CN105482810A - 一种对过氧化氢快速响应的荧光探针及其制备方法与应用 - Google Patents
一种对过氧化氢快速响应的荧光探针及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种对过氧化氢快速响应的荧光探针及其制备方法与应用,所述荧光探针的结构式如下:将原料TPE-Br和联硼酸频那醇酯溶于溶剂中,在催化剂和加热的条件下反应,得到上述探针,命名为TPE-BO。该探针分子具有聚集诱导发光性质,有效地降低了荧光探针与过氧化氢的响应时间(10min)。对活细胞的染色效果良好,染色时间较短,染色效率较高;合成步骤相对简单,产率较高且容易纯化。
Description
技术领域
本发明具体涉及一种对过氧化氢快速响应的荧光探针及其制备方法与应用,它具有响应速度快、高选择性等优点。
背景技术
过氧化氢是一类重要的活性氧,它作为第二信使在细胞信号传导中起着重要作用,在生物体的衰老和多种疾病中作为氧化应激标志物。生物新陈代谢过程中产生的过氧化氢是生命活动所必需的,如免疫细胞的活化、哺乳动物的血管重塑、植物中气孔的关闭和根的生长等。但是过量产生的过氧化氢会导致氧化损伤的积累,从而引起衰老和一系列的疾病,例如心血管疾病,糖尿病,癌症。因此,对体内过氧化氢的检测对于探究和理解过氧化氢在生命系统中的化学和生物作用具有非常重要的意义。
荧光法不仅具有操作简单易行、高灵敏度、高选择性、实时原位检测的特点,而且探针分子可以在不破坏生物活性的情况下进入细胞,与细胞内目标分子结合,并通过借助于激光共聚焦成像技术,实现细胞内某种生物分子的“可视化”,从而实现对它在生命体内进行“实时在线”的检测,荧光成像技术已经发展成为动态监控体内生物分子事件的最重要手段。目前,用于检测过氧化氢的荧光探针普遍存在的问题是:探针与过氧化氢的响应时间长,不能实现细胞内特定区域过氧化氢真实浓度的检测。主要原因是:(1)过氧化氢易穿膜;(2)过氧化氢与其他物质反应生成新的活性氧;(3)过氧化氢半衰期短。导致通常情况下稳态浓度极低,反应活性高,且生物体内多种氧化物和抗氧化物的存在都给过氧化氢的测定带来一定难度。因此,设计合成选择性好、快速响应、灵敏度高、具有生物兼容性的荧光探针用于检测活细胞内过氧化氢的真实浓度是生命化学学科发展中具有挑战性的前沿课题之一,尤其是对高选择性、快速响应的荧光探针的设计、合成还是非常具有挑战性的。
发明内容
本发明的目的是为了克服现有技术中存在的问题,提供一种对过氧化氢快速响应的荧光探针及其制备方法与应用。将其成功应用于活细胞中过氧化氢的成像分析,基本无背景荧光,效果良好。
本发明采取的技术方案为:
一种对过氧化氢快速响应的荧光探针,所述荧光探针的结构式如下:
上述荧光探针的合成方法,包括如下步骤:
将原料TPE-Br和联硼酸频那醇酯溶于溶剂中,加入碱后,在催化剂和加热的条件下反应,得到上述探针,命名为TPE-BO。
优选的,所述碱为乙酸钾、碳酸钾、碳酸钠或碳酸铯,催化剂为pd(dppf)Cl2、四三苯基磷钯或二茂铁三苯基磷氯化钯。提供碱性环境,反应需在碱性环境中进行。
优选的,TPE-Br、联硼酸频那醇酯、碱和催化剂的物质的量比为1-2:3-6:8-15:0.1-0.8。
优选的,所述溶剂为无水二甲基甲酰胺DMF、1,4-二氧六环或甲苯。
优选的,上述反应的反应温度为75-100℃,反应的时间为20-35h。
优选的,上述反应完毕后,将反应产物进行萃取、干燥后,重新溶解过柱分离。
进一步优选的,所述萃取所用的萃取剂为二氯甲烷和水的混合物,二氯甲烷和水的体积比为1:1-3:1。
进一步优选的,所述干燥使用的干燥剂为无水硫酸镁。
进一步优选的,过柱分离所用的洗脱剂为石油醚和乙酸乙酯的混合溶液,石油醚和乙酸乙酯的体积比为5:1-15:1。
上述荧光探针在活细胞成像中的应用。
一种对活细胞进行孵育染色的染色溶液,至少包括缓冲溶液和溶解在缓冲溶液中的上述荧光探针。
优选的,所述缓冲溶液为磷酸盐缓冲溶液;或染色溶液的pH值为7.0-7.8。
上述荧光探针对活细胞成像的方法,包括如下步骤:
1)将上述荧光探针溶解在水溶性有机溶剂中,然后加入缓冲溶液;或直接将所述荧光探针溶解在缓冲溶液中,得到染色溶液;
2)将所述染色溶液倒入培养好的细胞中进行孵育,孵育设定时间后,洗去染色溶液,然后进行激光共聚焦成像。
优选的,对孵育好的细胞进行洗涤,去除染色溶液。
本发明的荧光探针的合成方程式,如下所示:
本发明的有益效果为:
1、该探针分子具有聚集诱导发光性质,有效地降低了荧光探针与过氧化氢的响应时间(10min)。
2、本发明的荧光探针对活细胞的染色效果良好,染色时间较短,染色效率较高。
3、本发明的荧光探针合成步骤相对简单,产率较高且容易纯化。
附图说明
图1是本发明新型荧光探针的激发光谱及发射光谱谱图,横坐标为波长(nm),纵坐标为荧光强度;
图2(a)是本发明新型荧光探针与过氧化氢作用前后的荧光发射谱图;横坐标为波长(nm),纵坐标为荧光发射强度;
图2(b)是本发明新型荧光探针与过氧化氢作用时间的谱图;横坐标为反应时间(min),纵坐标为荧光发射强度;
图3是本发明荧光探针细胞成像图,即与巨噬细胞的荧光成像。
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。
荧光探针的合成
实施例1
在氩气保护下,将化合物TPE-Br(0.982g,2mmol)和联硼酸频那醇酯(1.27g,5mmol)溶于无水DMF20mL,加入无水乙酸钾(1.374g,14mmol)和pd(dppf)Cl2(0.08g,0.11mmol,85℃反应24h。反应完毕后,冷却至室温,将混合液用二氯甲烷和水萃取,将有机相用无水硫酸镁干燥过夜。用二氯甲烷重新溶解,柱层析分离(洗脱机为石油醚:乙酸乙酯15:1,V/V)得乳白色固体0.67g(57%)。
实施例2
在氩气保护下,将化合物TPE-Br(1mmol)和联硼酸频那醇酯(5mmol)溶于无水DMF20mL,加入无水碳酸钠(8mmol)和四三苯基磷钯(0.11mmol),100℃反应20h。反应完毕后,冷却至室温,将混合液用二氯甲烷和水萃取,将有机相用无水硫酸镁干燥过夜。用二氯甲烷重新溶解,柱层析分离(洗脱机为石油醚:乙酸乙酯15:1,V/V)得乳白色固体0.67g(57%)。
实施例3
在氩气保护下,将化合物TPE-Br(2mmol)和联硼酸频那醇酯(6mmol)溶于无水DMF20mL,加入无水碳酸铯(15mmol)和二茂铁三苯基磷氯化钯(0.11mmol),75℃反应35h。反应完毕后,冷却至室温,将混合液用二氯甲烷和水萃取,将有机相用无水硫酸镁干燥过夜。用二氯甲烷重新溶解,柱层析分离(洗脱机为石油醚:乙酸乙酯15:1,V/V)得乳白色固体0.67g(57%)。
核磁及质谱表征:
1HNMR(300MHz,CDCl3,δ):7.54(d,J=1.5Hz,2H),7.51(d,J=1.5Hz,2H),7.06-7.10(m,6H),7.05-6.99(m,8H),1.32(s,24H)
13CNMR(100MHz,CDCl3,δ):146.80,146.62,143.61,143.45,141.34,134.23,134.14,131.41,130.76,127.79,127.66,126.62,126.55,83.78,24.99
11BNMR(400MHz,CDCl3,δ)6.10
HRMS(m/z):([M+H]+)calcdforC38H42B2O4,585.3354;found,585.3328.
效果实验:
探针的荧光激发发射实验:
TPE-BO探针测量其荧光激发发射实验,激发和发射谱图显示于图1。图1横坐标为波长(nm),纵坐标表示荧光发射强度。荧光探针与过氧化氢作用前后的荧光发射谱图显示于图2(a);横坐标为波长(nm),纵坐标为荧光发射强度。荧光探针与过氧化氢作用时间的动力学谱图显示于图2(b);横坐标为时间(min),纵坐标为荧光发射强度。
探针对活细胞染色成像实验:
巨噬细胞由高糖的DMEM培养液培养,分别加入10μM的探针HEPES水溶液,37℃中孵育10分钟,然后将细胞进行激光共聚焦荧光成像,可以看出其对活细胞染色效果较好,由于其在水中基本无荧光,因此细胞背景较低(图3)。
上述虽然结合附图对本发明的具体实施方式进行了描述,但并非对发明保护范围的限制,所属领域技术人员应该明白,在本发明的技术方案的基础上,本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围内。
Claims (10)
1.一种对过氧化氢快速响应的荧光探针,其特征在于:所述荧光探针的结构式如下:
2.权利要求1所述荧光探针的合成方法,其特征在于:包括如下步骤:
将原料TPE-Br和联硼酸频那醇酯溶于溶剂中,加入碱后,在催化剂和加热的条件下反应,得到上述探针,命名为TPE-BO。
3.根据权利要求2所述的荧光探针的合成方法,其特征在于:所述碱为乙酸钾、碳酸钾、碳酸钠或碳酸铯;或催化剂为pd(dppf)Cl2、四三苯基磷钯或二茂铁三苯基磷氯化钯;或所述溶剂为无水二甲基甲酰胺、1,4-二氧六环或甲苯。
4.根据权利要求2所述的荧光探针的合成方法,其特征在于:TPE-Br、联硼酸频那醇酯、碱和催化剂的物质的量比为1-2:3-6:8-15:0.1-0.8。
5.根据权利要求2所述的荧光探针的合成方法,其特征在于:所述反应的反应温度为75-100℃,反应的时间为20-35h。
6.根据权利要求2所述的荧光探针的合成方法,其特征在于:所述反应完毕后,将反应产物进行萃取、干燥后,重新溶解过柱分离;或所述萃取所用的萃取剂为二氯甲烷和水的混合物,二氯甲烷和水的体积比为1:1-3:1;或所述干燥使用的干燥剂为无水硫酸镁;或过柱分离所用的洗脱剂为石油醚和乙酸乙酯的混合溶液,石油醚和乙酸乙酯的体积比为5:1-15:1。
7.权利要求1所述荧光探针在活细胞成像中的应用。
8.一种对活细胞进行孵育染色的染色溶液,其特征在于:至少包括缓冲溶液和溶解在缓冲溶液中的权利要求1所述荧光探针。
9.根据权利要求8所述的染色溶液,其特征在于:所述缓冲溶液为磷酸盐缓冲溶液;或所述染色溶液的pH值为7.0-7.8。
10.权利要求1所述荧光探针在活细胞成像的方法,其特征在于:包括如下步骤:
1)将上述荧光探针溶解在水溶性有机溶剂中,然后加入缓冲溶液;或直接将所述荧光探针溶解在缓冲溶液中,得到染色溶液;
2)将所述染色溶液倒入培养好的细胞中进行孵育,孵育设定时间后,洗去染色溶液,然后进行激光共聚焦成像。
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