CN109342384B - 一种检测氰离子的可视化比率荧光体系及其制备方法和应用 - Google Patents
一种检测氰离子的可视化比率荧光体系及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种检测氰离子的可视化比率荧光体系及其制备方法和应用,所述可视化比率荧光体系包括N‑乙酰‑L‑半胱氨酸修饰的CdTe量子点、碳点、铜离子和磷酸盐缓冲液;所述可视化比率荧光体系中,铜离子浓度为0.7~1.2μmol/L,N‑乙酰‑L‑半胱氨酸修饰的CdTe量子点浓度为20 nmol/L,在测试体系中,碳点的荧光强度与被铜离子猝灭的N‑乙酰‑L‑半胱氨酸修饰的CdTe量子点的荧光峰强度之比为25~35:1;所述可视化比率荧光体系的pH值为7~8.8。本发明所述的比率荧光系统制备简便且廉价,对氰离子具有很好的选择性和灵敏度,具有宽的线性范围,极低的检测限。
Description
技术领域
本发明涉及荧光化学检测领域,特别涉及一种检测氰离子的可视化比率荧光体系及其制备方法和应用。
背景技术
氰化物是剧毒物质,在日常生活中,木薯、果仁、香烟等食品或日用品中都含有较多的氰化物,在工业生产中,氰化物广泛应用于化工、冶金、制药等领域;近些年来,由于废物、废气、废液的非法排放,河水、湖水、自来水中可能都存在氰离子,这些都严重危害人类的健康。
氰化物的毒性来源于其与细胞色素氧化酶中铁的高亲和性,使人体内细胞不能及时得到氧气,从而导致头疼、昏迷、甚至死亡。此外氰化物除了直接进入人体,与皮肤接触也会导致人体产生不良反应,比如接触高浓度氰化物会导致甲状腺中毒。由于氰化物的剧毒性,世界卫生组织(WHO)规定:在饮用水中氰离子浓度不得超过1.9 μM。因此,人们必须研究高效检测氰离子的方法。
相比较于高效液相色谱、拉曼、电化学发光、比色等技术,利用荧光技术检测氰离子具有简单、廉价、快速、高灵敏度、高选择性的优点,因而受到研究者的青睐。目前利用荧光技术检测氰离子主要分为有机材料和无机材料两大类。荧光检测氰化物的有机材料可以分为吖嗪衍生物、香豆素衍生物、亚胺衍生物、偶氮衍生物、吲哚衍生物等荧光色素。它们的优点是检测限低、选择性高、大部分可用于细胞中检测氰离子;缺点是制备复杂、成本高、且制备时产生有机物污染、稳定性差,容易光漂白。
荧光检测氰化物的无机材料可以分为量子点、碳点、金簇、银簇、铜簇、纳米复合材料。他们的优点是制备简单,廉价、线性范围宽、稳定性好。他们的缺点是相对高的检测限,相对低的选择性,很难用于细胞中检测氰离子。
杰克逊州立大学的 Md. Alamgir Hossain于2014年报道了一种双核铜复合物,基于氰离子与铜的强结合能力使其荧光恢复来检测氰离子,检测限低至0.02 ppm。山西大学的韩辉最近报道了一种荧光染料吩噻嗪-半花菁,基于氰离子与其发生亲核加成反应使其荧光增强来检测氰离子,线性范围0 - 3μM,检测限低至20 nM。近10年来,也有许多报道利用铜离子猝灭量子点,随后氰离子的加入使其荧光恢复来检测氰离子,然而其灵敏度大多都低于荧光染料。
发明内容
针对现有技术存在的上述技术问题,本发明的目的在于提供一种检测氰离子的可视化比率荧光体系及其制备方法和应用,该检测系统具有廉价,线性范围宽,检测限低,选择性高,稳定性好的优点,在检测实际样品时也展现出它用于实际检测氰离子的可行性。
一种检测氰离子的可视化比率荧光体系,其特征在于包括N-乙酰-L-半胱氨酸修饰的CdTe量子点、碳点、铜离子和磷酸盐缓冲液;所述可视化比率荧光体系中,铜离子浓度为0.7~1.2μmol/L,N-乙酰-L-半胱氨酸修饰的CdTe量子点浓度为20 nmol/L,在测试体系中,碳点的荧光强度与被铜离子猝灭的N-乙酰-L-半胱氨酸修饰的CdTe量子点的荧光峰强度之比为25~35 : 1;所述可视化比率荧光体系的pH值为7~8.8。
所述的一种检测氰离子的可视化比率荧光体系,其特征在于铜离子浓度为0.94μmol/L。
所述的一种检测氰离子的可视化比率荧光体系,其特征在于碳点与被铜离子猝灭的N-乙酰-L-半胱氨酸修饰的CdTe量子点的荧光强度比为30 : 1。
所述的一种检测氰离子的可视化比率荧光体系的制备方法,其特征在于包括以下步骤:
1)制备N-乙酰-L-半胱氨酸修饰的CdTe量子点,置于磷酸盐缓冲液中,配成CdTe量子点溶液备用;制备发蓝色荧光的碳点,所述碳点用蒸馏水稀释,配成碳点溶液备用;
2)取步骤1)所得CdTe量子点溶液加入到磷酸盐缓冲液中,随后加入铜离子水溶液进行反应,铜离子猝灭N-乙酰-L-半胱氨酸修饰的CdTe量子点,然后加入步骤1)所得碳点溶液,混合均匀后冷藏保存,即制得所述可视化比率荧光体系。
所述的一种检测氰离子的可视化比率荧光体系的制备方法,其特征在于发蓝色荧光的碳点的制备过程为:向水热反应釜内加入蒸馏水、柠檬酸和乙二胺,于180~220℃下反应3~7h,反应结束后冷却至室温,取出反应产物并用透析袋进行提纯处理,即制得发蓝色荧光的碳点。
所述的一种检测氰离子的可视化比率荧光体系的制备方法,其特征在于柠檬酸的质量和乙二胺的体积比为1:3~8,优选为1:5,质量的单位是g,体积的单位是mL。
所述的一种检测氰离子的可视化比率荧光体系的制备方法,其特征在于步骤2)中,猝灭反应的时间为5~10min,优选为6min。
所述的可视化比率荧光体系在检测水样中的微量氰离子的应用。
相对于现有技术,本发明取得的有益效果是:
(1)本发明的荧光系统包括NAC修饰的CdTe量子点和碳点,碳点发蓝色荧光作为内标信号,荧光强度基本不变;CdTe量子点发红色荧光作为作为检测信号,荧光强度随加氰离子浓度的提高而增高;同时伴随着荧光颜色从蓝色到红色的变化。氰离子对NAC修饰的CdTe量子点有荧光增强作用,同时氰离子对被铜离子猝灭的NAC修饰的CdTe量子点具有荧光恢复作用。先通过铜离子猝灭CdTe量子点,再加入碳点构成比率荧光系统,氰离子加入后,量子点的荧光被恢复与增强。量子点被铜离子猝灭基于电荷转移机理,荧光恢复基于氰离子与铜离子具有极强的结合能力,与铜离子生成络合物Cu(CN)2− 或 Cu(CN)4 3−,铜离子从量子点上脱落,量子点的荧光强度得到恢复;氰离子对量子点的荧光增强作用基于氰离子与量子点上NAC的羰基发生亲核加成反应,进而发生配体-金属电荷转移(LMCT)促使NAC的能量向量子点转移,使其荧光增强。同时碳点发射的蓝色荧光作为参比信号,其荧光强度基本不变,随着量子点发射红色荧光的增强,检测系统展现出从蓝到红的变化。发明的机理过程如图1所示。
(2)本发明所述的比率荧光系统制备简便且廉价,对氰离子具有很好的选择性和灵敏度,具有宽的线性范围,极低的检测限。
(3)与单发射荧光系统相比较,比率荧光的应用避免了包括温度、噪音、仪器、光漂白等外界因素干扰及分析物浓度等因素引起的误差。同时荧光颜色变化明显,实现了可视化检测。
附图说明
图1为本发明的荧光体系检测氰离子的机理图;
图2为不同铜离子的可视化比率荧光体系被氰离子恢复后量子点荧光强度与碳点荧光强度比的数据图;
图3为0.94umol/L铜离子的可视化比率荧光体系被氰离子恢复后量子点荧光强度与碳点荧光强度比的数据图;
图4为考察可视化比率荧光体系的pH对量子点荧光恢复和荧光增强影响的数据图;
图5为考察CdTe量子点被铜离子猝灭的时间影响的数据图;
图6为考察可视化比率荧光体系被氰离子恢复的时间影响的数据图;
图7为考察可视化比率荧光体系被不同阴离子恢复影响的数据图。
具体实施方式
下面结合具体实施例对本发明作进一步说明,但本发明的保护范围并不限于此。
以下实施例中,铜离子水溶液的阴离子为Cl-1,氰离子水溶液的阳离子为Na+;荧光发射光谱的检测条件为:设定检测模式为发射光谱,激发波长为380 nm, 波长扫描范围为400-750nm。以下实施例中CdTe量子点溶液的荧光峰强度,是指NAC修饰的CdTe量子点的荧光峰强度。
实施例1:
制备一种检测氰离子的可视化比率荧光体系,步骤如下:
1)制备NAC修饰的CdTe量子点
首先,向100mL的0.004 mol/L的CdCl2水溶液中通氮气10min,随后向其中加入0.1371 g NAC(中文名称:N-乙酰-L-半胱氨酸),混合溶液的pH用1 mol/L NaOH溶液调节至11.0,然后按顺序依次加入0.2152 g二水合柠檬酸三钠、0.0177 g Na2TeO3、0.08 g NaBH4。在100℃条件下回流反应11 h,得到发红色荧光的NAC修饰的CdTe量子点溶液。取1mL 制备得到的NAC修饰的CdTe量子点溶液和2mL乙醇混合后,离心沉淀,取出沉淀物分散于1mL磷酸盐缓冲液(pH值为8)中,配成 CdTe量子点溶液置于冰箱中于4℃下保存待用。
2)制备发蓝色荧光的碳点
在40mL水热反应釜中加入25 mL蒸馏水,随后加入1 g柠檬酸,5 mL乙二胺,将水热反应釜拧紧置于烘箱中,设定温度200 ℃,反应5 h,反应结束后,待温度降低到室温,取出反应产物,反应产物用透析袋进行提纯处理,提纯后得到的碳点用蒸馏水稀释5倍,配成碳点溶液,放入冰箱中4℃保存待用。
3)比率荧光系统的制备
取步骤1)得到的CdTe量子点溶液14.1μL投入到2.926 mL磷酸盐缓冲液中(pH值为8),随后加入 0.1mol/L铜离子水溶液摇匀,等待6min后(即猝灭时间6min),加入步骤2)得到的碳点溶液31μL,混合均匀后,即制得所述可视化比率荧光体系,放入冰箱中4℃保存待用。
上述步骤3)加入的0.1mol/L铜离子水溶液的体积分别为21μL、27.6μL、28.2μL和36μL,分别制备得到的可视化比率荧光体系中的铜离子浓度分别为:0.7μmol/L、0.92μmol/L、0.94μmol/L和1.2μmol/L。
对制备得到的含有不同铜离子浓度的可视化比率荧光体系,加入不同量的氰离子(氰离子在可视化比率荧光体系中的浓度为0~100μmol/L),进行荧光光谱检测,荧光光谱检测的激发波长为380nm,检测到碳点荧光发射峰443nm,NAC修饰的CdTe量子点的荧光发射峰611 nm左右;以NAC修饰的CdTe量子点的荧光峰强度与碳点的荧光峰强度之比为纵坐标,氰离子在可视化比率荧光体系中的浓度为横坐标作图(见图2和图3),从图2可以看出,当可视化比率荧光体系中的铜离子浓度为0.94μmol/L时,检测不同浓度的氰离子具有较宽的线性恢复范围。从图3可看出,当检测的氰离子浓度在0.02~10μmol/L和15~80μmol/L时,线性较好;即当检测的氰离子浓度在0.02~10μmol/L内时,线性回归方程为y=0.03054+0.03079x,R2=0.9999。即当检测的氰离子浓度在15~80μmol/L内时,线性回归方程为y=0.27314+0.0078x,R2=0.9996。由此可以看出,检测氰离子浓度的准确率较高。
可视化比率荧光体系中,铜离子是作为猝灭剂的作用,对于基于荧光恢复的检测系统,猝灭剂的浓度会影响检测的效果,猝灭剂浓度过高,会导致恢复效果差,灵敏度低;猝灭剂浓度过低,会导致检测目标分析物的线性范围窄。
实施例2:
考察磷酸盐缓冲溶液的pH值的影响:
提前配制pH为6.0、6.4、6.8、7.0、7.4、7.6、8.0、8.4和8.8的磷酸盐缓冲液;
取实施例1步骤1)制备的CdTe量子点溶液14.1μL,投入到2.926 mL上述配制的不同pH的磷酸盐缓冲液中摇匀,进行荧光发射光谱检测,测试CdTe量子点溶液未加氰离子时荧光强度随pH变化曲线;随后加入30μL 的1 mmol/L氰离子水溶液,摇匀后等待15min,进行荧光发射光谱检测,测试CdTe量子点溶液加入10μmol/L氰离子后荧光强度随pH变化曲线。由此,以CdTe量子点溶液加入10μmol/L氰离子后的荧光峰强度与其未加氰离子时的荧光峰强度之比随pH变化曲线作图,结果如图4中的曲线b所示;
取实施例1步骤1)制备的CdTe量子点溶液14.1μL,投入到2.926 mL上述配制的不同pH的磷酸盐缓冲液中摇匀,随后加入28.2μL的0.1 mol/L的铜离子水溶液摇匀,等待6min后,加入31μL实施例1步骤2)制备的碳点溶液,摇匀后,进行荧光发射光谱检测,以NAC修饰的CdTe量子点的荧光峰强度与碳点荧光峰的强度之比随pH变化曲线作图,结果如图4中的曲线a所示;随后加入30μL的1 mmol/L氰离子水溶液,摇匀,进行荧光发射光谱检测,以NAC修饰的CdTe量子点的荧光峰强度与碳点的荧光峰强度之比随pH变化曲线作图,测试结果如图4中的曲线c所示;其中在图4中,曲线a和曲线c对应图4左边的纵坐标,曲线b对应图4右边的纵坐标。
以量子点与碳点荧光发射峰强度之比作纵坐标,磷酸盐缓冲溶液pH作为横坐标,加入氰离子恢复后与恢复前纵坐标差值最大的pH为最佳pH,从图3对比曲线a、曲线b和曲线c可以看出,在pH=8.0时,荧光恢复效果最好,pH=7.6和pH=8.0时,荧光增强效果较好。
实施例3:
考察猝灭时间的影响:
取实施例1中步骤1)得到的CdTe量子点溶液14.1μL投入到2.926 mL磷酸盐缓冲液中(pH值为8),进行荧光发射光谱检测,测试CdTe量子点溶液未加铜离子时的荧光峰强度;随后加入28.2μL的0.1mol/L铜离子水溶液摇匀,等待时间分别为2、4、6、8、10、20、40、60min后测其荧光发射光谱(加入铜离子后的等待时间即为猝灭时间),测试CdTe量子点溶液加入铜离子后的荧光峰强度随猝灭时间的变化曲线。以猝灭时间为横坐标,对应的CdTe量子点溶液加入铜离子后的荧光峰强度与其为未加铜离子时的荧光峰强度之比为纵坐标作图,结果如图5所示,从图5中可以看出,猝灭时间为6min时荧光强度趋于稳定,因此猝灭时间为6min效果最佳;
考察恢复时间的影响:
取实施例1中步骤1)得到的CdTe量子点溶液14.1μL投入到2.926 mL磷酸盐缓冲液中(pH值为8),随后加入28.2μL的0.1mol/L铜离子水溶液摇匀,等待6min后,加入80μL的3mmol/L氰离子水溶液摇匀,等待时间分别为2、4、6、8、10、15、20、40、60min后进行荧光发射光谱检测(加入氰离子后的等待时间即为恢复时间),检测到碳点荧光发射峰443nm,NAC修饰的CdTe量子点的荧光发射峰611 nm左右;以CdTe量子点溶液加入氰离子后的荧光峰强度与碳点的荧光峰强度之比随时间的变化曲线作图,结果如图6所示,从图6可以看出,恢复时间为15min时荧光峰强度之比最大,且趋于稳定,所以恢复时间为15min效果最佳。
实施例4:
考察实施例1制备的可视化比率荧光体系对检测阴离子的选择性识别性(本实施例配制阴离子的溶液时,阳离子均为Na+):
取实施例1中步骤1)得到的CdTe量子点溶液14.1μL投入到2.926 mL磷酸盐缓冲液中(pH值为8),随后加入28.2μL的0.1mol/L铜离子水溶液摇匀,等待6min后,加入80μL的3mmol/L氰离子水溶液摇匀,随后分别加入80 uL 用超纯水配制的不同阴离子的溶液,摇匀后等待15分钟,测其荧光发射光谱和荧光图片。
其中上述用超纯水配制的不同阴离子的溶液分别为:超纯水、3mmol/L氰离子、3mmol/L硝酸根离子、3 mmol/L亚硝酸根离子、3 mmol/L溴离子、3 mmol/L溴酸根离子、3mmol/L碳酸根离子、3 mmol/L碳酸氢根离子、3 mmol/L硫酸根离子、3 mmol/L硫酸氢根离子、3 mmol/L亚硫酸氢根离子、3 mmol/L草酸根离子、3 mmol/L硼酸根离子、3 mmol/L碘离子、3 mmol/L高碘酸根离子、3 mmol/L碘酸根离子、3 mmol/L氟离子、3 mmol/L氯离子、3mmol/L硫氰酸根离子。
以CdTe量子点溶液的荧光发射峰强度与碳点的荧光发射峰强度之比为纵坐标,加入的不同阴离子为横坐标作图,结果如图7所示,在图7内嵌入加入不同阴离子的荧光体系在365nm紫外灯下相应的荧光照片,从图7中可以看出,该可视化比率荧光体系对检测氰离子具有很好的选择性。
实施例5:
通过加标法验证可视化比率荧光体系对检测氰离子浓度的准确性:
加标法检测自来水、上塘河水、西湖水中的氰离子浓度,从自来水、上塘河水、西湖水中分别取得实际水样后,用滤纸过滤两次去除悬浮物,随后用0.22 um微孔滤膜过滤5次,取0.3 mL过滤后的水样与2.626 mL磷酸盐缓冲液(pH=8)混合,摇匀,即制得待测水溶液样品,所得待测水溶液样品分别准备若干份。随后向每份准备完成的待测水溶液样品中加入实施例1中步骤1)得到的CdTe量子点溶液14.1 uL,摇匀后加入28.2μL的1mmol/L铜离子水溶液,等待6分钟后,分别加入0.6μL的0.1 mmol/L氰离子水溶液、6μL 的1 mmol/L氰离子水溶液、24μL的1 mmol/L氰离子水溶液。等待15分钟后,进行荧光发射光谱测试,检测到碳点荧光发射峰443nm,NAC修饰的CdTe量子点的荧光发射峰611 nm左右,计算NAC修饰的CdTe量子点的荧光发射峰强度与碳点的荧光发射峰强度之比,带入实施例1得到的线性回归方程y=0.03054+0.03079x中,即可得出实际检测到的可视化比率荧光体系中的氰离子浓度,结果如表1所示。
表1 加标法检测自来水,上塘河水,西湖水中氰离子的结果(n = 5)
从表1中可以看出,本发明的可视化比率荧光体系检测氰离子浓度时,当氰离子浓度在0~8µmol/L,有较高的准确性。
本说明书所述的内容仅仅是对发明构思实现形式的列举,本发明的保护范围不应当被视为仅限于实施例所陈述的具体形式,本发明的保护范围也仅仅于本领域技术人员根据本发明构思所能够想到的等同技术手段。
Claims (10)
1.一种检测氰离子的可视化比率荧光体系,其特征在于包括N-乙酰-L-半胱氨酸修饰的CdTe量子点、碳点、铜离子和磷酸盐缓冲液;所述可视化比率荧光体系中,铜离子浓度为0.7~1.2μmol/L,N-乙酰-L-半胱氨酸修饰的CdTe量子点浓度为20 nmol/L,在测试体系中,碳点的荧光强度与被铜离子猝灭的N-乙酰-L-半胱氨酸修饰的CdTe量子点的荧光峰强度之比为25~35 : 1;所述可视化比率荧光体系的pH值为7.4~8.8。
2.根据权利要求1所述的一种检测氰离子的可视化比率荧光体系,其特征在于铜离子浓度为0.94μmol/L。
3.根据权利要求1所述的一种检测氰离子的可视化比率荧光体系,其特征在于碳点与被铜离子猝灭的N-乙酰-L-半胱氨酸修饰的CdTe量子点的荧光强度比为30 : 1。
4.根据权利要求1所述的一种检测氰离子的可视化比率荧光体系的制备方法,其特征在于包括以下步骤:
1)制备N-乙酰-L-半胱氨酸修饰的CdTe量子点,置于磷酸盐缓冲液中,配成CdTe量子点溶液备用;制备发蓝色荧光的碳点,所述碳点用蒸馏水稀释,配成碳点溶液备用;
2)取步骤1)所得CdTe量子点溶液加入到磷酸盐缓冲液中,随后加入铜离子水溶液进行反应,铜离子猝灭N-乙酰-L-半胱氨酸修饰的CdTe量子点,然后加入步骤1)所得碳点溶液,混合均匀后冷藏保存,即制得所述可视化比率荧光体系。
5.根据权利要求4所述的一种检测氰离子的可视化比率荧光体系的制备方法,其特征在于发蓝色荧光的碳点的制备过程为:向水热反应釜内加入蒸馏水、柠檬酸和乙二胺,于180~220℃下反应3~7h,反应结束后冷却至室温,取出反应产物并用透析袋进行提纯处理,即制得发蓝色荧光的碳点。
6.根据权利要求5所述的一种检测氰离子的可视化比率荧光体系的制备方法,其特征在于柠檬酸的质量和乙二胺的体积比为1:3~8,质量的单位是g,体积的单位是mL。
7.根据权利要求6所述的一种检测氰离子的可视化比率荧光体系的制备方法,其特征在于柠檬酸的质量和乙二胺的体积比为1:5,质量的单位是g,体积的单位是mL。
8.根据权利要求4所述的一种检测氰离子的可视化比率荧光体系的制备方法,其特征在于步骤2)中,猝灭反应的时间为5~10min。
9.根据权利要求8所述的一种检测氰离子的可视化比率荧光体系的制备方法,其特征在于步骤2)中,猝灭反应的时间为6min。
10.根据权利要求1所述的可视化比率荧光体系在检测水样中的微量氰离子的应用。
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