CN114605989B - 一种绿色荧光碳点及其制备方法和用途 - Google Patents
一种绿色荧光碳点及其制备方法和用途 Download PDFInfo
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Abstract
本发明提供了绿色荧光碳点及其制备方法和用途。所述荧光碳点是由牙刷树粉末和间苯二胺通过一步水热法制备。荧光碳点Mis‑mPD‑CDs具有对刚果红高灵敏度和高特异性的检测能力，能实现对溶液、细胞和动物体内的刚果红检测。此外，Mis‑mPD‑CDs具有良好的光稳定性和生物安全性。本发明为食品、工业废水、生物体内刚果红的检测提供了有效方法。
Description
技术领域
本发明属于纳米材料及制备方法和用途，特别涉及一种绿色荧光碳点及其制备方法和用途。
背景技术
由于印刷、农业和纺织工业产生有害污染物，环境承受着巨大压力。检测这些污染物是监测和控制环境污染的一项重要的全球任务(Journal of the Korean CeramicSociety.2020，1-11)。刚果红是一种衍生自联苯胺的偶氮染(Chemosphere.2005，61，492-501)，对环境和人类均显示出高毒性。在染料、纺织品、皮革、纸张、塑料、食品、化妆品和药品等行业中使用染料和颜料，产生了大量的含刚果红的废水。刚果红不易被微生物降解，并且在环境中高度稳定Journal of Environmental Management.2019，242，229-237)。而且刚果红进入人体后会转化为联苯胺，其有强烈的致癌作用(Chemical EngineeringResearch and Design.2016，109，824-834)。此外，刚果红对人体健康还有许多其他负面影响，引起呕吐，恶心，腹泻，过敏性问题Journal of colloid and interfacescience.2018，521，172-182)。然而，刚果红的检测并没有像其他的偶氮染料如亚甲基蓝，甲基橙，溴酚蓝和结晶紫一样受到关注Journal of Chromatography B.2005，826，244-251)。
当前，刚果红的检测方法包括传统的UV-可见分光光度法Journal of the KoreanCeramic Society.2020，1-11)，以及新近开发的分光光度法(Toxicological&Environmental Chemistry.2012，94，1886-1892)和电化学法(Aminabhavi，MicrochemicalJournal.2019，146，387-392)。紫外可见分光光度法对刚果红检测的灵敏度较低，需要采用不同策略对水样品中的刚果红进行预浓缩，这一过程非常耗时并且需要大量样品Journalof the Korean Ceramic Society.2020，1-11)。基于分光光度法的刚果红检测受到金属离子如Cu(II)，Fe(III)和Cr(VI)的干扰，而且使用到盐酸这种危险溶剂。电化学方法是通过在玻璃碳电极(Glassy carbon electrode)上制造的氧化石墨烯纳米颗粒记录刚果红在电化学降解过程中的循环伏安图来建立的(Aminabhavi，Microchemical Journal.2019，146，387-392)。这种方法会受到其他金属盐的干扰，例如氯化钡，硫酸铵，乙酸铜和硝酸钾(Aminabhavi，Microchemical Journal.2019，146，387-392)。所有这些方法都具有操作时间长，灵敏度低，选择性差，成本高和劳动强度大的缺点，且无法用于细胞或生物体的原位检测。因此，有必要开发新的低成本、高灵敏、高效的用于细胞和生物体内的刚果红检测方法。
碳点(Carbon Dots，CDs)是一种碳基荧光纳米材料，自从在单壁碳纳米管的处理过程中首次发现它们以来，近年来受到了广泛关注(Nano Today.2020，33，100879)。由于其在发光，稳定性，生物相容性和低成本方面的突出优点，碳点已被广泛作为荧光纳米探针(ACS nano.2014，8，4522-4529)、化学传感器和生物传感器(Chemical SocietyReviews.2015，44，362-381)、光电器件(Chemical communications.2012，48，2692-2694)、光催化反应剂(Angewandte Chemie International Edition.2016，55，12470-12474)和生物医药Journal of the American Chemical Society.2017，139，13147-13155)。近年来，碳点已经开始应用于环境污染物的检测，主要包括金属离子(Chemical EngineeringJournal.2020，126848)和染料(RSC advances.2019，9，29533-29540)的检测。然而，据我们所知，尚未有CD用于检测刚果红的报道。
发明内容
发明目的：本发明的目的是提供一种绿色、高光稳定性、高生物安全性的绿色荧光碳点。
本发明的另一目的是提供所述绿色荧光碳点的制备方法和用途。
技术方案：本发明的绿色荧光碳点Mis-mPD-CDs通过牙刷树以及间苯二胺mPD通过一步水热法制备得到。
具体包括如下步骤：
(1)将牙刷树粉末和间苯二胺溶解于去离子水中，得到混合溶液，160-200℃下反应6-18h；
(2)将步骤(1)中得到的反应产物离心，收集上清液，过滤；
(3)将所得的碳点进行透析，冷冻干燥，即可。
进一步地，步骤(1)中，牙刷树粉末和间苯二胺的浓度分别为1.5-5mg/mL，0.5-1.5mg/mL。
进一步地，步骤(1)中，所述水热釜为有聚四氟乙烯内衬的高温高压水热釜，容积为25-100mL。
进一步地，步骤(1)中，优选反应温度为180℃，最优反应时间为12h。
所述绿色荧光碳点Mis-mPD-CDs用于细胞内刚果红的的荧光检测。检测步骤如下：动物细胞、细菌或真菌与Mis-mPD-CDs共孵育一段时间后，用荧光显微镜进行荧光强度的观察，用流式细胞仪进行荧光强度的检测。通过定量细胞内的Mis-mPD-CDs荧光强度来检测细胞内刚果红。
其中，Mis-mPD-CD的浓度为10-1000μg/mL，孵育时间为2-60min。
绿色荧光碳点Mis-mPD-CDs用于生物内刚果红的的荧光检测。检测步骤如下：
生物体与Mis-mPD-CD共孵育一段时间后，。在具有10×物镜的荧光显微镜下，进行观察，通过荧光强度的检测用于定量分析生物体内的刚果红浓度。
其中，Mis-mPD-CD的浓度为50-1000μg/mL，孵育时间为30-120min。
本发明与现有技术相比，其显著优点为：绿色荧光碳点Mis-mPD-CDs具有对刚果红高灵敏度和高特异性的检测能力，检测限为5.8×10-8M，不受其它环境污染物如金属离子和燃料的干扰；Mis-mPD-CDs有很高的光稳定性，光照90min以内荧光强度几乎无影响；Mis-mPD-CDs生物安全性也很优异，在50μg/mL的浓度下只杀伤10％的细胞，且几乎无溶血性。更重要的是Mis-mPD-CDs不仅能能实现溶液中的刚果红检测，还可以实现细胞、生物体内的刚果红检测。Mis-mPD-CDs在食品、工业废水等情况下为对刚果红的快速检测提供了有效方法。
有益效果：本发明的荧光碳点绿色、高光稳定性、高生物安全性，实现细胞及生物体内刚果红的快速、简单、高灵敏性和高特异性的检测。
附图说明
图1为荧光碳点Mis-mPD-CDs的透射电镜成像图；
图2为荧光碳点Mis-mPD-CDs的zeta电位(Zeta potential)检测结果示意图；
图3为荧光碳点Mis-mPD-CDs的荧光谱图；
图4荧光碳点Mis-mPD-CDs的荧光随着刚果红的加入逐渐减弱的荧光谱图；
图5为荧光碳点Mis-mPD-CDs的刚果红检测能力评价；
图6为荧光碳点Mis-mPD-CDs的刚果红检测特异性评价；
图7荧光碳点Mis-mPD-CDs用于细胞内刚果红的检测结果；
图8为荧光碳点Mis-mPD-CDs用于斑马鱼的刚果红的检测结果；
图9为荧光碳点Mis-mPD-CDs的稳定性评价；
图10为Mis-mPD-CDs对动物细胞的细胞毒性(Cytotoxicity)评价；
图11为Mis-mPD-CDs对新鲜小鼠血红细胞的溶血性评价。
具体实施方式
实施例1荧光碳点Mis-mPD-CDs的制备
把牙刷树粉末和间苯二胺分别以1.5mg/mL和0.5mg/mL溶解于去离子水。将溶液转移至25mL水热反应器中，180℃下反应12h。冷却至室温后，离心取上清，用超纯水、透过分子量1kDa透析1d，过0.22μm滤膜，得到荧光碳点。
实施例2荧光碳点Mis-mPD-CDs的扫描电镜观察：
将过滤好的荧光碳点用去离子水稀释，取10μL滴在洁净的铜网上，用透射电镜(JEM-2100，JEOL Ltd，Japan)进行观察，如图1所示。透射电镜观察的结果显示该碳点Mis-mPD-CDs近似球形结构且分布均匀。
实施例3荧光碳点Mis-mPD-CDs的粒径分布检测
按比例尺测量图1扫描电镜观察得到的碳点Mis-mPD-CD的直径，统计其粒径分布，如图2所示，显示该碳点Mis-mPD-CDs的平均粒径约为19.6nm；
实施例4荧光碳点Mis-mPD-CDs的电位检测
将获得的荧光碳点置于zeta电位(Zeta potential)检测池中，使用纳米粒度和zeta电位分析仪(Malvern Instruments，Zetasizer Nano ZS，UK)测量其zeta电位，检测结果如图3所示。电位检测结果显示该Mis-mPD-CDs带负电(-6.7mV)；
实施例5荧光碳点Mis-mPD-CDs的刚果红检测能力的效果评价
16.6μL 3mg/mL碳点与100μM不同体积的刚果红(CR)混合，使用去离子水将每种混合物的最终体积调整为1ml，以达到不同的CR终浓度。在440nm激发下测量混合溶液的荧光发射光谱，如图4所示，随着刚果红浓度的提高，Mis-mPD-CDs的在510nm处的荧光逐渐减弱。工作曲线由log(F0-F)/F0和CR浓度绘制。F0和F分别是没有和有CR的碳点的荧光强度，如图5所示。Mis-mPD-CDs的(F0-F)/F0比值和刚果红浓度的线性检测范围为0.2-1.2μM。由检测限的计算公式获得Mis-mPD-CDs的刚果红检测限为5.8×10-8M。
检测限(LOD)的计算公式如下：
LOD＝3σ/K
其中K是线性范围的斜率，σ是空白的标准偏差(n＝11)。
实施例6荧光碳点Mis-mPD-CDs的荧光检测特异性评价
类似实施例5，将荧光碳点Mis-mPD-CD和其它其他环境污染物(亚甲基蓝MB、溴酚蓝BPB和结晶紫CV)、金属离子(Fe3+、Ca2+、Mg2+、K+、Mn2+、Zn2+、Cu2+、Hg2+、Fe2+、Ag+、和Cd2+)和重要生物分子(人血清白蛋白HSA、甘氨酸Gly、三聚氰胺Mel、多巴胺DA和谷胱甘肽GSH)进行混合，测得荧光碳点Mis-mPD-CD的荧光变化。如图6所示，荧光碳点Mis-mPD-CD的荧光不受其它环境污染物和重要生物分子的影响，表明Mis-mPD-CD对刚果红的检测具有高特异性。
实施例7荧光碳点Mis-mPD-CDs在实际样品中的刚果红检测
从鲳鱼和鸦片鱼中收集组织样本，切碎并冷冻。将10g解冻的组织溶解在含有30mL乙腈的50mL烧杯中，然后超声处理1h。8000rpm离心5min后收集上清液。上清液中加入无水硫酸钠。然后通过旋转蒸发仪蒸发乙腈，并使用磷酸盐缓冲盐水(PBS)分散样品。取样品与16.66μL的3mg/mL碳点Mis-mPD-CDs混合，最终体积调节至1 mL。在440nm激发下检测混合溶液的荧光发射光谱。使用碳点Mis-mPD-CDs检测工业废水中的CR与上述相同。如表1，在鱼类组织或工业废水中均未检测到CR。额外添加CR后，检出CR的准确率率为94％-109％，相对标准偏差(RSD)小于6.6％，足以在实际样品中进行定量测量。这些发现表明，基于Mis-mPD-CDs的荧光测量在检测真实样品中的CR方面具有巨大潜力。
表1为荧光碳点Mis-mPD-CDs对真实样品中的刚果红检测能力评价。
实施例8荧光碳点Mis-mPD-CDs在细胞中的刚果红检测
人肺癌细胞(A549)在DMEM中培养，将细胞以每孔5×103个细胞的密度接种于96孔板中；白色念珠菌(C.albicans)培养于YM培养基，离心收集；大肠杆菌(E.coli)和金黄色葡萄球菌(S.aureus)培养于LB培养基，离心收集。将对应的培养基更换为含有Mis-mPD-CDs(50μg/mL)的培养基，并在37℃下孵育不同时间(2、10、30、40和60min)。然后，使用具有63×油浸物镜的共聚焦激光扫描显微镜(CLSM，Leica SP8)检查细胞成像。在激发波长为488nm，荧光发射为500-550nm。如图7，在A459细胞、白色念珠菌、大肠杆菌和金黄色葡萄球菌中，随着刚果红浓度提高，Mis-mPD-CDs发出的荧光强度逐渐降低，表明荧光碳点Mis-mPD-CDs可用于动物、真菌和细菌细胞中的刚果红检测。
实施例9荧光碳点Mis-mPD-CDs在斑马鱼中的刚果红检测
使用标准斑马鱼E3培养基在2mL管中室温培养5天(受精后天数)的斑马鱼。首先，斑马鱼幼虫与0、5、15和20μM CR共孵育4h，然后用100μg/mL Mis-mPD-CD处理1h。然后在具有10×物镜的共聚焦激光扫描显微镜(CLSM，Leica SP8)上获得斑马鱼的共聚焦图像。激发波长为488nm，荧光发射为500--550nm。如图8，在斑马鱼体内，随着刚果红浓度提高，Mis-mPD-CDs发出的荧光强度逐渐降低，表明荧光碳点Mis-mPD-CDs可用于生物内的刚果红检测。
实施例10荧光碳点Mis-mPD-CDs的稳定性评价
使用365nm(20mW)的紫外灯照射碳点溶液不同的时间，并通过荧光分光光度计测量510nm(λex＝440nm)下照射的碳点溶液的相应荧光强度。同样，不同NaCl浓度(0.0、0.2、0.5、1.0、1.5、2.0和2.5M)和不同温(30、40、50、60、70、80、90和100℃)下的碳点溶液的荧光强度也被记录下来。如图9，在紫外灯照射30min以内、NaCl浓度2.5M以下、温度30-100℃以内，Mis-mPD-CDs的荧光强度都没有明显变化。
实施例11荧光碳点Mis-mPD-CDs对动物细胞的细胞毒性(Cytotoxicity)研究
在含有10％胎牛血清和100IU/mL青霉素的Dulbecco改良Eagle培养基(DMEM)中培养HPAEpiC(HP)细胞(正常人肺细胞)和Hep G2细胞(人肝癌细胞)，培养温度为37℃，5％CO2。将5×103个细胞转移到96孔板的每个孔中并生长24h。然后，将细胞与碳点孵育24h，然后加入10μL 5mg/mL的3-(4，5-二甲基噻唑-2-基)-2，5-二苯基溴化四唑(MTT)再孵育4h。除去培养基后加入150μL DMSO。最后，用酶标仪(Multiskan FC，Thermo Scientific，USA)测量波长570nm处的吸光度。结果如图10所示，该纳米粒子对人体细胞几乎无毒性。
实施例12荧光碳点Mis-mPD-CDs对新鲜小鼠血红细胞的溶血性研究
通过4000rpm离心5min，从小鼠全血中提取红细胞(RBC)，并重新悬浮在细胞用磷酸盐缓冲盐水(PBS)中。将分离的RBC与碳点在37℃下孵育1h，形成浓度梯度。在4000rpm离心5min后，将上清液转移到96孔板中，用酶标仪(Multiskan FC，Thermo FisherScientific，USA)记录405nm处的吸光度并计算溶血率。分别以Triton和无处理的红细胞作为阳性对照和阴性对照。结果如图11所示，该碳点对红细胞的溶血性可忽略不计。
Claims (8)
1.一种绿色荧光碳点，其特征在于：由牙刷树粉末和间苯二胺通过一步水热法制备获得；
所述牙刷树粉末和间苯二胺分别以1.5mg/mL 和0.5mg/mL 溶解于去离子水，在水热反应器中 180℃下反应 12h。
2.权利要求1所述的绿色荧光碳点的制备方法，其特征在于：
(1)将牙刷树粉末和间苯二胺溶解于去离子水中，得到混合溶液，180℃下反应12h；
(2)将步骤(1)中得到的反应产物冷却至室温后离心，收集上清液；
(3)将步骤(2)中得到的上清液透析过滤得到荧光碳点。
3.权利要求1所述的绿色荧光碳点在刚果红荧光检测中的应用。
4.根据权利要求3所述的应用，其特征在于：用于溶液、细胞和动物体内的刚果红的检测。
5.根据权利要求4所述的应用，其特征在于：所述溶液包括废水。
6.根据权利要求4所述的应用，其特征在于：所述细胞包括动物细胞、细菌和真菌。
7.根据权利要求4所述的应用，其特征在于：所述动物包括斑马鱼、老鼠、果蝇。
8.根据权利要求4所述的应用，其特征在于：检测的仪器包括荧光光谱仪、荧光共聚焦显微镜或流式细胞仪。
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