CN111647401B - 一种橙色荧光碳点及其在检测过氧亚硝酸根离子中的应用 - Google Patents
一种橙色荧光碳点及其在检测过氧亚硝酸根离子中的应用 Download PDFInfo
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Abstract
本发明公开了一种橙色荧光碳点及其在检测过氧亚硝酸根离子中的应用,所述的碳点(CDs)是以罗丹明B和对氨基酚为原料,通过一步水热合成法制备得到;该碳点是零维碳纳米材料,可以发出明亮的橙色荧光。本发明制备方法简便,制得的碳点水溶性好,荧光性质稳定。以碳点作为荧光探针能够快速灵敏地检测过氧亚硝酸根离子。当过氧亚硝酸根离子存在时,碳点的荧光被过氧亚硝酸根有效淬灭。同时,CDs溶液颜色由粉色变为蓝黑色。本发明提供的检测ONOO‑的分析方法与单纯的荧光分析法相比,双模式输出的方法在实际应用中更具有优势,预示着其在细胞生物学中探讨ONOO‑的作用具有潜在的应用前景。
Description
技术领域
本发明涉及碳纳米材料和过氧亚硝酸根离子的检测,具体涉及一种橙色荧光碳点及其制备方法,以及将该碳点用于荧光和比色双模式检测过氧亚硝酸根离子。
背景技术
过氧亚硝酸根(ONOO-)是由一氧化氮(NO)和超氧自由基(O2.-)扩散控制偶联自反应生成。研究表明,ONOO-以其酸的形式存在于质子化平衡中(ONOOH,pKa=6.8),它可以迅速变成硝酸盐。由于它寿命很短,稳态浓度相对较低和多种体内反应靶点的性质,人们称它是一种“隐形”的生物氧化剂。由于其强的氧化性和良好的亲核性,ONOO-可以与不同的生物分子反应,而且,ONOO-被认为与各种疾病的发生有关,例如炎症性疾病、阿尔茨海默病、心血管和神经退行性疾病等等。尽管ONOO-在人类健康和疾病中具有重要作用,但它们的生物学功能还没有得到充分的了解。主要原因之一是缺乏可靠的方法高灵敏高选择性监测体内ONOO-水平。此外,ONOO-的寿命极短,以及各种竞争性内源性分子的存在都使它的测定更具挑战性。
到目前为止,已经有许多检测过氧亚硝酸根的方法,如电子顺磁共振(EPR)光谱,电化学分析,紫外-可见吸收光谱,色谱法等等。然而,这些方法都有各自的缺点。例如,需要复杂的样品预处理,昂贵的仪器,灵敏度/选择性差,和不适合细胞内检测等等。因此,开发一种简便快速,选择性好,灵敏度高的分析技术检测识别过氧亚硝酸根具有重要意义。
荧光碳点作为新兴的荧光纳米材料在生化传感、生物成像、环境分析等领域具有良好的应用潜力。因此,本发明基于碳点优异的荧光性质和生物性能,将荧光分析和比色分析与纳米材料结合,开发了一种荧光和比色双模式检测过氧亚硝酸根的分析方法。与单纯的荧光变化相比,双输出模式检测方法在实际应用中更具有突出的优势,预示着其在细胞生物学中探讨ONOO-的作用具有潜在的应用前景。
发明内容
本发明的目的在于提供一种结构性质优异的荧光碳点及其制备方法,碳点的制备方法简便;所制备的碳点表面化学结构优异,可应用于荧光和比色双模式检测过氧亚硝酸根,并展现出高的选择性和灵敏度。
本发明提供的一种橙色荧光碳点的制备方法,包括以下步骤:
1)、将罗丹明B和对氨基苯酚置于玻璃烧杯中,加入乙醇,充分搅拌后超声溶解10-15分钟,罗丹明B和对氨基苯酚的质量比为:0.01-0.05∶0.03-0.1;
2)、将上述溶液转移至水热反应釜中,并置于烘箱内,180℃反应8-12小时;
3)、待反应停止后静置水热反应釜,冷却至室温,过滤去除不溶物,旋蒸得到深粉色溶液,用0.22μm滤膜过滤后得到纯净的碳点水溶液;
4)、将上述碳点溶液冷冻干燥后得到目标产物。
所述的罗丹明B和对氨基苯酚的质量比优选为:0.015-0.035∶0.04-0.06。
上述方法制备的碳点荧光性质稳定,具有良好的水溶性和分散性,合成方法简单有效,原料廉价易得,反应条件温和且环境友好,在一般实验室均能完成,易于推广。此外,该碳点表现出良好的选择性,在碳点溶液中,只有加入过氧亚硝酸根,使得溶液颜色由粉色变为蓝黑色,碳点的荧光淬灭,而加入还原剂(Cys和GSH)、氧化剂(H2O2、tBuO·、HO·和1O2)、一些离子(Zn2+、Mg2+、Ca2+、NO3 -、SO4 2-),溶液颜色无明显变化,并且碳点的荧光强度无明显变化。
本发明提供的一种荧光碳点荧光和比色双模式检测过氧亚硝酸根离子的方法,其特征在于步骤为:
1)、配置浓度为0.1mg/mL的上述方法制备的碳点溶液;
2)、向荧光杯中滴加11.7μL不同浓度(30-1070nM)的ONOO-溶液,使碳点的荧光逐渐淬灭,得到碳点/ONOO-溶液,溶液颜色由粉色变为蓝黑色;
4)、测定碳点反应前后的荧光强度,根据过氧亚硝酸根离子的浓度和相对荧光强度变化值之间的关系建立检测过氧亚硝酸根离子的标准曲线;进而实现荧光和比色双模式检测过氧亚硝酸根离子。
本发明具有以下有益技术效果:
(1)本发明通过一步水热法即可得到碳点溶液,合成方法简单有效,原料廉价易得,反应条件温和且环境友好,在一般实验室均能完成,易于推广。所制备的碳量子点作为探针可用于检测水溶液中的ONOO-,线性范围为30-480和670-1070nmol/L,检出限是15.45nmol/L。
(2)本发明所述的基于碳点的荧光探针与传统有机探针相比,制备步骤简单,无需后续添加强酸强碱或表面钝化剂进行处理,反应物在同一体系中进行碳化、聚合及表面修饰,即可得到目标碳点。
(3)本发明利用荧光分析和比色分析相结合的方法,与单一的检测方式相比,双输出检测模式在检测中更具有优越性,提高了分析方法的选择性和灵敏度,对待测物可实现准确的定性、定量分析。
附图说明
图1为实施例1制备的碳点的荧光发射光谱及紫外吸收光谱;
图2为实施例1制备的碳点的红外图谱图中横坐标为检测波长,纵坐标为透过率;
图3为实施例1制备的碳点检测ONOO-荧光发射光谱图;
图4为实施例1制备的碳点检测ONOO-的紫外吸收光谱;
图5为碳点、碳点/ONOO-溶液体系在日光灯和紫外灯下的照片;
图6为实施例1制备的碳点被ONOO-猝灭的激光共聚焦图,所述的细胞为PC-12细胞。
具体实施方式
下面结合实施例和附图对本发明做详细说明,实施例给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。
实施例1
碳点的制备方法:
1)、将0.03g罗丹明B和0.05g对氨基苯酚置于玻璃烧杯中,加入乙醇,充分搅拌后超声溶解15分钟;
2)、将上述溶液转移至水热反应釜中,并置于烘箱内,180℃反应10小时;
3)、待反应停止后静置水热反应釜,冷却至室温,过滤去除不溶物,旋蒸得到深粉色溶液,用0.22μm滤膜过滤后得到纯净的碳点水溶液;
4)、将上述碳点溶液冷冻干燥后得到目标产物。
实施例2
将实施例1制备的荧光碳点进行荧光激发、发射和紫外吸收光谱表征(见图1),碳点在221nm和295nm处出现的明显吸收峰可归因于芳香族苯环sp2的π-π*跃迁和C-N键n/π*跃迁。CDs的最佳激发和发射波长分别554nm和580nm。对该碳点进行红外表征(如图2),表面含有氨基、羧基、羟基基团。
实施例3
取实施例1制备的荧光碳点水溶液(0.1mg/mL)3mL置于荧光比色皿中,然后向荧光杯中滴加11.7μL不同浓度ONOO-(30-1070nM),混合均匀,在荧光光度计中扫描发射光谱(λex=554nm,λem=580nm),根据ONOO-的浓度和相对荧光强度变化值之间的关系,计算碳点对ONOO-的检测范围及检出限(见图3)。
实施例4
取实施例1制备的荧光碳点水溶液(0.1mg/mL)3mL置于荧光比色皿中,然后向荧光杯中滴加14μL不同浓度ONOO-(300-1000nM),混合均匀,在紫外光度计中扫描紫外吸收光谱(见图4)。
实施例5
图5为碳点、碳点/ONOO-溶液体系在日光灯和紫外灯下的照片;图中:第一排,将实施例1制备的荧光碳点水溶液置于玻璃瓶中,在日光灯下为粉色,加入ONOO-后变为蓝黑色;第二排,为第一排溶液在365nm紫外灯下的图片,碳点溶液为橙色荧光,加入ONOO-荧光淬灭(见图5)。
实施例6
实施例1制备的荧光碳点水溶液(0.1mg/mL)用于标记的PC-12细胞,如图6所示,细胞形态良好,可见碳点细胞毒性很低,可用于活细胞标记。图6为实施例1制备的碳点被ONOO-猝灭的激光共聚焦图,从左到右依次为:暗场细胞图(橙色),明场细胞图(橙色),明场和暗场叠加图。
Claims (3)
1.一种橙色荧光碳点在荧光和比色双模式检测ONOO-中的应用,所述橙色荧光碳点通过如下步骤的方法制备得到:
1)、将罗丹明B和对氨基苯酚置于玻璃烧杯中,加入乙醇,充分搅拌后超声溶解10-15分钟,罗丹明B和对氨基苯酚的质量比为:0.01-0.05∶0.03-0.1;
2)、将上述溶液转移至水热反应釜中,并置于烘箱内,180℃反应8-12小时;
3)、待反应停止后静置水热反应釜,冷却至室温,过滤去除不溶物,旋蒸得到深粉色溶液,用0.22μm滤膜过滤后得到纯净的碳点水溶液;
4)、将上述碳点溶液冷冻干燥后得到目标产物。
2.如权利要求1所述的应用,其特征在于,所述步骤1)中的罗丹明B和对氨基苯酚的质量比为:0.015-0.035∶0.04-0.06。
3.一种荧光和比色双模式检测过氧亚硝酸根离子的方法,其特征在于, 步骤为:
1)、配置浓度为0.1mg/mL的权利要求1中所述的橙色荧光碳点溶液;
2)、向荧光杯中滴加11.7μL不同浓度的ONOO-溶液;
3)、向碳点溶液中加入ONOO-,使碳点的荧光逐渐淬灭,得到碳点/ONOO-溶液,溶液颜色由粉色变为蓝黑色;
4)、测定碳点反应前后的荧光强度,根据ONOO-的浓度和相对荧光强度变化值之间的关系建立检测过氧亚硝酸根离子的标准曲线。
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