환원제를 포함하는 상기 형광화학센서에 시험물질과 구리이온 (Cu2+)을 동시에 또는 순차적으로 접촉시키는 단계 ; 및 (a) simultaneously or sequentially contacting a test substance and copper ions (Cu 2+ ) with the composition for detecting copper ions including a reducing agent or the fluorescent chemical sensor including a reducing agent; And
(b) 상기 형광체의 소광을 확인하는 단계 .  (b) confirming quenching of the phosphor.
본 발명의 또 다른 양태에 따르면， 본 발명은 다음의 단계를 포함하는 구리이온 킬레이트제의 킬레이트 효과 확인 방법을 제공한다:  According to another aspect of the present invention, the present invention provides a method for confirming the chelate effect of a copper ion chelating agent comprising the following steps:
(a) 환원제를 포함하는 상기 구리이온 검출용 조성물 또는 환원제를 포함하는 상기 형광화학센서에 구리이온 킬레이트제와 구리이온 (Cu2+)을 동시에 또는 순차적으로 접촉시키는 단계 ; 및 (a) simultaneously or sequentially contacting a copper ion chelating agent and a copper ion (Cu 2+ ) with the composition for detecting copper ions including a reducing agent or the fluorescent chemical sensor including a reducing agent; And
(b) 상기 형광체의 소광을 확인하는 단계.  (b) confirming quenching of the phosphor.
본 발명에 따르면， 시험물질이 구리이온 킬레이팅 효과를 보이지 않으면 형광체의 소광이 일어나게 된다. 따라서， 상기 형광체의 소광이 일어나면, 시험물질을 비-구리이은 킬레이트제로 판단할 수 있다. 반대로 시험물질이 구리이온 킬레이팅 효과를 보인다면 형광체의 형광세기에 변화가 없거나, 실질적으로 감소하지 않게 된다. 따라서, 상기 형광체의 형광세기에 변화가 없거나， 실질적으로 감소하지 않으면, 시험물질을 구리이온 킬레이트제로 판단할 수 있다. 본 명세서에서 사용된 용어, "실질적으로 감소하지 않는다 "의 의미는 시험물질에 의하여 형광체의 형광세기가 감소되긴 하나 감소 정도가 비-구리이온 킬레이트제에 의한 형광감소 정도에 비하여 작은 경우를 의미한다 .  According to the present invention, if the test material does not exhibit a copper ion chelating effect, the quenching of the phosphor occurs. Therefore, when quenching of the phosphor occurs, the test material can be determined as a non-copper silver chelating agent. In contrast, if the test material exhibits a copper ion chelating effect, there is no change in the fluorescence intensity of the phosphor or a substantial decrease. Therefore, if there is no change in the fluorescence intensity of the phosphor or does not substantially decrease, the test substance may be determined as a copper ion chelating agent. As used herein, the term "does not substantially reduce" means that the fluorescence intensity of the phosphor is reduced by the test substance, but the degree of reduction is small compared to the degree of fluorescence reduction by the non-copper chelating agent. .
따라서， 시험물질 또는 구리이온 킬레이트제 처리 후， 형광체의 소광올 정성적 또는 정량적으로 확인 및 분석함으로써 구리이온 킬레이트제를 '고속대량 스크리닝 (high throughput screening) 하거나, 기존의 구리이온 킬레이트제의 킬레이팅 효과를 평가할 수 있다. 【발명의 효과】 Therefore, after treating the test substance or the copper ion chelating agent, quaternary or quantitatively identifying and analyzing the matting oligomer of the phosphor, the ' high throughput screening ' of the copper ion chelating agent or the chelating of the existing copper ion chelating agent The effect can be evaluated. 【Effects of the Invention】
본 발명의 특징 및 이점을 요약하면 다음과 같다:  The features and advantages of the present invention are summarized as follows:
(i) 본 발명은 구리이온 (I)의 존재 하에서 선택적으로 결합하는 클릭케미스트리를 기반으로 높은 민감도로 간편하고 신속하게 구리이은을 검출할 수 있으며, 형광체-소광체 소광 시스템 (fluorophore dye-quehcer quenching system)을 사용하여 신호 대 '잡음 비율 (signal-to-noise ratio)과 민감도가 우수하다. (i) The present invention can detect copper silver easily and quickly with high sensitivity based on click chemistry selectively binding in the presence of copper ions (I), and a fluorophore dye-quehcer quenching system system) the sensitivity and signal-to "noise ratio (signal-to-noise ratio) is excellent, using a.
(ii) 본 발명은 수용액과 유기용매에 모두 용해되기 때문에 모든 용매에서 구리이온을 검출할 수 있으며, 강가， 호수， 바다 등의 수질과 혈장， 혈청 및 소변 등의 생체 샘플에서도 구리이은을 검출하는데 웅용이 가능하다.  (ii) In the present invention, copper ions can be detected in all solvents because they are dissolved in both aqueous solutions and organic solvents, and copper silver is also detected in biological samples such as plasma, serum, and urine, including water in rivers, lakes, and seas. You can use it.
(iii) 본 발명은 기존의 구리이온 킬레이트제의 효능 평가와 새로운 구리이온 킬레이트제 개발을 위한 고속대량 스크리닝 (high throughput screening)에 웅용가능하다.  (iii) The present invention is applicable to high throughput screening for evaluating the efficacy of existing copper ion chelating agents and developing new copper ion chelating agents.
(iv) 본 발명은 세포내 또는 체내 혈청에 존재하는 구리이온을 정성 및 정량적으로 검출이 가능하며， 질환의 진단키트로 응용가능하다.  (iv) The present invention can qualitatively and quantitatively detect copper ions present in intracellular or intracellular serum and can be applied as a diagnostic kit for diseases.
【도면의 간단한 설명】 [Brief Description of Drawings]
도 1은 본 발명의 검출원리의 일예를 보여준다.  1 shows an example of the detection principle of the present invention.
도 2는 본 발명의 센서가 다양한 금속이온 중 구리이온에만 선택적으로 반웅함을 보여준다.  2 shows that the sensor of the present invention selectively reacts only with copper ions among various metal ions.
도 3은 본 발명의 센서의 구리이온 검출에 소요되는 시간을 보여준다. 도 4는 본 발명의 센서가 금속 흔합물에서 구리이온에만 선택적으로 반웅함을 보여준다. 빨강 막대 : Li + , Ca2+, Ni+및 Hg2+의 흔합물; 녹색 막대 : 'Mg2+, K+, Pd2+ 및 Co2+의 혼합물; 파랑 막대: Mn2+, Cd2+, Fe2+ 및 Ag+의 흔합물. 3 shows the time required for copper ion detection of the sensor of the present invention. 4 shows that the sensor of the present invention selectively reacts only with copper ions in the metal complex. Red bars: a mixture of Li +, Ca 2+ , Ni + and Hg 2+ ; Green bar: ' Mg 2+ , K + , a mixture of Pd 2+ and Co 2+ ; Blue bars: a mixture of Mn 2+ , Cd 2+ , Fe 2+ and Ag + .
도 5는 본 발명의 센서의 검출한계를 보여준다.  5 shows the detection limit of the sensor of the present invention.
도 6은 본 발명의 센서로 구리 킬레이트제의 효능을 평가할 수 있음을 보여준다.  Figure 6 shows that the sensor of the present invention can evaluate the efficacy of the copper chelating agent.
도 7은 본 발명의 센서로 세포 내 구리이온을 검출할 수 있음을 보여준다. 도 8은 본 발명의 센서로 혈청 내 구리이온의 양을 정량할 수 있음을 보여준다. 7 shows that the sensor of the present invention can detect intracellular copper ions. Figure 8 shows that the sensor of the present invention can quantify the amount of copper ions in serum.
【발명을 실시하기 위한 구체작인 내용】 [Contents to be concrete to carry out invention]
이하， 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체작으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다. 실시예  Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. will be. Example
실시예 1. 아자이드 그룹을 포함하는 형광체 합성  Example 1 Phosphor Synthesis Containing Azide Groups
아자이드 그룹을 포함하는 형광체의 합성을 반웅식 5와 같이 실시하였다. 구체적으로， 11 tng의 Cy5.5 NHS 에스터와 40 nig의 아지도- PEG1C厂아민 (Azido-PEG10-amine)을 2 ^의 디클로로메탄 (didiloromethane)에 녹여 12시간 동안 반웅시킨 후， 유기용매를 회전증발기로 제거한 다음 얻어진 물질을 반분취 HPLC(semipreparative HPLC)를 통하여 정제하고， 합성된 화합물을 질량분석기로 확인하였다. 측정된 분자량은 1133.7이었으며, 순도는 98% 이상이었다. Synthesis of the phosphor containing an azide group was carried out as in Banung equation 5. Specifically, 11 tng of Cy5.5 NHS ester and 40 nig of azido- PEG 1C厂 amine (Azido-PEG 10 -amine) were dissolved in 2 ^ dichloromethane and reacted for 12 hours, followed by an organic solvent. Was removed by rotary evaporator, and the obtained material was purified by semipreparative HPLC, and the synthesized compound was confirmed by mass spectrometry. The molecular weight measured was 1133.7 and the purity was over 98%.
반웅식 5  Banungsik 5
Figure imgf000015_0001
실시예 2. 알킨 그룹을 포함하는 소광체 합성
Figure imgf000015_0001
Example 2. Quencher Synthesis Comprising Alkyne Groups
알킨 그룹을 포함하는 소광체의 합성을 반웅식 6과 같이 실시하였다. 구체적으로， 11 mg의 BHQ3 NHS 에스터와 8.9 ^의 프로파질아민 (propargylamine)을 1.5 in의 디클로로메탄에 녹여 12시간 동안 반웅시킨 후， 유기용매를 회전증발기로 제거한 다음 얻어진 물질을 반분취 HPUXsemipreparative HPLC)를 통하여 정제하고， 합성된 화합물을 질량분석기로 확인하였다. 측정된 분자량은 584.3이었으며， 순도는 98% 이상이었다. Synthesis of the quencher containing the alkyne group was carried out as described in Banungsik 6. Specifically, 11 mg of BHQ3 NHS ester and 8.9 ^ propargylamine were dissolved in 1.5 in dichloromethane for 12 hours. After the reaction, the organic solvent was removed by rotary evaporator, and the obtained material was purified by semipreparative HPUX semipreparative HPLC), and the synthesized compound was confirmed by mass spectrometry. The molecular weight measured was 584.3 and the purity was 98% or more.
반웅식 6  Banungsik 6
Figure imgf000016_0001
시험예 1. 구리이은 검출의 선택성 평가
Figure imgf000016_0001
Test Example 1 Evaluation of Selectivity of Copper Silver Detection
구리이온을 선택적으로 검출할 수 있는지를 평가하기 위하여, 아지도 -PEG1(厂 Cy5.5(25 μΜ)와 알킨 -BHQ3(25 μΜ), 소듐 아스코빅 (sodium ascorbate, 1 μΜ)이 함유된 PBS(pH 7.0) 용액에 Li + , Ca2+, Ni + , Η^+, Mg2+, K+, Na+, Pd2+, Co2+, Mn2+, Cd2+' Fe2+, Ag+ 또는 Cu2+ 이온을 첨가하여 2시간 동안 반웅시킨 후, 형광을 측정하였다. 이때, 구리이온 농도는 50 nM 이었으며, 다른 금속이은 농도는 0.5 mM이었다. To assess the selective detection of copper ions, azido-PEG 1 (厂 Cy5.5 (25 μΜ), alkyne -BHQ3 (25 μΜ), sodium ascorbate (1 μΜ) Li +, Ca 2+ , Ni + , Η ^ +, Mg 2+ , K + , Na + , Pd 2+ , Co 2+ , Mn 2+ , Cd 2+ 'Fe 2+ in PBS pH 7.0 solution After reacting for 2 hours with addition of Ag + or Cu 2+ ions, the fluorescence was measured, at which the copper ion concentration was 50 nM and the other metal silver concentration was 0.5 mM.
그 결과, 도 2에 나타난 바와 같이, 다른 금속이온의 첨가에 의해서는 형광이 감소되지 않았으나， 구리이온의 첨가에 의해서는 형광세기가 현저히 감소하였다. 이러한 결과는, 본 발명의 센서가 구리이온만을 선택적으로 검출할 수 있음을 보여준다. 시험예 2. 구리이은 검출의 반웅성 평가  As a result, as shown in Figure 2, the fluorescence was not reduced by the addition of other metal ions, the fluorescence intensity was significantly reduced by the addition of copper ions. These results show that the sensor of the present invention can selectively detect only copper ions. Test Example 2 Evaluation of Reaction of Copper-Silver Detection
구리이온을 얼마나 빠르게 검출할 수 있는지를 평가하기 위하여, 아지도 -PEG10—Cy5.5(25 iM)와 알킨 -BHQ3(25 μΜ), 소듐 아스코빅 (1 μΜ)이 함유된 PBS (ρΗ 7.0) 용액에 Cu2+ 이온 (50 nM)을 첨가하여 5， 10, 20， 30, 60 및 120분에 각각 의 형광세기를 측정하였다. To assess how fast copper ions can be detected, PBS containing azido-PEG 10 —Cy5.5 (25 iM), alkyne-BHQ3 (25 μΜ) and sodium ascorbic (1 μΜ) (ρΗ 7.0 ) Fluorescence intensities were measured at 5, 10, 20, 30, 60 and 120 minutes by adding Cu 2+ ions (50 nM) to the solution.
그 결과， 도 3에 나타난 바와 같이， 반응시간이 지남에 따라 형광의 세기가 현저히 감소하였으며, 반응시간 30분 이후에는 형광의 감소가 더 이상 큰 차이가 없었다. 이러한 결과는， 본 발명의 센서는 구리이온을 30분 정도에 빠르게 검출할 수 있음을 보여준다. 시험예 3. 구리이은 검출의 특이성 확인 As a result, as shown in Figure 3, the intensity of the fluorescence was significantly reduced as the reaction time passes, and after 30 minutes of the reaction time there was no significant difference in the decrease in fluorescence. These results show that the sensor of the present invention can quickly detect copper ions in about 30 minutes. Test Example 3 Confirmation of Specificity of Copper Silver Detection
구리이온 검출 센서가 다양한 종류의 금속이온의 흔합물에서도 구리이온을 특이적으로 검출할 수 있는지를 평가하였다. 아지도 -PEG10- Cy5.5(25 μΜ)와 알킨 -BHQ3(25 μΜ), 소듐 아스코빅 (1 μΜ)이 함유된 PBSCpH 7.0) 용액에 4종류의 금속흔합물 3 그룹 (그룹 1: Li Ca2+, Ni+, Hg2+; 그룹 2: Mg2+, K+, Pd2+, Co2+; 그룹 3: Mn2+, Cc , Fe2+, Ag+)에 각각 Cu2+ 아온 (50 nM)을 첨가한 경우와 무첨가한 경우로 나누어 30분 동안 반웅시킨 다음 형광을 관찰하였다. It was evaluated whether the copper ion detection sensor could specifically detect copper ions even in a mixture of various kinds of metal ions. Three groups of four metal compounds (Group 1: Li) in a solution of PBSCpH 7.0 containing azido-PEG 10 -Cy5.5 (25 μΜ), alkyne -BHQ3 (25 μΜ) and sodium ascorbic (1 μΜ) Ca 2+, Ni +, Hg 2+ ; group 2: Mg 2+, K +, Pd 2+, Co 2+; group 3: Cu 2+ respectively the Mn 2+, Cc, Fe 2+, Ag +) ahon ( 50 nM) was added to the case of addition and no addition and reacted for 30 minutes, followed by fluorescence.
그 결과, 도 4에 나타난 바와 같이， 구리이온이 혼합되지 않은 4종류의 금속흔합물에서는 형광이 감소하지 않았으나， 구리이온을 첨가한 경우에는 형광의 세기가 현저히 감소하였다. 이러한 결과는， 본 발명의 구리이은 검출 센서는 다양한 금속이온이 흔합되어 있더라도 Cu2+ 이온이 존재하지 않는 경우에는 반응을 나타내지 않으며, 구리이온에만 특이적으로 반웅할 수 있음을 보여준다. 시험예 4. 구리이온 검출의 검출한계 평가 As a result, as shown in Fig. 4, the fluorescence did not decrease in the four kinds of metal complexes in which copper ions were not mixed, but the intensity of fluorescence was remarkably decreased when copper ions were added. These results show that the copper silver detection sensor of the present invention does not react when Cu 2+ ions are present even though various metal ions are mixed, and can specifically react only to copper ions. Test Example 4 Evaluation of Detection Limit of Copper Ion Detection
구리이은 검출 센서의 구리이온 검출한계를 평가하기 위하여, 아지도 -PEG10-Cy5.5(25 μΜ)와 알킨 -BHQ3(25 μΜ), 소듐 아스코빅 (1 μΜ)이 함유된 PBS(pH 7,0) 용액에 서로 다른 농도의 Cu2+ 이온을 첨가한 후 30분간 반응시킨 후 형광의 변화를 관찰하였다. To evaluate the copper ion detection limit of the copper silver detection sensor, PBS containing azido-PEG 10 -Cy5.5 (25 μΜ), alkyne -BHQ3 (25 μΜ) and sodium ascorbic (1 μΜ) , 0) After the addition of different concentrations of Cu 2+ ions to the solution and reacted for 30 minutes, the change in fluorescence was observed.
그 결과, 도 5에 나타난 바와 같이, 구리이온을 1 nM에서 5 fM로 처리하였을 때 형광의 감소가 분명히 관찰되었다. 이러한 결과는， 본 발명의 구리이온 검출 센서는 fM 농도범위까지도 구리이온을 검출할 수 있음을 보여준다. 시험예 5. 구리 킬레이트제의 효능 평가  As a result, as shown in Fig. 5, a decrease in fluorescence was clearly observed when the copper ions were treated from 1 nM to 5 fM. These results show that the copper ion detection sensor of the present invention can detect copper ions up to the fM concentration range. Test Example 5 Evaluation of Efficacy of Copper Chelating Agent
구리이온 검출 센서를 이용하여 구리이온 킬레이트제의 효능을 평가하기 위하여， 아지도 -PEG10-Cy5.5(25 μ M)와 알킨 -BHQ3(25 μ M) , 소듐 아스코빅 (1 μΜ)과 구리이온 (50 nM)이 함유된 PBS(pH 7.0) 용액에 구리이온 킬레이팅 효과가 없는 시약인 시스테인 (Cysteine), 아세틸시스테인 (N- acetylcysteine) 또는 카페익산 (caffeic acid)과 구리이온 킬레이트제인 테트라에틸렌펜타민 (tetraethylenepentamine, TEPA) , 트리에틸렌테트라민 (triethylenetetramine, TETA), 에틸렌디아민테트라아세틱산To evaluate the efficacy of copper ion chelating agents using a copper ion detection sensor, azido-PEG 10 -Cy5.5 (25 μM), alkyne-BHQ3 (25 μM), sodium ascorbic (1 μΜ) and PBS (pH 7.0) solution containing copper ions (50 nM) is a cysteine, N-acetylcysteine or caffeic acid and a copper ion chelating agent that have no copper ion chelating effect. Tetraethylenepentamine (TEPA), triethylenetetramine (TETA), ethylenediaminetetraacetic acid
(ethylenediaminetetraacetic acid, EDTA) 또는 페니실아민 (penici 1 laraine, PA)을 각각 0.5 nM첨가한 후 30분간 반웅시킨 다음 형광을 관찰하였다. 그 결과， 도 6에 나타난 바와 같이, 구리이온 킬레이팅 효과가 없는 세 가지 화합물인 시스테인， 아세틸시스테인 및 카페익산을 첨가하였을 경우에는 형광이 현저히 감소하였으나, 구리 킬레이트제인 TEPA, ΤΕΤΑ, EDTA 및 ΡΑ를 첨가하였을 때에는 형광이 전혀 감소되지 않거나 조금 감소되었다. 이러한 결과는， 본 발명의 구리이온 검출 센서를 이용하여 구리 킬레이트제를 스크리닝 하거나, 구리이온 킬레이트제의 효능을 정성 또는 정략적으로 평가할 수 있음을 보여한다. 시험예 6. 세포 내 구리이은 검출 After adding 0.5 nM (ethylenediaminetetraacetic acid, EDTA) or penicylamine (penici 1 laraine, PA), they were reacted for 30 minutes and fluorescence was observed. As a result, as shown in Figure 6, the addition of three compounds without the copper ion chelating effect, cysteine, acetylcysteine and caffeic acid was significantly reduced fluorescence, but the copper chelating agents TEPA, ΤΕΤΑ, EDTA and ΡΑ When added, the fluorescence was not reduced at all or slightly decreased. These results show that the copper chelating agent can be screened using the copper ion detection sensor of the present invention, or the efficacy of the copper ion chelating agent can be evaluated qualitatively or politically. Test Example 6 Detection of Copper Silver in Cells
세포 내 존재하는 구리이온에 대한 검출효능을 평가하기 위하여， 배양된 ΗΕΚ 293Τ에 100 μΜ의 CuS04를 처리한 후 Cu2+이온이 Cu1+로 환원되는 층분한 시간인 12시간 동안 배양한 다음， 세포를 배양액으로 세척한 후 아지도 -PEG10-Cy5.5(25 μΜ)와 알킨— BHQ3(25 μΜ)를 20분간 처리하고， 다시 세포 배양액으로 세척한 뒤 4시간동안 더 배양하였다. 세포 내 구리이온 검출은 공초점 주사 레이저 현미경을 통하여 관찰하였다. In order to evaluate the detection effect on the copper ions present in the cells, the cultured ΗΕΚ 293Τ was treated with 100 μΜ of CuS0 4 , followed by incubation for 12 hours, which is a time when Cu 2+ ions are reduced to Cu 1+ . ， Cells were washed with culture medium and treated with azido-PEG 10 -Cy5.5 (25 μΜ) and alkyne—BHQ3 (25 μΜ) for 20 minutes, and then washed with cell culture and incubated for 4 hours. Intracellular copper ion detection was observed by confocal scanning laser microscope.
그 결과, 도 7에 나타난 바와 같이， 구리이온 검출용 센서만 처리된 그룹에서는 강한 형광이 관찰되었으나 구리이온과 센서가 모두 처리된 그룹에서는 형광의 세기가 감소하였다. 이는， 세포 내로 흡수된 아지도- PEG10-Cy5.5와 알킨 -BHQ3가 세포 내에 존재하는 구리이온의 촉매역할을 통해 화학적 결합이 되어 형광의 세기가 감소한 것이며, 이러한 결과는 본 발명의 구리이온 검출용 센서가 세포 내 존재하는 구리이온올 검출할 수 있음을 보여준다. 시험예 7. 혈청에서 구리이은의 정량 As a result, as shown in FIG. 7, strong fluorescence was observed in the group treated only with the copper ion detection sensor, but the intensity of fluorescence decreased in the group treated with both the copper ion and the sensor. This is because the azido- PEG 10 -Cy5.5 and alkyne-BHQ3 absorbed into the cell are chemically bonded through the catalytic role of copper ions present in the cell, thereby reducing the intensity of fluorescence. It shows that the sensor for detection can detect the copper ion present in the cell. Test Example 7 Determination of Copper and Silver in Serum
혈청에서 구이이온을 검출할 수 있는지를 평가하기 위하여， BALB/C 마우스에 5 mg/kg 용량으로 꼬리정맥을 통하여 CuS04를 투여한 후， Cu2+ 이온이 Cu1+로 환원되는 충분한 시간인 2시간 후에 마우스의 대정맥에서 P T/KR2013/010418 In order to assess whether the serum can detect guine ion, BALB / C mice were administered CuS0 4 via the tail vein at a dose of 5 mg / kg, which is a sufficient time for Cu 2+ ions to be reduced to Cu 1+ . After 2 hours in the vena cava of the mouse PT / KR2013 / 010418
혈액을 채취하였다. 채취한 혈액은 실은에서 1시간 동안 방치한 후， 13,000 rpm으로 10분간 원심분리하여 혈청을 얻었다. 혈청에서 구리이온을 정량하기 위하여, 50배 희석된 혈청과 아지도 -PEG10-Cy5, 5(25 μΜ)와 알킨- BHQ3C25 μΜ)를 30분 동안 반웅시킨 후 형광세기를 측정하였다. 혈청에서의 구리이은 농도는 10 ηΜ 에서 60 ηΜ 범위에서 얻어진 구리이온 표준곡선 (standard curve)으로부터 정량하였다. Blood was collected. The collected blood was left for 1 hour at the actual silver, centrifuged at 13,000 rpm for 10 minutes to obtain a serum. In order to quantify copper ions in serum, 50-fold diluted serum and azido-PEG 10 -Cy5, 5 (25 μΜ) and alkyne-BHQ3C25 μΜ) were reacted for 30 minutes and then fluorescence intensity was measured. The copper silver concentration in the serum was quantified from the copper ion standard curve obtained in the range of 10 ηΜ to 60 ηΜ.
그 결과， 도 8에 나타난 바와 같이， 정상 마우스와 구리이온이 투여된 마우스의 혈청에서 검출된 구리이은의 양은 0.85 士 0.32 iiM 및 1.74 土 0.29 ιιΜ로 측정되었다. 이러한 결과는, 본 발명의 구리이온 검출 센서를 이용하여 혈청의 구리이은 농도를 알아낼 수 있음을 보여준다. 이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.  As a result, as shown in Figure 8, the amount of copper silver detected in the serum of normal mice and mice administered copper ions were measured as 0.85 0.3 0.32 iiM and 1.74 土 0.29 ιιΜ. These results show that the copper silver concentration of serum can be determined using the copper ion detection sensor of the present invention. Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

[특허청구범위】 [Patent Claims]
【청구항 1】  [Claim 1]
형광체 ; 및 상기 형광체의 발광효과를 흡수하여 소광효과를 나타내는 소광체를 포함하고， 상기 형광체 및 소광체는 각각 아자이드 (azide) 그룹 또는 알킨 (alkyne) 그룹을 포함하되, 형광체가 아자이드 그룹 또는 알킨그룹을 포함하는 경우 소광체는 알킨그룹 또는 아자이드 그룹을 포함하는 것을 특징으로 하는 구리이온 검출용 조성물.  Phosphor; And a quencher which absorbs the luminous effect of the phosphor and exhibits a quencher effect, wherein the phosphor and the quencher include an azide group or an alkyne group, respectively, wherein the phosphor is an azide group or an alkyne group. When containing a quencher composition for detecting copper ions, characterized in that it comprises an alkyne group or an azide group.
【청구항 2] [Claim 2]
제 1 항에 있어서， 상기 형광체는 적색 또는 근적외선의 형광을 발광하는 것을 특징으로 하는 조성물.  The composition of claim 1, wherein the phosphor emits red or near infrared fluorescence.
【청구항 3】 [Claim 3]
제 1 항에 있어서， 상기 형광체는 시아닌 (Cyanine), 플루오레신 (fluorescein), 보디피 (B0DIPY), 테트라메틸로드아민 The method of claim 1, wherein the phosphor is cyanine, fluorescein, fluorine (B0DIPY), tetramethylrodamine
(Trtramethylrhodamine), 알렉사 (Alexa) 및 알로피코시아닌(Trtramethylrhodamine), Alexa and allopicocyanine
(allopicocyanine)으로 구성된 군으로부터 선택되는 것을 특징으로 하는 조성물. (allopicocyanine) is selected from the group consisting of.
【청구항 4】 [Claim 4]
제 1 항에 있어서 , 상기 소광체는 블랙홀 소광체 (blackhole quencher , BHQ) 또는 블랙베리 소광체 (blackberry quencher , BBQ)인 것을 특징으로 하는 조성물.  The composition of claim 1, wherein the quencher is a blackhole quencher (BHQ) or a blackberry quencher (BBQ).
【청구항 5】 [Claim 5]
제 1 항 내지 제 4 항 중 어느 한 항에 따른 구리이온 검출용 조성물을 포함하는 구리이온 검출용 형광화학센서 .  A copper ion sensor for detecting copper ions comprising the composition for detecting copper ions according to any one of claims 1 to 4.
【청구항 6] [Claim 6]
다음의 단계를 포함하는 구리이온 검출용 조성물의 제조방법:  Method for producing a composition for detecting copper ions comprising the following steps:
(a) 아자이드 그룹 또는 알킨 그룹이 도입된 형광체를 얻는 단계; (b) 알킨 그룹 또는 아자이드 그룹이 도입된 소광체를 얻는 단계; 및(a) obtaining a phosphor into which an azide group or an alkyne group is introduced; (b) obtaining a quencher in which an alkyne group or an azide group is introduced; And
(c) 단계 (a)에서 얻은 아자이드 그룹이 도입된 형광체와 단계 (b)에서 얻은 알킨 그룹이 도입된 소광체， 또는 단계 (a)에서 얻은 알킨 그룹이 도입된 형광체와 단계 (b)에서 얻은 아자이드 그룹이 도입된 소광체를 흔합하는 단계. (c) the phosphor to which the azide group obtained in step (a) is introduced and the quencher to which the alkyne group obtained in step (b) is introduced, or the phosphor to which the alkyne group obtained in step (a) is introduced and in step (b) Mixing the quencher into which the obtained azide group was introduced.
【청구항 7】 [Claim 7]
다음의 단계를 포함하는 시료 내 구리이온의 선택적 검출방법:  Selective detection of copper ions in a sample comprising the following steps:
(a) 제 1 항의 구리이온 검출용 조성물 또는 제 6 항의 구리이온 검출용 형광화학센서에 시료를 접촉시키는 단계 ; 및  (a) contacting the sample with the composition for detecting copper ions of claim 1 or the fluorescent chemical sensor for detecting copper ions of claim 6; And
(b) 형광체의 소광을 확인하는 단계 .  (b) confirming quenching of the phosphor.
【청구항 8】 [Claim 8]
제 7 항에 있어서, 상기 형광체의 소광은 시료 내에 구리이온이 존재하는 경우에 발생하는 것을 특징으로 하는 검출방법.  8. The detection method according to claim 7, wherein the quenching of the phosphor occurs when copper ions are present in the sample.
【청구항 9】 [Claim 9]
제 7 항에 있어서， 상기 시료는 수용액， 유기용액, 세포， 세포 용해물 (cell lysate), 혈액， 혈장， 혈청， 림프， 타액， 뇨， 정액 및 복수로 구성 ¾ 군으로부터 선택되는 것을 특징으로 하는 검출방법.  The method of claim 7, wherein the sample is selected from the group ¾ consisting of aqueous solution, organic solution, cells, cell lysate, blood, plasma, serum, lymph, saliva, urine, semen and ascites Detection method.
【청구항 10] [Claim 10]
다음의 단계를 포함하는 구리이온 킬레이트제의 스크리닝 방법 : Screening method of copper ion chelating agent comprising the following steps:
(a) 환원제를 포함하는 제 1 항의 구리이온 검출용 조성물 또는 환원제를 포함하는 제 6 항의 구리이온 검출용 형광화학센서에 시험물질과 구리이온 (Cu2+)을 동시에 또는 순차적으로 접촉시키는 단계 ; 및 (a) simultaneously or sequentially contacting a test substance and copper ions (Cu 2+ ) with the copper ion detecting composition of claim 1 comprising a reducing agent or the fluorescent chemical sensor for detecting copper ions of claim 6 comprising a reducing agent; And
(b) 상기 형광체의 소광을 확인하는 단계 .  (b) confirming quenching of the phosphor.
【청구항 111 [Claim 111]
다음의 단계를 포함하는 구리이온 킬레이트제의 킬레이트 효과 확인 방법： (a) 환원제를 포함하는 제 1 항의 구리이온 검출용 조성물 또는 환원제를 포함하는 제 6 항의 구리이온 검출용 형광화학센서에 구리이온 킬레이트제와 구리이온 (Cu2+)을 동시에 또는 순차적으로 접촉시키는 단계; (b) 상기 형광체의 소광을 확인하는 단계. Method for confirming chelation effect of copper ion chelating agent comprising the following steps: (a) contacting the copper ion chelating agent and the copper ion (Cu 2+ ) simultaneously or sequentially with the copper ion detecting composition of claim 1 comprising the reducing agent or the fluorescent chemical sensor for detecting copper ion comprising the reducing agent step; (b) confirming quenching of the phosphor.
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