WO2022242130A1 - 纳米荧光探针及其制备方法和应用 - Google Patents

纳米荧光探针及其制备方法和应用 Download PDF

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WO2022242130A1
WO2022242130A1 PCT/CN2021/138026 CN2021138026W WO2022242130A1 WO 2022242130 A1 WO2022242130 A1 WO 2022242130A1 CN 2021138026 W CN2021138026 W CN 2021138026W WO 2022242130 A1 WO2022242130 A1 WO 2022242130A1
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solution
fluorescent probe
nano
preparation
nano fluorescent
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王国成
慈乔乔
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深圳先进技术研究院
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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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • 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"
    • 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

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  • the invention belongs to the technical field of nano biosensing, and in particular relates to a nano fluorescent probe and its preparation method and application.
  • Fluorescence-based microscopy is one of the most widely used techniques for visualizing biological samples, as it enables non-invasive real-time monitoring of cells with high spatiotemporal resolution.
  • a variety of fluorescent probes have been developed to detect biologically important ions/species or microenvironmental fluctuations in cells.
  • pH-sensitive fluorescent probes have been used to visualize intracellular compartments with acidic or basic pH values.
  • a variety of physiological and pathological processes can cause pH changes in tissues and cells, including cell proliferation, apoptosis, receptor-mediated signal transduction, ion transport, muscle contraction, inflammation, and tumor growth.
  • the pH microenvironment can regulate Structure and function of all biologically active macromolecules.
  • Fluorescent probes are an effective method for monitoring changes in cell pH.
  • the existing fluorescent probes for detecting cell pH generally have shortcomings such as complex synthesis, poor photostability, poor water solubility, and poor biocompatibility.
  • the earth limits its application in living organisms.
  • Chinese patent application (CN111138639A) discloses a pH-responsive conjugated polymer nanoparticle and its preparation method and application, using polythiophene derivatives as the main raw material to prepare a pH-responsive conjugated polymer nanoparticle aqueous solution, which has a biological phase With good capacity, high fluorescence intensity and low toxicity, it can be used for accurate measurement of weakly acidic pH in aqueous solution, sensitive detection and fluorescence imaging of tumor cell microenvironmental responses.
  • the response range of the nanoparticles prepared by this method to pH is small (5-8), which cannot achieve a wide range of pH detection, and the raw materials used are organic small molecule materials, which have certain toxicity.
  • the present invention provides a nano fluorescent probe and its preparation method and application to solve the problem that the existing nano particle fluorescent probe has a small response range to pH.
  • the present invention adopts the following technical solutions:
  • the particle size of the composite nanoparticles is 10 nm to 100 nm.
  • Another aspect of the present invention provides the preparation method of nanometer fluorescent probe as above, it comprises:
  • Step S10 dissolving o-phenylenediamine, catecholamine and ferric chloride in a solvent to prepare a first solution and adjusting its pH to 1-6 with an acidic reagent;
  • Step S20 placing the first solution in a reactor and heating to react to form a second solution
  • Step S30 adding a surfactant to the second solution, placing it in a stirring container for a stirring reaction to form a third solution;
  • Step S40 purifying the third solution and drying it to form a composite nanoparticle solid powder to obtain the nano fluorescent probe.
  • the concentration of o-phenylenediamine is 1 g/L ⁇ 10 g/L
  • the concentration of catecholamines is 1 g/L ⁇ 10 g/L
  • the concentration of ferric chloride is 0.5 g/L ⁇ 5 g/L.
  • the catecholamine is selected from any one of dopamine, epinephrine or norepinephrine, and the solvent is selected from any of deionized water, ethanol, methanol or dimethyl sulfoxide
  • the acidic reagent is concentrated hydrochloric acid.
  • the reaction temperature of the heating reaction is 70° C. to 200° C.
  • the reaction time is 6 h to 36 h.
  • the concentration of the surfactant is 0.1 g/L ⁇ 2 g/L
  • the surfactant is selected from any one of four-arm polyethylene glycol amino, polyethylene glycol-400 or polyethylene glycol-800.
  • the stirring reaction is carried out in a magnetic stirrer, and the stirring time is 6 h to 36 h.
  • the step S40 specifically includes: placing the third solution in a dialysis container for dialysis for more than 3 days, and changing the dialysis fluid at least 3 times during the period; Granular solid powder forms the nano fluorescent probe.
  • the present invention also provides the application of the above-mentioned nano fluorescent probes in the preparation of cell fluorescence imaging materials.
  • the fluorescent nanoprobes provided in the embodiments of the present invention have different color responses to different pHs, and the response range of pH is 2 to 10, which has a relatively large response range of pH; the preparation method of the fluorescent nanoprobes provided in the embodiments of the present invention has With the advantages of simple process and green raw materials, the prepared fluorescent nanoprobe has good biocompatibility.
  • Fig. 1 is the process flow chart of the preparation method of the fluorescent nanoprobe in the embodiment of the present invention
  • Fig. 2 is the transmission electron micrograph of the nano fluorescent probe that embodiment 1 prepares to obtain;
  • Fig. 3 is the high-resolution transmission electron microscope picture of the nanometer fluorescent probe that embodiment 1 prepares;
  • Fig. 4 is the Fourier transform infrared spectrogram of the nano fluorescent probe that embodiment 1 prepares to obtain;
  • Fig. 5 is the X-ray photoelectron energy spectrogram of the nano fluorescent probe that embodiment 1 prepares;
  • Fig. 6 is the photo illustration of the color of nano fluorescent probe under different pH value conditions in embodiment 2;
  • Fig. 7 is the fluorescence spectrogram of nano fluorescent probe under different pH value conditions in embodiment 3;
  • Fig. 8 is in embodiment 3, at the wavelength of 720 nm, the fluorescence intensity line graph of the nano fluorescent probe under different pH conditions;
  • Fig. 9 is in embodiment 3, at 720 nm wavelength place, the linear relationship figure of the fluorescent intensity of nano fluorescent probe and pH;
  • Fig. 10 is the fluorescence intensity line chart of nanometer fluorescent probe to pH change cycle detection in embodiment 4.
  • Fig. 11 is the fluorescent kinetic figure of nanometer fluorescent probe to pH detection in embodiment 5;
  • Fig. 12 is the result figure of the toxicity test of nano fluorescent probe to 293T cells in embodiment 6;
  • Fig. 13 is a graph showing the results of toxicity test of the Mi fluorescent probe on 4T1 cells in Example 6.
  • the particle size of the composite nanoparticles is 10 nm to 100 nm.
  • the embodiment of the present invention provides the preparation method of nano fluorescent probe as described above, referring to Fig. 1, the preparation method comprises:
  • Step S10 dissolving o-phenylenediamine (o-PD), catecholamine (CA) and ferric chloride (FeCl 3 ) in a solvent to prepare a first solution and adjusting its pH to 1-6 with an acidic reagent.
  • o-PD o-phenylenediamine
  • CA catecholamine
  • FeCl 3 ferric chloride
  • the concentration of o-phenylenediamine is 1 g/L ⁇ 10 g/L
  • the concentration of catecholamines is 1 g/L ⁇ 10 g/L
  • the concentration of ferric chloride is 0.5 g/L ⁇ 5 g/L.
  • the catecholamine is selected from any one of dopamine (DA), epinephrine (Ad) or norepinephrine (NA), and the solvent is selected from deionized water, ethanol, methanol or dimethyl any one of the base sulfoxides, and the acidic reagent is concentrated hydrochloric acid.
  • DA dopamine
  • Ad epinephrine
  • NA norepinephrine
  • the solvent is selected from deionized water, ethanol, methanol or dimethyl any one of the base sulfoxides
  • the acidic reagent is concentrated hydrochloric acid.
  • Step S20 placing the first solution in a reactor and heating to react to form a second solution.
  • the reaction temperature of the heating reaction is 70° C. to 200° C.
  • the reaction time is 6 h to 36 h.
  • Step S30 adding a surfactant to the second solution, placing it in a stirring container for a stirring reaction to form a third solution.
  • the concentration of the surfactant is 0.1 g/L to 2 g/L, and the surfactant is selected from four-arm polyethylene glycol amino ( 4ARM-PEG-NH 2 ), polyethylene glycol-400 or polyethylene glycol-800.
  • the stirring reaction is carried out in a magnetic stirrer, and the stirring time is 6 h to 36 h.
  • Step S40 purifying the third solution and drying it to form a composite nanoparticle solid powder to obtain the nano fluorescent probe.
  • the step S40 specifically includes: placing the third solution in a dialysis container (such as a dialysis bag) for dialysis for more than 3 days, and changing the dialysate at least 3 times (preferably 3 to 5 times) during the period After the dialysis is completed, take the solution in the dialysis container and freeze-dry to obtain a composite nanoparticle solid powder to form the nano fluorescent probe.
  • a dialysis container such as a dialysis bag
  • the embodiment of the present invention also provides the application of the above-mentioned nano fluorescent probes in the preparation of cell fluorescence imaging materials.
  • This embodiment provides a nano fluorescent probe and its preparation method, specifically as follows:
  • Fig. 2 is a transmission electron micrograph (TEM) of the nano-fluorescent probe prepared in this example
  • Fig. 3 is a high-resolution transmission electron micrograph (HRTEM) of the nano-fluorescent probe prepared in this example. It can be seen from Figure 2 and Figure 3 that the size of the nano-fluorescent probe (composite nanoparticle) prepared in this example is about 20 nm, and has an obvious lattice structure with a lattice spacing of 0.21 nm.
  • Figure 5 is the X-ray photoelectron energy spectrum diagram of the nano fluorescent probe prepared in this example, it can be seen from the figure that the nano fluorescent probe mainly contains four elements C, N, O and Cl, and C, N, O The contents of the four elements, Cl and Cl are respectively: 70.47%, 10.01%, 15.78%, and 3.74%.
  • Example 1 The nano-fluorescent probe solid powder prepared in Example 1 was prepared with deionized water to make a probe solution with a concentration of 1 mg/mL for later use.
  • Tris-HCl buffer solutions with a concentration of 10 mM and different pH (pH 2, 3, 4, 5, 6, 6.5, 7, 8, 9 and 10) were prepared.
  • Figure 6 is a photo illustration of the colors of the nano-fluorescent probes in this embodiment under different pH conditions.
  • the color has high response sensitivity, the response range of pH is 2 ⁇ 10, and the response range of pH is relatively large.
  • Example 1 The nano-fluorescent probe solid powder prepared in Example 1 was prepared with deionized water to make a probe solution with a concentration of 1 mg/mL for later use.
  • Fig. 7 is the fluorescence spectrum diagram of the nano fluorescent probe in this embodiment at different pH.
  • Fig. 8 is a line graph of the fluorescence intensity of the nano-fluorescent probe in this embodiment at a wavelength of 720 nm under different pH conditions. It can be known from Fig. 7 and Fig. 8 that the fluorescence intensity of the nano fluorescent probe provided by the present invention decreases with the increase of pH, and has a high response sensitivity and a large pH response range.
  • Fig. 9 is a graph showing the linear relationship between the fluorescence intensity and pH of the nano fluorescent probe in this embodiment at a wavelength of 720 nm. It can be seen from FIG. 9 that, when the pH is between 3 and 7, there is a good linear relationship between the fluorescence of the fluorescent nanoprobe provided by the present invention and the pH, so the quantitative detection of pH can be realized.
  • Example 1 The nano-fluorescent probe solid powder prepared in Example 1 was prepared with deionized water to make a probe solution with a concentration of 1 mg/mL for later use.
  • step (3) Use a high-concentration Tris solution to adjust the pH of the reaction solution in step (2) to 6, shake it well, and record the fluorescence intensity.
  • Fig. 10 is a line graph of the fluorescent intensity of the nano-fluorescent probes in this embodiment for cyclic detection of pH changes. It can be seen from FIG. 10 that the nano fluorescent probe provided by the present invention has a stable cycle detection performance between pH 2 and pH 6.
  • Example 1 The nano-fluorescent probe solid powder prepared in Example 1 was prepared with deionized water to make a probe solution with a concentration of 1 mg/mL for later use.
  • Fig. 11 is the fluorescence kinetics diagram of the nano-fluorescent probe for pH detection in this embodiment. It can be known from Fig. 11 that the fluorescence stability of the nano-fluorescent probe of the present invention is good at different pHs, and the fluorescence intensity remains stable at 2 h. Has good chemical stability.
  • Example 1 (1) Prepare the nano-fluorescent probe solid powder prepared in Example 1 with deionized water to a concentration of 0 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 0.75 mg/mL, 1 mg/mL, 1.5 mg/mL, and 3 mg/mL probe solutions for later use.
  • step (1) When the cells proliferate to 70% ⁇ 80%, add different concentrations of step (1) (0/0.25/0.75/0.5/1/1.5/3 mg/mL) of the probe solution, incubated together for 24h, and then the cytotoxicity test (MTT test) was performed to detect the cytotoxicity of the nano-fluorescent probe.
  • MTT test cytotoxicity test
  • Fig. 12 is a graph showing the results of the toxicity test of the nano-fluorescent probes on 293T cells in this example
  • Fig. 13 is a graph showing the results of the toxicity tests of the nano-fluorescent probes on 4T1 cells in this embodiment. It can be known from Fig. 12 and Fig. 13 that the nano fluorescent probe provided by the present invention has little toxicity to cells and has excellent biocompatibility.
  • the fluorescent nanoprobes provided by the embodiments of the present invention have different color responses to different pHs, have high response sensitivity, and have a pH response range of 2 to 10, which has a relatively large pH response range; the present invention
  • the preparation method of the fluorescent nanoprobe provided in the example has the advantages of simple process, green raw materials, etc., and the prepared fluorescent nanoprobe has good chemical stability and biocompatibility.

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Abstract

本发明提供了一种纳米荧光探针，所述纳米荧光探针为复合纳米颗粒，所述复合纳米颗粒为主要由C、N、O和Cl四种元素构成纳米晶体，其中包含有-OH官能团、-NH 2官能团和C=C官能团。其制备方法包括：步骤S10、将邻苯二胺、儿茶酚胺和氯化铁溶解于溶剂中配制形成第一溶液并使用酸性试剂将其pH调节至1~6；步骤S20、将所述第一溶液置于反应釜中加热反应，形成第二溶液；步骤S30、向所述第二溶液中加入表面活性剂，置于搅拌容器中进行搅拌反应，形成第三溶液；步骤S40、将所述第三溶液进行纯化处理，干燥后形成复合纳米颗粒固体粉末，获得所述纳米荧光探针。本发明提供的荧光纳米探针对不同pH有不同颜色响应，并且具有良好的化学稳定性和生物相容性。

Description

纳米荧光探针及其制备方法和应用 技术领域
本发明属于纳米生物传感技术领域，具体涉及一种纳米荧光探针及其制备方法和应用。
 背景技术
基于荧光的显微技术是最广泛使用的可视化生物样品的技术之一，因为它可以实现对细胞的非侵入性实时监测，具有高的时空分辨率。多种荧光探针已被开发用于检测生物学上重要的离子/物质或细胞中的微环境波动。在这些技术中，pH敏感荧光探针已被用于可视化具有酸性或碱性pH值的细胞内腔室。多种生理和病理过程均会引起组织和细胞内pH的变化，包括细胞增殖、凋亡，受体介导的信号转导、离子转运、肌肉收缩、炎症和肿瘤生长等，pH微环境能调节所有具有生物活性的大分子的结构和功能。细胞和组织中的异常与pH值的变化密切相关，如癌症和老年痴呆症之类的疾病。此外，细胞内的pH值分布是不同的，各个细胞器内特征化的pH值环境保障了细胞器的功能正常。例如，溶酶体和内体是酸性的pH值（4.7-6.5），细胞质和细胞核是中性的pH值（7.2-7.4），线粒体pH值大约是8。因此，利用有效的方法监测细胞pH值变化对更好地认识细胞以及为早期疾病的诊断提供关键的帮助具有重要意义。
荧光探针是一种有效的监测细胞pH值变化得方法，目前已有的检测细胞pH值的荧光探针普遍存在合成复杂、光稳定性差、水溶性差和生物相容性较差等不足，极大地限制了其在生物体内得应用。中国专利申请（CN111138639A）公开了一种pH响应型共轭聚合物纳米粒子及其制备方法和应用，利用聚噻吩衍生物为主要原料制备得到pH响应型共轭聚合物纳米颗粒水溶液，具有生物相容性良好、荧光强度高和毒性低等特性，可用于水溶液中弱酸性pH值的精确测量，也可用于肿瘤细胞微环境响应的灵敏检测和荧光成像。但是该方法制备的纳米颗粒对pH的响应区间较小（5~8），无法达到一个较大范围的pH检测，并且其使用的原料为有机小分子材料，具有一定的毒性。
 技术问题
鉴于现有技术存在的不足，本发明提供一种纳米荧光探针及其制备方法和应用，以解决现有的纳米颗粒荧光探针对pH的响应区间较小的问题。
为了解决以上所述的问题，本发明采用了如下的技术方案：
本发明的一方面提供了一种纳米荧光探针，所述纳米荧光探针为复合纳米颗粒，所述复合纳米颗粒为主要由C、N、O和Cl四种元素构成纳米晶体，其中包含有-OH官能团、-NH 2官能团和C=C官能团。
具体地，所述复合纳米颗粒的粒径为10 nm~100 nm。
本发明的另一方面是提供如上所述的纳米荧光探针的制备方法，其包括：
步骤S10、将邻苯二胺、儿茶酚胺和氯化铁溶解于溶剂中配制形成第一溶液并使用酸性试剂将其pH调节至1~6；
步骤S20、将所述第一溶液置于反应釜中加热反应，形成第二溶液；
步骤S30、向所述第二溶液中加入表面活性剂，置于搅拌容器中进行搅拌反应，形成第三溶液；
步骤S40、将所述第三溶液进行纯化处理，干燥后形成复合纳米颗粒固体粉末，获得所述纳米荧光探针。
具体地，所述步骤S10中，配制形成的第一溶液中，邻苯二胺的浓度为1 g/L ~10 g/L，儿茶酚胺的浓度为1 g/L~10 g/L，氯化铁的浓度为0.5 g/L ~5 g/L。
具体地，所述步骤S10中，所述儿茶酚胺选自多巴胺、肾上腺素或去甲肾上腺素中的任意一种，所述溶剂选自去离子水、乙醇、甲醇或二甲基亚砜中的任意一种，所述酸性试剂为浓盐酸。
具体地，所述步骤S20中，所述加热反应的反应温度为70℃~200℃，反应时间为6 h~36 h。
具体地，所述步骤S30形成的第三溶液中，所述表面活性剂的浓度为0.1 g/L ~2 g/L，所述表面活性剂选自四臂聚乙二醇氨基、聚乙二醇-400或聚乙二醇-800中的任意一种。
具体地，所述步骤S30中，所述搅拌反应是在磁力搅拌器中进行，搅拌时间为6 h~36 h。
具体地，所述步骤S40具体包括：将所述第三溶液置于透析容器中透析3天以上，并且期间至少更换3次透析液；透析完成后取透析容器中的溶液进行冷冻干燥获得复合纳米颗粒固体粉末，形成所述纳米荧光探针。
本发明还提供如上所述纳米荧光探针在制备细胞荧光成像材料中的应用。
本发明实施例提供的荧光纳米探针对不同pH有不同颜色响应，pH的响应区间为2~10，具有较大的pH的响应区间；本发明实施例提供的荧光纳米探针的制备方法具有工艺简单、原材料绿色环保的优点，制备获得的荧光纳米探针具有良好的生物相容性。
 附图说明
图1是本发明实施例中的荧光纳米探针的制备方法的工艺流程图；
图2是实施例1制备获得的纳米荧光探针的透射电镜图；
图3是实施例1制备获得的纳米荧光探针的高分辨率透射电镜图；
图4是实施例1制备获得的纳米荧光探针的傅里叶红外光谱图；
图5是实施例1制备获得的纳米荧光探针的X射线光电子能谱图；
图6是实施例2中纳米荧光探针在不同pH值条件下的颜色的照片图示；
图7是实施例3中纳米荧光探针在不同pH值条件下的荧光光谱图；
图8是实施例3中，在720 nm波长处，纳米荧光探针在不同pH值条件下的荧光强度折线图；
图9是实施例3中，在720 nm波长处，纳米荧光探针的荧光强度和pH的线性关系图；
图10是实施例4中纳米荧光探针对pH变化循环检测的荧光强度折线图；
图11是实施例5中纳米荧光探针对pH检测的荧光动力学图；
图12是实施例6中纳米荧光探针对293T细胞的毒性试验结果图；
图13是实施例6中米荧光探针对4T1细胞的毒性试验结果图。
 本发明的实施方式
为使本发明的目的、技术方案和优点更加清楚，下面结合附图对本发明的具体实施方式进行详细说明。这些优选实施方式的示例在附图中进行了例示。附图中所示和根据附图描述的本发明的实施方式仅仅是示例性的，并且本发明并不限于这些实施方式。
在此，还需要说明的是，为了避免因不必要的细节而模糊了本发明，在附图中仅仅示出了与本发明的方案密切相关的结构和/或处理步骤，而省略了与本发明关系不大的其他细节。
本发明实施例首先提供了一种纳米荧光探针，所述纳米荧光探针为复合纳米颗粒，所述复合纳米颗粒为主要由C、N、O和Cl四种元素构成纳米晶体，其中包含有-OH官能团、-NH 2官能团和C=C官能团。
其中，所述复合纳米颗粒的粒径为10 nm~100 nm。
本发明实施例提供了如上所述的纳米荧光探针的制备方法，参阅图1，所述制备方法其包括：
步骤S10、将邻苯二胺（o-PD）、儿茶酚胺（CA）和氯化铁（FeCl 3）溶解于溶剂中配制形成第一溶液并使用酸性试剂将其pH调节至1~6。
在优选的方案中，配制形成的第一溶液中，邻苯二胺的浓度为1 g/L ~10 g/L，儿茶酚胺的浓度为1 g/L~10 g/L，氯化铁的浓度为0.5 g/L ~5 g/L。
在优选的方案中，所述儿茶酚胺选自多巴胺（DA）、肾上腺素（Ad）或去甲肾上腺素（NA）中的任意一种，所述溶剂选自去离子水、乙醇、甲醇或二甲基亚砜中的任意一种，所述酸性试剂为浓盐酸。
步骤S20、将所述第一溶液置于反应釜中加热反应，形成第二溶液。
在优选的方案中，所述加热反应的反应温度为70℃~200℃，反应时间为6 h~36 h。
步骤S30、向所述第二溶液中加入表面活性剂，置于搅拌容器中进行搅拌反应，形成第三溶液。
在优选的方案中，所述步骤S30形成的第三溶液中，所述表面活性剂的浓度为0.1 g/L ~2 g/L，所述表面活性剂选自四臂聚乙二醇氨基（4ARM-PEG-NH 2）、聚乙二醇-400或聚乙二醇-800中的任意一种。
在优选的方案中，所述搅拌反应是在磁力搅拌器中进行，搅拌的时间为6 h~36 h。
步骤S40、将所述第三溶液进行纯化处理，干燥后形成复合纳米颗粒固体粉末，获得所述纳米荧光探针。
在优选的方案中，所述步骤S40具体包括：将所述第三溶液置于透析容器（例如透析袋）中透析3天以上，并且期间至少更换3次（优选为3~5次）透析液；透析完成后取透析容器中的溶液进行冷冻干燥获得复合纳米颗粒固体粉末，形成所述纳米荧光探针。
本发明实施例还提供如上所述纳米荧光探针在制备细胞荧光成像材料中的应用。
实施例1：
本实施例提供一种纳米荧光探针及其制备方法，具体如下：
（1）称取邻苯二胺0.10 g，多巴胺0.10 g和氯化铁0.05 g溶解于15 mL的去离子水中形成混合溶液，将混合溶液置入烧杯中，用浓盐酸（1 M）调节混合溶液的pH为3.0。需要说明的是，在另外的一些实施例中，多巴胺可替换为使用肾上腺素或去甲肾上腺素。
（2）将上述混合溶液倒入容积为50 mL的干净的反应釜内胆中，接着将内胆置于反应釜中，旋紧盖子，置于烘箱中，在180℃下反应12 h。反应完毕后，取出反应釜，自然冷却至室温，打开盖子，将内胆中的反应物倒入烧杯中。
（3）取上述反应物15 mL加入15 mg四臂聚乙二醇氨基，再置于磁力搅拌器上剧烈搅拌24 h。需要说明的是，在另外的一些实施例中，四臂聚乙二醇氨基可替换为使用聚乙二醇-400或聚乙二醇-800。
（4）将上述反应物置于透析袋中，用去离子水透析3天，期间换水3次以上，透析完毕后取出透析液置于50 mL的离心管中进行冷冻干燥，得到固体粉末即为复合纳米颗粒，获得所述纳米荧光探针。
图2为本实施例制备获得的纳米荧光探针的透射电镜图（TEM），图3为本实施例制备获得的纳米荧光探针的高分辨率透射电镜图（HRTEM）。从图2和图3可以获知，本实施例制备获得的纳米荧光探针（复合纳米颗粒）的尺寸为20 nm左右，有明显的晶格结构，晶格间距为0.21 nm。
图4为本实施例制备获得的纳米荧光探针的傅里叶红外光谱图，从图中可以看到纳米荧光探针含有-OH官能团、-NH 2官能团和C=C官能团。
图5为本实施例制备获得的纳米荧光探针的X射线光电子能谱图，从图中可以看到纳米荧光探针主要含有C、N、O、Cl四种元素，且C、N、O、Cl四种元素的含量依次分别为：70.47%、10.01%、15.78%、3.74%。
实施例2
（1）将实施例1制备得到的纳米荧光探针固体粉末，用去离子水配制成浓度为1 mg/mL的探针溶液，备用。
（2）配置浓度为10 mM不同pH（pH分别为2、3、4、5、6、6.5、7、8、9和10）的Tris-HCl缓冲溶液。
（3）室温下，取0.5 mL每一pH值的Tris-HCl缓冲溶液置于96孔板中，再分别加入10 μL步骤（1）所述的探针溶液，充分震荡摇匀，观察96孔板各孔中溶液的颜色变化。
图6是本实施例中纳米荧光探针在不同pH值条件下的颜色的照片图示，从图6可以看出，本发明提供的纳米荧光探针在不同pH下可呈现出肉眼可见的不同的颜色，具有很高的响应灵敏度，pH的响应区间为2~10，具有较大的pH的响应区间。
实施例3
（1）将实施例1制备得到的纳米荧光探针固体粉末，用去离子水配制成浓度为1 mg/mL的探针溶液，备用。
（2）配置浓度为10 mM不同pH（pH分别为2、3、4、5、6、7、8、9和10）的Tris-HCl缓冲溶液。
（3）室温下，取0.5 mL每一pH值的Tris-HCl缓冲溶液置于1mL的离心管中，再分别加入10 μL步骤（1）所述的探针溶液，充分震荡摇匀，通过荧光分光光度计测量各个离心管中反应液的荧光强度并绘制荧光强度图。
图7是本实施例中纳米荧光探针在不同pH下的荧光光谱图。图8是在720 nm波长处，本实施例中纳米荧光探针在不同pH值条件下的荧光强度折线图。由图7和图8可以获知，本发明提供的纳米荧光探针的荧光强度随着pH的增大而减小，具有很高的响应灵敏度和较大的pH的响应区间。
图9为在720 nm波长处，本实施例中纳米荧光探针的荧光强度和pH的线性关系图。由图9可知，在pH为3~7之间，本发明提供的纳米荧光探针的荧光和pH之间具有很好的线性关系，因此可实现对pH的定量检测。
实施例4
（1）将实施例1制备得到的纳米荧光探针固体粉末，用去离子水配制成浓度为1 mg/mL的探针溶液，备用。
（2）配置浓度为10 mM的pH为2的Tris-HCl缓冲溶液，取0.5 mL置于1mL的离心管中，加入10 μL步骤（1）所述的探针溶液，充分震荡摇匀，参照实施例3的方式记录反应液的荧光强度。
（3）用高浓度的Tris溶液调节步骤（2）的反应液pH至6，充分震荡摇匀，记录其荧光强度。
（4）用高浓度的HCl溶液调节上述反应液pH至2，充分震荡摇匀，记录其荧光强度。
（5）将第（3）步和第（4）步重复5次。
图10是本实施例中的纳米荧光探针对pH变化循环检测的荧光强度折线图。从图10可以获知，本发明提供的纳米荧光探针在pH为2和pH为6之间的循环检测性能稳定。
实施例5
（1）将实施例1制备得到的纳米荧光探针固体粉末，用去离子水配制成浓度为1 mg/mL的探针溶液，备用。
（2）配置浓度为10 mM的pH分别为2、4和6的Tris-HCl缓冲溶液。
（3）室温下，取0.5 mL每一pH值的Tris-HCl缓冲溶液置于1 mL的离心管中，再分别加入10 μL步骤（1）所述的探针溶液，充分震荡摇匀，测量反应液的荧光强度。
（4）在不同的时间段下，测量并记录各个反应液的荧光强度，监测时间为2h，绘制荧光动力学图。
图11是本实施例中纳米荧光探针对pH检测的荧光动力学图，从图11可以获知，本发明的纳米荧光探针在不同pH下的荧光稳定性良好，荧光强度在2h保持稳定，具有良好的化学稳定性。
实施例6
（1）将实施例1制备得到的纳米荧光探针固体粉末，用去离子水配制成浓度为0 mg/mL、0.25 mg/mL、0.5 mg/mL、0.75 mg/mL、1 mg/mL、1.5 mg/mL和、3 mg/mL的探针溶液，备用。
（2）在37℃、5% CO 2条件下，分别培养人肾上皮细胞（293T）和乳腺癌细胞（4T1）于96孔板中。
（3）待细胞增殖至70%~80%，向96孔板各孔中加入步骤（1）不同浓度（0/0.25/0.75/0.5/1/1.5/3 mg/mL）的探针溶液，共同孵育24h，然后进行细胞毒性测试（MTT测试），检测纳米荧光探针的细胞毒性。
图12是本实施例中的纳米荧光探针对293T细胞的毒性试验结果图，图13是本实施例中的纳米荧光探针对4T1细胞的毒性试验结果图。从图12和图13可以获知，本发明提供的纳米荧光探针对细胞的毒性很小，具有优异的生物相容性。
综上所述，本发明实施例提供的荧光纳米探针对不同pH有不同颜色响应，具有很高的响应灵敏度，pH的响应区间为2~10，具有较大的pH的响应区间；本发明实施例提供的荧光纳米探针的制备方法具有工艺简单、原材料绿色环保等优点，制备获得的荧光纳米探针具有良好的化学稳定性和生物相容性。
以上所述仅是本申请的具体实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本申请原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本申请的保护范围。

Claims (10)

  1. 一种纳米荧光探针，其特征在于，所述纳米荧光探针为复合纳米颗粒，所述复合纳米颗粒为主要由C、N、O和Cl四种元素构成纳米晶体，其中包含有-OH官能团、-NH 2官能团和C=C官能团。
  2. 根据权利要求1所述的纳米荧光探针，其特征在于，所述复合纳米颗粒的粒径为10 nm~100 nm。
  3. 如权利要求1或2所述的纳米荧光探针的制备方法，其特征在于，包括：
    步骤S10、将邻苯二胺、儿茶酚胺和氯化铁溶解于溶剂中配制形成第一溶液并使用酸性试剂将其pH调节至1~6；
    步骤S20、将所述第一溶液置于反应釜中加热反应，形成第二溶液；
    步骤S30、向所述第二溶液中加入表面活性剂，置于搅拌容器中进行搅拌反应，形成第三溶液；
    步骤S40、将所述第三溶液进行纯化处理，干燥后形成复合纳米颗粒固体粉末，获得所述纳米荧光探针。
  4. 根据权利要求3所述的纳米荧光探针的制备方法，其特征在于，所述步骤S10配制形成的第一溶液中，邻苯二胺的浓度为1 g/L ~10 g/L，儿茶酚胺的浓度为1 g/L~10 g/L，氯化铁的浓度为0.5 g/L ~5 g/L。
  5. 根据权利要求4所述的纳米荧光探针的制备方法，其特征在于，所述步骤S10中，所述儿茶酚胺选自多巴胺、肾上腺素或去甲肾上腺素中的任意一种，所述溶剂选自去离子水、乙醇、甲醇或二甲基亚砜中的任意一种，所述酸性试剂为浓盐酸。
  6. 根据权利要求3所述的纳米荧光探针的制备方法，其特征在于，所述步骤S20中，所述加热反应的反应温度为70℃~200℃，反应时间为6 h~36 h。
  7. 根据权利要求3所述的纳米荧光探针的制备方法，其特征在于，所述步骤S30形成的第三溶液中，所述表面活性剂的浓度为0.1 g/L ~2 g/L，所述表面活性剂选自四臂聚乙二醇氨基、聚乙二醇-400或聚乙二醇-800中的任意一种。
  8. 根据权利要求7所述的纳米荧光探针的制备方法，其特征在于，所述步骤S30中，所述搅拌反应是在磁力搅拌器中进行，搅拌时间为6 h~36 h。
  9. 根据权利要求3所述的纳米荧光探针的制备方法，其特征在于，所述步骤S40具体包括：将所述第三溶液置于透析容器中透析3天以上，并且期间至少更换3次透析液；透析完成后取透析容器中的溶液进行冷冻干燥获得复合纳米颗粒固体粉末，形成所述纳米荧光探针。
  10. 根据权利要求1或2所述的纳米荧光探针在制备细胞荧光成像材料中的应用。
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