CN106525782B - 3,4-二(3-吲哚)-3-吡咯啉-2,5-二酮-1-(2-羟基苯基)腙作为荧光探针在检测铜离子中的应用 - Google Patents
3,4-二(3-吲哚)-3-吡咯啉-2,5-二酮-1-(2-羟基苯基)腙作为荧光探针在检测铜离子中的应用 Download PDFInfo
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Abstract
本发明公开了3,4‑二(3‑吲哚)‑3‑吡咯啉‑2,5‑二酮‑1‑(2‑羟基苯基)腙作为荧光探针在检测铜离子中的应用,取30μL探针化合物标准溶液分别与3 mL不同浓度的铜离子储备液混合,室温下静置8 min,分别测混合液在波长568 nm处的荧光发射强度,以铜离子的浓度为横坐标,以荧光强度为纵坐标绘制标准曲线,或再拟合得到标准直线方程;将30μL摩尔浓度为0.1 mmol/L的探针化合物标准溶液加入到3 mL待测样品溶液中,室温下静置8 min,测定被检测溶液在波长568 nm处的荧光发射强度,再与标准曲线对比或代入标准直线方程计算,得到待测样品溶液中铜离子的浓度。本发明的探针化合物对铜离子具有高度专一性,可在生理环境条件下工作,具有应用于生物荧光成像的前景。
Description
技术领域
本发明属于环境分析、生物分析检测的荧光探针技术领域,具体涉及3,4-二(3-吲哚)-3-吡咯啉-2,5-二酮-1-(2-羟基苯基)腙作为荧光探针在检测铜离子中的应用。
背景技术
铜作为一种动植物体内所必需的微量元素和必需的营养素,在生物体的基本代谢过程中发挥着重要作用。铜离子作为如多巴胺β-羟化酶、过氧化物歧化酶和铜蓝蛋白等二十多种金属酶的催化辅助因子,广泛参与了生物体内的酶反应、酶转录以及一些氧化还原等一系列过程。虽然铜在生物体内的总量很小,但当生物体内铜离子浓度超出或低于细胞所需的浓度范围时会产生细胞中毒,生物体的正常活动将被扰乱,从而引起严重的铜代谢障碍疾病。例如肝豆状核变性疾病、阿耳茨海默氏早老性痴呆病和威尔森氏综合症等疾病。环境中微量的铜离子污染,可以通过生物链作用而产生富集,当人饮用或食用受铜离子污染的水和食物,导致体内铜离子含量过高时,便会引发各种疾病之症。
环境中铜污染的来源主要有城市生活垃圾,含铜农药化肥的施用以及含铜饲料添加剂的广泛应用以及工业“三废”的排放等。传统的检测方法主要有紫外分光光度法、原子吸收/发射法、生物传感器法、电感耦合等离子体质谱法和电化学法(伏安法和极谱法)等,上述几种检测方法虽然可以完成对铜离子的检测,但上述方法所用仪器价格昂贵,检测过程较为繁琐,且需要待测样品量很大,不适合大批量检测和实时检测,这些方法的局限性制约着对各种待测样品特别是生物体内的即时检测与痕量分析。 但这些检测方法需要昂贵复杂的检测设备。因此,开发一种快速有效的铜离子检测方法在环境治理、生物研究和医学诊断中具有重要的意义。
荧光探针检测法因其高灵敏度,较好的选择性,测量方法简单,并且可通过荧光成像技术对生物体内的重金属离子进行实时检测而具有极大的优势。常见的铜离子荧光探针机理有基于光诱导电子转移(photoinduced electron transfer,PET)机理的荧光探针,基于C-O键断裂过程的荧光探针,基于胺及酰胺配位作用的荧光探针,基于荧光共振能量转移(fluorescence resonanceenergy transfer,FRET)作用的荧光探针以及基于核磁共振技术的双功能探针。荧光探针检测技术作为一种新型高效简便的检测手段,被广泛的应用于铜离子的检测中。本发明以双吲哚马来酰肼与邻羟基苯甲醛为原料,合成了一种双吲哚马来酰腙类铜离子荧光探针并将其用于铜离子的检测。
发明内容
本发明解决的技术问题是提供了一种方便快捷且灵敏度较高的3,4-二(3-吲哚)-3-吡咯啉-2,5-二酮-1-(2-羟基苯基)腙(HOZ)作为荧光探针在检测铜离子中的应用。
本发明为解决上述技术问题采用如下技术方案,3,4-二(3-吲哚)-3-吡咯啉-2,5-二酮-1-(2-羟基苯基)腙作为荧光探针在检测铜离子中的应用,其特征在于具体过程为:
(1)将3,4-二(3-吲哚)-3-吡咯啉-2,5-二酮-1-(2-羟基苯基)腙溶于二甲基亚砜溶液中配成摩尔浓度为0.1 mmol/L的探针化合物标准溶液,用N-(2-羟乙基)哌嗪-N'-2-乙烷磺酸的缓冲溶液配制不同浓度的铜离子储备液,取30 μL探针化合物标准溶液分别与3mL不同浓度的铜离子储备液混合,室温下静置8 min,分别测混合液在波长568 nm处的荧光发射强度,以铜离子的浓度为横坐标,以荧光强度为纵坐标绘制标准曲线,或再拟合得到标准直线方程;
(2)将30 μL摩尔浓度为0.1 mmol/L的探针化合物标准溶液加入到3 mL待测样品溶液中,室温下静置8 min,测定被检测溶液在波长568 nm处的荧光发射强度,再与标准曲线对比或代入标准直线方程计算,得到待测样品溶液中铜离子的浓度;
测试条件为:室温,激发狭缝宽度3.0nm,发射狭缝宽度3.0nm,激发波长λex为568nm,灵敏度为1,响应时间1s。
进一步优选,所述3,4-二(3-吲哚)-3-吡咯啉-2,5-二酮-1-(2-羟基苯基)腙的具体合成过程为:在25 mL圆底烧瓶中加入102 mg 1-氨基-3,4-二(3-吲哚)-3-吡咯啉-2,5-二酮和5 mL无水乙醇,再加入邻羟基苯甲醛73.2 mg和乙酸20 μL,回流条件下反应,薄层色谱分析跟踪监测,反应结束后,降至室温,加水出现大量沉淀,抽滤,水洗,用体积比为1:1的氯仿与石油醚的混合溶液清洗,柱层析分离得到探针化合物3,4-二(3-吲哚)-3-吡咯啉-2,5-二酮-1-(2-羟基苯基)腙。
进一步优选,所述待测样品溶液中铜离子的浓度范围为0.25×10-6-6×10-6mol/L。
本发明与现有技术相比具有以下有益效果:本发明提供的荧光探针化合物,合成步骤简单,反应时间短,易于提纯且收率较高,该探针化合物是铜离子的荧光增强型探针,对铜离子具有高度专一性,可在生理环境条件下工作,具有应用于生物荧光成像的前景。
附图说明
图1是不同溶剂对荧光探针HOZ荧光光谱的影响曲线;
图2是pH对荧光探针HOZ及HOZ+Cu2+荧光光谱的影响曲线;
图3是配位时间对荧光探针HOZ及HOZ+Cu2+荧光光谱的影响曲线;
图4是荧光探针HOZ对不同金属离子的选择性识别曲线;
图5是共存离子对荧光探针HOZ检测铜离子的影响图;
图6是荧光探针HOZ对铜离子的荧光测试曲线;
图7是荧光探针HOZ对铜离子络合的工作曲线。
具体实施方式
以下通过实施例对本发明的上述内容做进一步详细说明,但不应该将此理解为本发明上述主题的范围仅限于以下的实施例,凡基于本发明上述内容实现的技术均属于本发明的范围。
实施例1
3,4-二(3-吲哚)-3-吡咯啉-2,5-二酮-1-(2-羟基苯基)腙的合成
具体反应步骤为:
在25 mL圆底烧瓶中加入102 mg(0.3 mmol)1-氨基-3,4-二(3-吲哚)-3-吡咯啉-2,5-二酮和5 mL无水乙醇,再加入邻羟基苯甲醛73.2 mg(0.6 mmol)和乙酸20 μL(0.002mmol),回流条件下反应,TLC跟踪监测,反应结束后,降至室温,加入水约10 mL,出现大量沉淀,抽滤,水洗,用体积比为1:1的氯仿与石油醚的混合溶液清洗,柱层析分离(体积比石油醚:乙酸乙酯=2:1)得到探针化合物3,4-二(3-吲哚)-3-吡咯啉-2,5-二酮-1-(2-羟基苯基)腙。
具体测定过程为:
将3,4-二(3-吲哚)-3-吡咯啉-2,5-二酮-1-(2-羟基苯基)腙溶于二甲基亚砜溶液中配成摩尔浓度为0.1 mmol/L的探针化合物标准溶液,用N-(2-羟乙基)哌嗪-N'-2-乙烷磺酸的缓冲溶液配制不同浓度的铜离子储备液,取30 μL探针化合物标准溶液分别与3 mL不同浓度的铜离子储备液混合,室温下静置8 min,分别测混合液在波长568 nm处的荧光发射强度,以铜离子的浓度为横坐标,以荧光强度为纵坐标绘制标准曲线,或再拟合得到标准直线方程;
将30 μL摩尔浓度为0.1 mmol/L的探针化合物标准溶液加入到3 mL待测样品溶液中,室温下静置8 min,测定被检测溶液在波长568 nm处的荧光发射强度,再与标准曲线对比或代入标准直线方程计算,得到待测样品溶液中铜离子的浓度;
测试条件:室温,样品池为1 cm×1 cm×4 cm石英比色皿,激发狭缝宽度3.0 nm,发射狭缝宽度3.0 nm,激发波长λex为568 nm,灵敏度为1,响应时间1 s,波长范围500-700nm。
实施例2
溶剂的选择
为检测不同溶剂体系对化合物紫外及荧光光谱是否有影响,在不同10 mL比色管内用移液枪分别移取50 μL摩尔浓度为2.0×10-3 mol/L的探针化合物HOZ后分别用CH3CN、DMSO、EtOH标定至刻度线。其紫外及荧光光谱如下,在测定荧光光谱时结果表明当CH3CN、EtOH作溶剂测定荧光时探针化合物HOZ都有不同程度的淬灭现象(图1),故探针化合物HOZ用DMSO配制成1×10-5mol/L的溶液,金属盐用二次去离子水配成1×10-4mol/L的溶液.移取探针化合物HOZ的溶液1mL置于比色管(10mL)中,然后分别加入当量的金属离子溶液,用纯净水稀释至刻度,测定其荧光发射光谱,其中pH由不同体系缓冲溶液调节。
pH对探针化合物HOZ探针荧光光谱的影响
为了将探针化合物HOZ更好地应用于有机体或者环境等复杂体系中,配制不同的缓冲体系如HAc-NaAc、HEPES(N-(2-羟乙基)哌嗪-N'-2-乙烷磺酸)、NaHCO3-Na2CO3等调整不同的pH值,测定了不同pH对探针化合物HOZ以及HOZ+Cu2+在其最大发射波长(568nm)处荧光强度的影响,如图2所示,可以看出,在pH为6-7.4的范围内,HOZ+Cu2+的荧光强度几乎不变。因此,在pH为6-7.4的范围内,探针化合物HOZ对Cu2+的检测不受pH的影响,有利于其在活细胞及生物体内Cu2+的检测。所以实验选择生理条件的HEPES缓冲体系(pH=7.20)作为测定介质的缓冲溶液。
配位时间和稳定性
将配置好的浓度同为1×10-5mol/L的空白HOZ探针溶液迅速加入比色皿中,从第1min开始测其荧光强度随时间的变化,测试完毕后再对浓度同为1×10-5mol/L的铜离子及探针化合物HOZ混合溶液进行测试,结果表明,空白体系的荧光强度基本不随时间变化而变化,当加入铜离子以后,其荧光强度淬灭迅速,在5 min后即趋于稳定,到8 min后荧光强度变化很小。故选择在所有试剂混合作用8 min后进行测定(图3)。
实施例3
探针化合物HOZ对各种金属离子的光谱响应
分别选择过渡金属(Cu2+、Hg2+、Fe3+、Ni2+、Fe2+、Zn2+、Cr3+、Mn2+、Cd2+、Ag+、Co2+),碱金属(K+、Na+)和碱土金属(Ba2+、Ca2+、Mg2+)为识别对象。如图4中所示,除细胞内浓度较大的Na+、Mg2+、Ca2+的浓度为20 μM外,其余离子及探针浓度均为1 μM。静置15 min后进行测定。如图4所示,当同样浓度的等当量各种金属离子分别与作用时,Cu2+的加入使体系在 568 nm处荧光强度出现明显的淬灭,除Ag+、Co2+略使探针出现部分荧光淬灭外,其它金属离子与探针分子作用时,相比较于空白试液,其荧光强度无明显变化。这表明,探针化合物HOZ对铜离子的识别具有较好的选择性。
共存离子的影响
进一步考察了探针化合物HOZ对铜离子传感的荧光选择性,尝试了污染水样中有可能存在的不同金属离子与铜离子共存时对体系荧光强度的影响。在图5中,标记为Cu2+的组分为仅含1.0×10-5 mol/L铜离子时体系的荧光强度,其余为同等浓度铜离子与各种不同浓度倍数的其它金属离子共存时体系的荧光强度,由左至右金属离子浓度为铜离子浓度的倍数分别如下:K+ (100倍),Na+ (100倍),Ca2+ (100倍),Mg2+ (100倍),Mn2+ ( 10倍),Zn2+(10倍),Ba2+ ( 10倍),Fe3+ (10倍),Co2+ (10倍),Cd2+ (20倍),Ni2+ (10倍),Ag+ (10倍),Hg2+ (10倍)。由图可知,探针化合物HOZ在 Zn2+、Ag+和Co2+浓度较高浓度时对铜离子的响应有一定的影响。
实施例4
探针化合物HOZ对Cu2+离子工作曲线的测定:
测定在不同Cu2+浓度条件下探针化合物HOZ的荧光发射光谱(图6),在探针化合物HOZ的最大发射波长(568nm)处,随着Cu2+浓度的逐渐增大,其最大荧光强度不断降低,表明探针化合物HOZ与Cu2+之间存在着良好的络合作用.进一步研究发现,如图7所示,Cu2+浓度在0.25×10 -6-6×10-6mol/L范围内与探针化合物HOZ的荧光强度呈良好的线性关系,曲线方程为y=386.688-54.687x,相关系数R=0.970,这表明探针化合物HOZ可以在此范围内定量地检测此范围内Cu2+的含量。
以上实施例描述了本发明的基本原理、主要特征及优点,本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明原理的范围下,本发明还会有各种变化和改进,这些变化和改进均落入本发明保护的范围内。
Claims (3)
1.3,4-二(3-吲哚)-3-吡咯啉-2,5-二酮-1-(2-羟基苯基)腙作为荧光探针在检测铜离子中的应用,其特征在于具体过程为:
(1)将3,4-二(3-吲哚)-3-吡咯啉-2,5-二酮-1-(2-羟基苯基)腙溶于二甲基亚砜溶液中配成摩尔浓度为0.1 mmol/L的探针化合物标准溶液,用N-(2-羟乙基)哌嗪-N'-2-乙烷磺酸的缓冲溶液配制不同浓度的铜离子储备液,取30 μL探针化合物标准溶液分别与3 mL不同浓度的铜离子储备液混合,室温下静置8 min,分别测混合液在波长568 nm处的荧光发射强度,以铜离子的浓度为横坐标,以荧光强度为纵坐标绘制标准曲线,或再拟合得到标准直线方程;
(2)将30 μL摩尔浓度为0.1 mmol/L的探针化合物标准溶液加入到3 mL待测样品溶液中,室温下静置8 min,测定被检测溶液在波长568 nm处的荧光发射强度,再与标准曲线对比或代入标准直线方程计算,得到待测样品溶液中铜离子的浓度;
测试条件为:室温,激发狭缝宽度3.0nm,发射狭缝宽度3.0nm,激发波长λex为568 nm,灵敏度为1,响应时间1s。
2.根据权利要求1所述的3,4-二(3-吲哚)-3-吡咯啉-2,5-二酮-1-(2-羟基苯基)腙作为荧光探针在检测铜离子中的应用,其特征在于所述3,4-二(3-吲哚)-3-吡咯啉-2,5-二酮-1-(2-羟基苯基)腙的具体合成过程为:在25 mL圆底烧瓶中加入102 mg 1-氨基-3,4-二(3-吲哚)-3-吡咯啉-2,5-二酮和5 mL无水乙醇,再加入邻羟基苯甲醛73.2 mg和乙酸20 μL,回流条件下反应,薄层色谱分析跟踪监测,反应结束后,降至室温,加水出现大量沉淀,抽滤,水洗,用体积比为1:1的氯仿与石油醚的混合溶液清洗,柱层析分离得到探针化合物3,4-二(3-吲哚)-3-吡咯啉-2,5-二酮-1-(2-羟基苯基)腙。
3.根据权利要求1所述的3,4-二(3-吲哚)-3-吡咯啉-2,5-二酮-1-(2-羟基苯基)腙作为荧光探针在检测铜离子中的应用,其特征在于:所述待测样品溶液中铜离子的浓度范围为0.25×10-6-6×10-6mol/L。
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