CN113061140A - 一种含羟基脲结构的六元螺环罗丹明铜离子荧光探针及其制备方法和应用 - Google Patents
一种含羟基脲结构的六元螺环罗丹明铜离子荧光探针及其制备方法和应用 Download PDFInfo
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- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 title claims abstract description 43
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- WTDHULULXKLSOZ-UHFFFAOYSA-N Hydroxylamine hydrochloride Chemical compound Cl.ON WTDHULULXKLSOZ-UHFFFAOYSA-N 0.000 claims abstract description 16
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims abstract description 16
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- -1 rhodamine compound Chemical class 0.000 claims description 3
- WGTODYJZXSJIAG-UHFFFAOYSA-N tetramethylrhodamine chloride Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C(O)=O WGTODYJZXSJIAG-UHFFFAOYSA-N 0.000 claims description 3
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Abstract
本发明公开一种含羟基脲结构的六元螺环罗丹明铜离子荧光探针及其制备方法和应用。所述含羟基脲结构的六元螺环罗丹明铜离子荧光探针具有如(Ⅰ)所示的结构通式。制备方法包括如下步骤：取罗丹明类化合物、三氯氧磷和干燥的1，2‑二氯乙烷，于90℃下反应4h后，冷却至室温，减压蒸馏去除溶剂，所得固体溶于干燥的乙腈中，加入叠氮化钠，室温下搅拌过夜，加干燥剂干燥，过滤，所得滤液于82℃反应40min，冷却至室温，加入盐酸羟胺和三乙胺的混合液，反应得到目标产物。本发明的荧光探针对铜离子具有良好的灵敏度，能定性和定量地检测溶液中的铜离子。
Description
技术领域
本发明涉及一种含羟基脲结构的六元螺环罗丹明铜离子荧光探针。
背景技术
荧光信号显示出快速的响应时间、出色的空间分辨率和高的信噪比，荧光染色具有高灵敏度，并且在显微镜下具有对样品无损且对细胞损伤小的优点。罗丹明荧光染料因其高吸光系数，在可见光区域具有较宽的荧光，高荧光量子产率和光稳定性而被广泛用作荧光探针，用于检测重金属离子。在以往的研究中，罗丹明类荧光探针几乎都含有五元螺内酰胺环结构，鲜有设计合成六元环的罗丹明荧光探针。但是，五元螺内酰胺部分，可能水溶性差，荧光稳定时间短，pH依赖性强。已报道的六元环罗丹明探针表明，六元环探针具有更高的灵敏度，在生物成像方面比五元环更有优势。因此六元环结构的罗丹明荧光指示剂拥有广阔的应用前景。
Cu2+是动物和人类体内重要的微量元素之一，也是许多氧化还原酶所需的必要因子，这些酶参与了各种关键的生理过程，在细胞呼吸，能量产生，色素沉着，神经递质合成，代谢排毒，抗氧化防御和结缔组织形成等方面起到至关重要的作用。然而，铜虽然被认为是各种代谢功能所必需的过渡金属离子之一，当人体内的金属铜离子的浓度过高时，铜也会引起和加剧与衰老和疾病相关的氧化应激和损害事件，包括严重的神经退行性疾病，例如Menkes和Wilson病，肌萎缩性侧索硬化症和Alzheimer病等。为了研究疾病的起源并评估潜在药物的临床应用，诸如铜的细胞浓度和分布等信息是至关重要的信息。因此，开发操作简便、选择性好、灵敏度高且成本低廉的铜离子荧光探针有非常重要的意义。
发明内容
本发明的目的是提供一种可以快速响应、高灵敏度检测铜离子的荧光探针。
本发明采用的技术方案是：一种含羟基脲结构的六元螺环罗丹明铜离子荧光探针，所述含羟基脲结构的六元螺环罗丹明铜离子荧光探针为RhX-OH，具有如(Ⅰ)所示的结构通式：
其中，
R1＝R2＝-CH3，R3＝R4＝H；
或R1＝R2＝-CH2CH3，R3＝R4＝H；
或R1至R4构成-(CH2)3-，R2至R3构成-(CH2)3-
一种含羟基脲结构的六元螺环罗丹明铜离子荧光探针的制备方法，包括如下步骤：称取罗丹明类化合物、三氯氧磷和干燥的1,2-二氯乙烷，于90℃下反应4h后，冷却至室温，减压蒸馏，所得固体溶于干燥的乙腈溶液中，加入叠氮化钠，室温下搅拌过夜，干燥过滤，滤液于82℃加热40min得到异氰酸酯；将所得异氰酸酯溶液与盐酸羟胺和三乙胺的混合液，混合均匀，室温搅拌，所得产物减压蒸馏浓缩后，得含羟基脲结构的六元螺环罗丹明铜离子荧光探针。反应式如下：
优选的，上述的制备方法，所述罗丹明类化合物为罗丹明B、四甲基罗丹明TMR或罗丹明101。
优选的，上述的制备方法，按摩尔比，罗丹明类化合物:三氯氧磷:叠氮化钠:盐酸羟胺:三乙胺＝1:3:1.3:3:3。
本发明提供的一种含羟基脲结构的六元螺环罗丹明铜离子荧光探针在检测铜离子中的应用。本发明的含羟基脲结构的六元螺环罗丹明铜离子荧光探针的机理为铜离子诱导的络合作用，使六元螺环开环生成亮红色和强荧光罗丹明，从而实现了铜离子的快速检测。
优选的，含羟基脲结构的六元螺环罗丹明铜离子荧光探针定性检测铜金属离子。方法如下：将含羟基脲结构的六元螺环罗丹明铜离子荧光探针溶解于乙腈水溶液中，配制浓度为2×10-5～3×10-5mol/L、pH为7.4的测试液，将待测溶液加入到测试液中，观察溶液颜色的变化，溶液由无色变为红色即可定性该待测溶液中含有铜离子。
优选的，含羟基脲结构的六元螺环罗丹明铜离子荧光探针定量检测金属铜离子。方法如下：
1)配制测试液：将权利要求1所述的含羟基脲结构的六元螺环罗丹明铜离子荧光探针溶解于乙腈水溶液中，配制浓度为2×10-5～3×10-5mol/L、pH为7.4的测试液；
2)绘制标准曲线：取测试液和已知浓度的铜离子溶液，混合均匀后，分别进行紫外-可见吸收光谱和荧光光谱测试，并绘制铜离子的紫外-可见吸收光谱和荧光光谱的标准曲线；
3)测试：取测试液和含有铜金属离子的待测溶液，混合均匀后分别进行紫外-可见吸收光谱和荧光光谱测试，利用铜金属离子的紫外-可见吸收光谱和荧光光谱的标准曲线计算含有铜金属离子的待测溶液中铜离子的浓度。
优选的，乙腈水溶液中，按体积比，乙腈:水＝4:6。
本发明的有益效果是：本发明构建的含羟基脲结构的六元螺环罗丹明铜离子荧光探针，六元环结构在遇到铜离子后即可发生络合反应，生成的强荧光亮红色的罗丹明，溶液由无颜色无荧光变成肉眼可见的亮红色和强荧光，从而实现对铜金属离子的紫外-可见分光光度法和荧光分光光度法检测。
附图说明
图1是实施例1制备的RhB-OH对Cu2+的吸收和荧光颜色响应；
其中，加入铜离子前后，a:紫外可见吸收颜色变化；b:荧光颜色的变化。
图2是实施例1制备的RhB-OH对阳离子的紫外可见吸收光谱的选择性。
图3是实施例1制备的RhB-OH对阳离子的荧光光谱的选择性。
图4是实施例1制备的RhB-OH对铜离子浓度滴定中紫外可见吸收峰随铜离子浓度的变化趋势(吸收--标准曲线)。
图5是实施例1制备的RhB-OH对铜离子浓度滴定中荧光峰随铜离子浓度的变化趋势(荧光--标准曲线)。
具体实施方法
实施例1含羟基脲结构的六元螺环罗丹明铜离子荧光探针——RhB-OH
反应式如下：
于圆底烧瓶中，将1mol的罗丹明B和3mol的POCl3加入到干燥的120mL的1,2-二氯乙烷中，90℃油浴反应4h，冷却至室温后，减压蒸馏去除溶剂，所得固体物用120mL干燥的乙腈溶解，加入1.3mol叠氮化钠，室温下搅拌过夜，将反应液加无水硫酸镁干燥后过滤，滤液于82℃加热40min得到异氰酸酯，将所得异氰酸酯溶液与3mol盐酸羟胺和3mol三乙胺的混合液混合均匀，室温搅拌，反应60min，所得产物减压蒸馏浓缩后，得到含羟基脲结构的六元螺环罗丹明铜离子荧光探针RhB-OH。HRMS：472.2474。
实施例2含羟基脲结构的六元螺环罗丹明铜离子荧光探针——RhTMR-OH
于圆底烧瓶中，将1mol的四甲基罗丹明TMR和3mol的POCl3加入到干燥的120mL的1,2-二氯乙烷中，90℃油浴反应4h，冷却至室温后，减压蒸馏去除溶剂，所得固体物用120mL干燥的乙腈溶解，加入1.3mol叠氮化钠，室温下搅拌过夜，将反应液加无水硫酸镁干燥后过滤，滤液于82℃加热40min得到异氰酸酯，将所得异氰酸酯溶液与3mol盐酸羟胺和3mol三乙胺的混合液混合均匀，反应60min，所得产物减压蒸馏浓缩后，得到含羟基脲结构的六元螺环罗丹明铜离子荧光探针RhTMR-OH。HRMS：416.1848。
实施例3含羟基脲结构的六元螺环罗丹明铜离子荧光探针——Rh101-OH
于圆底烧瓶中，将1mol的罗丹明101和3mol的POCl3加入到干燥的120mL的1,2-二氯乙烷中，90℃油浴反应4h，冷却至室温后，减压蒸馏去除溶剂，所得固体物用120mL干燥的乙腈溶解，加入1.3mol叠氮化钠，室温下搅拌过夜，将反应液加无水硫酸镁干燥后过滤，滤液于82℃加热40min得到异氰酸酯，将所得异氰酸酯溶液与3mol盐酸羟胺和3mol三乙胺的混合液混合均匀，反应60min，所得产物减压蒸馏浓缩后得到含羟基脲结构的六元螺环罗丹明铜离子荧光探针RH101-OH。HRMS：520.2474。
实施例4应用试验
本应用试验采用实施例1制备的RhB-OH荧光探针进行。
(一)定性检测
1、配制RhB-OH测试液：将RhB-OH溶解于乙腈水溶液(按体积比，乙腈:水＝4:6)中，得浓度为2×10-5mol/L，pH＝7.4的RhB-OH测试液。
2、分别取RhB-OH测试液3mL，分别加入30uL浓度为2×10-2mol/L的不同阳离子Fe2 +、Fe3+、Al3+、Mg2+、Na+、Ca2+、Hg2+、K+、Mn2+、Cr3+、Zn2+、Pb2+、Cd2+分别进行紫外可见光谱和荧光光谱检测。
如图1中(a)所示，当RhB-OH遇到铜离子后溶液颜色由无色变为红色。而加入其他阳离子对紫外可见吸收光谱无影响，如图2所示。
如图1中(b)所示，当RhB-OH遇到铜离子后溶液荧光颜色由无色变为红色。而加入其他阳离子对荧光光谱无影响，如图3所示。
本发明的含羟基脲结构的六元螺环罗丹明铜离子荧光探针检测铜离子的机理为络合作用，使六元螺环开环生成亮红色和强荧光罗丹明，从而实现了铜离子的快速检测。RhB-OH能够对铜离子实现肉眼可见的、快速的和高灵敏的检测。其他阳离子均未引起紫外可见光谱和荧光光谱的变化。
(二)定量检测
1、配制RhB-OH测试液：将RhB-OH溶解于乙腈水溶液(按体积比，乙腈:水＝4:6)中，得浓度为2×10-5mol/L，pH＝7.4的RhB-OH测试液。
2、绘制标准曲线：取3mL RhB-NH2测试液和30uL 0-4×10-2mol/L浓度范围内已知浓度的铜离子溶液，混合均匀后，分别进行紫外可见光谱和荧光光谱检测。
选取紫外可见光谱中最大吸收峰563nm处的吸光度为纵坐标，铜离子浓度为横坐标绘制标准曲线。如图4所示，在吸收标准曲线中铜离子浓度在0-2.5×10-5mol/L范围内呈现线性关系。
选取荧光光谱中最大荧光发射峰592nm处的强度为纵坐标，铜离子浓度为横坐标绘制标准曲线。如图5所示，在荧光标准曲线中铜离子浓度在0-2.5×10-5mol/L范围内呈线性关系。
3、取3mL RhB-OH测试液和30uL未知浓度的含有铜离子的待测溶液，混合均匀后，分别测试紫外可见光谱和荧光光谱，分别测定紫外可见光谱中最大吸收峰563nm处的吸光度和荧光光谱中最大荧光发射峰592nm处的强度，然后通过标准曲线计算待测溶液中铜离子的浓度。
Claims (8)
1.一种含羟基脲结构的六元螺环罗丹明铜离子荧光探针，其特征在于，所述含羟基脲结构的六元螺环罗丹明铜离子荧光探针为RhX-OH，具有如(Ⅰ)所示的结构通式：
其中，
R1＝R2＝-CH3，R3＝R4＝H；
或R1＝R2＝-CH2CH3，R3＝R4＝H；
或R1至R4构成-(CH2)3-，R2至R3构成-(CH2)3-。
2.权利要求1所述的一种含羟基脲结构的六元螺环罗丹明铜离子荧光探针的制备方法，其特征在于，包括如下步骤：称取罗丹明类化合物、三氯氧磷和干燥的1,2-二氯乙烷，于90℃下反应4h后，冷却至室温，减压蒸馏，所得固体溶于干燥的乙腈溶液中，加入叠氮化钠，室温下搅拌过夜，干燥过滤，滤液于82℃加热40min得到异氰酸酯；将所得异氰酸酯溶液与盐酸羟胺和三乙胺的混合液，混合均匀，室温搅拌，所得产物减压蒸馏浓缩后，得含羟基脲结构的六元螺环罗丹明铜离子荧光探针。
3.根据权利要求1所述的制备方法，其特征在于，所述罗丹明类化合物为罗丹明B、四甲基罗丹明TMR或罗丹明101。
4.根据权利要求1所述的制备方法，其特征在于，按摩尔比，罗丹明类化合物:三氯氧磷:叠氮化钠:盐酸羟胺:三乙胺＝1:3:1.3:3:3。
5.权利要求1所述的一种含羟基脲结构的六元螺环罗丹明铜离子荧光探针在检测铜离子中的应用。
6.根据权利要求5所述的应用，其特征在于，含羟基脲结构的六元螺环罗丹明铜离子荧光探针定性检测铜金属离子，方法如下：将权利要求1所述的含羟基脲结构的六元螺环罗丹明铜离子荧光探针溶解于乙腈水溶液中，配制浓度为2×10-5～3×10-5mol/L、pH为7.4的测试液，将待测溶液加入到测试液中，观察溶液颜色的变化。
7.根据权利要求5所述的应用，其特征在于，含羟基脲结构的六元螺环罗丹明铜离子荧光探针定量检测金属铜离子，方法如下：
1)配制测试液：将权利要求1所述的含羟基脲结构的六元螺环罗丹明铜离子荧光探针溶解于乙腈水溶液中，配制浓度为2×10-5～3×10-5mol/L、pH为7.4的测试液；
2)绘制标准曲线：取测试液和已知浓度的铜离子溶液，混合均匀后，分别进行紫外-可见吸收光谱和荧光光谱测试，并绘制铜离子的紫外-可见吸收光谱和荧光光谱的标准曲线；
3)测试：取测试液和含有铜金属离子的待测溶液，混合均匀后分别进行紫外-可见吸收光谱和荧光光谱测试，利用铜金属离子的紫外-可见吸收光谱和荧光光谱的标准曲线计算含有铜金属离子的待测溶液中铜离子的浓度。
8.根据权利要求6或7所述的应用，其特征在于，乙腈水溶液中，按体积比，乙腈:水＝4:6。
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