CN113603701B - 一种检测次氯酸根离子的比色/荧光探针及其制备方法与应用 - Google Patents
一种检测次氯酸根离子的比色/荧光探针及其制备方法与应用 Download PDFInfo
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Abstract
一种检测次氯酸根离子的比色/荧光探针及其制备方法与应用,荧光探针命名为TPE‑acylamide‑Rh,其化学结构式如式I所示;由1‑(4‑甲酸苯基)‑1,2,2‑三苯乙烯溶于氯化亚砜中,加热反应后,在惰性气氛和催化作用下与罗丹明B酰肼反应合成、纯化得到。该探针在溶液状态和聚集状态下对ClO‑识别性能表现出优良的选择性、抗离子干扰能力以及灵敏度。另外,该探针在聚集状态形成纳米颗粒,容易细胞染色,有利于细胞成像应用。Hela细胞成像研究表明:TPE‑acylamide‑Rh表现出对细胞中ClO‑优异的识别特性。该探针制备工艺简单、合成路线短。将探针应用于检测次氯酸根离子中,拓宽了探针的应用范围。
Description
技术领域
本发明属于有机荧光分子探针领域,具体涉及一种检测次氯酸根离子的比色/荧光探针 及其制备方法与应用。
背景技术
次氯酸,作为一种强氧化剂,在日常生活中广泛的应用于自来水消毒和衣物漂白等; 也是生物体中一种重要的活性氧小分子,参与生物体的免疫调节、抗原响应、信号传导以 及细胞凋零等过程。打破体内ClO-平衡会引发各种心血管疾病、组织炎症甚至癌症。因此, 为了防止来自ClO-的威胁,研究快速、有效、准确的检测环境中以及生物体内ClO-的方法 有着迫切需求。
与传统的ClO-检测方法(碘量法、库伦法、极谱法、电化学法、电势法等)相比,荧光探针显示出快速、灵敏、选择性好、原位实时检测等优点和潜力而得到广泛应用,并且 在细胞中次氯酸显影成像技术表现出巨大优势。ClO-既没有金属离子的配位能力,也没有 CN-的强的亲核性质或F-的强的电负性,因此,ClO-荧光探针的设计合成具有一定的挑战。
近年来,研究人员设计合成了很多基于不同检测机制的ClO-荧光探针,如荧光淬灭型、 OFF-ON型、比率型等。与荧光淬灭型探针相比,OFF-ON型型癸光探针虽在检测灵敏度上 有一定的提高,易于实现可视化检测,但由于检测是在单一波长下进行,易于受检测设备 本身光强度、光漂白及背景巧光的干扰;检测过程中,探针样品自身浓度,其所受的微环境(如测试样品均匀度、粘度、溢度)的改变都可能给实验结果造成偏差;而比率型荧光 探针有两个相关的发射信号,可避免仪器和环境因素造成的检测误差,可表现出更好的灵 敏度和动态响应范围等。另外,目前设计和报道的一些用于ClO-荧光成像的探针,少有可 实现对生物系统中ClO-的实时定量荧光成像。因此,设计合成具有能够实时定量荧光成像、高灵敏度的比率型ClO-荧光探针,并将其应用于生命体中ClO-的实时定量荧光成像依然是ClO-探针研究工作的重心。
发明内容
本发明的目的之一是提供一种检测次氯酸根离子的比色/荧光探针,该探针灵敏度高, 能够实现实时定量荧光成像。
本发明的目的之二是提供上述检测次氯酸根离子的比色/荧光探针的制备方法,该制备 工艺简单,合成路线短。
本发明的目的之三是提供上述检测次氯酸根离子的比色/荧光探针的应用,拓宽探针的 应用范围。
为实现上述目的,本发明采用的技术方案如下:一种检测次氯酸根离子的比色/荧光探 针,所述荧光探针命名为TPE-acylamide-Rh,对应的化学结构式如式I所示:
上述检测次氯酸根离子的比色/荧光探针的制备方法,包括以下步骤:
将1-(4-甲酸苯基)-1,2,2-三苯乙烯溶于氯化亚砜中,在60-80℃下加热搅拌4-8h后, 减压蒸馏除去氯化亚砜,冷却至室温后,在惰性气氛保护下,依次加入乙腈和三乙胺,最 后加入罗丹明B酰肼,在40-85℃下加热搅拌10-18h;1-(4-甲酸苯基)-1,2,2-三苯乙烯与罗丹明B酰肼的摩尔比为1:1.03;反应完毕后,将反应液进行萃取、合并有机相,最后 干燥得到粗产品,粗产品再通过硅胶柱色谱法纯化得到TPE-acylamide-Rh;所述罗丹明B 酰肼的结构式为
进一步的,反应完毕后,将反应液倒入去离子水中,再用二氯甲烷萃取三次。
优选的,柱色谱法纯化采用的洗脱剂为体积比30:1的二氯甲烷/甲醇溶液。
具体的合成路线如下:
本发明还提供上述荧光探针在检测次氯酸根离子含量中的应用。
本发明中四苯乙烯是具有聚集诱导发光机制(AIE)性质的分子,罗丹明的红光特性使 其成为优异的荧光基团,在两者相互耦合作用下,不仅可以在溶液状态下表现出比色/荧光 双通道响应,而且在聚集状态下,可获得比率型荧光探针。该荧光探针在溶液状态下可以 通过比色和荧光双通道校准;也可以在聚集状态下,通过双波长响应,排除干扰。本发明 将四苯乙烯作为荧光基团,修饰、调控罗丹明的不同位置,设计合成反应性ClO-荧光探针 TPE-acylamide-Rh。由于四苯乙烯的聚集诱导效应,该荧光探针在聚集状态下,表现出比率 型荧光探针性质,且识别位点均为罗丹明的酰胺结构,这种酰胺结构在ClO-强氧化性作用 下,发生分解反应,进而导致罗丹明“开环反应”,引起探针溶液的比色和荧光响应。
与现有技术相比,本发明具有以下优点:
本发明以罗丹明酰胺基团为识别反应位点,设计合成了四苯乙烯耦合罗丹明类反应的 比率型ClO-比色/荧光双通道荧光探针TPE-acylamide-Rh,该探针表现出AIE性质,并且在 溶液状态下(50%乙醇-水体系中),表现出比色/荧光双通道响应,在ClO-作用下,溶液颜 色发生“裸眼”可见的响应(由无色变成红色);在365nm紫外灯激发下,发出橙黄色荧光;并且表现出较好的灵敏度(紫外吸收光谱最低检出限1.3μM;荧光光谱最低检出限2.5 μM);在聚集状态下(60%乙醇-水体系中),表现出比率型荧光特性,识别ClO-也具有良 好的灵敏度(荧光光谱最低检出限2.8μM)。因此,该探针在溶液状态和聚集状态下,对 ClO-识别性能表现出优良的选择性、抗离子干扰能力以及灵敏度,实现实时定量荧光成像。 另外,该探针在聚集状态形成纳米颗粒,容易细胞染色,有利于细胞成像应用。Hela细胞 成像研究表明TPE-acylamide-Rh表现出对细胞中ClO-优异的识别特性,因此,该探针成功 的应用于活细胞内ClO-荧光成像技术。该荧光探针的制备工艺简单、合成路线短。将该探 针应用于检测次氯酸根离子中,从而拓宽了探针的应用范围。
附图说明
图1是化合物TPE-acylamide-Rh的核磁氢谱图;
图2是化合物TPE-acylamide-Rh的质谱图;
图3是TPE-acylamide-Rh(2μM)在不同含水量的乙醇-水溶液中荧光峰值随含水量的变 化趋势图;
图4是TPE-acylamide-Rh(2μM)在不同含水量的乙醇-水溶液中的荧光发射谱图;
图5是TPE-acylamide-Rh(2μM)在不同含水量的乙醇-水溶液中对ClO-荧光光谱图;
图6是TPE-acylamide-Rh(2μM)在50%乙醇-水体系下对不同检测物的紫外吸收光谱 图;
图7是TPE-acylamide-Rh(2μM)在50%乙醇-水体系下对不同检测物和加入ClO-后的 紫外吸收光谱图;
图8是TPE-acylamide-Rh(2μM)在50%乙醇-水体系下对不同检测物的荧光光谱图;
图9是TPE-acylamide-Rh(2μM)在50%乙醇-水体系下对不同检测物和加入ClO-后的 荧光光谱图;
图10是TPE-acylamide-Rh(2μM)在60%乙醇-水体系下对不同检测物的荧光光谱图;
图11是TPE-acylamide-Rh(2μM)在60%乙醇-水体系下对不同检测物和加入ClO-后 的荧光光谱图;
图12是TPE-acylamide-Rh(2μM)在50%乙醇-水体系下荧光滴定光谱图;
图13是TPE-acylamide-Rh(2μM)在50%乙醇-水体系下荧光强度与ClO-浓度的线性 关系图;
图14是TPE-acylamide-Rh(2μM)在60%乙醇-水体系下荧光滴定光谱图;
图15是TPE-acylamide-Rh(2μM)在60%乙醇-水体系下荧光强度比值与ClO-浓度的 线性关系图;
图16是TPE-acylamide-Rh(2μM)在50%乙醇-水体系下紫外吸收滴定光谱图;
图17是TPE-acylamide-Rh(2μM)在50%乙醇-水体系下紫外吸收强度与ClO-浓度的 线性关系图;
图18是TPE-acylamide-Rh的HeLa细胞毒性实验结果统计图;
图19是TPE-acylamide-Rh的HeLa细胞荧光成像图:(a)HeLa细胞在TPE-acylamide-Rh 明场中细胞成像图;(b)HeLa细胞在TPE-acylamide-Rh绿色通道中细胞成像图;(c)HeLa 细胞在TPE-acylamide-Rh和20μM ClO-红色通道中细胞成像图;(d)图b和图c叠加图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明。
以下实施例中所用的原料和试剂,如无特殊说明,均为市售商品,纯度为分析纯及以 上。
实施例1:探针化合物TPE-acylamide-Rh的合成
称量1-(4-甲酸苯基)-1,2,2-三苯乙烯0.50g(1.33mmol)加入到5mL氯化亚砜溶液, 加热回流4h;反应完毕后,减压蒸馏蒸出氯化亚砜溶液。待温度降到室温,加入30mL乙腈溶液,再加入1mL三乙胺;最后称量罗丹明B酰肼0.62g(1.37mmol)加入反应液中, 氮气保护,加热回流,反应过夜。反应完毕后,反应液倒入水中,二氯甲烷萃取三次,合 并有机相,无水硫酸镁干燥,旋干得到粗产品,用二氯甲烷:甲醇体积比为30:1作为流动 相,过硅胶柱得到0.35g纯品,产率:31.4%,熔点:173-175℃。氢谱如图1所示,质谱 如图2所示,1H NMR(600MHz,DMSO-d6)δ10.10(s,1H),7.84(d,J=7.3Hz,1H),7.63–7.51 (m,2H),7.40(d,J=8.1Hz,2H),7.19–7.05(m,10H),7.01–6.91(m,8H),6.55(d,J=8.8Hz, 2H),6.35–6.25(m,4H),3.33–3.19(m,8H),1.06(t,J=7.0Hz,12H).13C NMR(151MHz, Chloroform-d)δ156.87.,153.15,151.88,148.75,143.83,140.48,136.04,133.80,133.05, 131.24,131.22,131.18,128.91,128.10,128.02,127.63,127.60,127.52,126.34,126.12,126.04,123.75,123.47,123.26,110.08,107.89,106.26,97.95,65.84,55.37,44.32,12.67. MALDI-MS:m/z calcd for C55H50N4O3:815.0105,found:814.9627[M]+.
实施例2:探针TPE-acylamide-Rh的比率型荧光特性与AIE性质
探针TPE-acylamide-Rh是由四苯乙烯通过酰胺键与罗丹明B连接的具有AIE特性的 ClO-荧光探针。探针TPE-acylamide-Rh的识别位点是罗丹明酰胺结构,其在ClO-强氧化性 作用下,酰胺键被打断,罗丹明螺环发生“开环反应”,探针比色/荧光均发生变化。探针TPE-acylamide-Rh在聚集状态下,表现出四苯乙烯的荧光,在与ClO-反应后,探针的四苯乙烯的聚集诱导发光消失,显示出罗丹明结构的荧光,成为一种典型的比率型荧光探针。
探针TPE-acylamide-Rh由于具有“螺旋桨”结构的四苯乙烯基团,因此表现出典型的 聚集诱导发光性质(如图3和图4所示)。探针TPE-acylamide-Rh在乙醇-水体系下溶解性较好,溶液含水量在0~50%之间时,探针TPE-acylamide-Rh处于溶解状态,无荧光发射;当溶液含水量达到60%,TPE-acylamide-Rh出现纳米聚集(平均粒径为396nm),在波长487nm处出现明显的荧光发射峰,其为典型的四苯乙烯荧光发射峰;溶液含水量在70%时,荧光强度达到最大值;然后,随着溶液含水量的增加,荧光强度逐渐下降。
实施例3:探针TPE-acylamide-Rh的溶剂配比选择
TPE-acylamide-Rh是一种反应性ClO-荧光探针,溶剂配比的选择影响着探针灵敏度。 测试在不同含水量的乙醇-水体系中加入ClO-的荧光强度来探究最佳溶剂配比,结果如图5 所示。TPE-acylamide-Rh溶液含水量在0%到50%之间,处于溶解状态,无荧光发射,在 ClO-作用下,583nm处出现荧光发射峰,且在含水量50%时发射峰强度最大,表明: TPE-acylamide-Rh在溶液状态下,对ClO-具有良好的灵敏度,在含水量50%的乙醇-水体系 下,对ClO-响应灵敏度最高。因此,TPE-acylamide-Rh在溶液状态下对ClO-识别的溶剂配 比选择50%乙醇-水体系。当溶液含水量达到60%时,探针处于纳米聚集状态,在583nm 和478nm处同时出现发射峰,表现出典型的比率型荧光特性,当含水量大于70%时,在583 nm处的荧光发射峰强度非常微弱,几乎对ClO-离子没有响应,因此可知,探针 TPE-acylamide-Rh在聚集态的溶剂配比最优条件为:60%乙醇-水体系。
实施例4:探针TPE-acylamide-Rh对ClO-识别的选择性和抗离子干扰能力
TPE-acylamide-Rh溶液的制备:TPE-acylamide-Rh的2.0×10-3mol L-1溶液是用色谱纯的 乙醇作为溶剂,称量计算量实施例1制备的探针加入到3mL容量瓶中,用乙醇定容,超声, 直至样品完全溶解,密封保存,以备使用。
ClO-以及干扰离子溶液的配制:用NO2 -、NO3 -、HCO3 -、ACO-、Br-、ClO4 -、SO4 2-、 S2-、H2O2、NO、ONOO-、DTBP和ClO-溶解于去离子水中,配制离子溶液。离子溶液浓 度为1.0×10- 2mol L-1,称量计算量的离子盐放入5mL容量瓶中,用去离子水定容到容量品 刻度线,摇晃、超声,使各个离子盐完全溶解,密封保存,以备使用。
如图6所示,探针TPE-acylamide-Rh在50%乙醇-水体系中处于溶液状态,除了在ClO-存在下溶液由无色变为红色,其他干扰离子都没有明显变化。同时,在其他离子存在下, 继续加入ClO-,如图7所示,探针TPE-acylamide-Rh表现出良好的抗离子干扰能力。同样, 探针TPE-acylamide-Rh在荧光光谱中也表现出优异的选择性与抗离子干扰能力(如图8-9 所示)。
探针TPE-acylamide-Rh在含水量60%乙醇-水体系中,表现出比率型荧光探针性质。探 针TPE-acylamide-Rh(2μM)的60%乙醇-水中,分别加入10倍当量的测试离子,由荧光 光谱强度研究探针的选择性。如图10所示,TPE-acylamide-Rh在60%乙醇-水体系中,由于探针处于聚集状态,加入测试离子之前,荧光光谱在487nm出现四苯乙烯的荧光发射峰,当加入测试离子后,只有加入ClO-荧光探针溶液中荧光发出橙红色荧光,荧光发射波长为583nm;其他离子溶液在487nm处的发射荧光强度仅有微小变化,表明探针 TPE-acylamide-Rh对于ClO-的识别具有较高的选择性。同时,在其他离子存在下,继续加 入ClO-,如图11所示,探针TPE-acylamide-Rh表现出较好的抗离子干扰能力。
实施例5:探针TPE-acylamide-Rh对ClO-荧光光谱滴定
探针TPE-acylamide-Rh在溶液状态下(50%乙醇-水),荧光强度与ClO-浓度的变化趋 势如图12所示。TPE-acylamide-Rh空白溶液荧光较弱;加入ClO-后,荧光光谱在583nm处出现新的荧光发射峰,随着ClO-浓度的增加,荧光强度逐渐增强,当ClO-浓度达到30μM,荧光强度增速放缓,ClO-浓度达到饱和状态。由图13可知,探针TPE-acylamide-Rh荧光强度在ClO-在0.5μM到30μM呈现出良好的线性关系。经过线性拟合,得到荧光强度与ClO-浓度关系为:y=14680.71x+106666.31,R2=0.993。由最低检出限公式LOD=3σ/k可计算出 探针TPE-acylamide-Rh在50%乙醇-水体系中的最低检出限为:1.3μM。
探针TPE-acylamide-Rh在聚集状态下(60%乙醇-水),荧光强度与ClO-浓度的变化趋 势如图14所示。TPE-acylamide-Rh空白溶液在365nm激发下,溶液产生很强的四苯乙烯 荧光发射峰(最大发射波长:476nm),加入ClO-后,荧光光谱在583nm处出现新的荧光 发射峰,随着ClO-浓度的增加,476nm处荧光强度逐渐减小,583nm处荧光强度逐渐增强; 当ClO-浓度达到30μM两处荧光强度几乎不再变化。由图15可知,探针TPE-acylamide-Rh 在583nm和476nm处荧光强度比值在ClO-在0.5μM到30μM呈现出良好的线性关系。 经过线性拟合,得到I579nm/I485nm与ClO-浓度关系为:y=0.08394x+0.16389,R2=0.998。由最 低检出限公式LOD=3σ/k计算可知探针TPE-acylamide-Rh在60%乙醇-水体系中的最低检出 限为:2.8μM。
实施例6:探针TPE-acylamide-Rh对ClO-紫外吸收光谱滴定
探针TPE-acylamide-Rh是基于罗丹明结构设计合成,因此,探针TPE-acylamide-Rh在 识别ClO-后,不仅会有荧光响应,同时也会引起比色响应。如图16所示,TPE-acylamide-Rh 空白溶液没有紫外吸收峰,溶液呈现出无色,加入ClO-后,在556nm处出现新的吸收峰紫 外吸收光谱,随着ClO-浓度的增加,吸收峰强度逐渐增强,当ClO-浓度达到30μM以后, 紫外吸收峰强度停止增加。如图17所示,探针TPE-acylamide-Rh紫外吸收强度在ClO-浓 度为2μM到7μM之间时呈现出良好的线性关系。经过线性拟合,得到紫外吸收光谱强度 与ClO-浓度关系为:y=0.02688x-0.17201,R2=0.978。由最低检出限公式LOD=3σ/k可计算 出探针TPE-acylamide-Rh在50%乙醇-水体系中的最低检出限为:2.5μM。
实施例7:探针TPE-acylamide-Rh的细胞毒性研究
细胞培养:细胞成像的宫颈癌细胞(Hela细胞)购置于生物公司,Hela细胞培养过程: 将购买的Hela细胞转移至装有2mL的10%胎牛血清的DMEM培养液的培养皿中,将培养皿放入二氧化碳含量保持在5%、37℃恒温、无菌的培养箱中,培养24h,以备用于细胞 成像。
细胞毒性测试:将Hela细胞转移至96孔的含有100μL培养液的培养皿中,在二氧化碳含量5%、37℃恒温的环境中孵育24h,然后,用上述培养液配制100μL的0μM、5μM、 10μM、20μM和30μM浓度的探针溶液,分别放入有标记的96孔培养皿中,为了实验的 准确性,每个浓度的探针都做5组平行实验,继续培养24h。然后在每个实验孔内加入20μL 的MTT(mg/mL),在培养4h。最后,加入100μL的二甲基亚砜溶液混合均匀后,最后 加入酶标仪,通过测定其在570nm处的紫外吸收强度来算出细胞存活率,进而反映出荧光 探针的毒性强弱。
探针TPE-acylamide-Rh经过Hela细胞毒性试验(MTT)结果如图18所示。Hela细胞在不同浓度(0μM、5μM、10μM、20μM和30μM)探针TPE-acylamide-Rh中孵育相同 时间,观察细胞存活率。细胞存活率随着TPE-acylamide-Rh浓度的增加而小幅降低,当探 针浓度达到30μM,细胞存活率95%以上。结果表明低浓度TPE-acylamide-Rh探针对细胞 毒性较小,适合用于细胞成像技术。
实施例8:探针TPE-acylamide-Rh的细胞成像
细胞成像主要用于检测外源性ClO-细胞成像实验。分两组用于细胞成像。第一组将Hela 细胞放入含有培养液的培养皿中培养24h,用pH=7.4的PBS缓冲溶液洗涤三次,然后将 20μL的荧光探针标准溶液加入到细胞培养液中,继续培养1h,最后用pH=7.4的PBS缓冲溶液洗涤三次,洗去细胞代谢物以及死亡的细胞,剩下的细胞用于细胞成像;第二组将Hela细胞放入含有培养液的培养皿中培养24h,用pH=7.4的PBS缓冲溶液洗涤三次,然 后将20μL荧光探针标准溶液加入到细胞培养液中,继续培养1h,加入10μL的ClO-标准 溶液,继续培养1h,用pH=7.4的PBS缓冲溶液洗涤三次,洗去细胞代谢物以及死亡的细 胞,剩下的细胞用于细胞成像。
如图19所示,探针TPE-Acylamide-Rh溶液加入到Hela细胞培养液中培养后,将细胞 转移至共聚焦显微镜上,用405nm紫外激发,通过观察蓝色通道(425nm-475nm)可以 得到探针TPE-Acylamide-Rh容易进入细胞内,并且发出微弱的黄绿色荧光(如图19-b所示)。当加入20μM ClO-继续培养后,再次放入共聚焦显微镜下拍照观察,发现细胞发出红色荧光(如图19-c所示)。通过探针TPE-Acylamide-Rh细胞成像实验表明,探针在细胞中对 ClO-识别效果(细胞成像中发出红色荧光)要比在60%乙醇-水体系中(溶液发出橙黄色荧 光)更加明显。这是因为共聚焦显微镜具有双通道(蓝光通道和红光通道),其中蓝光通 道激发波长为405nm,红光通道(570-620nm)的激发波长561nm,细胞成像实验中,探 针与离子反应后所得到的图片是在在561nm激发下通过红色通道收集的,因此要比荧光滴 定溶液显示的荧光要强。结果表明探针TPE-Acylamide-Rh在细胞内可以灵敏的检测ClO-。 因此,探针TPE-Acylamide-Rh能够实现对Hela活细胞内ClO-检测,并且可以实现比率型 细胞荧光成像。
Claims (5)
3.根据权利要求2所述的检测次氯酸根离子的比色/荧光探针的制备方法,其特征在于,反应完毕后,将反应液倒入去离子水中,再用二氯甲烷萃取三次。
4.根据权利要求2或3所述的检测次氯酸根离子的比色/荧光探针的制备方法,其特征在于,柱色谱法纯化采用的洗脱剂为体积比30:1的二氯甲烷/甲醇溶液。
5.权利要求1所述的荧光探针在检测次氯酸根离子含量中的应用。
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