CN111830000A - 一种对纳米颗粒加载探针作用于斑马鱼胚胎/幼鱼的方法 - Google Patents
一种对纳米颗粒加载探针作用于斑马鱼胚胎/幼鱼的方法 Download PDFInfo
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Abstract
发明公开一种对纳米颗粒加载探针并作用于斑马鱼胚胎/幼鱼的方法，步骤如下：(1)将纳米颗粒分散于去离子水中；(2)使用去离子水分散Cy7荧光探针；(3)将纳米颗粒分散液与Cy7荧光探针混合摇匀；(4)将负载了Cy7荧光探针的纳米颗粒分散液透析；(5)使斑马鱼胚胎/幼鱼暴露于加载探针的纳米颗粒分散液中；(6)使用小动物成像观察斑马鱼胚胎/幼鱼荧光值。本发明优点为：该纳米颗粒加载荧光探针的方法简便、准确、易于实现，用于天然纳米材料对斑马鱼暴露的毒性实验中研究材料在斑马鱼体内外的分布，辅助判断颗粒在毒性实验中对斑马鱼的作用位点和作用方式。
Description
技术领域
本专利涉及生态毒理学，特别是环境中纳米材料对斑马鱼胚胎的毒理作用。
背景技术
对于纳米材料对生物体的作用，无论是天然存在还是人工合成的纳米颗粒，其是否进入生物体、在生物体内存在的位置都是相关毒性和机理研究的重点。使用荧光探针标记纳米材料，示踪其在生物组织的分布是近年来纳米毒理研究中的而重点之一。然而，生物组织在200-600nm波长下会出现自发荧光信号，会对纳米颗粒分布的结果造成干扰；当波长在700nm以上时，生物的自发荧光信号会大大减弱。
发明内容：
本发明的目的是针对上述技术分析，提供一种天然纳米胶体的标记方法及毒理学应用，该负载方法工艺简单、易于实施，用于天然纳米材料对斑马鱼暴露的毒性实验中研究材料在斑马鱼体内外的分布，辅助判断颗粒在毒性实验中对斑马鱼的作用位点和作用方式。
本发明的技术方案：一种对纳米颗粒加载探针并作用于斑马鱼胚胎/幼鱼的方法，步骤如下：
(1)分散天然纳米颗粒，使用时为使颗粒更好地分散，应在40kw功率下冰浴超声30min；
(2)配制sulfo-cyanine 7 NHS ester(Cy7)荧光探针分散液；
(3)Cy7荧光探针负载：取纳米颗粒分散液，加入Cy7荧光探针，密闭摇匀；
(4)取出摇匀的分散液，置入透析袋，对分散液进行透析，直至透析水无荧光；
(5)取出分散液，使用10x E3培养液以纳米颗粒浓度为准，将加载Cy7探针的纳米颗粒分散液配制为浓度适宜的纳米颗粒E3分散液备用；
(6)准备斑马鱼幼鱼胚胎，产卵12h后将其置入96孔板，每孔包含1个胚胎，使用纳米颗粒E3分散液培养斑马鱼胚胎；
(7)胚胎暴露后取出斑马鱼胚胎，进行红外摄像，判断颗粒粘附位置。
所述步骤(1)纳米颗粒分散粒径在20-100nm，分散液浓度为50mg/L，介质为去离子水。
所述步骤(2)Cy7分散液浓度为1mg/mL。
所述步骤(3)纳米颗粒分散液体积为20mL，Cy7分散液体积为100μL。
所述步骤(4)透析袋孔径为5kDa，透析水为高纯去离子水。
所述步骤(5)，10x E3培养液的配比为50mmol/L NaCl，1.7mmol/L KCl，3.3mmol/LCaCl2，3.3mmol/L MgSO4，pH＝7.4，E3培养液的配比为5mmol/L NaCl，0.17mmol/L KCl，0.33mmol/L CaCl2，0.33mmol/L MgSO4。
所述步骤(6)，纳米颗粒的暴露浓度为45mg/L。
一种对纳米颗粒负载染色并使其加载于斑马鱼幼鱼表面的方法的应用，用于检测纳米材料在斑马鱼幼鱼中的分布和吸收。
本发明的优点是：该纳米颗粒荧光探针负载和分析方法简单、准确，易于实施，用于斑马鱼幼鱼暴露于天然纳米材料实验，可提高纳米颗粒在斑马鱼幼鱼体表和体内的分布的检测水平。
本发明使用近红外荧光探针对天然纳米颗粒物进行负载标记，助力研究纳米颗粒对生物毒性位点的判断和分析。
本发明所使用的受试生物为斑马鱼胚胎/幼鱼。斑马鱼具有体型小、易于养殖繁殖以及成本低等优点，在生态毒理等领域的使用十分广泛。且斑马鱼胚胎/幼鱼组织透明，可以更好的呈现荧光信号。
附图说明
图1暴露于负载探针纳米颗粒24h后斑马鱼胚胎白光及荧光图像：
其中：1-A和1-B分别为斑马鱼胚胎白光下未清洗和清洗后的图像，1-a和1-b分别为1-A和1-B在荧光下的图像。
图2暴露于负载探针纳米颗粒120h后斑马鱼幼鱼白光及荧光图像：
其中：2-A和2-B分别为斑马鱼幼鱼白光下未清洗和清洗后的图像，2-a和2-b分别为图2-A和图2-B在荧光下的图像。
具体实施方式
以下结合附图和具体实施例来对本发明作进一步的说明。
实施例1
(1)分散粒径在20-100nm的天然纳米颗粒，纳米颗粒浓度为50mg/L，纳米颗粒分散液介质为去离子水，使用前在40kw功率下冰浴超声30min；
(2)配制Cy7荧光探针分散液，其介质为去离子水，其浓度为1mg/mL；
(3)Cy7荧光探针负载：取20mL纳米颗粒分散液，加入100μL Cy7荧光探针分散液，密闭摇匀后，置入60r/min的摇床，摇动12h；
(4)取出摇匀的分散液，置入5kDa孔径透析袋，以高纯水为介质，对分散液进行透析24h，其中每4h更换一次透析水，直至透析水无荧光；
(5)取出加载探针的纳米颗粒分散液，使用10xE3培养液将其稀释为纳米颗粒浓度45mg/L的分散液备用；
(6)准备斑马鱼幼鱼胚胎，产卵12h后将其置入96孔板，每孔包含1个胚胎，注入200μL45mg/L加载探针的纳米颗粒分散液；
(7)胚胎暴露24h后取出，分两组，一组取出后直接进行红外摄像，另一组使用E3培养液清洗三遍后进行红外摄像，判断荧光值。
分析仪器为小动物活体成像系统NightOWL ⅡLB983，滤光片激发波长750nm，发射波长800nm。
实施例2
(1)分散天然纳米颗粒，纳米颗粒浓度为50mg/L，纳米颗粒分散液介质为去离子水，使用前在40kw功率下冰浴超声30min；
(2)配制Cy7荧光探针分散液，其介质为去离子水，其浓度为1mg/mL；
(3)Cy7荧光探针负载：取20mL纳米颗粒分散液，加入100μL Cy7荧光探针分散液，密闭摇匀后，置入60r/min的摇床，摇动12h；
(4)取出摇匀的分散液，置入5kDa孔径透析袋，以高纯水为介质，对分散液进行透析24h，其中每4h更换一次透析水，直至透析水无荧光；
(5)分散液取出，使用E3培养液分散，以纳米颗粒浓度为准，将加载Cy7探针的分散液配制为45mg/L的分散液备用；
(6)准备斑马鱼胚胎，产卵12h后将其置入96孔板，每孔包含1个胚胎。将幼鱼胚胎分为两组：分别为对照组45mg/L组；
(7)斑马鱼胚胎培养12h后，对照组每孔注入200μLE3培养液进行斑马鱼胚胎培养，45mg/L组每孔注入200μL45mg/L加载探针的纳米颗粒分散液进行斑马鱼胚胎培养，期间两组斑马鱼胚胎均将孵化为斑马鱼幼鱼；
(8)斑马鱼胚胎/幼鱼暴露120h后取出5条，直接进行红外摄像，观察荧光情况；
(9)斑马鱼胚胎/幼鱼暴露120h后取出5条，使用E3培养液冲洗三遍后，进行红外摄像，观察荧光情况。
分析仪器为小动物活体成像系统NightOWL ⅡLB983，滤光片激发波长750nm，发射波长800nm
结果如图1和图2。图1中的1-A和1-B分别为斑马鱼胚胎白光下未清洗和清洗后的图像，1-a和1-b分别为1-A和1-B在荧光下的图像。结果显示，在加载荧光探针的纳米颗粒分散液中培养24h后，荧光结果显示斑马鱼胚胎表面均有纳米颗粒的粘附，且经过清洗后，斑马鱼胚胎表面的荧光值并未减弱，说明纳米颗粒在斑马鱼胚胎表面的粘附是十分稳固的。
在图2中的2-A和图2-B分别为斑马鱼幼鱼白光下未清洗和清洗后的图像，2-a和2-b分别为2-A和2-B在荧光下的图像。结果显示，在加载荧光探针的纳米颗粒分散液中培养120h后，荧光结果显示斑马鱼幼鱼表面有纳米颗粒的粘附，但经过清洗后，斑马鱼幼鱼荧光信号消失，说明纳米颗粒对斑马鱼幼鱼的作用位置为皮肤。
以上结果表明，该发明简便、易行，且可以准确地判断纳米颗粒对斑马鱼胚胎/幼鱼的作用方式。
Claims (8)
1.一种对纳米颗粒加载探针并作用于斑马鱼胚胎/幼鱼的方法，其特征在于，步骤如下：
(1)分散天然纳米颗粒，获得纳米颗粒分散液；
(2)配制Cy7 NHS ester即Cy7荧光探针分散液；
(3)Cy7荧光探针负载：取纳米颗粒分散液，加入Cy7荧光探针，密闭摇匀；
(4)取出摇匀的分散液，置入透析袋，对分散液进行透析，直至透析水无荧光；
(5)取出分散液，使用10x E3培养液以纳米颗粒浓度为准，将加载Cy7探针的纳米颗粒分散液配制为纳米颗粒E3分散液备用；
(6)准备斑马鱼胚胎，产卵12h后将其置入孔板，使用所述步骤(5)的纳米颗粒E3分散液培养斑马鱼胚胎；
(7)胚胎暴露后取出斑马鱼胚胎，进行红外摄像，判断颗粒粘附位置。
2.根据权利要求1所述的对纳米颗粒加载探针并作用于斑马鱼胚胎/幼鱼的方法，其特征在于：所述步骤1)纳米颗粒分散粒径在20-100nm，分散液浓度为50mg/L，介质为去离子水。
3.根据权利要求1所述对纳米颗粒加载探针并作用于斑马鱼胚胎/幼鱼的方法，其特征在于：所述步骤2)Cy7分散液浓度为1mg/mL。
4.根据权利要求1所述对纳米颗粒加载探针并作用于斑马鱼胚胎/幼鱼的方法，其特征在于：所述步骤3)纳米颗粒分散液体积为20mL，Cy7分散液体积为100μL。
5.根据权利要求1所述对纳米颗粒加载探针并作用于斑马鱼胚胎/幼鱼的方法，其特征在于：所述步骤4)透析袋孔径为5kDa，透析水为高纯去离子水。
6.根据权利要求1所述对纳米颗粒加载探针并作用于斑马鱼胚胎/幼鱼的方法，其特征在于：所述步骤5)10x E3培养液的配比为50mmol/L NaCl，1.7mmol/L KCl，3.3mmol/LCaCl2，3.3mmol/L MgSO4，pH＝7.4，E3培养液的配比为5mmol/L NaCl，0.17mmol/L KCl，0.33mmol/L CaCl2，0.33mmol/L MgSO4。
7.根据权利要求1所述对纳米颗粒加载探针并作用于斑马鱼胚胎/幼鱼的方法，其特征在于：所述步骤6)纳米颗粒的暴露浓度为45mg/L。
8.根据权利要求1所述的对纳米颗粒加载探针并作用于斑马鱼胚胎/幼鱼的方法，其特征在于：所述步骤1)分散天然纳米颗粒在40kw功率下冰浴超声30min。
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