CN110846026B - 基于锌掺杂黑磷量子点的谷胱甘肽荧光纳米探针制备方法 - Google Patents

基于锌掺杂黑磷量子点的谷胱甘肽荧光纳米探针制备方法 Download PDF

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CN110846026B
CN110846026B CN201911210474.8A CN201911210474A CN110846026B CN 110846026 B CN110846026 B CN 110846026B CN 201911210474 A CN201911210474 A CN 201911210474A CN 110846026 B CN110846026 B CN 110846026B
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桂日军
姜晓文
金辉
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Abstract

本发明公开了基于锌掺杂黑磷量子点(Zn‑BPQDs)的谷胱甘肽(GSH)荧光纳米探针的制备方法，采用超声和溶剂热法制备Zn‑BPQDs，采用液相超声和溶液反应制备羟基氧化钴纳米片(CoOOH NSs)，通过超声辅助在溶液中自组装制备Zn‑BPQDs/CoOOH NSs复合物的水分散液。由于荧光共振能量转移(FRET)效应，Zn‑BPQDs荧光被淬灭，谷胱甘肽GSH将CoOOH NSs还原成Co2+，导致片层结构破坏，抑制了FRET效应，引起Zn‑BPQDs荧光恢复。拟合混合分散液的荧光发射峰强度与GSH浓度之间的线性关系，构建荧光纳米探针，该探针可用于生物医学样品中GSH的高灵敏和选择性检测。

Description

基于锌掺杂黑磷量子点的谷胱甘肽荧光纳米探针制备方法
技术领域
本发明属于功能纳米材料和荧光纳米探针的制备技术领域，具体涉及一种基于锌掺杂黑磷量子点的谷胱甘肽荧光纳米探针的制备方法，其制备的探针可用于生物医学样品中谷胱甘肽的高灵敏和选择性定量检测。
背景技术
谷胱甘肽(GSH)是一种含γ-酰胺键和巯基的三态，由谷氨酸、半胱氨酸及甘氨酸组成。GSH几乎存在于身体的每一个细胞中，它能帮助保持正常的免疫系统功能，具有抗氧化和整合解毒作用。半胱氨酸上的巯基为其活性基团，可与某些药物、毒素等结合，使其具有整合解毒作用。谷胱甘肽不仅可用于药物，也可作为功能性食品的基料，在延缓衰老、增强免疫力、抗肿瘤等功能性食品领域具有广阔的应用前景。GSH作为一种细胞内重要的调节代谢物质，参与体内三羧酸循环及糖代谢，并能激活多种酶，从而促进糖类、脂肪和蛋白质代谢。GSH分子含有活性巯基，可参与机体多种重要的生化反应，保护体内重要酶蛋白巯基不被氧化、灭活，保证能量代谢、细胞利用等。同时，其通过巯基与体内的自由基结合，可直接使自由基还原成酸性物质，加速自由基的排泄，并对抗自由基对重要脏器的损害。GSH含量的降低是一种潜在的凋亡早期激活信号，随后产生的氧自由基促使细胞发生凋亡。
黑磷量子点(BPQDs)是将黑磷块体通过液相超声剥离和溶剂热处理制备的零维黑磷纳米材料，相比其它维度的黑磷材料，BPQDs具有更高的能带隙，更小的尺寸，更大的比表面积，单位质量上更多边缘活性位点等。黑磷材料随着层数减少，变得越不稳定，在空气中或水中易被氧化而降解。锌原子的空轨道可与黑磷的孤对电子配位，配位后的磷原子其孤对电子被占据，不在与氧气反应，有效防止了黑磷的氧化和降解。本发明制备了锌离子掺杂的黑磷量子点(Zn-BPQDs)，其具有优异的胶体和荧光稳定性，将其与羟基氧化钴纳米片(CoOOHNSs)复合，可发生荧光共振能量转移(FRET)，引起Zn-BPQDs荧光淬灭。GSH可将CoOOHNSs还原成Co2+，引起CoOOHNSs结构的破坏，进而使FRET被抑制，故Zn-BPQDs荧光恢复。在制备的Zn-BPQDs/CoOOH NSs复合物中，外加一定量GSH，拟合Zn-BPQDs荧光强度与GSH浓度之间的线性关系，构建定量检测GSH的荧光纳米探针。
经文献检索发现，Gu等制备BPQDs用于构建比率荧光探针检测汞离子(Wei Gu,Xueyu Pei,Yuxiao Cheng,Cuiling Zhang,Jidong Zhang,Yinghan Yan,Caiping Ding,Yuezhong Xian,Black phosphorus quantum dots as the ratiometric fluorescenceprobe for trace mercury ion detection based on inner filter effect,ACSSensors,2017,2,576-582)；Gu等制备荧光BPQDs用作免标记传感探针用于乙酰胆碱酯酶活性评估(Wei Gu,Yinghan Yan,Xueyu Pei,Cuiling Zhang,Caiping Ding,Yuezhong Xian,Fluorescent black phosphorus quantum dots as label-free sensingprobes forevaluation of acetyl cholinesterase activity,Sensors and Actuators B:Chemical,2017,250,601–607)；李晓春等发明了“一种基于适体链-黑磷纳米片荧光能量共振转移的砷离子检测方法”(李晓春,李海琴,张校亮,于化忠.公开号：CN109239040A)；李桢等发明了“基于黑磷的近红外二区荧光纳米探针及其制备和应用”(李桢,徐一帆,赵崇军.公开号：CN109913201A)。截止目前，尚未检索到基于锌掺杂黑磷量子点的荧光纳米探针及其谷胱甘肽检测的国内外文献和专利的报道。
发明内容
本发明的目在于克服上述现有技术存在的不足，设计一种方法简便、高灵敏和高选择性的一种基于锌掺杂黑磷量子点的谷胱甘肽荧光纳米探针制备方法。
为了实现上述目的，本发明涉及的一种基于锌掺杂黑磷量子点的谷胱甘肽荧光纳米探针，其制备方法具体包括以下步骤：
1.基于锌掺杂黑磷量子点的谷胱甘肽荧光纳米探针制备方法，其特征在于，该方法具体包括以下步骤：
(1)Zn-BPQDs的制备：将20mg黑磷块体加入30mL氮甲基吡咯烷酮中，再加入1mmol/L的硝酸锌水溶液，用细胞粉碎机超声4h，然后用水浴超声10小时。将超声分散液转入高压反应釜中，在N2保护下加热至140℃，连续搅拌反应12h。反应混合物在3500rpm转速下离心20min，然后取上清液在12000rpm转速下离心20min，将沉淀物用乙醇和蒸馏水洗涤3次，真空干燥后得到Zn-BPQDs。
(2)CoOOH NSs的制备：分别配制1mol/L、10mmol/L、1mol/L的氢氧化钠、氯化钴和次氯酸钠水溶液，量取1mL氢氧化钠水溶液加入10mL氯化钴水溶液中，水浴超声5min，在4000rpm转速下离心20min，得到深黄色沉淀，将其分散在50mL蒸馏水中。在磁力搅拌下，向其中逐滴加入2.5mL次氯酸钠水溶液，水浴超声20min，在12000rpm转速下离心10min，得到棕色沉淀，用乙醇和蒸馏水洗涤3次，真空干燥后得到CoOOHNSs。
(3)制备Zn-BPQDs/CoOOH NSs复合物：在磁力搅拌下，配制0.5mg/mL Zn-BPQDs水分散液，配制1mmol/L CoOOH NSs水分散液，向1～10mL CoOOH NSs水分散液逐滴加入0.1～1mLZn-BPQDs水分散液，连续搅拌20min后形成Zn-BPQDs/CoOOH NSs复合物的水分散液。
(4)向上述复合物的水分散液中加入GSH水溶液，搅拌均匀以形成均质混合液，在避光处孵育5min后，测定不同GSH浓度下，均质混合液的荧光发射光谱，拟合荧光发射峰强度与GSH浓度之间的线性关系，构建定量检测GSH的荧光纳米探针。其中GSH浓度的线性范围0.01～1μmol/L，检测限0.01～0.05μmol/L。
本发明的效果是：报道了一种基于锌掺杂黑磷量子点的谷胱甘肽荧光纳米探针制备方法，用于定量检测谷胱甘肽。采用超声和溶剂热的一锅法制备了Zn-BPQDs，采用液相超声和溶液反应制备了CoOOH NSs，通过超声辅助在溶液中自组装制备了Zn-BPQDs/CoOOHNSs复合物的水分散液。由于FRET效应，Zn-BPQDs荧光被CoOOHNSs淬灭；外加的GSH可将CoOOH NSs还原成Co2+，导致片层结构破坏，抑制了FRET效应，进而引起Zn-BPQDs荧光恢复。拟合混合分散液的荧光发射峰强度与GSH浓度之间的线性关系，构建定量检测GSH的荧光纳米探针。与现有技术相比，本发明方法操作简便，灵敏度高和选择性好，可作为一种新颖的荧光纳米探针用于生物医学样品中谷胱甘肽的高灵敏和选择性检测。
附图说明
图1为基于锌掺杂黑磷量子点的谷胱甘肽荧光纳米探针制备方法的原理示意图；
图2为测定不同谷胱甘肽浓度下该荧光纳米探针体系的荧光发射光谱；
图3为不同谷胱甘肽浓度所对应的荧光发射峰相对强度F/F0(F0和F分别表示谷胱甘肽加入前和加入后的荧光发射峰强度)，拟合不同F/F0与谷胱甘肽浓度之间的线性关系。
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
本实施例涉及的基于锌掺杂黑磷量子点的谷胱甘肽荧光纳米探针制备方法，其制备步骤和荧光检测谷胱甘肽的原理示意图，如图1所示，具体制备步骤如下：
Zn-BPQDs的制备：将20mg黑磷块体加入30mL氮甲基吡咯烷酮中，再加入1mmol/L的硝酸锌水溶液，用细胞粉碎机超声4h，然后用水浴超声10小时；将超声分散液转入高压反应釜中，在N2保护下加热至140℃，连续搅拌反应12h；反应混合物在3500rpm转速下离心20min，然后取上清液在12000rpm转速下离心20min，将沉淀物用乙醇和蒸馏水洗涤3次，真空干燥后得到Zn-BPQDs；
CoOOH NSs的制备：分别配制1mol/L、10mmol/L、1mol/L的氢氧化钠、氯化钴和次氯酸钠水溶液，量取1mL氢氧化钠水溶液加入10mL氯化钴水溶液中，水浴超声5min，在4000rpm转速下离心20min，得到深黄色沉淀，将其分散在50mL蒸馏水中；在磁力搅拌下，向其中逐滴加入2.5mL次氯酸钠水溶液，水浴超声20min，在12000rpm转速下离心10min，得到棕色沉淀，用乙醇和蒸馏水洗涤3次，真空干燥后得到CoOOHNSs；
制备Zn-BPQDs/CoOOH NSs复合物：在磁力搅拌下，配制0.5mg/mL Zn-BPQDs水分散液，配制1mmol/L CoOOHNSs水分散液，向2mL CoOOH NSs水分散液逐滴加入0.1mL Zn-BPQDs水分散液，连续搅拌20min后形成Zn-BPQDs/CoOOH NSs复合物的水分散液；
向上述复合物的水分散液中加入GSH水溶液，搅拌均匀以形成均质混合液，在避光处孵育5min后，测定不同GSH浓度下，均质混合液的荧光发射光谱(如图2所示)，拟合荧光发射峰强度与GSH浓度之间的线性关系(如图3所示)，构建定量检测GSH的荧光纳米探针；其中GSH浓度的线性范围0.01～0.4μmol/L，检测限0.01μmol/L。
实施例2
本实施例涉及的Zn-BPQDs和CoOOH NSs的制备步骤同实施例1，其它具体步骤如下：制备Zn-BPQDs/CoOOH NSs复合物：在磁力搅拌下，配制0.5mg/mL Zn-BPQDs水分散液，配制1mmol/L CoOOHNSs水分散液，向5mL CoOOH NSs水分散液逐滴加入0.2mL Zn-BPQDs水分散液，连续搅拌20min后形成Zn-BPQDs/CoOOH NSs复合物的水分散液；向上述复合物的水分散液中加入GSH水溶液，搅拌均匀以形成均质混合液，在避光处孵育5min后，测定不同GSH浓度下，均质混合液的荧光发射光谱，拟合荧光发射峰强度与GSH浓度之间的线性关系，构建定量检测GSH的荧光纳米探针；其中GSH浓度的线性范围0.05～0.5μmol/L，检测限0.03μmol/L。
实施例3
本实施例涉及的Zn-BPQDs和CoOOH NSs的制备步骤同实施例1，其它具体步骤如下：制备Zn-BPQDs/CoOOH NSs复合物：在磁力搅拌下，配制0.5mg/mL Zn-BPQDs水分散液，配制1mmol/L CoOOHNSs水分散液，向8mL CoOOH NSs水分散液逐滴加入0.5mL Zn-BPQDs水分散液，连续搅拌20min后形成Zn-BPQDs/CoOOH NSs复合物的水分散液；向上述复合物的水分散液中加入GSH水溶液，搅拌均匀以形成均质混合液，在避光处孵育5min后，测定不同GSH浓度下，均质混合液的荧光发射光谱，拟合荧光发射峰强度与GSH浓度之间的线性关系，构建定量检测GSH的荧光纳米探针；其中GSH浓度的线性范围0.05～1μmol/L，检测限0.04μmol/L。

Claims (1)

1.基于锌掺杂黑磷量子点的谷胱甘肽荧光纳米探针制备方法，其特征在于，该方法具体包括以下步骤：
(1)锌掺杂黑磷量子点Zn-BPQDs的制备：将20mg黑磷块体加入30mL氮甲基吡咯烷酮中，再加入1mmol/L的硝酸锌水溶液，用细胞粉碎机超声4h，然后用水浴超声10小时；将超声分散液转入高压反应釜中，在N2保护下加热至140℃，连续搅拌反应12h；反应混合物在3500rpm转速下离心20min，然后取上清液在12000rpm转速下离心20min，将沉淀物用乙醇和蒸馏水洗涤3次，真空干燥后得到Zn-BPQDs；
(2)羟基氧化钴纳米片CoOOH NSs的制备：分别配制1mol/L、10mmol/L、1mol/L的氢氧化钠、氯化钴和次氯酸钠水溶液，量取1mL氢氧化钠水溶液加入10mL氯化钴水溶液中，水浴超声5min，在4000rpm转速下离心20min，得到深黄色沉淀，将其分散在50mL蒸馏水中；在磁力搅拌下，向其中逐滴加入2.5mL次氯酸钠水溶液，水浴超声20min，在12000rpm转速下离心10min，得到棕色沉淀，用乙醇和蒸馏水洗涤3次，真空干燥后得到CoOOHNSs；
(3)制备锌掺杂黑磷量子点/羟基氧化钴纳米片Zn-BPQDs/CoOOH NSs复合物：在磁力搅拌下，配制0.5mg/mL Zn-BPQDs水分散液，配制1mmol/L CoOOH NSs水分散液，向1～10mLCoOOH NSs水分散液逐滴加入0.1～1mL Zn-BPQDs水分散液，连续搅拌20min后形成Zn-BPQDs/CoOOH NSs复合物的水分散液；
(4)向上述复合物的水分散液中加入谷胱甘肽GSH水溶液，搅拌均匀以形成均质混合液，在避光处孵育5min后，测定不同GSH浓度下，均质混合液的荧光发射光谱，拟合荧光发射峰强度与GSH浓度之间的线性关系，构建定量检测GSH的荧光纳米探针；其中GSH浓度的线性范围0.01～1μmol/L，检测限0.01～0.05μmol/L。
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