CN115340867B - 绿色荧光碳点GB-CDs制备方法及在检测线粒体中Fe3+和ATP的应用 - Google Patents
绿色荧光碳点GB-CDs制备方法及在检测线粒体中Fe3+和ATP的应用 Download PDFInfo
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- CN115340867B CN115340867B CN202211059377.5A CN202211059377A CN115340867B CN 115340867 B CN115340867 B CN 115340867B CN 202211059377 A CN202211059377 A CN 202211059377A CN 115340867 B CN115340867 B CN 115340867B
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Abstract
本发明公开了绿色荧光碳点GB‑CDs制备方法及在检测线粒体中Fe3+和ATP的应用，以绿豆(Green bean，GB)为原材料，通过一步水热法获得荧光碳点GB‑CDs。GB‑CDs可以用来检测溶液中的Fe3+，而GB‑CDs+Fe3+用于检测胎牛血清中的ATP。此外，GB‑CDs可以进入细胞，靶向线粒体进行成像，具有良好生物相容性和优异的光稳定性。因此，GB‑CDs和GB‑CDs+Fe3+可以分别用于追踪癌细胞线粒体中的Fe3+和ATP。本发明为原位实时检测线粒体中的Fe3+和ATP提供了强有力的工具。
Description
技术领域
本发明涉及纳米材料与生物技术，特别涉及绿色荧光碳点GB-CDs制备方法及在检测线粒体中Fe3+和ATP的应用。
背景技术
碳点(CDs)作为一种非晶或纳米晶结构的准球形碳基荧光纳米材料，在生物成像、传感器、催化、生物医学、荧光墨水和光电子学等领域有着巨大的应用潜力(AnalyticaChimica Acta 2022，339672)。CDs具有很多的优点，包括易于合成和功能化，具有化学惰性和低毒性等(Journal ofColloid and Interface Science 2020，579，96-108)。基于CDs的细胞Fe3+(Nanoscale research letters 2019，14，1-13)和ATP(Analytical Chemistry2020，92，7940-7946)的生物传感器也得到了很好的发展。然而，用于检测亚细胞器中的Fe3+或ATP探针的CDs很少见。据我们所知，目前只有一种检测溶酶体ATP的CDs：Xin等人通过微波介导的方法制备了一种新型CDs，用于溶酶体中的ATP检测(Analytical Chemistry2020，92，7940-7946)。利用CDs检测线粒体中的Fe3+和ATP仍未见报道。
铁是生物体中不可缺少的微量元素，在许多生化过程中起着至关重要的作用(Nature 2005，437，769-772)。线粒体是唯一可以合成血红素的部位(Proceedings of theNational Academy of Sciences 2009，106，16381-16386)，而Fe3+是线粒体中含量最丰富的金属离子之一(Chemical reviews 2009，109，4708-4721)。线粒体Fe3+的失调可导致与线粒体相关的疾病，如弗里德赖希共济失调和铁硫簇缺乏症(Journal of BiologicalChemistry 2006，281，22493-22502)。因此，了解Fe3+稳态维持线粒体功能的生理过程是很有必要的，这需要可靠的技术来检测线粒体Fe3+。然而，用于检测线粒体Fe3+的生物探针也很罕见。目前有一种用于测量活细胞中线粒体Fe3+水平的荧光探针(RNP1)，它是通过将烷基三苯基鏻与连接到有萘发色团罗丹明主骨架上得到的(Dalton Transactions 2013，42，10093-10096)。然而，罗丹明和萘对细胞和人体都有剧毒(Water Research 2021，197，117109)，并且RNP1的制备过程复杂。
三磷酸腺苷(ATP)是生物体内必需的多功能核苷酸(Sensors and Actuators B：Chemical 2020，310，127851)，主要通过线粒体的氧化磷酸化产生(Nature 2006，443，787-795)。线粒体ATP的波动通常与许多疾病有关，例如恶性肿瘤、帕金森病、缺血和血管性心脏病(Nature 2005，436，108-111)。关于线粒体ATP检测的方法较少。例如，Tan等人合成了一种名为Mito-Rh的荧光探针用于实时检测线粒体中的ATP浓度(Analytical chemistry2017，89，1749-1756)。Srivastava等人开发了分子支架/荧光探针来检测线粒体中的ATP(Biosensors and Bioelectronics 2015，69，179-185)。然而，这些已有的探针表现出低选择性，不能有效地区分ATP与其他具有相似结构的核苷多磷酸盐，如二磷酸腺苷(Journalof the American Chemical Society 2012，134，18779-18789)。更不利的是，Mito-Rh等材料原料昂贵、制备繁琐(Journal of the American Chemical Society 2012，134，18779-18789)。因此，开发一种易制备、高选择性的生物传感器，用于实时测量线粒体ATP变化的分析方法是非常有意义的。
发明内容
发明目的：本发明的目的是提供绿色荧光碳点GB-CDs制备方法。
本发明另一目的是提供所述绿色荧光碳点GB-CDs在检测线粒体中Fe3+和ATP的应用。
技术方案：所述绿色荧光碳点GB-CDs在检测线粒体中Fe3+和ATP的应用。
所述绿色荧光碳点GB-CDs以绿豆(GB)为原料制备得到。
绿豆首先打磨成粉并溶解于水中。然后通过一步水热法，形成绿色荧光碳点GC-CDs。GC-CDs在溶液中可以检测Fe3+，而GC-CDs+Fe3+可以检测胎牛血清中的ATP。同时GC-CDs可用于动物细胞成像以及线粒体Fe3+和ATP的检测。
其中，GB-CDs通过以下步骤制备得到：
(1)将干绿豆打磨成粉末状；
(2)将步骤(1)中得到的绿豆粉溶解于超纯水中；
(3)取步骤(2)中得到的溶液，转移入水热釜中反应；
(4)将步骤(3)中得到的反应产物离心，取上清，过滤，得到荧光碳点GB-CDs。
所述绿豆的用量为0.1-1g，超纯水的用量为10-100mL。
所述的步骤(3)中的反应条件为120-240℃、6-18h。
Fe3+的检测方法包括：
(1)将待测样品稀释至Fe3+浓度约在10-60μM；
(2)将步骤(1)中稀释好的样品与GB-CDs混合；
(3)将步骤(2)的得到的混合溶液在360nm激发下测量在440nm处的荧光强度；
(4)将步骤(3)得到的荧光强度带入标准曲线公式，再乘以稀释倍数即可获得待测样品的Fe3+浓度。
ATP的检测方法包括：
(1)将待测样品稀释至ATP浓度约在50-600μM；
(2)将步骤(1)中稀释好的样品与GB-CDs和Fe3+混合；
(3)将步骤(2)的得到的混合溶液在360nm激发下测量在440nm处的荧光强度；
(4)将步骤(3)得到的荧光强度带入标准曲线公式，在乘以稀释倍数即可获得待测样品的ATP浓度。
检测对象还包括溶液、水体、胎牛血清或动物细胞。
有益效果：本发明与现有技术相比，具有如下优势：
1、荧光碳点GB-CDs能实现亚细胞水平的成像和关键物质检测。
2、荧光碳点GB-CDs仅使用绿豆为原材料，通过简便的一步水热法合成，无需任何添加剂，制备简易。
3、GB-CDs对于Fe3+和ATP的检测具有高选择性和高灵敏度。此外，GB-CDs具有高稳定性和良好的生物相容性。
4、GB-CDs可以进行细胞线粒体成像，并用于检测线粒体内的铁离子和ATP。
附图说明
图1为荧光碳点的透射电镜成像图；
图2为荧光碳点的粒径分布频率示意图；
图3为荧光碳点的荧光图谱图；
图4为荧光碳点检测Fe3+能力的评价；
图5为荧光碳点检测Fe3+能力的特异性评价；
图6为荧光碳点检测ATP能力的评价；
图7为荧光碳点检测ATP能力的特异性评价；
图8为荧光碳点染色线粒体的能力评价；
图9为荧光碳点检测线粒体内Fe3+的能力评价；
图10为荧光碳点检测线粒体内ATP的能力评价；
图11为荧光碳点的稳定性评价；
图12为荧光碳点对动物细胞的细胞毒性评价；
图13为荧光碳点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 GB-CDs荧光碳点的制备
干绿豆研磨成细粉后，将0.5g绿豆粉溶解在40mL蒸馏水中。将所得溶液移入80mL聚四氟乙烯衬里的不锈钢高压釜中，并在180℃下加热12h。冷却至室温后，将溶液以15000rpm离心15min以收集上清液，进一步通过0.22μm滤膜过滤。最后将所得GB-CDs溶液4℃保存备用。
实施例2 GB-CDs荧光碳点的形态观察
将过滤好的GB-CDs荧光碳点用去离子水稀释，取10μL滴在铜网上，用透射电镜(Malvern Instruments，Nano ZS，United Kingdom)进行观察，如图1所示。透射电镜观察的结果显示该荧光碳点近似球形结构且分布均匀。
实施例3 GB-CDs荧光碳点粒径分布检测
按比例尺测量图1透射电镜观察得到的荧光碳点的直径，统计其粒径分布，如图2所示，显示该荧光碳点的平均粒径约为14nm。
实施例4 GB-CDs荧光碳点荧光光谱检测
通过荧光分光光度计(RF-5301PC，Shimadzu，Japan)检测GB-CDs在水中的荧光光谱。如图3所示，显示该GB-CDs具有绿色用光，激发波长为360nm时，在440nm处有最强荧光。
实施例5 GB-CDs荧光碳点的检测Fe3+的能力评价
将150μL(1mg/mL)GB-CDs与Fe3+混合，在加入超纯水将每种混合物的最终体积调整为1mL，使Fe3+的最终浓度为10、20、30、40、50、和60μM。在360nm激发下测量混合溶液在440nm处的荧光吸收强度。如图4，随着Fe3+浓度的增加，GB-CDs的荧光强度逐步减弱，二者在Fe3+浓度为10-60nM范围内成线性关系，最低检出限(LOD)为3.6nM，表明具有GB-CDs检测Fe3+的能力。
实施例6 GB-CDs荧光碳点的检测Fe3+的特异性评价
类似实施例5，分别评价GB-CDs对Fe3+以外的其他金属离子(Cu2+、Ni2+、Mn2+、Hg2+、Fe2+、Zn2+、Cd2+、Ag+、Ca2+和K+)的荧光检测性。在360nm激发下测量混合溶液在440nm处的荧光吸收强度。如图5所示，Fe3+以外的金属离子对GB-CDs的荧光强度没有影响，所以GB-CDs检测Fe3+具有特异性。
实施例7 GB-CDs+Fe3+检测ATP的能力评价
将150μL(1mg/mL)GB-CD与Fe3+混合后，再加入ATP，加入超纯水将每种混合物的最终体积调整为1mL，使Fe3+的最终浓度都为500μM，ATP的最终浓度为100、200、300、400、500和600μM。在360nm激发下测量混合溶液在440nm处的荧光吸收强度。如图6，随着ATP浓度的增加，GB-CDs+Fe3+溶液的荧光强度逐渐增强，’二者在ATP浓度为50-600nM范围内成线性关系，最低检出限(LOD)为60nM，表明GB-CDs具有检测ATP的能力。
实施例8 GB-CDs+Fe3+检测ATP的特异性评价
类似实施例7，分别评价ATP类似物如：三磷酸尿苷(UTP)、三磷酸鸟苷(GTP)、三磷酸胞苷(CTP)、焦磷酸盐(Ppi)、二磷酸腺苷(ADP)、一磷酸腺苷(AMP)和环磷酸腺苷(cAMP)；以及其他生物分子如：人血清白蛋白(HAS)、谷胱甘肽(GSH)、酪氨酸(Try)、胱氨酸(Cys)、多巴胺(DA)、甘氨酸(Gly)、谷氨酸(Glu)、硫胺素(Thi)、赖氨酸(Lys)、精氨酸(Arg)和丙氨酸(Ala)对GB-CDs+Fe3+检测ATP能力的干扰作用。在360nm激发下测量混合溶液在440nm处的荧光吸收强度。如图7所示，ATP的类似物对GB-CDs+Fe3+的荧光强度没有影响，所以GB-CDs+Fe3 +检测ATP具有特异性。
实施例9 GB-CDs荧光碳点的染色线粒体的能力评价
用GB-CDs与A549细胞孵育2h后，用荧光共聚焦显微镜观察细胞；另外同时使用GB-CDs和专用于线粒体的商业染料Mitotracker Red孵育A549细胞后，用荧光共聚焦显微镜观察细胞。由图8中可知，A549显示绿色荧光，表明GB-CDs可以渗透到动物细胞中。此外，A549中的绿色荧光与Mitotracker Red的红色荧光很好地重叠。这表明GB-CDs在进入细胞后在线粒体中积累，可用于细胞线粒体的成像。
实施例10 GB-CDs荧光碳点检测线粒体内Fe3+的评价
A549细胞先用0、100、200、300或400μM Fe3+处理2h，然后与GB-CDs(100μg/mL)一起孵育。然后使用流式细胞仪检测。如图9所示，随着Fe3+浓度的增加，A549细胞中GB-CDs荧光的强度降低。GB-CDs可用于细胞内线粒体Fe3+监测。
实施例11 GB-CDs荧光碳点的检测线粒体内ATP的评价
A549细胞用ATP(400M)处理2h，然后用GB-CDs+Fe3+孵育。通过流式细胞仪测定样品的荧光强度。如图10所示，A549细胞中GB-CDs的荧光强度在Fe3+存在时降低，但在有ATP的时候荧光恢复，表明GB-CDs+Fe3+系统适用于线粒体ATP的实时监测。
实施例12 GB-CDs荧光碳点的稳定性评价
通过使GB-CDs溶液处在不同pH值(1-11)下，紫外灯照射的不同时间后，不同NaCl浓度(0.0、0.5、1.0、1.5、2.0、和2.5M)下和不同温度(30、40、50、60、70、80、和90℃)下，都通过荧光分光光度计记录这些溶液在440nm(λex＝360nm)的荧光强度。由图11可知，GB-CDs在pH值3-9、紫外照射90min、溶液NaCl浓度升高2.5M、不同温度90℃处理之后，仍然具有较好的稳定性。
实施例13 GB-CDs荧光碳点对动物细胞的细胞毒性研究
在含有10％胎牛血清和100IU/mL青霉素的DMEM培养基中培养HPAEpiC(HP)细胞(正常人肺细胞)，A549细胞(人肺癌细胞)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GB-CDs荧光碳点孵育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，ThermoScientific，USA)测量波长570nm处的吸光度。结果如图12所示，该荧光碳点对人体细胞几乎无毒性。
实施例14 GB-CDs荧光碳点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CMCS-PEI-PpIX荧光碳点GB-CDs在37℃下孵育1h，其最终浓度分别为1、2、4、和10μM。在4000rpm离心5min后，将上清液转移到96孔板中，用酶标仪(Multiskan FC，Thermo Fisher Scientific，USA)记录405nm处的吸光度并计算溶血率。分别以超纯水和细胞PBS处理的红细胞作为阳性对照和阴性对照。结果如图13所示，该荧光碳点对红细胞的溶血性可忽略不计。表明GB-CDs具有很好的生物安全性。
表1为荧光碳点检测自来水中Fe3+和胎牛血清中ATP的结果。
Claims (6)
1.一种绿色荧光碳点GB-CDs在检测线粒体中Fe3+和ATP的应用，所述应用不以疾病的诊断和治疗为目的；所述绿色荧光碳点GB-CDs以绿豆为原料制备得到；
所述绿色荧光碳点GB-CDs制备方法包括：
（1）将干绿豆打磨成粉末状；
（2）将步骤（1）中得到的绿豆粉溶解于超纯水中；
（3）取步骤（2）中得到的溶液，转移入水热釜中反应；
（4）将步骤（3）中得到的反应产物离心，取上清，过滤，得到荧光碳点GB-CDs。
2.根据权利要求1所述的应用，其特征在于：所述绿豆的用量为0.1-1 g，超纯水的用量为10-100 mL。
3.根据权利要求1所述的应用，其特征在于：所述的步骤（3）中的反应条件为120-240℃、6-18 h。
4.根据权利要求1所述的应用，其特征在于：Fe3+的检测方法包括：
（1）将待测样品稀释至Fe3+浓度在10-60 μM；
（2）将步骤（1）中稀释好的样品与GB-CDs混合；
（3）将步骤（2）的得到的混合溶液在360 nm激发下测量在440 nm处的荧光强度；
（4）将步骤（3）得到的荧光强度带入标准曲线公式，再乘以稀释倍数即可获得待测样品的Fe3+浓度。
5.根据权利要求1所述的应用，其特征在于：ATP的检测方法包括：
（1）将待测样品稀释至ATP浓度在50-600 μM；
（2）将步骤（1）中稀释好的样品与 GB-CDs和Fe3+混合；
（3）将步骤（2）的得到的混合溶液在360 nm激发下测量在440 nm处的荧光强度；
（4）将步骤（3）得到的荧光强度带入标准曲线公式，再乘以稀释倍数即可获得待测样品的ATP浓度。
6.根据权利要求1所述的应用，其特征在于：检测对象还包括溶液、水体、胎牛血清或动物细胞。
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