WO2019237769A1 - 基于银纳米簇复合物的三聚氰胺比率荧光探针的制备方法 - Google Patents

基于银纳米簇复合物的三聚氰胺比率荧光探针的制备方法 Download PDF

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WO2019237769A1
WO2019237769A1 PCT/CN2019/076942 CN2019076942W WO2019237769A1 WO 2019237769 A1 WO2019237769 A1 WO 2019237769A1 CN 2019076942 W CN2019076942 W CN 2019076942W WO 2019237769 A1 WO2019237769 A1 WO 2019237769A1
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melamine
rhodamine
solution
silver
complex
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French (fr)
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桂日军
付永鑫
金辉
卜祥宁
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青岛大学
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    • 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"
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    • C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
    • 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"
    • G01N2021/6432—Quenching

Definitions

  • the invention belongs to the technical field of preparation of precious metal nanomaterials and ratio fluorescent probes, and particularly relates to a method for preparing a melamine ratio fluorescent probe based on a DNA-stabilized silver nanocluster and a rhodamine 6G complex.
  • the prepared probe can be used for Highly sensitive detection of melamine.
  • Melamine is a triazine nitrogen-containing heterocyclic organic compound with a nitrogen content of up to 66%. It is commonly used in many industries such as plastics, pesticides and fertilizers. Adding 1% melamine can increase the protein content in food by more than 4%. Some companies use melamine instead of protein to obtain high profits.
  • the US Food and Drug Administration and the Chinese Ministry of Health have a safety limit of 1 mg / kg for infant formula and 2.5 mg / kg for adult food. Detection of melamine in daily food is of great significance to people's healthy growth.
  • the detection methods of melamine mainly include high performance liquid chromatography, gas chromatography and mass spectrometry, spectrophotometry, surface enhanced Raman scattering and so on. These traditional detection methods generally have the problems of time-consuming sample preparation, complicated operation, expensive instruments, and high detection costs.
  • the development of a low-cost, simple, fast, and efficient melamine detection method has become a key technical problem that needs to be solved urgently.
  • Fluorescence method is an analysis method that uses the substance itself or its surface-modified functional group to react with the detection substance to cause changes in the fluorescence intensity or the position of the spectral line, thereby performing substance identification and content determination.
  • This method has the advantages of simple operation and high sensitivity.
  • Literature search shows that Wu et al.
  • the ratio fluorescence method is a method that uses a dual-emission fluorescent probe to react with a detection substance and quantitatively detects the ratio of fluorescence intensity at two different wavelengths.
  • the ratio fluorescence method has a self-calibration function, which can eliminate the fluorescence interference caused by the system itself and environmental factors, effectively improving the accuracy and reliability of the detection results of the target.
  • Silver nanoclusters have unique physical and chemical properties, such as strong fluorescence emission, good light stability, high biocompatibility, and sub-nano size. They can be used in many fields such as nanomedicine, bioimaging, drug delivery, and biochemical sensing.
  • Rhodamine 6G is a widely used organic dye with the advantages of high light stability, pH insensitivity, and high fluorescence quantum yield.
  • the present invention first constructs a dual-emission fluorescent probe based on a DNA-stabilized silver nanocluster / rhodamine 6G complex and applies it to the ratio fluorescence detection of melamine in actual samples. So far, no domestic or foreign literature and patent reports on melamine ratio fluorescent probes based on DNA-stabilized silver nanoclusters and rhodamine 6G complexes have been published.
  • the purpose of the present invention is to overcome the shortcomings of the prior art described above, and to design a melamine ratio fluorescent probe based on a DNA-stabilized silver nanocluster and rhodamine 6G complex that is simple and easy to obtain, low cost, and high sensitivity.
  • the present invention relates to a method for preparing a melamine ratio fluorescent probe based on a silver nanocluster complex.
  • the preparation process includes the following steps:
  • step (3) Disperse the complex prepared in step (2) in 100 microliters of citrate water buffer, incubate in a dark place for 30 minutes, and measure the homogeneous solution of the complex under different rhodamine 6G molar concentrations. Fluorescence emission spectrum, optimize the intensity of the dual emission fluorescence peaks to determine the ratio of silver nanoclusters to rhodamine 6G;
  • the size of the silver nanoclusters described in step (1) according to the present invention is 6 to 12 nanometers, and the molar concentrations of silver nitrate, DNA, and sodium borohydride are 5 to 10 mmol / L and 200 to 600 ⁇ mol / L, respectively.
  • the pH of the citrate aqueous buffer described in step (2) is 5.5 to 6.5, and the molar concentration of rhodamine 6G is 0.1 to 2 nanomol / L; as described in step (3)
  • the mass concentration of the complex solution is 1 to 5 mg / ml; the concentration range of the melamine in step (4) is 0.5 to 15 micromoles / liter, and the detection limit is 0.05 to 0.2 micromoles / liter.
  • the present invention uses electrostatic self-assembly technology to construct a DNA-stabilized silver nanocluster / rhodamine 6G complex.
  • the absorption spectrum of rhodamine 6G and the fluorescence emission spectrum of the silver nanocluster partially overlap, and fluorescence occurs between the two.
  • Resonance energy transfer caused the fluorescence quenching of silver nanoclusters, while the rhodamine 6G fluorescence increased.
  • the added melamine can form a strong hydrogen bond with thymine on the surface of the DNA-stabilized silver nanocluster, dissociate rhodamine 6G from the surface of the silver nanocluster, increase the distance between the donor and the acceptor, and weaken the fluorescence resonance energy transfer.
  • a melamine ratio fluorescent probe can be constructed.
  • the preparation process of the probe is simple, the cost is low, and the product sensitivity is high.
  • the probe can be developed into a novel melamine ratio fluorescent probe for effective detection of melamine in actual samples.
  • FIG. 1 is a schematic diagram of a method for preparing a melamine ratio fluorescent probe based on a DNA-stabilized silver nanocluster and a rhodamine 6G complex according to the present invention
  • FIG. 2 shows the response to the fluorescence emission peak intensity of a DNA-stabilized silver nanocluster and rhodamine 6G complex as the molar concentration of rhodamine 6G increases;
  • FIG. 4 is a linear relationship between the fluorescence peak intensity ratio of the DNA-stabilized silver nanocluster and the rhodamine 6G complex and the molar concentration of melamine.
  • This embodiment relates to a method for preparing a melamine ratio fluorescent probe based on a DNA-stabilized silver nanocluster and a rhodamine 6G complex.
  • the preparation process and principle of melamine ratio fluorescence detection are shown in FIG. 1. The specific process steps are as follows:
  • DNA-stable silver nanoclusters At 0 ° C, add a certain amount of silver nitrate solution and DNA solution to 1 ml of secondary distilled water, magnetically stir for 20 minutes to form a homogeneous mixed solution, and then add freshly prepared sodium borohydride The solution was stirred and reacted for 20 minutes in a dark place.
  • the molar concentrations of silver nitrate, DNA and sodium borohydride were 5 mmol / L, 200 ⁇ mol / L and 5 mmol / L, respectively.
  • the obtained product solution was filtered through a 0.4 micron filter membrane, and the filtrate was dialyzed by a dialysis bag with a molecular weight cut-off of 5000 Daltons to remove unreacted experimental raw materials.
  • the solution in the dialysis bag was subjected to a rotary evaporation treatment to remove 90% of the solvent, and then Freeze-dried to obtain dry samples of silver nanoclusters, stored at 4 ° C and protected from light;
  • Preparation of silver nanocluster / rhodamine 6G complex dissolve the prepared silver nanocluster in 200 microliters of secondary distilled water, add 100 microliters of citrate water buffer (pH 5.5) and mix, and then add 100 microliters A rhodamine 6G solution of 0.1 to 0.5 nanomole / liter, the mixed solution is reacted in a dark place for 30 minutes, and the product solution is centrifuged, washed with ethanol, and dried under vacuum to obtain a silver nanocluster / rhodamine 6G complex;
  • the prepared complex was dispersed in 100 microliters of citrate water buffer solution, the mass concentration of the complex was 1 to 2 mg / ml, and incubated in a dark place for 30 minutes, and the rhodamine 6G molar concentrations were measured. Fluorescence emission spectrum of the homogeneous solution of the complex, and optimize the intensity of the dual emission fluorescence peaks to determine the ratio of silver nanoclusters to rhodamine 6G (see Figure 2);
  • the specific process steps for preparing DNA-stabilized silver nanoclusters in this example are the same as in Example 1.
  • the molar concentrations of silver nitrate, DNA, and sodium borohydride are 8 mmol / L, 400 ⁇ mol / L, and 7 mmol, respectively. /Rise.
  • the prepared silver nanoclusters were dissolved in 200 microliters of secondary distilled water, 100 microliters of citrate water buffer solution (pH 6.0) was added, and 100 microliters of 0.2 to 1 nanomole / liter of rhodamine 6G were added. The solution and the mixed solution were reacted in a dark place for 30 minutes.
  • the product solution was centrifuged, washed with ethanol, and dried under vacuum to obtain a silver nanocluster / rhodamine 6G complex.
  • the fluorescence emission spectrum of the homogeneous solution of the complex was optimized to optimize the intensity of the dual emission fluorescence peaks to determine the ratio of silver nanoclusters to rhodamine 6G.
  • the specific process steps for preparing DNA-stabilized silver nanoclusters in this example are the same as in Example 1.
  • the molar concentrations of silver nitrate, DNA, and sodium borohydride are 10 mmol / L, 600 ⁇ mol / L, and 10 mmol, respectively. /Rise.
  • the prepared silver nanoclusters were dissolved in 200 microliters of secondary distilled water, 100 microliters of citrate water buffer (pH 6.5) was added and mixed, and then 100 microliters of 0.2 to 2 nanomole / liter of rhodamine 6G were added. The solution and the mixed solution were reacted in a dark place for 30 minutes.
  • the product solution was centrifuged, washed with ethanol, and dried under vacuum to obtain a silver nanocluster / rhodamine 6G complex.
  • the fluorescence emission spectrum of the homogeneous solution of the complex was optimized to optimize the intensity of the dual emission fluorescence peaks to determine the ratio of silver nanoclusters to rhodamine 6G.
  • This embodiment relates to the application of the melamine ratio fluorescent probe prepared in Example 1, which is used for the detection of melamine in an actual sample such as milk.
  • Milk is mixed with melamine and acetonitrile with different concentrations, and the mixture is prepared by sonication for 30 minutes, and then centrifuged at 14000 rpm for 15 minutes. The supernatant is filtered and the filtrate is further diluted 25 times and collected for further testing.
  • the detection range of the melamine molar concentration is 0.5 to 15 micromoles / liter, the detection limit is 0.09 micromoles / liter, the detection recovery rate is 99.8 to 100.5%, and the relative standard deviation is 1.1 to 2.1%.
  • ratio fluorescence method is used to detect melamine, and the detection recovery of melamine in milk samples is It is 94.0 to 102.0%, and the relative standard deviation is 1.2 to 3.2%.
  • the ratio fluorescence probe of the invention has higher detection recovery rate, lower relative standard deviation, simple preparation process, low cost and high product sensitivity, and can be developed into a novel melamine ratio fluorescence probe, which is suitable for different practical samples. Efficient detection of melamine.

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Abstract

本发明属于贵金属纳米材料和比率荧光探针的制备技术领域，具体涉及一种基于DNA稳定的银纳米簇与罗丹明6G复合物的三聚氰胺比率荧光探针的制备方法。采用静电自组装构建了银纳米簇/罗丹明6G复合物；三聚氰胺与银纳米簇表面DNA中胸腺嘧啶形成氢键，使罗丹明6G从银纳米簇表面解离，荧光共振能量转移被破坏，银纳米簇荧光恢复。此过程对罗丹明6G荧光的影响甚微，作为参比信号，银纳米簇荧光作为响应信号。拟合银纳米簇与罗丹明6G荧光发射峰强度比率与三聚氰胺摩尔浓度之间的线性关系，构建出三聚氰胺比率荧光探针。该探针的制备工艺简单、成本低、产品灵敏度高，可发展为一种新颖的三聚氰胺比率荧光探针，用于实际样品中三聚氰胺的有效检测。

Description

基于银纳米簇复合物的三聚氰胺比率荧光探针的制备方法 技术领域
本发明属于贵金属纳米材料和比率荧光探针的制备技术领域，具体涉及一种基于DNA稳定的银纳米簇与罗丹明6G复合物的三聚氰胺比率荧光探针的制备方法，其制备的探针可用于三聚氰胺的高灵敏度检测。
背景技术
三聚氰胺是一种三嗪含氮杂环有机化合物，氮含量高达66％，常用于塑料、杀虫剂和肥料等诸多行业。添加1％的三聚氰胺可在食品中增加4％以上的蛋白含量，某些企业使用三聚氰胺代替蛋白质来获得高额利润。美国食品和药物管理局和中国卫生部对婴幼儿配方奶粉的安全限值为1毫克/千克，成人食品为2.5毫克/千克。检测日常食品中三聚氰胺的含量对人们的健康成长具有重要的意义。目前，检测三聚氰胺的方法主要包括高效液相色谱法、气相色谱与质谱联用法、分光光度法、表面增强拉曼散射法等。这些传统的检测方法普遍存在样品制备耗时、操作复杂、仪器昂贵、检测成本高等问题，发展一种低成本、简单、快速和高效的三聚氰胺检测方法已成为当前亟待解决的关键技术问题。
荧光法是利用物质本身或其表面修饰的官能团与检测物反应引起荧光强度或谱线位置发生变化，从而进行物质鉴别和含量测定的分析方法。该方法具有操作简便、灵敏度高等优点。文献检索表明：Wu等利用上转换纳米粒与金纳米粒之间荧光共振能量转移检测三聚氰胺(An upconversion fluorescence resonance energy transfer nanosensor for one step detection ofmelamine in raw milk,Qiongqiong Wu,Qian Long,Haitao Li,Youyu Zhang,Shouzhuo Yao,Talanta,2015,136,47-53)；Kalaiyarasa等基于金纳米簇设计了三聚氰胺荧光传感器(Melamine dependent fluorescence of glutathione protected gold nanoclusters and ratiometric quantification of melamine in commercial cow milk and infant formula, Gopi Kalaiyarasan,Anusuya K,James Joseph,Appl.Surf.Sci.,2017,420,963-969)；姜翠凤等报道了一种基于双光子激发荧光检测三聚氰胺的方法(专利公开号CN105158225A)。
比率荧光法是一种利用双发射荧光探针与检测物反应，以两个不同波长处荧光强度比值来定量检测的方法。比率荧光法具有自校准功能，可消除体系本身和环境因素产生的荧光干扰，有效提升了目标物检测结果的准确性和可靠性。银纳米簇具有独特的理化特性，如荧光发射强，光稳定性好，生物相容性高、亚纳米尺寸等优点，可用于纳米医学、生物成像、药物运载、生化传感等诸多领域。罗丹明6G是一种广泛使用的有机染料，具有光稳定性高、pH不敏感和高荧光量子产率等优点。尽管有关三聚氰胺荧光检测的文献已有报道，本发明首次构建了基于DNA稳定银纳米簇/罗丹明6G复合物的双发射荧光探针，并将其应用于实际样品中三聚氰胺的比率荧光检测。迄今，尚未有基于DNA稳定的银纳米簇与罗丹明6G复合物的三聚氰胺比率荧光探针的国内外文献和专利报道。
发明内容
本发明的目的在于克服上述现有技术存在的不足，设计一种方法简单易得、成本低廉、灵敏度高的基于DNA稳定的银纳米簇与罗丹明6G复合物的三聚氰胺比率荧光探针。
为了实现上述目的，本发明涉及的一种基于银纳米簇复合物的三聚氰胺比率荧光探针的制备方法其制备工艺包括以下步骤：
(1)制备DNA稳定的银纳米簇：在0℃下，向1毫升二次蒸馏水中加入一定量的硝酸银溶液与DNA溶液，磁力搅拌20分钟以形成均质混合液，然后加入新配制的硼氢化钠溶液，在避光处剧烈搅拌反应20分钟。所得产物溶液采用0.4微米的滤膜过滤，滤液经截留分子量为5000道尔顿的透析袋透析处理，以去除未反应的实验原料，将透析袋中溶液进行旋蒸处理以去除90％溶剂，再进行冷冻干燥得到银纳米簇干样，在 4℃避光条件下储存备用；
(2)制备银纳米簇/罗丹明6G复合物：将步骤(1)中制得的银纳米簇溶解在200微升二次蒸馏水中，加入100微升柠檬酸盐水缓冲液混匀，再加入100微升不同浓度的罗丹明6G溶液，混合液在避光处反应30分钟，产物溶液采用离心分离、乙醇洗涤和真空干燥处理，得到银纳米簇/罗丹明6G复合物；
(3)将步骤(2)中制得的复合物分散在100微升柠檬酸盐水缓冲液中，在避光处孵化30分钟，分别测定不同罗丹明6G摩尔浓度下，复合物均质溶液的荧光发射光谱，优化双发射荧光峰强度以确定银纳米簇与罗丹明6G的配比；
(4)在室温和缓慢磁力搅拌下，向步骤(3)中制得的优化配比复合物均质溶液中加入15微升不同浓度的三聚氰胺溶液，继续搅拌5分钟，使其充分反应形成复合物与三聚氰胺的均质溶液，测定不同的三聚氰胺摩尔浓度下，复合物与三聚氰胺均质溶液的荧光发射光谱，拟合银纳米簇与罗丹明6G荧光发射峰强度比率与三聚氰胺摩尔浓度之间的线性关系，构建三聚氰胺比率荧光探针。
本发明涉及的步骤(1)中所述的银纳米簇尺寸为6～12纳米，硝酸银、DNA和硼氢化钠的摩尔浓度分别为5～10毫摩尔/升、200～600微摩尔/升和5～10毫摩尔/升；步骤(2)中所述的柠檬酸盐水缓冲液pH为5.5～6.5，罗丹明6G摩尔浓度为0.1～2纳摩尔/升；步骤(3)中所述的复合物溶液质量浓度为1～5毫克/毫升；步骤(4)中所述的三聚氰胺的浓度范围为0.5～15微摩尔/升，检测限为0.05～0.2微摩尔/升。
本发明与现有技术相比，采用静电自组装技术构建了DNA稳定的银纳米簇/罗丹明6G复合物，罗丹明6G的吸收光谱与银纳米簇的荧光发射光谱部分重叠，二者发生荧光共振能量转移，引起银纳米簇荧光猝灭，而罗丹明6G荧光增强。外加的三聚氰胺可与DNA稳定的银纳米簇表面胸腺嘧啶形成强烈的氢键作用，使罗丹明6G从银纳米簇表面解 离，供体与受体间距离增大，荧光共振能量转移减弱，从而使银纳米簇荧光恢复。此过程对罗丹明6G荧光的影响甚微，可作为参比信号，而银纳米簇荧光作为响应信号。通过拟合银纳米簇与罗丹明6G荧光发射峰强度比率与三聚氰胺摩尔浓度之间的线性关系，可构建出三聚氰胺比率荧光探针。该探针的制备工艺简单、成本低、产品灵敏度高，可发展为一种新颖的三聚氰胺比率荧光探针，用于实际样品中三聚氰胺的有效检测。
附图说明
图1为本发明涉及的一种基于DNA稳定的银纳米簇与罗丹明6G复合物的三聚氰胺比率荧光探针的制备方法示意图；
图2为随着罗丹明6G摩尔浓度增大对DNA稳定的银纳米簇与罗丹明6G复合物荧光发射峰强度的响应；
图3为随着三聚氰胺摩尔浓度增大对DNA稳定的银纳米簇与罗丹明6G复合物荧光发射峰强度的响应；
图4为DNA稳定的银纳米簇与罗丹明6G复合物的荧光峰强度比率与三聚氰胺摩尔浓度之间拟合的线性关系。
具体实施方式
下面结合附图并通过具体实施例对本发明进行详细说明。
实施例1
本实施例涉及的一种基于DNA稳定的银纳米簇与罗丹明6G复合物的三聚氰胺比率荧光探针的制备方法，其制备工艺与三聚氰胺比率荧光检测原理参见图1所示，具体工艺步骤为：
制备DNA稳定的银纳米簇：在0℃下，向1毫升二次蒸馏水中加入一定量的硝酸银溶液与DNA溶液，磁力搅拌20分钟以形成均质混合液，然后加入新配制的硼氢化钠溶液，在避光处剧烈搅拌反应20分钟，其中硝酸银、DNA和硼氢化钠的摩尔浓度分别为5毫摩尔/升、200微摩尔/升和5毫摩尔/升。所得产物溶液采用0.4微米的滤膜过滤，滤液经 截留分子量为5000道尔顿的透析袋透析处理，以去除未反应的实验原料，将透析袋中溶液进行旋蒸处理以去除90％溶剂，再进行冷冻干燥得到银纳米簇干样，在4℃避光条件下储存备用；
制备银纳米簇/罗丹明6G复合物：将制得的银纳米簇溶解在200微升二次蒸馏水中，加入100微升柠檬酸盐水缓冲液(pH 5.5)混匀，再加入100微升0.1～0.5纳摩尔/升的罗丹明6G溶液，混合液在避光处反应30分钟，产物溶液采用离心分离、乙醇洗涤和真空干燥处理，得到银纳米簇/罗丹明6G复合物；
将制得的复合物分散在100微升柠檬酸盐水缓冲液中，复合物的质量浓度为1～2毫克/毫升，在避光处孵化30分钟，分别测定不同罗丹明6G摩尔浓度下，复合物均质溶液的荧光发射光谱，优化双发射荧光峰强度以确定银纳米簇与罗丹明6G的配比(参见图2)；
在室温和缓慢磁力搅拌下，向制得的优化配比复合物均质溶液中加入15微升不同浓度的三聚氰胺溶液，继续搅拌5分钟，使其充分反应形成复合物与三聚氰胺的均质溶液，测定不同的三聚氰胺摩尔浓度下，复合物与三聚氰胺均质溶液的荧光发射光谱(参见图3)，拟合银纳米簇与罗丹明6G荧光发射峰强度比率F DNA-AgNCs/F Rh6G与三聚氰胺摩尔浓度C MA之间的线性关系为：F DNA-AgNCs/F Rh6G＝0.06014C MA+0.5612(R 2＝0.9959)(参见图4)，构建三聚氰胺比率荧光探针，其中检测三聚氰胺的线性浓度范围为2～10微摩尔/升，三聚氰胺的检测极限为0.2微摩尔/升。
实施例2
本实施例中的制备DNA稳定的银纳米簇其具体工艺步骤同实施例1，其中硝酸银、DNA和硼氢化钠的摩尔浓度分别为8毫摩尔/升、400微摩尔/升和7毫摩尔/升。将制得的银纳米簇溶解在200微升二次蒸馏水中，加入100微升柠檬酸盐水缓冲液(pH 6.0)混匀，再加入100微升0.2～1纳摩尔/升的罗丹明6G溶液，混合液在避光处反应30分钟，产物 溶液采用离心分离、乙醇洗涤和真空干燥处理，得到银纳米簇/罗丹明6G复合物。将制得的复合物分散在100微升柠檬酸盐水缓冲液中，复合物的质量浓度为1～4毫克/毫升，在避光处孵化30分钟，分别测定不同罗丹明6G摩尔浓度下，复合物均质溶液的荧光发射光谱，优化双发射荧光峰强度以确定银纳米簇与罗丹明6G的配比。在室温和缓慢磁力搅拌下，向制得的优化配比复合物均质溶液中加入15微升不同浓度的三聚氰胺溶液，继续搅拌5分钟，使其充分反应形成复合物与三聚氰胺的均质溶液，测定不同的三聚氰胺摩尔浓度下，复合物与三聚氰胺均质溶液的荧光发射光谱，拟合银纳米簇与罗丹明6G荧光发射峰强度比率与三聚氰胺摩尔浓度之间的线性关系，构建三聚氰胺比率荧光探针，其中检测三聚氰胺的线性浓度范围为0.5～10微摩尔/升，三聚氰胺的检测极限为0.05微摩尔/升。
实施例3
本实施例中的制备DNA稳定的银纳米簇其具体工艺步骤同实施例1，其中硝酸银、DNA和硼氢化钠的摩尔浓度分别为10毫摩尔/升、600微摩尔/升和10毫摩尔/升。将制得的银纳米簇溶解在200微升二次蒸馏水中，加入100微升柠檬酸盐水缓冲液(pH 6.5)混匀，再加入100微升0.2～2纳摩尔/升的罗丹明6G溶液，混合液在避光处反应30分钟，产物溶液采用离心分离、乙醇洗涤和真空干燥处理，得到银纳米簇/罗丹明6G复合物。将制得的复合物分散在100微升柠檬酸盐水缓冲液中，复合物的质量浓度为2～5毫克/毫升，在避光处孵化30分钟，分别测定不同罗丹明6G摩尔浓度下，复合物均质溶液的荧光发射光谱，优化双发射荧光峰强度以确定银纳米簇与罗丹明6G的配比。在室温和缓慢磁力搅拌下，向制得的优化配比复合物均质溶液中加入15微升不同浓度的三聚氰胺溶液，继续搅拌5分钟，使其充分反应形成复合物与三聚氰胺的均质溶液，测定不同的三聚氰胺摩尔浓度下，复合物与三聚氰胺均质溶液的荧光发射光谱，拟合银纳米簇与罗丹明6G荧光发射峰强度比率 与三聚氰胺摩尔浓度之间的线性关系，构建三聚氰胺比率荧光探针，其中检测三聚氰胺的线性浓度范围为2～15微摩尔/升，三聚氰胺的检测极限为0.1微摩尔/升。
实施例4
本实施例涉及到实施例1制备的三聚氰胺比率荧光探针的应用，将其用于实际样品如牛奶中三聚氰胺的检测。将牛奶与不同浓度的三聚氰胺和乙腈混合，30分钟超声处理后制得混合物，然后以14000转/分钟的速度离心15分钟，取上清液过滤，滤液进一步稀释25倍并收集用于进一步检测，其中三聚氰胺摩尔浓度检测范围为0.5～15微摩尔/升，检测限达0.09微摩尔/升，检测回收率99.8～100.5％，相对标准偏差1.1～2.1％。与现有技术相比，如先前文献Talanta,2015,136,47-53和Appl.Surf.Sci.,2017,420,963-969采用单一或比率荧光方法检测三聚氰胺，对牛奶样品中三聚氰胺的检测回收率为94.0～102.0％，相对标准偏差为1.2～3.2％。本发明比率荧光探针的检测回收率更高，相对标准偏差更低，且制备工艺简便、成本低、产品灵敏度高，能够发展成为一种新颖的三聚氰胺比率荧光探针，适用于不同实际样品中三聚氰胺的高效检测。

Claims (1)

  1. 一种基于银纳米簇复合物的三聚氰胺比率荧光探针的制备方法，其特征在于，该方法具体包括以下步骤：
    (1)制备DNA稳定的银纳米簇：在0℃下，向1毫升二次蒸馏水中加入一定量的硝酸银溶液与DNA溶液，磁力搅拌20分钟以形成均质混合液，然后加入新配制的硼氢化钠溶液，在避光处剧烈搅拌反应20分钟。所得产物溶液采用0.4微米的滤膜过滤，滤液经截留分子量为5000道尔顿的透析袋透析处理，以去除未反应的实验原料，将透析袋中溶液进行旋蒸处理以去除90％溶剂，再进行冷冻干燥得到银纳米簇干样，在4℃避光条件下储存备用；
    (2)制备银纳米簇/罗丹明6G复合物：将步骤(1)中制得的银纳米簇溶解在200微升二次蒸馏水中，加入100微升柠檬酸盐水缓冲液混匀，再加入100微升不同浓度的罗丹明6G溶液，混合液在避光处反应30分钟，产物溶液采用离心分离、乙醇洗涤和真空干燥处理，得到银纳米簇/罗丹明6G复合物；
    (3)将步骤(2)中制得的复合物分散在100微升柠檬酸盐水缓冲液中，在避光处孵化30分钟，分别测定不同罗丹明6G摩尔浓度下，复合物均质溶液的荧光发射光谱，优化双发射荧光峰强度以确定银纳米簇与罗丹明6G的配比；
    (4)在室温和缓慢磁力搅拌下，向步骤(3)中制得的优化配比复合物均质溶液中加入15微升不同浓度的三聚氰胺溶液，继续搅拌5分钟，使其充分反应形成复合物与三聚氰胺的均质溶液，测定不同的三聚氰胺摩尔浓度下，复合物与三聚氰胺均质溶液的荧光发射光谱，拟合银纳米簇与罗丹明6G荧光发射峰强度比率与三聚氰胺摩尔浓度之间的线性关系，构建三聚氰胺比率荧光探针。
    本发明涉及的步骤(1)中所述的银纳米簇尺寸为6～12纳米，硝酸银、DNA和硼氢化钠的摩尔浓度分别为5～10毫摩尔/升、200～600微摩尔/升和5～10毫摩尔/升；步骤(2)中所述的柠檬酸盐水缓冲液pH为5.5～6.5，罗丹明6G摩尔浓度为0.1～2纳摩尔/升；步骤(3)中所述的复 合物溶液质量浓度为1～5毫克/毫升；步骤(4)中所述的三聚氰胺的浓度范围为0.5～15微摩尔/升，检测限为0.05～0.2微摩尔/升。
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