WO2015106466A1 - 一种基于核苷等生物分子的原位快速合成金银荧光纳米材料及其制备方法和应用 - Google Patents

一种基于核苷等生物分子的原位快速合成金银荧光纳米材料及其制备方法和应用 Download PDF

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WO2015106466A1
WO2015106466A1 PCT/CN2014/071547 CN2014071547W WO2015106466A1 WO 2015106466 A1 WO2015106466 A1 WO 2015106466A1 CN 2014071547 W CN2014071547 W CN 2014071547W WO 2015106466 A1 WO2015106466 A1 WO 2015106466A1
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gold
silver
reagent
nucleoside
solution
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French (fr)
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王雪梅
张园园
姜晖
革伟
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东南大学
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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
    • C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/58—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing copper, silver or gold
    • 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
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures

Definitions

  • the invention relates to a preparation method of in situ rapid synthesis of gold and silver fluorescent nano materials based on biomolecules such as nucleosides and the application thereof in body fluid detection of diseases such as cancer. Specifically, it relates to the preparation of a fluorescent-enhanced gold-silver nano-alloy material using biomolecules such as nucleosides as a template, and the use of the method for preparing a kit for detecting body fluids of diseases such as cancer. Background technique
  • the organism contains a cis-dihydroxy biomolecule such as glycoproteins and glycopeptides, modified nucleosides, and monosaccharides and polysaccharides, which play an extremely important role in many physiological processes.
  • a cis-dihydroxy biomolecule such as glycoproteins and glycopeptides, modified nucleosides, and monosaccharides and polysaccharides, which play an extremely important role in many physiological processes.
  • the monitoring of such molecules in the human body can reflect the health of the human body to a certain extent.
  • Nucleosides are components of the RNA structure that release modified nucleosides when the transport RNA degrades. These modified nucleosides are excreted in the urine. When the body becomes cancerous, it accelerates the degradation of the transport RNA, resulting in an increase in the concentration of modified nucleosides in the urine. Therefore, the level of modified nucleosides in urine reflects the physiological health of the human body to some extent.
  • Nanomaterials have attracted much attention in research such as cancer detection because of their special properties.
  • precious metal nanomaterials such as gold and silver have good biocompatibility and stable fluorescence characteristics, and have broad application prospects in biomedicine: for example, in the early diagnosis and treatment of diseases such as tumors, they can be applied to the analysis and detection of tumor evolution processes in vivo or Molecular imaging; improve the targeting of drug treatment, avoid biological or physiological barriers in the body; can also monitor the treatment effect in real time.
  • Early diagnosis of diseases such as cancer is one of the important ways to achieve effective treatment. Therefore, it is of great value and potential to develop rapid and effective detection and diagnosis methods by virtue of the excellent properties of nanomaterials such as gold and silver with excellent biocompatibility. Social and economic benefits. Summary of the invention
  • the present invention provides a method for preparing in-situ rapid synthesis of gold-silver fluorescent nanomaterials based on biomolecules such as nucleosides, and an application thereof, which has excellent biocompatibility and stable fluorescence characteristics, and can be realized. Highly sensitive and rapid detection of body fluids (such as saliva, blood, urine, etc.) in normal people and cancer patients.
  • body fluids such as saliva, blood, urine, etc.
  • the solution containing a cis-dihydroxy biomolecule includes a glycoprotein, a glycopeptide, a nucleoside, a monosaccharide or a polysaccharide solution.
  • the nucleoside includes any one of adenosine nucleoside, guanosine nucleoside, cytidine nucleoside, thymidine, and uridine.
  • the human body fluid includes saliva, urine, and serum.
  • the gold reagent includes any one of chloroauric acid, tetrakisium octachloride, gold chloride, gold trichloride, and gold monochloride, or a mixed solution of any two.
  • the silver reagent is a silver ammonia solution or a silver nitrate solution or a mixed solution of the two.
  • the ratio of the amount of the substance of the cis-dihydroxy biomolecule to the amount of the gold ion is 0. 4 ⁇ 20 : 1
  • the amount of silver reagent added is the same as the amount of gold ions added.
  • a method for preparing the nucleoside-based in situ rapid synthesis of gold and silver fluorescent nanomaterials the steps are as follows: In the first step, a solution containing a cis-dihydroxy biomolecule or a human body fluid is mixed with an aqueous solution of gold reagent, and added The sodium citrate buffer solution is controlled to pH 6-7 and then thoroughly mixed, heated in a water bath or placed at room temperature;
  • the second step after the reaction for a period of time, a solution of 0. 025 mmol/L of the silver reagent is added to the mixed solution, and the gold-silver fluorescent nanomaterial is obtained by cooling.
  • the reaction condition in the second step is 37 .
  • C Incubate for 3 h or 80 °0 water bath for 5 minutes.
  • the nucleoside-based in situ rapid synthesis of gold and silver fluorescent nanomaterials is used in the preparation of a test kit for a body fluid test for a cancer patient.
  • the organism contains many cis-dihydroxy biomolecules, such as glycoproteins and glycopeptides, modified nucleosides, and monosaccharides and polysaccharides. These compounds play an extremely important role in many physiological processes. When the physiological health of the human body occurs, the content of these cis-dihydroxy biomolecules in the body fluid changes. Using these cis-dihydroxy biomolecules as templates, under the action of reducing agents, gold and silver ions are reduced, and gold and silver are used. It is an excellent fluorescent property of nanomaterials, and monitors cis-dihydroxy biomolecules in biological fluids to achieve diagnosis of diseases.
  • nucleoside solution Different concentrations of nucleoside solution were mixed with a fixed concentration of gold reagent and sodium citrate buffer, and then reacted with a fixed concentration of silver reagent to obtain gold and silver fluorescent nanomaterials. The obtained fluorescence intensity and color were recorded to establish a standard library.
  • the normal human body fluid was mixed with a fixed concentration of gold reagent and sodium citrate buffer, and then reacted with a fixed concentration of silver reagent to obtain gold and silver fluorescent nanomaterials, and the obtained fluorescence intensity and color were recorded to establish a standard library.
  • the body fluids of different cancer patients are separately mixed with a fixed concentration of gold reagent and sodium citrate buffer, and then reacted with a fixed concentration of silver reagent to obtain gold and silver fluorescent nanomaterials, and the obtained fluorescence intensity, color and other characteristics are collected and collected before.
  • the intensity, color, etc. in the standard library are compared, and the general indication of the patient can be qualitatively determined. It is similar to the application of pH test paper.
  • nucleoside biomolecules Different concentrations of nucleoside biomolecules, gold reagent and sodium citrate buffer are mixed. After a certain period of time, gold nanomaterials with weak fluorescence are formed, and then silver reagent is added to immediately produce a stable and intuitive fluorescence visible to the naked eye under ultraviolet light. .
  • serum such as cancer and normal human serum replaces the biomolecules such as nucleosides in the above experiments, and obtains different fluorescence emission spectra, thereby realizing the detection and differentiation of physiological indicators of cancer patients and normal people.
  • the invention discovers and develops in-situ synthetic precious metal gold and silver nano-alloy materials using biomolecules such as nucleosides as templates, and utilizes the fluorescent characteristics to realize differentiation and detection of cancer patients and normal human body fluids. Because of its simple operation, intuitive results, high sensitivity and low cost, this method has potential application prospects in the early diagnosis of diseases.
  • this method uses biomolecules such as nucleosides as templates to rapidly prepare precious metal gold-silver nano-alloy materials and is used for rapid detection of physiological indicators of patients with cancer and other diseases.
  • the invention can realize the non-invasive, rapid, high-sensitivity, simple and intuitive detection of physiological indexes of diseases such as cancer, and further combines fluorescence, Raman, ultrasound, CT and nuclear magnetic, etc., and can perform multi-modal and multi-modal simultaneous diagnosis. And accurate targeting and treatment, has broad medical application prospects.
  • Figure 1 is an optical diagram of Example 1 of the present invention and a fluorescence diagram excited by 365 nm light, and a corresponding fluorescence emission spectrum at 370 nm excitation;
  • Figure A Cytidine/gold nanomaterial;
  • B Cytidine/ Gold and silver nanomaterials.
  • Fig. 2 is a diagram showing the results of the method of the present invention, which is used in the detection of serum of patients such as cancer and leukemia and normal humans.
  • a histogram drawn by fluorescence emission peak position and fluorescence intensity can distinguish between cancer patients and normal human serum.
  • Biomolecules such as nucleosides in this experiment were purchased from Sigma-Aldrich, chloroauric acid, tetra-gold tetrachloride, gold chloride, gold trichloride, gold monochloride, silver nitrate, silver ammonia solution, sodium citrate and Reagents such as citric acid were purchased from Sinopharm Chemical Reagent Co., Ltd.
  • a method for preparing in situ rapid synthesis of gold and silver fluorescent nanomaterials based on biomolecules such as nucleosides which is characterized by the following steps:
  • a certain concentration of a solution containing a cis-dihydroxy biomolecule such as a glycoprotein, a glycopeptide, a nucleoside (adenosine, guanosine, cytosine, thymidine, uracil) is prepared. Any one of the nucleosides), a monosaccharide or a polysaccharide solution.
  • aqueous solution of gold reagent and silver reagent is prepared in the first step.
  • the solution containing the cis-dihydroxy biomolecule or the human body fluid and the gold reagent are mixed according to the cis and the molar ratio of the hydroxy biomolecule to the gold ion is 0. 4 ⁇ 20:1, and a certain amount of citric acid is added.
  • Sodium solution adjust the pH to 6 ⁇ 7, mix the three thoroughly, then heat in a water bath or room temperature.
  • the third step after incubating at 37 ° C for 3 hours or 80 ° 0 water bath for 5 minutes, adding a silver reagent equivalent to the gold reagent to the mixed solution, cooling to obtain a gold-silver nano-alloy excellent in fluorescence; wherein heating time, heating The ratio of temperature, nucleoside biomolecules and gold reagents, and silver reagents can be adjusted, except that the fluorescent color of the material is different.
  • the gold reagent is a mixed solution of chloroauric acid, tetrakisium octachloride, gold chloride, gold trichloride, gold monochloride or any two;
  • the silver reagent is a silver ammonia solution, nitric acid One or a mixture of two in a silver solution.
  • a method for preparing in situ rapid synthesis of gold and silver fluorescent nanomaterials based on biomolecules such as nucleosides is prepared by the following steps:
  • a certain concentration of cytidine, gold reagent and silver reagent aqueous solution is prepared.
  • the cytosine nucleoside and the gold reagent are mixed according to the ratio of the nucleoside to the gold ion in a molar ratio of 16:1, and the sodium citrate solution is added to adjust the pH value of 6 to 7, the three are thoroughly mixed, and then heated in a water bath or Place at room temperature.
  • the third step after incubating at 37 ° C for 3 hours or at 80 ° C for 5 minutes, a silver reagent equivalent to gold reagent is added to the mixed solution, and cooling is performed to obtain a gold-silver nano-alloy excellent in fluorescence.
  • the gold reagent is chloroauric acid; the silver reagent is a silver nitrate solution.
  • the resulting material is tested under UV light. The specific test results are shown in Figure 1.
  • cytosine nucleoside biomolecule a certain concentration of cytosine nucleoside biomolecule, gold reagent and silver reagent aqueous solution are prepared.
  • the cytidine nucleoside and the gold reagent are mixed at a molar ratio of 16:1, and a certain amount of sodium citrate solution is added to adjust the pH value of 6 to 7, and the three are thoroughly mixed, and then heated in a water bath or at room temperature.
  • the third step after incubating at 37 ° C for 3 hours or at 80 ° C for 5 minutes, an equivalent amount of silver reagent is added to the mixed solution, and cooling is performed to obtain a gold-silver nano-alloy excellent in fluorescence; wherein heating time, heating temperature
  • the ratio of cytosine nucleoside and gold reagent, silver reagent can be adjusted, only the fluorescent color of the material is different.
  • the gold reagent is tetrakisium octachloride; the silver reagent is a silver nitrate solution.
  • a certain concentration of cytidine nucleoside, gold reagent and silver reagent aqueous solution are prepared.
  • the cytosine nucleoside and the gold reagent are mixed in a ratio of 16:1, and a certain amount of sodium citrate solution is added to adjust the pH value of 6 to 7, and the three are thoroughly mixed, and then heated in a water bath or at room temperature.
  • the third step after incubating at 37 ° C for 3 hours or at 80 ° C for 5 minutes, an equivalent amount of silver reagent is added to the mixed solution, and cooling is performed to obtain a gold-silver nano-alloy excellent in fluorescence; wherein heating time, heating temperature
  • the ratio of cytosine nucleoside and gold reagent, silver reagent can be adjusted, only the fluorescent color of the material is different.
  • the gold reagent is gold chloride; the silver reagent is a silver nitrate solution.
  • a certain concentration of cytidine nucleoside, gold reagent and silver reagent aqueous solution are prepared.
  • the cytosine nucleoside and the gold reagent are mixed in a ratio of 16:1, and a certain amount of sodium citrate solution is added to adjust the pH value of 6 to 7, and the three are thoroughly mixed, and then heated in a water bath or at room temperature.
  • the third step after incubating at 37 ° C for 3 hours or at 80 ° C for 5 minutes, an equivalent amount of silver reagent is added to the mixed solution, and cooling is performed to obtain a gold-silver nano-alloy excellent in fluorescence; wherein heating time, heating temperature
  • the ratio of cytosine nucleoside and gold reagent, silver reagent can be adjusted, only the fluorescent color of the material is different.
  • the gold reagent is gold trichloride; the silver reagent is a silver nitrate solution.
  • a certain concentration of cytidine nucleoside, gold reagent and silver reagent aqueous solution are prepared.
  • the cytidine nucleoside and the gold reagent are mixed in a ratio of 1:1, and a certain amount of sodium citrate solution is added to adjust the pH value of 6 to 7, and the three are thoroughly mixed, and then heated in a water bath or at room temperature.
  • a certain amount of sodium citrate solution is added to adjust the pH value of 6 to 7, and the three are thoroughly mixed, and then heated in a water bath or at room temperature.
  • an equivalent amount of silver reagent is added to the mixed solution, and cooling is performed to obtain a gold-silver nano-alloy excellent in fluorescence; wherein heating time, heating temperature
  • the ratio of cytosine nucleoside and gold reagent, silver reagent can be adjusted, only the fluorescent color of the material is different.
  • the gold reagent is gold monochloride; the silver reagent is a silver nitrate solution.
  • a certain concentration of cytidine nucleoside, gold reagent and silver reagent aqueous solution are prepared.
  • the cytosine nucleoside and the gold reagent are mixed in a ratio of 16:1, and a certain amount of sodium citrate solution is added to adjust the pH value of 6 to 7, and the three are thoroughly mixed, and then heated in a water bath or at room temperature.
  • the third step after incubating at 37 ° C for 3 hours or at 80 ° C for 5 minutes, an equivalent amount of silver reagent is added to the mixed solution, and cooling is performed to obtain a gold-silver nano-alloy excellent in fluorescence; wherein heating time, heating temperature
  • the ratio of cytosine nucleoside and gold reagent, silver reagent can be adjusted, only the fluorescent color of the material is different.
  • the gold reagent is chloroauric acid; the silver reagent is a silver ammonia solution.
  • a certain concentration of cytidine nucleoside, gold reagent and silver reagent aqueous solution are prepared.
  • the cytosine nucleoside and the gold reagent are mixed in a ratio of 16:1, and a certain amount of sodium citrate solution is added to adjust the pH value of 6 to 7, and the three are thoroughly mixed, and then heated in a water bath or at room temperature.
  • the third step after incubating at 37 ° C for 3 hours or at 80 ° C for 5 minutes, an equivalent amount of silver reagent is added to the mixed solution, and cooling is performed to obtain a gold-silver nano-alloy excellent in fluorescence; wherein heating time, heating temperature
  • the ratio of cytosine nucleoside and gold reagent, silver reagent can be adjusted, only the fluorescent color of the material is different.
  • the gold reagent is tetrakisium octachloride; the silver reagent is a silver ammonia solution.
  • a certain concentration of cytidine nucleoside, gold reagent and silver reagent aqueous solution are prepared.
  • the cytosine nucleoside and the gold reagent are mixed in a ratio of 16:1, and a certain amount of sodium citrate solution is added to adjust the pH value of 6 to 7, and the three are thoroughly mixed, and then heated in a water bath or at room temperature.
  • the third step after incubating at 37 ° C for 3 hours or at 80 ° C for 5 minutes, an equivalent amount of silver reagent is added to the mixed solution, and cooling is performed to obtain a gold-silver nano-alloy excellent in fluorescence; wherein heating time, heating temperature
  • the ratio of cytosine nucleoside and gold reagent, silver reagent can be adjusted, only the fluorescent color of the material is different.
  • the gold reagent is gold chloride; the silver reagent is a silver ammonia solution.
  • Example 9 In the first step, a certain concentration of cytidine nucleoside, gold reagent and silver reagent aqueous solution are prepared.
  • the cytosine nucleoside and the gold reagent are mixed in a ratio of 16:1, and a certain amount of sodium citrate solution is added to adjust the pH value of 6 to 7, and the three are thoroughly mixed, and then heated in a water bath or at room temperature.
  • the third step after incubating at 37 ° C for 3 hours or at 80 ° C for 5 minutes, an equivalent amount of silver reagent is added to the mixed solution, and cooling is performed to obtain a gold-silver nano-alloy excellent in fluorescence; wherein heating time, heating temperature
  • the ratio of cytosine nucleoside and gold reagent, silver reagent can be adjusted, only the fluorescent color of the material is different.
  • the gold reagent is gold trichloride; the silver reagent is a silver ammonia solution.
  • a certain concentration of cytidine nucleoside, gold reagent and silver reagent aqueous solution are prepared.
  • the cytosine nucleoside and the gold reagent are mixed in a ratio of 16:1, and a certain amount of sodium citrate solution is added to adjust the pH value of 6 to 7, and the three are thoroughly mixed, and then heated in a water bath or at room temperature.
  • the third step after incubating at 37 ° C for 3 hours or at 80 ° C for 5 minutes, an equivalent amount of silver reagent is added to the mixed solution, and cooling is performed to obtain a gold-silver nano-alloy excellent in fluorescence; wherein heating time, heating temperature
  • the ratio of cytosine nucleoside and gold reagent, silver reagent can be adjusted, only the fluorescent color of the material is different.
  • the gold reagent is monochlorinated gold; the silver reagent is a silver ammonia solution.
  • the serum of 40 ⁇ cancer and other diseases and the gold reagent with a concentration of 0.025 mmol/L were mixed according to the ratio of nucleoside to gold ion molar ratio of 16:1, and a certain amount of sodium citrate solution was added to adjust the pH value of 6 ⁇ 7.
  • the three were thoroughly mixed and then heated in a 37 ° C water bath. After incubating for 3 hours, a silver reagent containing silver ions in an amount such as gold ions was added to the mixed solution, and cooling was performed to obtain a gold-silver nanoalloy excellent in fluorescence; the results of the obtained material under a fluorometer are shown in Fig. 2.
  • the gold reagent is chloroauric acid; the silver reagent is a silver nitrate solution.
  • the gold reagent is chloroauric acid; the silver reagent is a silver nitrate solution.
  • Example 13 The ratio of the amount of the nucleoside-containing nucleoside-containing substance to the amount of the gold-ion substance is 0.44: Mix 1 in proportion, add a certain amount of sodium citrate solution to adjust the pH value of 6 ⁇ 7, mix the three thoroughly, and then heat in a 37 °C water bath. After incubating for 3 hours, the same amount of silver reagent as the gold reagent is added to the mixed solution, and cooling is performed to obtain a gold-silver nano-alloy excellent in fluorescence; wherein the heating time, the heating temperature, the ratio of the gold reagent and the silver reagent can be adjusted, but only The fluorescent colors of the materials are different.
  • the gold reagent is tetrakisium octachloride; the silver reagent is a silver ammonia solution.
  • the ratio of the ratio of the amount of the nucleoside-containing substance in the urine to the amount of the gold ion in the ratio of the amount of the nucleoside-containing substance to the amount of the gold ion is 20:1, and the concentration of 1. 44 mL of normal human urine is mixed with the concentration of 0.025 mmol/L of the gold reagent. Add a certain amount of sodium citrate solution to adjust the pH value of 6 ⁇ 7, mix the three thoroughly, and then heat in a 37 °C water bath.
  • the same amount of silver reagent as the gold reagent is added to the mixed solution, and cooling is performed to obtain a gold-silver nano-alloy excellent in fluorescence; wherein the heating time, the heating temperature, the ratio of the gold reagent and the silver reagent can be adjusted, but only The fluorescent colors of the materials are different.
  • the gold reagent is gold chloride; the silver reagent is a mixture of a silver nitrate solution and a silver ammonia solution.
  • the saliva of a patient with a disease such as 40 ⁇ cancer and a gold reagent having a concentration of 0.025 mmol/L are mixed according to the ratio of the amount of the nucleoside substance contained in the saliva to the amount of the gold ion substance in a ratio of 4:1.
  • a certain amount of sodium citrate solution was adjusted to pH 6 to 7, and the three were thoroughly mixed and then heated in a 37 ° C water bath. After incubating for 3 hours, an equivalent amount of silver reagent is added to the mixed solution, and cooling is performed to obtain a gold-silver nano-alloy excellent in fluorescence; wherein the heating time, heating temperature, gold reagent, and silver reagent ratio can be adjusted, but only The fluorescent colors of the materials are different.
  • the gold reagent is a mixture of chloroauric acid and gold trichloride; the silver reagent is a silver nitrate solution.
  • 40 ⁇ of normal human saliva and a concentration of 0.025 mmol/L of gold reagent are mixed according to the ratio of the amount of the nucleoside substance contained in the saliva to the amount of the gold ion substance of 10:1, and a certain amount is added.
  • the sodium citrate solution, the three were thoroughly mixed and then heated in a 37 ° 0 water bath. After incubating for 3 hours, the same amount of silver reagent as the gold reagent is added to the mixed solution, and cooling is performed to obtain a gold-silver nano-alloy excellent in fluorescence; wherein the heating time, the heating temperature, the ratio of the gold reagent and the silver reagent can be adjusted, but only The fluorescent colors of the materials are different.
  • the gold reagent is chloroauric acid; the silver reagent is a silver nitrate solution.
  • the invention synthesizes fluorescence-enhanced gold-silver nano-alloy in situ using cytidine nucleoside as template and applies the method to cancer
  • the detection of body fluids in patients with diseases can achieve an intuitive and rapid diagnosis of their physiological indicators.
  • This method is the first to be applied in the detection of body fluids in patients with cancer and other diseases.
  • the method is simple, rapid, intuitive, and low in cost, and provides an efficient and feasible method for cancer diagnosis.

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Abstract

　本发明公开了一种基于核苷等生物分子的原位快速合成金银荧光纳米材料及其制备方法和应用。本发明通过核苷等生物分子作为模板原位快速生成金银纳米合金，该纳米材料具有优良的生物相容性，稳定的荧光特性，可以实现对正常人与癌症等疾病患者体液的高灵敏快速检测，从而达到对相关生理指标的诊断；本发明的方法可对癌症等病人体液中的核苷及其他相关生物物质进行检测，实现对癌症等病人生理指标的快速、高灵敏、低成本、直观检测；本发明的方法应用简便易行、毒性低、灵敏度高、特异性强，可同时进行多形态与多模态的实时动态临床监测，具有广阔的医学临床应用价值和前景。

Description

一种基于核苷等生物分子的原位快速合成金银荧光纳米材料及其制备方法 和应用
技术领域
本发明涉及基于核苷等生物分子的原位快速合成金银荧光纳米材料的制备方法及其 在癌症等疾病患者体液检测中的应用。 具体涉及以核苷等生物分子为模板， 荧光增强的 金银纳米合金材料的制备， 以及该方法用于制备对癌症等疾病患者体液的检测的试剂盒 中的应用。 背景技术
众所周知， 癌症早期症状不明显， 很难被及时发现， 且现行的常规组织病理学诊断 方法成本高， 过程冗余且很难检测病变组织以及细胞早期所发生的生化和分子异常， 因 此寻找方便、 快捷、 准确的癌症早期诊断方法意义重大， 所以研究更先进的诊断方法， 来实现准确、 及时的癌症早期诊断及治疗一直是人们努力的方向之一。
生物体中含有一种顺式二羟基生物分子， 如糖蛋白和糖肽， 修饰核苷以及单糖和多 糖等， 这类化合物在许多生理过程中发挥着极其重要的作用。 当人体生理健康状况发生 病变时， 这些顺式二羟基生物分子在体液中的含量就会发生改变。 对人体中此类分子的 监测， 在一定程度上可以反映人体的健康状况。 核苷是 RNA 结构的组成单元， 当转运 RNA发生降解时会释放修饰核苷， 这些修饰核苷随尿液排出体外。 当人体发生癌变时会 加速转运 RNA的降解， 从而导致尿液中修饰核苷浓度增大。 因此尿液中修饰核苷的水平 一定程度上反映了人体的生理健康状况。
纳米材料因其自身具备的一些特殊性质在癌症检测等研究中备受瞩目。 尤其是金银 等贵金属纳米材料， 生物相容性好， 荧光特性稳定， 在生物医学中具有广阔的应用前景： 如在肿瘤等疾病的早期诊治中， 可应用于体内肿瘤演化过程的分析检测或分子显像； 提 高药物治疗的靶向性， 避开体内的生物或生理学屏障； 还可以对治疗效果进行实时监测。 癌症等疾病的早期诊断是实现其有效治疗的重要途径之一， 因而， 借助具有优良生物相 容性的金银等纳米材料的优良特性， 发展快速有效的检测诊断方法具有重要的价值和潜 在的社会经济效益。 发明内容
发明目的： 本发明提供了一种基于核苷等生物分子的原位快速合成金银荧光纳米材料的 制备方法及其应用， 该纳米材料具有优良的生物相容性， 稳定的荧光特性， 可以实现对 正常人与癌症等疾病患者体液 （如唾液， 血液， 尿液等） 的高灵敏快速检测。
技术方案： 一种基于核苷等生物分子的原位快速合成金银荧光纳米材料， 以含有顺式二 羟基生物分子的溶液或人体体液与金试剂水溶液混合， 加入柠檬酸钠缓冲溶液反应得到 具有微弱荧光的金纳米材料， 再加入银试剂水溶液继续反应后得到荧光增强的金银荧光 纳米材料。
所述的含有顺式二羟基生物分子的溶液包括糖蛋白、 糖肽、 核苷、 单糖或多糖溶液。 所述的核苷包括腺嘌呤核苷， 鸟嘌呤核苷， 胞嘧啶核苷， 胸腺嘧啶核苷， 尿嘧啶核 苷中的任一种。
所述的人体体液包括唾液， 尿液， 血清。
所述的金试剂包括氯金酸、 八氯化四金、 氯化金、 三氯化金、 一氯化金中的任意一 种或者任意二种的混合溶液。
所述的银试剂为银氨溶液或硝酸银溶液或者二种的混合溶液。
含有顺式二羟基生物分子的溶液或人体体液与金试剂水溶液混合时按照其中含有的顺式 二羟基生物分子的物质的量与金离子的物质的量之比为 0. 4〜20 : 1 的比例混合， 银试剂 的加入量与金离子的加入量相同。
一种制备所述的基于核苷的原位快速合成的金银荧光纳米材料的方法， 步骤为： 第一步， 将含有顺式二羟基生物分子的溶液或人体体液与金试剂水溶液混合， 加入 柠檬酸钠缓冲溶液控制 PH6-7然后再充分混合， 水浴加热或者室温放置；
第二步， 反应一段时间后， 向混合溶液中加入 0. 025 mmol/L的银试剂水溶液， 冷却 即得到金银荧光纳米材料。
第二步中反应条件为 37 。C 孵育 3h或者 80 °0水浴加热 5分钟。 所述的基于核苷的原位快速合成的金银荧光纳米材料在制备对癌症疾病患者体液检 测试剂盒中的应用。
原理说明： 生物体中含有诸多顺式二羟基生物分子， 如糖蛋白和糖肽， 修饰核苷以 及单糖和多糖等， 这类化合物在许多生理过程中发挥着极其重要的作用。 当人体生理健 康状况发生病变时， 这些顺式二羟基生物分子在体液中的含量就会发生改变。 以这些顺 式二羟基生物分子作为模板， 在还原剂的作用下， 金、 银离子被还原， 利用金银等贵金 属纳米材料的优良荧光特性， 对生物体体液中的顺式二羟基生物分子进行监测， 达到对 疾病的诊断。
以不同浓度的核苷溶液与固定浓度的金试剂、 柠檬酸钠缓冲液混合， 再和固定浓度 的银试剂反应得到金银荧光纳米材料， 记录得到的荧光强度、 颜色等特征， 建立标准库。
分别以正常人的体液与与固定浓度的金试剂、 柠檬酸钠缓冲液混合， 再和固定浓度 的银试剂反应得到金银荧光纳米材料， 记录得到的荧光强度、 颜色等特征， 建立标准库。
再针对不同癌症患者的体液分别与固定浓度的金试剂、 柠檬酸钠缓冲液混合， 再和 固定浓度的银试剂反应得到金银荧光纳米材料， 记录得到的荧光强度、 颜色等特征与之 前采集到的标准库内的强度、 颜色等进行比对， 即可定性的判断病人的大体指征。 就类 似于 pH试纸的应用。
有益效果：
将不同浓度的核苷生物分子， 金试剂和柠檬酸钠缓冲液混合， 一定时间后， 形成具 有微弱荧光的金纳米材料， 然后加入银试剂， 立刻产生稳定直观的紫外灯下裸眼可视的 荧光。 用癌症等疾病患者和正常人的血清替代上述实验中的核苷等生物分子， 得到不同 的荧光发射谱， 实现了对癌症等疾病患者和正常人生理指标的检测区分。 本发明发现和 研制了核苷等生物分子为模板的原位合成贵金属金、 银纳米合金材料， 并利用其荧光特 性实现对癌症等疾病患者和正常人体液的区分和检测。 由于该方法具有操作简便、 结果 直观、 灵敏度高、 成本低等优点， 在疾病的早期诊断中具有潜在的应用前景。
该方法首次以核苷等生物分子为模板， 快速制备贵金属金银纳米合金材料并用于癌 症等疾病患者生理指标的快速检测。
该发明可实现对癌症等疾病患者生理指标的无损伤、 快速、 高灵敏、 简便、 直观检 测， 进一步结合荧光、 拉曼、 超声、 CT和核磁等， 可进行多形态与多模态的同步诊断及 准确靶向定位与治疗， 具有广阔的医学应用前景。 附图说明
图 1是本发明实施例 1光学图与在 365 nm光激发下的荧光图， 以及相对应的 370 nm 激发下的荧光发射图谱； 图中 A :胞苷 /金纳米材料； B :胞苷 /金银纳米材料。
图 2是本发明实施例 11， 12该方法用于癌症等疾病患者如白血病人与正常人血清检 测区分中的结果图。 通过荧光发射峰位置和荧光强度进行绘制的柱形图可以达到对癌症 患者和正常人血清的区分。
具体实施方式 下面结合附图说明本发明的实施例。 本发明保护范围不以实施例为限。 本实验的核苷等生物分子购自 Sigma-Aldrich公司， 氯金酸、 八氯化四金、 氯化金、 三氯化金、 一氯化金、 硝酸银、 银氨溶液、 柠檬酸钠和柠檬酸等试剂购于国药集团化学 试剂有限公司。
一种基于核苷等生物分子的原位快速合成金银荧光纳米材料的制备方法， 其特征在 于由如下步骤制得：
第一步， 配制一定浓度的含有顺式二羟基生物分子的溶液， 如糖蛋白、 糖肽、 核苷 (腺嘌呤核苷， 鸟嘌呤核苷， 胞嘧啶核苷， 胸腺嘧啶核苷， 尿嘧啶核苷中任一种）、 单糖 或多糖溶液。 金试剂和银试剂水溶液。
第二步， 将含有顺式二羟基生物分子的溶液或人体体液与金试剂按照顺式而羟基生 物分子与金离子摩尔比为 0. 4〜20 : 1的浓度混合， 加入一定量的柠檬酸钠溶液， 调节 pH 值为 6〜7， 将三者充分混合， 然后水浴加热或者室温放置。
第三步， 37°C孵育 3小时或者 80 °0水浴加热 5分钟后， 向混合溶液中加入与金试剂 等量的银试剂， 冷却即得到荧光优良的金银纳米合金； 其中加热时间， 加热温度， 核苷 生物分子和金试剂， 银试剂的比例都可以调节， 只是得到材料的荧光颜色不同。
所述金试剂是氯金酸、 八氯化四金、 氯化金、 三氯化金、 一氯化金中的一种或者任 意二种的混合溶液； 所述银试剂是银氨溶液、硝酸银溶液中的一种或者二种的混合溶液。
所述的一种基于核苷 （腺嘌呤核苷， 鸟嘌呤核苷， 胞嘧啶核苷， 胸腺嘧啶核苷， 尿 嘧啶核苷中任一种） 生物分子的原位快速合成金银荧光纳米材料方法以及该方法在癌症 等检测中的应用
实施例 1
一种基于核苷等生物分子的原位快速合成金银荧光纳米材料的制备方法， 由如下步 骤制得：
第一步， 配制一定浓度的胞嘧啶核苷、 金试剂和银试剂水溶液。
第二步， 将胞嘧啶核苷和金试剂按照核苷与金离子摩尔比为 16 : 1的比例混合， 加入 柠檬酸钠溶液调节 pH值 6〜7， 将三者充分混合， 然后水浴加热或者室温放置。
第三步， 37 °C孵育 3小时或者 80 °C加热 5分钟后， 向混合溶液中加入与金试剂等 量的银试剂， 冷却即得到荧光优良的金银纳米合金。
所述金试剂是氯金酸； 所述银试剂是硝酸银溶液。 所得材料在紫外灯下进行检测， 具体检测结果见图 1。
实施例 2
第一步， 配制一定浓度的胞嘧啶核苷生物分子， 金试剂和银试剂水溶液。
第二步， 将胞嘧啶核苷和金试剂按照摩尔比 16 : 1的比例混合， 加入一定量的柠檬酸 钠溶液调节 pH值 6〜7， 将三者充分混合， 然后水浴加热或者室温放置。
第三步， 37 °C孵育 3小时或者 80 °C加热 5分钟后， 向混合溶液中加入与金试剂等 量的银试剂， 冷却即得到荧光优良的金银纳米合金； 其中加热时间， 加热温度， 胞嘧啶 核苷和金试剂， 银试剂的比例都可以调节， 只是得到材料的荧光颜色不同。
所述金试剂是八氯化四金； 所述银试剂是硝酸银溶液。
实施例 3
第一步， 配制一定浓度的胞嘧啶核苷， 金试剂和银试剂水溶液。
第二步， 将胞嘧啶核苷和金试剂按照 16 : 1的比例混合， 加入一定量的柠檬酸钠溶液 调节 pH值 6〜7， 将三者充分混合， 然后水浴加热或者室温放置。
第三步， 37 °C孵育 3小时或者 80 °C加热 5分钟后， 向混合溶液中加入与金试剂等 量的银试剂， 冷却即得到荧光优良的金银纳米合金； 其中加热时间， 加热温度， 胞嘧啶 核苷和金试剂， 银试剂的比例都可以调节， 只是得到材料的荧光颜色不同。
所述金试剂是氯化金； 所述银试剂是硝酸银溶液。
实施例 4
第一步， 配制一定浓度的胞嘧啶核苷， 金试剂和银试剂水溶液。
第二步， 将胞嘧啶核苷和金试剂按照 16 : 1的比例混合， 加入一定量的柠檬酸钠溶液 调节 pH值 6〜7， 将三者充分混合， 然后水浴加热或者室温放置。
第三步， 37 °C孵育 3小时或者 80 °C加热 5分钟后， 向混合溶液中加入与金试剂等 量的银试剂， 冷却即得到荧光优良的金银纳米合金； 其中加热时间， 加热温度， 胞嘧啶 核苷和金试剂， 银试剂的比例都可以调节， 只是得到材料的荧光颜色不同。
所述金试剂是三氯化金； 所述银试剂是硝酸银溶液。
实施例 5
第一步， 配制一定浓度的胞嘧啶核苷， 金试剂和银试剂水溶液。
第二步， 将胞嘧啶核苷和金试剂按照 16 : 1的比例混合， 加入一定量的柠檬酸钠溶液 调节 pH值 6〜7， 将三者充分混合， 然后水浴加热或者室温放置。 第三步， 37 °C孵育 3小时或者 80 °C加热 5分钟后， 向混合溶液中加入与金试剂等 量的银试剂， 冷却即得到荧光优良的金银纳米合金； 其中加热时间， 加热温度， 胞嘧啶 核苷和金试剂， 银试剂的比例都可以调节， 只是得到材料的荧光颜色不同。
所述金试剂是一氯化金； 所述银试剂是硝酸银溶液。
实施例 6
第一步， 配制一定浓度的胞嘧啶核苷， 金试剂和银试剂水溶液。
第二步， 将胞嘧啶核苷和金试剂按照 16 : 1的比例混合， 加入一定量的柠檬酸钠溶液 调节 pH值 6〜7， 将三者充分混合， 然后水浴加热或者室温放置。
第三步， 37 °C孵育 3小时或者 80 °C加热 5分钟后， 向混合溶液中加入与金试剂等 量的银试剂， 冷却即得到荧光优良的金银纳米合金； 其中加热时间， 加热温度， 胞嘧啶 核苷和金试剂， 银试剂的比例都可以调节， 只是得到材料的荧光颜色不同。
所述金试剂是氯金酸； 所述银试剂是银氨溶液。
实施例 7
第一步， 配制一定浓度的胞嘧啶核苷， 金试剂和银试剂水溶液。
第二步， 将胞嘧啶核苷和金试剂按照 16 : 1的比例混合， 加入一定量的柠檬酸钠溶液 调节 pH值 6〜7， 将三者充分混合， 然后水浴加热或者室温放置。
第三步， 37 °C孵育 3小时或者 80 °C加热 5分钟后， 向混合溶液中加入与金试剂等 量的银试剂， 冷却即得到荧光优良的金银纳米合金； 其中加热时间， 加热温度， 胞嘧啶 核苷和金试剂， 银试剂的比例都可以调节， 只是得到材料的荧光颜色不同。
所述金试剂是八氯化四金； 所述银试剂是银氨溶液。
实施例 8
第一步， 配制一定浓度的胞嘧啶核苷， 金试剂和银试剂水溶液。
第二步， 将胞嘧啶核苷和金试剂按照 16 : 1的比例混合， 加入一定量的柠檬酸钠溶液 调节 pH值 6〜7， 将三者充分混合， 然后水浴加热或者室温放置。
第三步， 37 °C孵育 3小时或者 80 °C加热 5分钟后， 向混合溶液中加入与金试剂等 量的银试剂， 冷却即得到荧光优良的金银纳米合金； 其中加热时间， 加热温度， 胞嘧啶 核苷和金试剂， 银试剂的比例都可以调节， 只是得到材料的荧光颜色不同。
所述金试剂是氯化金； 所述银试剂是银氨溶液。
实施例 9 第一步， 配制一定浓度的胞嘧啶核苷， 金试剂和银试剂水溶液。
第二步， 将胞嘧啶核苷和金试剂按照 16 : 1的比例混合， 加入一定量的柠檬酸钠溶液 调节 pH值 6〜7， 将三者充分混合， 然后水浴加热或者室温放置。
第三步， 37 °C孵育 3小时或者 80 °C加热 5分钟后， 向混合溶液中加入与金试剂等 量的银试剂， 冷却即得到荧光优良的金银纳米合金； 其中加热时间， 加热温度， 胞嘧啶 核苷和金试剂， 银试剂的比例都可以调节， 只是得到材料的荧光颜色不同。
所述金试剂是三氯化金； 所述银试剂是银氨溶液。
实施例 10
第一步， 配制一定浓度的胞嘧啶核苷， 金试剂和银试剂水溶液。
第二步， 将胞嘧啶核苷和金试剂按照 16 : 1的比例混合， 加入一定量的柠檬酸钠溶液 调节 pH值 6〜7， 将三者充分混合， 然后水浴加热或者室温放置。
第三步， 37 °C孵育 3小时或者 80 °C加热 5分钟后， 向混合溶液中加入与金试剂等 量的银试剂， 冷却即得到荧光优良的金银纳米合金； 其中加热时间， 加热温度， 胞嘧啶 核苷和金试剂， 银试剂的比例都可以调节， 只是得到材料的荧光颜色不同。
所述金试剂是一氯化金； 所述银试剂是银氨溶液。
实施例 11 生物样本检测
将 40 μΐ癌症等疾病患者的血清与浓度为 0. 025 mmol/L的金试剂按照核苷与金离子 摩尔比 16: 1的比例混合，加入一定量的柠檬酸钠溶液调节 pH值 6〜7，将三者充分混合， 然后 37°C水浴加热。 孵育 3小时后， 向混合溶液中加入含有与金离子等物质的量的银离 子的银试剂，冷却即得到荧光优良的金银纳米合金;所得材料在荧光仪下检测结果见图 2。
所述金试剂是氯金酸； 所述银试剂是硝酸银溶液。
实施例 12
将 40 μΐ正常人的血清与浓度为 0. 025 mmol/L的金试剂混合， 按照核苷与金离子摩 尔比 16: 1的比例混合， 加入一定量的柠檬酸钠溶液调节 pH值 6〜7， 将三者充分混合， 然后 37 °0水浴加热。孵育 3小时后， 向混合溶液中加入与金试剂等量的银试剂， 冷却即 得到荧光优良的金银纳米合金； 其中加热时间， 加热温度， 金试剂， 银试剂的比例都可 以调节， 只是得到材料的荧光颜色不同。
所述金试剂是氯金酸； 所述银试剂是硝酸银溶液。
实施例 13 将 1. 44 mL癌症等疾病患者的尿液与浓度为 0. 025 mmol/L的金试剂按照尿液中含有 的核苷的物质的量与金离子的物质的量之比为 0. 4 : 1 的比例混合， 加入一定量的柠檬酸 钠溶液调节 pH值 6〜7， 将三者充分混合， 然后 37 °C水浴加热。 孵育 3小时后， 向混合 溶液中加入与金试剂等量的银试剂， 冷却即得到荧光优良的金银纳米合金； 其中加热时 间， 加热温度， 金试剂， 银试剂的比例都可以调节， 只是得到材料的荧光颜色不同。
所述金试剂是八氯化四金； 所述银试剂是银氨溶液。
实施例 14
将 1. 44 mL正常人的尿液与浓度为 0. 025 mmol/L的金试剂按照尿液含有的核苷的物 质的量与金离子的物质的量之比为 20 : 1的比例混合，加入一定量的柠檬酸钠溶液调节 pH 值 6〜7， 将三者充分混合， 然后 37 °C水浴加热。 孵育 3小时后， 向混合溶液中加入与 金试剂等量的银试剂， 冷却即得到荧光优良的金银纳米合金； 其中加热时间， 加热温度， 金试剂， 银试剂的比例都可以调节， 只是得到材料的荧光颜色不同。
所述金试剂是氯化金； 所述银试剂是硝酸银溶液与银氨溶液的混合液。
实施例 15
将 40 μΐ癌症等疾病患者的唾液与浓度为 0. 025 mmol/L的金试剂按照唾液中含有的 核苷的物质的量与金离子的物质的量之比为 4 : 1 的比例混合， 加入一定量的柠檬酸钠溶 液调节 pH值 6〜7， 将三者充分混合， 然后 37°C水浴加热。 孵育 3小时后， 向混合溶液 中加入与金试剂等量的银试剂， 冷却即得到荧光优良的金银纳米合金； 其中加热时间， 加热温度， 金试剂， 银试剂的比例都可以调节， 只是得到材料的荧光颜色不同。
所述金试剂是氯金酸与三氯化金混合物； 所述银试剂是硝酸银溶液。
实施例 16
将 40 μΐ正常人的唾液与浓度为 0. 025 mmol/L的金试剂按照唾液中含有的核苷的物 质的量与金离子的物质的量之比为 10 : 1的比例混合， 加入一定量的柠檬酸钠溶液， 将三 者充分混合， 然后 37 °0水浴加热。孵育 3小时后， 向混合溶液中加入与金试剂等量的银 试剂， 冷却即得到荧光优良的金银纳米合金； 其中加热时间， 加热温度， 金试剂， 银试 剂的比例都可以调节， 只是得到材料的荧光颜色不同。
所述金试剂是氯金酸； 所述银试剂是硝酸银溶液。
由以上具体实例结果表明： 以核苷为模板的荧光增强的金银混合溶液的制备对癌症 患者通过体液对其生理指标进行检测具有良好的效果。
本发明以胞嘧啶核苷为模板原位合成荧光增强的金银纳米合金并把该方法用于癌症 等疾病患者体液的检测， 实现对其生理指标的直观快速诊断。 该方法是首次在癌症等疾 病患者体液检测中应用， 方法简单， 快速， 直观， 成本低， 为癌症的诊断提供了高效可 行的方法。
以上所述仅是本发明的优选实施方式， 应当指出： 对于本技术领域的技术人员来说， 在不脱离本发明原理的前提下， 还可以做出若干改进和润饰， 这些改进和润饰也应视为 本发明的保护范围。

Claims

权利要求书
1. 一种基于核苷等生物分子的原位快速合成金银荧光纳米材料， 其特征在于， 以含有顺 式二羟基生物分子的溶液或人体体液与金试剂水溶液混合， 加入柠檬酸钠缓冲溶液反应 得到具有微弱荧光的金纳米材料， 再加入银试剂水溶液继续反应后得到荧光增强的金银 荧光纳米材料。
2.如权利要求 1 所述的基于核苷的原位快速合成的金银荧光纳米材料， 其特征在于， 所 述的含有顺式二羟基生物分子的溶液包括糖蛋白、 糖肽、 核苷、 单糖或多糖溶液。
3.如权利要求 1 所述的基于核苷的原位快速合成的金银荧光纳米材料， 其特征在于， 所 述的核苷包括腺嘌呤核苷， 鸟嘌呤核苷， 胞嘧啶核苷， 胸腺嘧啶核苷， 尿嘧啶核苷中的 任一种。
4. 如权利要求 1 所述的基于核苷的原位快速合成的金银荧光纳米材料， 其特征在于， 所 述的人体体液包括唾液， 尿液， 血清。
5. 如权利要求 1 所述的基于核苷的原位快速合成的金银荧光纳米材料， 其特征在于， 所 述的金试剂包括氯金酸、 八氯化四金、 氯化金、 三氯化金、 一氯化金中的任意一种或者 任意二种的混合溶液。
6. 如权利要求 1 所述的基于核苷的原位快速合成的金银荧光纳米材料， 其特征在于， 所 述的银试剂为银氨溶液或硝酸银溶液或者二种的混合溶液。
7. 如权利要求 1 所述的基于核苷的原位快速合成的金银荧光纳米材料， 其特征在于， 含 有顺式二羟基生物分子的溶液或人体体液与金试剂水溶液混合时按照其中含有的顺式二 羟基生物分子的物质的量与金离子的物质的量之比为 0. 4〜20 : 1 的比例混合， 银试剂的 加入量与金离子的加入量相同。
8.一种制备权利要求 1〜7 任一所述的基于核苷的原位快速合成的金银荧光纳米材料的方 法， 其特征在于， 步骤为：
第一步， 将含有顺式二羟基生物分子的溶液或人体体液与金试剂水溶液混合， 加入 柠檬酸钠缓冲溶液控制 PH6-7然后再充分混合， 水浴加热或者室温放置；
第二步， 反应一段时间后， 向混合溶液中加入 0. 025 mmol/L 的银试剂水溶液， 冷却 即得到金银荧光纳米材料。
9.如权利要求 8 所述的制备基于核苷的原位快速合成的金银荧光纳米材料的方法， 其特 征在于， 第二步中反应条件为 37 。C 孵育 3h或者 80 °0水浴加热 5分钟。
10.权利要求 1〜7 任一所述的基于核苷的原位快速合成的金银荧光纳米材料在制备对癌 症疾病患者体液检测试剂盒中的应用。
PCT/CN2014/071547 2014-01-16 2014-01-27 一种基于核苷等生物分子的原位快速合成金银荧光纳米材料及其制备方法和应用 WO2015106466A1 (zh)

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CN110202128A (zh) * 2019-06-25 2019-09-06 重庆科技学院 一种金银复合纳米簇、制备工艺及在生物硫醇检测中的应用
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Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CAO, Y.C. ET AL.: "Nanoparticles with Raman Spectroscopic Fingerprints for DNA and RNA Detection", SCIENCE, vol. 29, 30 August 2002 (2002-08-30) *
LI, LI ET AL.: "Sensitive and selective detection of cysteine using gold nanoparticles as colorimetric probes", ANALYST, vol. 134, no. 7, 2 April 2009 (2009-04-02) *
LIU, XIAOLAN ET AL.: "Application of Nanogold-based Colorimetry in Nucleic Acid Detection", JOURNAL OF MEDICAL MOLECULAR BIOLOGY, vol. 8, no. 6, 31 December 2011 (2011-12-31) *
ZHANG, WEIMIN ET AL.: "Determination of 6-Mercaptopurine with Gold Nanoparticles-based Colorimetric Method", CHINESE JOURNAL OF SPECTROSCOPY LABORATORY, vol. 30, no. 4, 31 July 2013 (2013-07-31) *
ZHOU, HUALAN ET AL.: "Nanoparticle-based Colorimetric Sensing Assay and Its Applications in Chemical and Biological Detections", JOURNAL OF INSTRUMENTAL ANALYSIS, vol. 31, no. 5, 31 May 2012 (2012-05-31) *

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