CN112592716B - 一种可快速标记大肠杆菌的碳量子点及其制备方法和应用 - Google Patents
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Abstract
本发明公开了一种可快速标记大肠杆菌的碳量子点的制备方法，包括以下步骤：将单糖分子和苯二胺加入水中混合均匀；在水热反应釜中进行水热反应，反应温度为150℃～240℃，反应时间为1h～5h；反应结束后，所得产物先进行离心，取上清液透析，干燥，得到发射黄色荧光的碳量子点。本发明所制备的发射黄色荧光的碳量子点可以快速结合大肠杆菌，实现对大肠杆菌的标记。
Description
技术领域
本发明属于生物医用材料领域，涉及一种可快速标记大肠杆菌的碳量子点及其制备方法和应用。
背景技术
细菌感染目前已经成为威胁人类健康的杀手，而随着抗生素的滥用，必然会引起细菌耐药性的产生，例如近年来不断出现的超级耐药菌，已经逐渐成为世界性的治疗难题。因此实现细菌的特异性标记，可以为人类进行针对性的使用抗菌药物提供有利条件。目前对细菌进行标记和区分常用的是染色法，包括单染色法和复染色法，但是操作步骤复杂，因此设计合成一种生物相容性良好的纳米材料，来实现细菌的特异性快速标记，具有很重要的意义。
发明内容
有鉴于此，本发明提供一种可快速标记大肠杆菌的碳量子点及其制备方法和应用。本发明具体提供了如下的技术方案：
1、一种可快速标记大肠杆菌的碳量子点的制备方法，包括以下步骤：
1)将单糖分子和苯二胺加入水中混合均匀；
2)在水热反应釜中进行水热反应，反应温度为150℃～240℃，反应时间为1h～5h；
3)反应结束后，所得产物先进行离心，取上清液透析，干燥，得到发射黄色荧光的碳量子点。
进一步，所述的单糖分子为甘露糖、葡萄糖、阿拉伯糖、来苏糖、果糖或半乳糖中的一种。
进一步，所述的苯二胺为邻苯二胺、间苯二胺或对苯二胺中的一种。
进一步，所述的单糖分子为甘露糖。
进一步，所述的苯二胺为间苯二胺。
进一步，所述的单糖分子和苯二胺的投料摩尔比为7:3。
进一步，步骤2)所述的反应温度为180℃，反应时间为2h。
2、上述制备方法制备得到的一种可快速标记大肠杆菌的碳量子点。
3、上述一种可快速标记大肠杆菌的碳量子点在快速标记大肠杆菌上的应用。
本发明的有益效果在于：本发明通过水热反应成功制备了发射黄色荧光的碳量子点，这些碳量子点对动物细胞具有良好的生物相容性，并且可以快速结合大肠杆菌，实现对大肠杆菌的快速标记。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图：
图1是发射黄色荧光的碳量子点的制备及快速标记大肠杆菌(E.coli)的示意图；
图2是发射黄色荧光的碳量子点的透射电镜图(TEM)；
图3是发射黄色荧光的碳量子点的激发光谱与发射光谱；
图4是发射黄色荧光的碳量子点的细胞相容性结果；
图5是发射黄色荧光的碳量子点对大肠杆菌(E.coli)的标记结果；
具体实施方式
下面结合附图，对本发明的优选实施例进行详细的描述。
实施例1可快速标记大肠杆菌的碳量子点的制备
图1是可快速标记大肠杆菌的碳量子点的制备及应用示意图，可快速标记大肠杆菌的碳量子点的制备包括以下步骤：
将单糖分子、苯二胺和水在搅拌下混合均匀。其中，单糖分子为甘露糖(Man)，苯二胺为间苯二胺(m-PD)。两者的投料摩尔比为7:3，总物质的量为0.5mmol。然后加入10mL水，均匀混合于水热反应釜中，在水热条件下，180℃反应2h。反应结束后，所得产物先进行离心(10000rpm,5min)，然后取上清液置于透析袋中(MWCO＝200Da),透析2h，最后冷冻干燥，得到发射黄色荧光的碳量子点,命名为CDs。
实施例2碳量子点的TEM表征
将实施例1中合成的碳量子点CDs配成溶液(0.25mg/mL),取一滴加入到铜网上，待样品干燥后，放置于透射电镜下进行扫描，得到TEM图，如图2所示。由图2可知，具有球形形状的碳量子点已经成功合成，并且大小均一，形貌规整。
实施例3碳量子点的荧光光谱图
将实施例1中合成的的碳量子点CDs进行冷冻干燥，然后配成碳量子点溶液(浓度为0.025mg/mL),取2mL加入荧光比色皿中，然后置于荧光分光光度计中进行荧光测试，扫描得到碳量子点的激发光谱与发射光谱，如图3所示。其中荧光分光光度计的狭缝宽度设为5nm,光谱扫描速度设为600nm/min。由图3可知，碳量子点的最佳激发峰位于445nm，最佳发射峰位于525nm,进一步证明发光碳量子点的合成。
实施例4碳量子点的细胞相容性实验
将实施例1中合成的的碳量子点CDs进行冷冻干燥，与培养基混合配成含有不同碳量子点浓度(5,25,50，100and200μg/mL)的溶液。以动物成前骨细胞MC3T3为例，将含有不同碳量子点浓度的培养基加入到MC3T3中，培养24h。然后将培养基吸出，在黑暗条件下将新鲜培养基以及CCK-8溶液加入细胞中，继续培养2.5h后，然后置于酶标仪上测试。所得到的结果如图4所示。从图4可知，碳量子点对MC3T3具有较好的细胞相容性。
实施例5碳量子点用于大肠杆菌标记
将碳量子点分散到生理盐水中，配成浓度为5mg/mL的碳量子点溶液，取20μL加入到2mL大肠杆菌(E.coli,106CFU/mL)的菌液中，放置于37℃恒温摇床上，10min后离心(5000rpm,3min)得到大肠杆菌与碳量子点的沉淀，然后用生理盐水清洗三次后，再重新分散到生理盐水中，置于激光共聚焦显微镜下观察，其中不加碳量子点的大肠杆菌作为对照组，如图5所示。由图5可知，加入碳量子点的大肠杆菌在激光共聚焦显微镜下呈现很明显的荧光，证明碳量子点可以很好的实现大肠杆菌的快速标记。
综上，碳量子点可以快速标记大肠杆菌，因此在细菌标记与区分中具有潜在的应用价值，为抗生素药物的选择提供依据，对生物医学领域具有重要的意义。
最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本发明权利要求书所限定的范围。
Claims (6)
1.一种可快速标记大肠杆菌的碳量子点的制备方法，其特征在于，包括以下步骤：
1)将单糖分子和苯二胺加入水中混合均匀；所述的单糖分子为甘露糖；所述的单糖分子和苯二胺的投料摩尔比为7:3；
2)在水热反应釜中进行水热反应，反应温度为150℃～240℃，反应时间为1h～5h；
3)反应结束后，所得产物先进行离心，取上清液透析，干燥，得到发射黄色荧光的碳量子点。
2.根据权利要求1所述的一种可快速标记大肠杆菌的碳量子点的制备方法，其特征在于，所述的苯二胺为邻苯二胺、间苯二胺或对苯二胺中的一种。
3.根据权利要求1所述的一种可快速标记大肠杆菌的碳量子点的制备方法，其特征在于，所述的苯二胺为间苯二胺。
4.根据权利要求1所述的一种可快速标记大肠杆菌的碳量子点的制备方法，其特征在于，步骤2)所述的反应温度为180℃，反应时间为2h。
5.根据权利要求1-4任一所述的制备方法制备得到的一种可快速标记大肠杆菌的碳量子点。
6.根据权利要求5所述的一种可快速标记大肠杆菌的碳量子点在快速标记大肠杆菌上的应用。
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