WO2020186503A1 - 一种用于可视化检测槲皮素的纳米复合物荧光探针及其制备方法 - Google Patents

一种用于可视化检测槲皮素的纳米复合物荧光探针及其制备方法 Download PDF

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WO2020186503A1
WO2020186503A1 PCT/CN2019/078996 CN2019078996W WO2020186503A1 WO 2020186503 A1 WO2020186503 A1 WO 2020186503A1 CN 2019078996 W CN2019078996 W CN 2019078996W WO 2020186503 A1 WO2020186503 A1 WO 2020186503A1
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quercetin
preparation
fluorescence
rhodamine
nanocomposite
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金辉
桂日军
卜祥宁
付永鑫
姜晓文
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青岛大学
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    • 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
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    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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    • 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

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  • the invention belongs to the technical field of preparation of nanocomposites and visual fluorescent probes, and in particular relates to a nanocomposite fluorescent probe for visually detecting quercetin and a preparation method thereof.
  • the prepared probe can be used for quercetin in biological samples. Fluorescence visual detection of cortin.
  • Quercetin (3,3',4',5,7-quercetin) is a common flavonol. As a natural compound, it is widely present in vegetables, fruits, beverages and other nutritious foods. Among the nutrients obtained through diet, the human intake of flavonols is about 2.6-38.2 mg/day. Clinical medicine has confirmed that quercetin is beneficial to human health. It can prevent diabetes, inhibit headache shock, reduce capillary fragility, induce cell apoptosis, and have functions such as anti-inflammatory drugs. Quercetin has an important impact on life-sustaining activities, has specific biomedical effects, and has its unique contribution to promoting human health. Therefore, the development of a simple and efficient quercetin detection method is important for human health monitoring Meaning.
  • the currently reported detection methods for quercetin mainly include spectrophotometry, capillary electrophoresis, high performance liquid chromatography, electrochemistry, etc.
  • Gai Ying et al. used capillary electrophoresis-mass spectrometry analysis to identify dihydroquercetin in larch (Chinese Invention Patent Publication No. CN108802160A);
  • Zhang Ling et al. used thin-layer chromatography and high performance liquid chromatography to analyze quercetin in Ganhuang Quban Capsules (Chinese Invention Patent Publication No.
  • the present invention designs a fluorescent probe based on carbon dot/rhodamine 6G nanocomposite, and visually detects quercetin based on the fluorescent color type of the probe.
  • the purpose of the present invention is to overcome the above-mentioned defects in the prior art, and to design a nanocomposite fluorescent probe for the visual detection of quercetin with a simple method, low cost, and high sensitivity.
  • the preparation of a nanocomposite fluorescent probe for visually detecting quercetin includes the following steps:
  • a nanocomposite fluorescent probe for visually detecting quercetin and a preparation method thereof characterized in that the method specifically includes the following steps:
  • the mass concentration ratio of sodium citrate and ammonium carbonate in step (1) is 1:2 ⁇ 1:10, the reaction temperature is 120-200°C, and the reaction time is 2-10h;
  • step (2) the mass concentration of carbon dots is 1-10 mg mL -1 , the molar concentration of rhodamine 6G is 1-50 ⁇ M, the molar concentration of phosphate is 10-50 mM, and the buffer pH is 7-8;
  • the molar concentration of quercetin described in step (3) is 1-100 ⁇ M
  • the fluorescent color types described in step (4) include deep red, orange red, orange yellow, dark yellow, yellow green, light green, dark green, and transition colors between the two colors.
  • the invention utilizes the excellent electron-obtaining effect of quercetin to cause light-induced electron transfer of the fluorescent body under photon excitation to quench the fluorescent body, and is used for fluorescence detection of quercetin.
  • quercetin simultaneously causes fluorescence quenching of carbon dots and rhodamine 6G, in which the carbon dots emit green fluorescence and rhodamine 6G emits red fluorescence.
  • the fluorescence emission spectrum of the complex shows green fluorescence and red fluorescence simultaneously and gradually quenches, and the fluorescence color of the complex solution (in the centrifuge tube and dripped on the filter paper) appears from deep red, orange, and orange. , Dark yellow, yellow-green, light green to dark green gradually.
  • a method for visually detecting quercetin based on the type of fluorescence color was constructed.
  • the probe of the invention has simple preparation, low cost and high sensitivity, and can be used for the fluorescent visual detection of quercetin in biological samples.
  • Figure 1 A schematic diagram of the preparation and principle of a nanocomposite fluorescent probe for visual detection of quercetin
  • the probe solution contains different coexisting concentrations of quercetin.
  • the present invention relates to a nanocomposite fluorescent probe for visually detecting quercetin and a preparation method thereof.
  • the preparation process and principle are shown in Fig. 1, and the specific preparation steps are as follows:
  • the carbon dot mass concentration is 2 mg mL -1
  • the molar concentration of rhodamine 6G is 10 ⁇ M
  • the phosphate concentration is 10 mM
  • the buffer pH is 7.0, keep the ultrasonic and magnetic stirring for 30 minutes.
  • the aqueous solution of quercetin was added dropwise to the aqueous dispersion of the nanocomposite, and a homogeneous mixture was formed under ultrasonic and magnetic stirring. The mixture was allowed to stand in a dark place and incubated for 10 minutes.
  • the concentration of quercetin was determined at 1-50 ⁇ M. Fluorescence emission spectrum of the mixed liquid (as shown in Figure 2). Prepare mixed solutions containing different coexisting concentrations of quercetin. Pipette part of the mixed solution into a centrifuge tube and drip on the filter paper. Under the excitation of 365 nm, the mixture in the centrifuge tube and on the filter paper will show different fluorescent colors. Use a digital camera Shoot (as shown in Figure 3). As the concentration of quercetin increases, the fluorescence color of the complex probe changes from dark red, light red, light yellow, yellow-green, and light green to dark green. The fluorescent color type of the probe solution is constructed to quantify the visual detection method of quercetin. .
  • the mass concentration of carbon dots is 4 mg mL -1
  • the molar concentration of rhodamine 6G is 20 ⁇ M
  • the phosphate concentration is 20 mM.
  • the buffer pH is 7.4, keep the ultrasonic and magnetic stirring for 30 minutes.
  • the aqueous solution of quercetin was added dropwise to the aqueous dispersion of the nanocomposite, and a homogeneous mixture was formed under ultrasonic and magnetic stirring. The mixture was allowed to stand in a dark place and incubated for 10 minutes.
  • the concentration of quercetin at 1 ⁇ 80 ⁇ M was measured. Fluorescence emission spectrum of the mixed liquid. Prepare mixed solutions containing different coexisting concentrations of quercetin. Pipette part of the mixed solution into a centrifuge tube and drip on the filter paper. Under the excitation of 365 nm, the mixture in the centrifuge tube and on the filter paper will show different fluorescent colors. Use a digital camera Shoot. As the concentration of quercetin increases, the fluorescence color of the complex probe changes from dark red, orange, dark yellow, light yellow, yellow-green, and light green to dark green. The fluorescence color type of the complex probe solution is quantified. The visual detection method of element.
  • the mass concentration of carbon dots is 5 mg mL -1
  • the molar concentration of rhodamine 6G is 50 ⁇ M
  • the phosphate concentration is 30 mM.
  • the pH of the buffer solution is 8.0
  • the mixed solution is kept under ultrasonic and magnetic stirring for 30 minutes.
  • the aqueous solution of quercetin was added dropwise to the aqueous dispersion of the nanocomposite to form a homogeneous mixture under ultrasonic and magnetic stirring. Incubate for 10 minutes in a dark place.

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Abstract

本发明属于纳米复合物和可视化荧光探针的制备技术领域，具体涉及一种用于可视化检测槲皮素的纳米复合物荧光探针及其制备方法。槲皮素优异的得电子效应可引起光子激发下荧光体发生光诱导的电子转移而发生淬灭，在静电吸附自组装的碳点/罗丹明6G纳米复合物中，槲皮素同时引起碳点绿荧光和罗丹明6G红荧光淬灭。随槲皮素浓度增大，离心管内和滴涂在滤纸上的复合物探针溶液荧光颜色呈现从深红、橘红、橘黄、深黄、黄绿、浅绿到深绿的逐渐变化。建立槲皮素不同浓度与探针溶液荧光颜色之间的关联，基于荧光颜色类型来可视化检测槲皮素。与现有技术相比，本发明探针其制备简单，成本低廉，灵敏度高，可用于生物样品中槲皮素的荧光可视化检测。

Description

一种用于可视化检测槲皮素的纳米复合物荧光探针及其制备方法 技术领域：
本发明属于纳米复合物和可视化荧光探针的制备技术领域，具体涉及一种用于可视化检测槲皮素的纳米复合物荧光探针及其制备方法，其制备的探针可用于生物样品中槲皮素的荧光可视化检测。
背景技术：
槲皮素(3,3',4',5,7-五羟黄酮)是一种常见的黄酮醇，作为一种天然化合物广泛存在于蔬菜、水果、饮料以及其它营养食品中。在通过饮食获取的营养物中，人类对黄酮醇的摄入量约为2.6～38.2毫克/天。临床医学证实了槲皮素对人体健康是有益的，可预防糖尿病，抑制头痛休克，降低毛细血管脆弱，诱导细胞凋亡，具备消炎药的作用等功能。槲皮素对于维持生命活动产生重要的影响，具备特定的生物医学功效，在促进人体健康方面有其独特的贡献，因此发展一种简单和高效的槲皮素检测方法对于人体健康的监测具有重要的意义。
当前报道的槲皮素检测方法主要包括分光光度法、毛细管电泳法、高效液相色谱法、电化学法等。盖颖等采用毛细管电泳-质谱联用分析鉴定落叶松中二氢槲皮素(中国发明专利公开号CN108802160A)；张玲等采用薄层色谱法和高效液相色谱法对赶黄祛斑胶囊中槲皮素进行检测(中国发明专利公开号CN103115974A)；吴宏伟等利用离子液体-加速溶剂萃取高效液相色谱化学发光法来检测槲皮素(中国发明专利公开号CN102539572A)；王宗花等利用石墨烯修饰电极发展了槲皮素电化学检测方法(中国发明专利公开号CN102288669A)；师赛鸽等基于缬氨酸和多壁碳纳米管修饰的玻碳电极发展了测定槲皮素的电化学传感器(中国发明专利公开号CN106680343A)。Kan等构筑了碳点/金纳米粒/多壁碳纳米管复合物改性的玻碳电极用作槲皮素电化学传感器(Xianwen Kan,Tingting Zhang,Min Zhong,et al.CD/AuNPs/MWCNTs based electrochemical sensor for quercetin dual-signal detection.Biosens.Bioelectron.2016,77,638)；Chen等基于金纳米簇开发了槲皮素荧光传感器 (Zhanguang Chen,Sihua Qian,Junhui Chen,et al.Highly fluorescent gold nanoclusters based sensor for the detection of quercetin.J.Nanopart.Res.2012,14,1264)。
常规的仪器分析方法普遍存在一些不足如仪器昂贵，成本高，样品预处理复杂，操作严格，灵敏度不高，特异性差等。相比之下，化学或生物传感器器方法尤其是电化学和荧光传感器具备操作简单，灵敏性高，选择性好等优势，在生化分析领域展现出广阔的应用前景。当前，电化学传感器已被用于高灵敏检测槲皮素，荧光传感器可实现裸眼可视的槲皮素检测。裸眼可视化荧光检测是一种十分便捷的分析方法，相比单一荧光的强弱，人体裸眼更容易区分荧光颜色的类型，依据荧光颜色的类型来确定样品中目标物的浓度，可对目标物进行简单和高效的检测。经文献检索，尽管已有文献报道了基于荧光的强弱来定量槲皮素的方法，但目前尚未有基于荧光颜色类型来定量槲皮素的荧光可视化检测方法的国内外文献和专利的报道。基于此，本发明设计了一种基于碳点/罗丹明6G纳米复合物的荧光探针，基于该探针的荧光颜色类型来可视化检测槲皮素。
发明内容：
本发明的目的在于克服上述现有技术存在的缺陷，设计一种方法简单、成本低廉、灵敏度高的用于可视化检测槲皮素的纳米复合物荧光探针。
为了实现上述目的，本发明涉及的一种用于可视化检测槲皮素的纳米复合物荧光探针的制备包括以下步骤：
1.一种用于可视化检测槲皮素的纳米复合物荧光探针及其制备方法，其特征在于，该方法具体包括以下步骤：
(1)碳点的制备：称取柠檬酸钠和碳酸铵，在磁力搅拌下溶于二次蒸馏水，混合液转入附有聚四氟乙烯内衬的高压反应釜中，在室温下搅拌，在特定温度下反应。冷却产物溶液，得深棕色悬浮液，用滤膜过滤后收集滤液，采用旋转蒸发除去大部分溶剂，残留滤液经离心处理，沉淀物用乙醇洗涤，对纯化后的碳点进行真空干燥，得到干燥的碳点粉末。
(2)碳点/罗丹明6G纳米复合物的制备：将干燥的碳点粉末分散于磷酸盐水缓冲液中，在超声和磁力搅拌下将罗丹明6G水溶液逐滴加入其中，保持超声和磁 力搅拌一段时间，使碳点与罗丹明6G充分结合。
(3)向碳点/罗丹明6G纳米复合物的水分散液中，逐滴加入槲皮素的水溶液，在超声和磁力搅拌下形成均质混合液，在避光处静置孵育10分钟，然后测定不同槲皮素浓度下均质混合液的荧光发射光谱。
(4)配制含槲皮素不同共存浓度的一组复合物混合液，分别移取一定体积的混合液于离心管中和滴涂在常规定性滤纸上，在紫外灯365纳米激发下呈现一组荧光颜色，用数码相机拍摄，建立槲皮素的不同共存浓度与混合液荧光颜色类型之间的关联，构建基于荧光颜色类型来定量槲皮素的荧光可视化检测方法。
步骤(1)中所述的柠檬酸钠和碳酸铵的质量浓度比为1:2～1:10，反应温度为120～200℃，反应时间为2～10h；
步骤(2)中所述的碳点的质量浓度为1～10mg mL -1，罗丹明6G的摩尔浓度为1～50μM，磷酸盐摩尔浓度为10～50mM，缓冲液pH为7～8；
步骤(3)中所述的槲皮素摩尔浓度为1～100μM；
步骤(4)中所述的荧光颜色类型包括深红、橘红、橘黄、深黄、黄绿、浅绿、深绿，以及两色之间的过度色等。
本发明利用槲皮素优异的得电子效应，引起光子激发下荧光体发生光诱导的电子转移使荧光体发生淬灭，用于槲皮素的荧光检测。在静电吸附自组装的碳点/罗丹明6G纳米复合物中，槲皮素同时引起碳点和罗丹明6G荧光淬灭，其中碳点发射绿荧光，罗丹明6G发射红荧光。随着槲皮素浓度增大，复合物荧光发射光谱呈现绿荧光和红荧光同步逐渐淬灭，复合物溶液(在离心管内和滴涂在滤纸上)荧光颜色类型呈现从深红、橘红、橘黄、深黄、黄绿、浅绿到深绿的逐渐变化。基于槲皮素不同浓度与复合物(探针)溶液荧光颜色类型之间的关联，构建基于荧光颜色类型来可视化检测槲皮素的方法。与现有技术相比，本发明探针其制备简单，成本低廉，灵敏度高，可用于生物样品中槲皮素的荧光可视化检测。
附图说明：
图1.一种用于可视化检测槲皮素的纳米复合物荧光探针的制备及原理示意图；
图2.碳点/罗丹明6G纳米复合物探针在不同槲皮素共存浓度下测定的荧光发 射光谱图；
图3.探针溶液中含有不同共存浓度的槲皮素，在紫外灯365纳米激发下，探针溶液在离心管内和滤纸上呈现的荧光颜色。
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：
本发明涉及的一种用于可视化检测槲皮素的纳米复合物荧光探针及其制备方法，其制备过程与原理如图1所示，具体制备步骤如下：
称取1g柠檬酸钠和2g碳酸铵，在磁力搅拌下溶于30mL二次蒸馏水，混合液转入50mL附有聚四氟乙烯内衬的高压反应釜中，在室温下搅拌30min，升温至120℃后反应12h。冷却产物溶液，得深棕色悬浮液，用0.22μm滤膜过滤后收集滤液，采用旋转蒸发除去大部分溶剂，残留滤液经离心处理，沉淀物用乙醇洗涤，纯化后的碳点在60℃真空干燥制得碳点粉末。将碳点粉末分散于磷酸盐水缓冲液中，在超声和磁力搅拌下逐滴加入罗丹明6G水溶液，其中碳点质量浓度为2mg mL -1，罗丹明6G摩尔浓度为10μM，磷酸盐浓度为10mM，缓冲液pH为7.0，保持继续超声和磁力搅拌30min。向纳米复合物的水分散液中逐滴加入槲皮素的水溶液，在超声和磁力搅拌下形成均质混合液，在避光处静置孵育10分钟，测定1～50μM槲皮素浓度下均质混合液的荧光发射光谱(如图2所示)。配制含槲皮素不同共存浓度的混合液，分别移取部分混合液于离心管中和滴涂在滤纸上，在365纳米激发下离心管内和滤纸上混合液呈现不同的荧光颜色，用数码相机拍摄(如图3所示)。随槲皮素浓度增大，复合物探针荧光颜色类型呈现从深红、浅红、浅黄、黄绿、浅绿变成深绿，构建探针溶液荧光颜色类型定量槲皮素的可视化检测方法。
实施例2：
称取1g柠檬酸钠和4g碳酸铵，在磁力搅拌下溶于30mL二次蒸馏水，混合液转入50mL附有聚四氟乙烯内衬的高压反应釜中，在室温下搅拌30min，升温至150℃后反应10h。冷却产物溶液，得深棕色悬浮液，用0.22μm滤膜过滤后收集滤液，采用旋转蒸发除去大部分溶剂，残留滤液经离心处理，沉淀物用乙醇洗涤， 纯化后的碳点在60℃真空干燥制得碳点粉末。将碳点粉末分散于磷酸盐水缓冲液中，在超声和磁力搅拌下逐滴加入罗丹明6G水溶液，其中碳点质量浓度为4mg mL -1，罗丹明6G摩尔浓度为20μM，磷酸盐浓度为20mM，缓冲液pH为7.4，保持继续超声和磁力搅拌30min。向纳米复合物的水分散液中逐滴加入槲皮素的水溶液，在超声和磁力搅拌下形成均质混合液，在避光处静置孵育10分钟，测定1～80μM槲皮素浓度下均质混合液的荧光发射光谱。配制含槲皮素不同共存浓度的混合液，分别移取部分混合液于离心管中和滴涂在滤纸上，在365纳米激发下离心管内和滤纸上混合液呈现不同的荧光颜色，用数码相机拍摄。随槲皮素浓度增大，复合物探针荧光颜色类型呈现从深红、橘红、深黄、淡黄、黄绿、浅绿变成深绿，构建复合物探针溶液荧光颜色类型定量槲皮素的可视化检测方法。
实施例3：
称取1g柠檬酸钠和5g碳酸铵，在磁力搅拌下溶于30mL二次蒸馏水，混合液转入50mL附有聚四氟乙烯内衬的高压反应釜中，在室温下搅拌30min，升温至180℃后反应8h。冷却产物溶液，得深棕色悬浮液，用0.22μm滤膜过滤后收集滤液，采用旋转蒸发除去大部分溶剂，残留滤液经离心处理，沉淀物用乙醇洗涤，纯化后的碳点在60℃真空干燥制得碳点粉末。将碳点粉末分散于磷酸盐水缓冲液中，在超声和磁力搅拌下逐滴加入罗丹明6G水溶液，其中碳点质量浓度为5mg mL -1，罗丹明6G摩尔浓度为50μM，磷酸盐浓度为30mM，缓冲液pH为8.0，混合液保持继续超声和磁力搅拌30min。向纳米复合物的水分散液中逐滴加入槲皮素的水溶液，在超声和磁力搅拌下形成均质混合液，在避光处静置孵育10分钟，测定1～100μM槲皮素浓度下均质混合液的荧光发射光谱。配制含槲皮素不同共存浓度的混合液，分别移取部分混合液于离心管中和滴涂在滤纸上，在365纳米激发下离心管内和滤纸上混合液呈现不同的荧光颜色，用数码相机拍摄。随槲皮素浓度增大，复合物探针荧光颜色类型呈现从深红、浅红、橘红、橘黄、深黄、淡黄、黄绿变成浅绿，构建基于复合物探针溶液的荧光颜色类型定量槲皮素的可视化检测方法。

Claims (5)

  1. 一种用于可视化检测槲皮素的纳米复合物荧光探针及其制备方法，其特征在于，该方法具体包括以下步骤：
    (1)碳点的制备：称取柠檬酸钠和碳酸铵，在磁力搅拌下溶于二次蒸馏水，混合液转入附有聚四氟乙烯内衬的高压反应釜中，在室温下搅拌，在特定温度下反应；冷却产物溶液，得深棕色悬浮液，用滤膜过滤后收集滤液，采用旋转蒸发除去大部分溶剂，残留滤液经离心处理，沉淀物用乙醇洗涤，对纯化后的碳点进行真空干燥，得到干燥的碳点粉末；
    (2)碳点/罗丹明6G纳米复合物的制备：将干燥的碳点粉末分散于磷酸盐水缓冲液中，在超声和磁力搅拌下将罗丹明6G水溶液逐滴加入其中，保持超声和磁力搅拌一段时间，使碳点与罗丹明6G充分结合；
    (3)向碳点/罗丹明6G纳米复合物的水分散液中，逐滴加入槲皮素的水溶液，在超声和磁力搅拌下形成均质混合液，在避光处静置孵育10分钟，然后测定不同槲皮素浓度下均质混合液的荧光发射光谱；
    (4)配制含槲皮素不同共存浓度的一组复合物混合液，分别移取一定体积的混合液于离心管中和滴涂在常规定性滤纸上，在紫外灯365纳米激发下呈现一组荧光颜色，用数码相机拍摄，建立槲皮素的不同共存浓度与混合液荧光颜色类型之间的关联，构建基于荧光颜色类型来定量槲皮素的荧光可视化检测方法。
  2. 一种用于可视化检测槲皮素的纳米复合物荧光探针及其制备方法，其特征在于，步骤(1)中所述的柠檬酸钠和碳酸铵的质量浓度比为1:2～1:10，反应温度为120～200℃，反应时间为2～10h。
  3. 一种用于可视化检测槲皮素的纳米复合物荧光探针及其制备方法，其特征在于，步骤(2)中所述的碳点的质量浓度为1～10mg mL -1，罗丹明6G的摩尔浓度为1～50μM，磷酸盐摩尔浓度为10～50mM，缓冲液pH为7～8。
  4. 一种用于可视化检测槲皮素的纳米复合物荧光探针及其制备方法，其特征在于，步骤(3)中所述的槲皮素摩尔浓度为1～100μM。
  5. 一种用于可视化检测槲皮素的纳米复合物荧光探针及其制备方法，其特征在于，步骤(4)中所述的荧光颜色类型包括深红、橘红、橘黄、深黄、黄绿、浅绿、深绿，以及两色之间的过度色等。
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