CN106366131A - 生理pH传感双核钌配合物的制备方法和应用 - Google Patents
生理pH传感双核钌配合物的制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种双核钌配合物的制备方法及其在水相和细胞内pH传感中的应用。配合物的细胞毒性小,可见光激发,生理pH响应,可比率磷光传感生物细胞,实时检测生物细胞中pH的变化。
Description
技术领域
本发明属于荧光传感器和生物无机化学领域,涉及双核钌配合物制备方法及其在非细胞环境下的水溶液和细胞内pH传感相关领域中的应用。
背景技术
细胞内pH在钙的调节、细胞的成长与分化、细胞内吞作用、细胞粘连、离子的运输及肌肉的收缩等过程中都扮演着重要角色(H.Izumi,T.Torigoe,H.Ishiguchi,H.Uramoto,Y.Yoshida,M.Tanabe,Cellular pH regulators:potentially promising moleculartargets for cancer chemotherapy.,Cancer Treat.Rev.,2003,29,541;R.J.Gillies,N.Raghunand,M.L.Garcia-Martin,R.A.Gatenby,A review of pH measurement methodsand applications in cancers.IEEE Eng.Med.Biol.Mag.2004,23,57;N.M.Walker,J.E.Simpson,R.C.Levitt,K.T.Boyle,L.L.Clarke,J.Pharmacol.Exp.Ther.,2006,317,275.)。同一个细胞,不同细胞器内的pH值也不相同,如溶酶体内的pH值就比较低。非正常的pH可能会导致蛋白质发生变性,或刺激酶和功能蛋白的活性,会导致心肺和神经问题(如癌症和老年痴呆症)。pH的实时监测可为疾病的诊断、治疗提供更多的依据,因此实现显得尤为重要。因此,细胞内pH的监测对理解细胞内生理机能的调节机制至关重要。检测细胞内pH值的方法有很多,如微电极与核磁共振等,但很难使用这些方法检测生物体系的pH,特别是活细胞内的pH。pH荧光探针技术灵敏、方便,可以实现活细胞内pH的动态成像和区域成像,成为检测化学体系和生物体系中pH的重要工具(J.Han,K.Burgess,Fluorescentindicators for intracellular pH,Chem.Rev.,2010,110,2709.)。因此,开发新型的荧光细胞pH探针是是一项非常有价值的工作。带有可质子化/去质子化的钌基金属配合物具有pH可调控的诱人的基态和激发态光物理/光化学性质,较纯有机pH探针有更好的光稳定性和可见光激发的优点,但文献报道的钌基金属配合物在传感细胞内生理pH方面的应用极为少见(R.Wang,C.Yu,F.Yu,L.Chen,Molecular fluorescent probes for monitoring pHchanges in living cells,Trends in Analytical Chemistry,2010,.29,1004.)。
发明内容
本发明的目的是公开一个新型双核钌配合物的制备方法。
本发明的目的是公开该配合物具有生理pH磷光传感性质。
本发明的另一个目的是公开该配合物作为水溶液中生理pH传感器性质。
本发明还有一个目的是公开该配合物合物作为细胞的pH传感器性质。
本发明的技术方案如下:
本实验中的双核钌配合物是由阳离子和阴离子组成,所述阳离子为[(bpy)2Ru(HL)Ru(bpy)2]4+,结构式如下式所示:
配制Britton-Robinson(简称BR)缓冲溶液,酸碱滴定在该缓冲溶液中进行。配制2μM的配合物待测液,测定pH连续变化时配合物的紫外可见吸收和发射光谱的变化。每间隔0.2个pH测定一个数据,再根据不同pH时的紫外可见吸收光谱468nm吸光度和发射光谱的积分强度,计算出发光量子效率,绘制标准工作曲线。进而,通过测定未知水样的紫外可见吸收光谱和发射光谱,计算出发光量子效率与标准工作曲线作对比,从而得到未知水样的pH值。还可通过建立环境下配合物在438nm激发和380nm激发下的发光强度比对pH作图的工作曲线,测定细胞的pH。
与现有技术相比,本发明的优势在于:
本发明所制备的双核钌配合物结构稳定,具有一定的水溶性。该配合物中桥联配体中的 中性咪唑环的去质子化能导致配合物的磷光发射大幅度增强,绘制发光量子效率对pH作图得标准工作曲线,用于检测生理pH范围水样的pH(pH=6.3-8.5);还可通过双激发波长和细胞内pH,简单方便,易于操作。
与现有技术相比,本发明的有益效果在于:
大部分已报道的钌配合物pH荧光探针不在生理pH范围,且荧光探针对被分析物质的荧光响应只是改变单一波长处的荧光强度。而本发明的探针的pH范围在生理pH区,且可实现比率荧光测量。在单波长处荧光强度响应的荧光探针的主要弊端是它的响应信号容易受到周围检测环境、探针浓度、激发光强、光渗漏等因素的干扰,而产生荧光检测信号人工假象,从而极大的影响对被分析分子的定量识别。与之相比较,比率荧光探针与被分析分子作用后,在不同波长处使荧光强度同时发生改变,通过计算荧光强度比率值(即比率法测量),可以有效的消除上述因素的影响,并且可以提高荧光探针识别被分析分子的动态范围。
附图说明
图1(a)是配合物BR缓冲水溶液(2μM)在随pH由1.2增加到5.2时吸收光谱的变化;图1(b)是配合物BR缓冲水溶液(2μM)在随pH由5.2增加到9.6时吸收光谱的变化;图1(c)是是配合物BR缓冲水溶液(2μM)在380nm处的吸光度随pH的变化。
图2(a)是配合物缓冲的水溶液(2μM)在随pH由1.5增加到10.0时发射光谱的变化;图2(b)是发射峰强度随pH的变化;图2(c)是配合物的发光量子效率随pH的变化。
图3 HeLa细胞在不同浓度的配合物和顺铂作用下的存活百分比
图4配合物在HEK-MT(第一行图)、MCF-7(第二行图)、HeLa(第三行图)细胞中成像
图5配合物在HeLa、MCF-7细胞中与DAPI共定位成像
图6配合物在HEK-WT细胞中荧光比率变化曲线
图7配合物在MCF细胞中荧光比率变化曲线
图8配合物和BCECF在HEK-WT细胞中荧光比率随pH变化的标准曲线
图9配合物和BCECF在MCF-7细胞中荧光比率随pH变化的标准曲线
具体实施方式
下面通过实施例对本发明进一步说明。
实施例一、钌配合物的制备
1.1配体HL的制备:
按照下式所示的途径制备配体HL:
150mg吡嗪并[2,3-f][1,10]邻菲咯啉-2-羧酸、112mg 1,10-邻菲咯啉-5,6-二胺、和22g聚磷酸加入25mL三口瓶,通氮气保护,机械搅拌使之混合均匀。先加热至120℃,然后升温至190℃反应7小时,混合物由红色透明状变为深绿色。氮气保护下冷却至室温,将所得粘稠液体倾倒入冰水中,得到浅黄绿色沉淀,用氨水中和,沉淀颜色加深。抽滤收集沉淀,经水洗和甲醇索式提取,真空干燥后得HL固体139mg(57.4%)。红外光谱(KBr,cm–1):3402,3273,3059,1638,1578,1554,1517,1471,1428,1396,1369,1317,1275,1260,1245,1218,1196,1169,1138,1116,1083,812.
1.2钌(II)配合物[(bpy)2Ru(HL)Ru(bpy)2](ClO4)4·2H2O的制备:
Ru(bpy)2Cl2·2H2O(105mg,0.20mmol)和HL(44.6mg,0.10mmol)在60mL乙醇/水(v/v,2/1)中,氮气保护下,加热回流10小时,溶液由紫色变为澄清红色。停止反应后冷却抽滤,浓缩滤液,加入饱和高氯酸钠水溶液,析出红色沉淀。乙腈-乙醚扩散重结晶后用柱层析(展开剂:乙腈/水/饱和硝酸钾溶液=40/4/1,体积比)分离,收集橙色荧光带,旋蒸除去大部分乙腈后,加入高氯酸钠水溶液,析出橙红色沉淀。沉淀用乙腈/乙醚扩散重结晶,真空干燥后得橙红色粉末0.064g(49.92%)。1H NMR(500MHz,DMSO-d6,298K):10.26(s,1H),10.00(d,J=8.5Hz,1H),9.61(d,J=8Hz,1H),9.41(d,J=8Hz,1H),9.24(d,J=8Hz,1H),8.87(m,9H),8.33(s,2H),8.24(s,4H),8.15(t,J=7.5Hz,7H),8.04(m,3H),7.86(d,J=4.0Hz,4H),7.75(dd,J1=15.5Hz,J2=5.0Hz,2H),7.67(s,1H),7.61(s,4H),7.38(m,4H)。元素分析C67H46Cl4N16O16Ru2·2H2O(F.W.=1711.15)]计算值:C,47.03;H,2.95;N,13.09%;实测值:C,47.46;H,3.49;N,13.07%。MALDI-TOF MS:m/z:425.7(425.7)[M-4ClO4 -+H+]3+;687.8(688.0)[M-3ClO4 -+H+]2+;738(738.1)[M-2ClO4 -]2+.
实施例二、pH变化对钌配合物的Britton-Roberson(BR)缓冲溶液的紫外-可见吸收光谱和光致发光光谱的影响
紫外-可见吸收光谱在UV-2600紫外-可见分光光度仪上测定,测定时以BR缓冲溶液作为参比液。荧光发射光谱在Cary Eclipse荧光分光光度计上测定。发光量子效率的求得是以 三联吡啶钌[Ru(bpy)3]2+做标准物(Φstd=0.028),测浓度为1.0×10-6mol/L的[Ru(bpy)3]2+水溶液的紫外可见吸收光谱和发射光谱,读取紫外可见吸收光谱468nm处的吸光度Astd和发射光谱的积分强度Istd,根据公式(1):
Φ=Φstd(Astd/A)(I/Istd) (1)
Φ和Φstd分别为待测物和标准物的发光量子效率,A和Astd是待测物和标准物激发波长处的吸光度,I和Istd是未待测物和标准样的发光积分强度。读取不同pH时配合物紫外可见吸收光谱468nm处的吸光度A和发射光谱的积分强度I,根据公式算出不同pH时的发光量子效率Φ。以pH为横坐标,发光量子效率为纵坐标,绘制标准工作曲线。
从图1(a)可见,从pH1.2增加到5.2的过程中,各吸收峰吸光度以及峰位均无明显变化,由此可知,配合物中桥联配体芳环氮原子的质子化并未引起配合物吸收性质的明显变化。从图1(b)可见,当pH从5.2增加到9.6的过程中,仅当pH从5.2增加到9.6,引起配合物380nm处的吸收峰较大程度的减弱,其它吸收峰无明显变化。光谱的变化,可归因于桥联配体中咪唑环的去质子化所致。模拟图1(b)数据,得到配合物的基态酸解离常数pKa=7.14,处于生理pH范围,这在钌配合物的pH传感器中极为少见。从图2(a)可以看到,当pH从1.5增加到10.0的过程中,仅当pH从5.2增加到9.6,引起配合物由几乎不发光到强发光(激发波长468nm),荧光增强约100倍(图2(b)),峰位基本没有变化,是一个在近生理pH值驱动优良的“off-on”型pH光开关。由光致发光量子效率(φ)对pH作图得到的图2(c)可知,配合物pH传感的线性pH范围为6.3-8.5,该图即为测定水样pH值的工作曲线。
实施例三、未知水样pH的测定
取一定量未知水样,向其中加入氯化钠至浓度为0.1M,并加入定量配合物使其浓度为2μM,测水样的紫外吸收和发射光谱,并根据公式(1)计算配合物的发光量子产率,根据标准曲线(图2(c)),读取该量子产率时对应的pH,从而得知未知水样的pH。
实施例四、钌配合物的某些细胞生物学行为和对细胞pH的传感
4.1配合物的细胞毒性
将HeLa细胞培养在含10%小牛血清的RPMI1640培养基中,内含青霉素100U/ml,链霉素100μg/ml,于37℃,5%CO2培养箱中传代培养。0.3%胰酶0.6ml消化贴壁的肿瘤细胞,含10%小牛血清的RPMI 1640培养液配制细胞悬液。在96孔板上,每孔加入HeLa细胞悬液0.1ml培养24h后,在单层细胞上分别加入不同浓度的钌配合物溶液和顺铂溶液,每种浓度重复4孔,并设正常细胞对照,置于37℃5%CO2培养箱中培养48h后,弃上清培养液,每孔加入含5mg/ml:噻唑蓝(MTT),继续培养3h后弃MTT上 清,磷酸缓冲盐溶液(PBS)洗3次,每孔加裂解液,振荡5min待结晶完全溶解,用酶标仪测570nm处的吸光度值。计算出存活百分比并作图。由图3可知,当配合物浓度达到80μM时,细胞的存活率仍高于90%,说明该配合物对细胞存活无抑制作用,可作为荧光探针。
4.2.配合物在细胞中吸收情况
培养于圆形载玻片上的HEK-MT、MCF-7、HeLa细胞,分别用含10μM[(bpy)2Ru(HL)Ru(bpy)2](ClO4)4(图4以及以后中标记为KZW86)的DMEM培养液在培养箱内孵育过夜,在ZEISSLSM710荧光共聚焦显微镜下观察吸收情况。由图4可知,该配合物均能够进入HEK-WT、MCF-7、HeLa细胞中,发出红色荧光。
4.3配合物在细胞中共定位检测
培养于圆形载玻片上的MCF-7、HeLa细胞,分别用含10μM KZW86的DMEM培养液在培养箱内孵育过夜,用4',6-二脒基-2-苯基吲哚(DAPI)染料染核30min,在ZEISS LSM710荧光共聚焦显微镜下观察吸收情况。由图5可知,配合物均能够进入HeLa、MCF-7细胞的细胞核和细胞质,均匀的分布于整个细胞中。
4.4配合物在细胞中比率荧光的测定
在共聚焦显微镜下,分别用波长380nm和434nm的光激发,收集波长大于509nm的相应的配合物发出的荧光(F380和F438),最后以两者之间的比值F438/F380来表示配合物的信号,并与已经商业化的对pH敏感的荧光探针BCECF-AM做了比较,结果示于图6到8图。由图6和图8可知,在HEK-WT细胞中,钌配合物与BCECF-AM的pH响应行为类似,能感应细胞内pH变化,pH梯度变化依次为9,8.2,7.7,7.2,6.7,6.2,4,随pH的增大荧光比值增加大,在pH=8.2时荧光比值达最大,可作为HEK-WT细胞内pH磷光探针。如图7和图9所示,在MCF-7细胞中,钌配合物与BCECF-AM的pH响应行为类似,能感应细胞内pH变化,pH梯度变化依次为9,8.2,7.7,7.2,6.7,6.2,4,随pH的增大荧光比值增加大,在pH=8.2时荧光比值达最大,可作为MCF-7细胞内细胞内pH磷光探针。
Claims (5)
1.钌配合物[(bpy)2Ru(HL)Ru(bpy)2](ClO4)4,其中bpy、HL和钌配合物阳离子的结构如下式所示:
2.如权利要求1所述的桥联配体,其特征在于,所述的桥联配体HL的制备方法包括:将吡嗪并[2,3-f][1,10]邻菲咯啉-2-羧酸与,10-邻菲咯啉-5,6-二胺在聚磷酸存在下缩合后经氨水中和,水洗后得到桥联配体HL粗产品,粗产品经水洗和甲醇索式提取后可直接用于钌配合物的合成。
3.如权利要求1所述的钌配合物,其特征在于,所述钌配合物的制备方法包括:将Ru(bpy)2Cl2·2H2O和HL在乙醇/水反应后沉淀的粗产品在乙腈-乙醚扩散重结晶后用柱层析(展开剂:乙腈/水/饱和硝酸钾溶液)分离。
4.如权利要求1所述的双核钌配合物,在水相pH传感方面的应用。
5.如权利要求1所述的双核钌配合物,在细胞成像及细胞内环境下传感方面的应用。
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