CN114479832B - 用于检测活细胞内次氯酸的比率型纳米荧光探针及其制备方法 - Google Patents
用于检测活细胞内次氯酸的比率型纳米荧光探针及其制备方法 Download PDFInfo
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Classifications
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
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- C—CHEMISTRY; METALLURGY
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
- C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
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- C—CHEMISTRY; METALLURGY
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
- C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
- C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
- C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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Abstract
本发明公开了用于检测活细胞内次氯酸的比率型纳米荧光探针及其制备方法,属于荧光探针技术领域。本发明的荧光探针由两亲性共聚物为外壳包裹有机染料分子构成,所述的有机染料分子包括香豆素衍生物和1,8‑萘酰亚胺衍生物,所述的香豆素衍生物为香豆素苯并噻唑衍生物或香豆素吡啶盐衍生物。该探针利用肟基作为识别位点,与ClO‑发生氧化反应,形成羰基,释放出前体荧光信号,制备得到的比率型荧光探针具有良好的灵敏性、特异的选择性及超快的响应速度,在较宽的pH范围内稳定且不受其它相关离子的干扰,是一种检测次氯酸的良好荧光分子探针,可应用于活细胞中外源性/内源性的ClO‑荧光成像检测。
Description
技术领域
本发明属于荧光探针技术领域,更具体地说,涉及用于检测活细胞内次氯酸的比率型纳米荧光探针及其制备方法。
背景技术
活性氧(Reactive oxygen species, ROS)在所有生命进行生理活动时具有重要的作用,不同的ROS承担不同的生理作用。次氯酸(Hypochlorous acid,HClO)是一种重要的ROS,在生理活动中担任信号分子,抗菌等作用。次氯酸的pKa值为7.5左右,在生理条件下与ClO‒达到平衡。在生命体内,ClO‒是吞噬细胞中氯化物和过氧化氢由髓过氧化酶催化得到的产物,具有膜渗透性。ClO‒具有很强的氧化能力,能氧化DNA、RNA、脂肪酸、蛋白质、胆固醇等生物分子。生理浓度的ClO‒可以杀死广泛的微生物,但是当人体内的ClO‒过高时,又可能会通过体内复杂的通道导致组织损伤、心脏病、神经退行性变、癌症、类风湿性关节炎和帕金森病。因此,需要对生命系统中的ClO‒进行检测。
虽然已知ClO‒具有多种生物学功能和作用,但是参与ClO‒分布的具体细胞器仍然未知,且由于ClO‒浓度低、氧化性强、寿命短,在亚细胞水平快速检测ClO‒仍然是一个主要的挑战。目前检测ClO‒的主要方法是荧光探针法。现有技术的探针已应用在体外和体内成像,但它们还存在一些问题,如选择性差,水溶性差,氧化反应时间长,自氧化和光漂白稳定性差,很大程度上限制了其应用。此外,其中大多数是基于合成染料的小分子荧光探针,由于缺乏内部参考,应用中常常受到诸如仪器参数波动、探针分子周围微环境变化和光漂白等问题的干扰。相比之下,比率型荧光探针可以引起分析物在两个或两个以上不同波长下的荧光强度发生变化,从而提供内置的自校准,排除各种与分析物无关的因素,并增加荧光的测量范围。像许多其他分析方法中使用的内标一样,比率型传感器特性减小了信噪比,因此可以更可靠地量化分析物。
然而,比率型荧光探针大多具有较差的选择性,阻碍了对单个活性氧物种分析的实际应用。此外,分析物在灵敏的共轭体系荧光团和纳米颗粒基质之间的光漂白速率有很大差异。
因此,需要开发一种响应迅速、灵敏度高且选择性高的比率型荧光探针。
发明内容
针对现有技术存在的上述问题,本发明的目的在于提供基于荧光能量共振转移的比率型纳米荧光探针,用于活细胞中ClO‒成像。本发明的另一目的在于提供该荧光探针的制备方法。
为了解决上述问题,本发明所采用的技术方案如下:
用于检测活细胞内次氯酸的比率型纳米荧光探针,由两亲性共聚物为外壳包裹有机染料分子构成,所述的有机染料分子为香豆素衍生物和1,8-萘酰亚胺衍生物,所述的香豆素衍生物为香豆素苯并噻唑衍生物或香豆素吡啶盐衍生物,
所述的香豆素苯并噻唑衍生物的结构式为
,其中R为-H或-OCH3;
所述的香豆素吡啶盐衍生物的结构式为
,其中R为-H或-OCH3。
优选的,所述的香豆素衍生物的结构式为
。
优选的,所述的1,8-萘酰亚胺衍生物的结构式为
。
优选的,所述的两亲性共聚物为F-127。
所述的香豆素苯并噻唑衍生物的制备方法为:
(1)向反应器中加入水杨醛或5-甲氧基水杨醛和苯并噻唑-2-乙腈,加入乙醇溶解,再加入哌啶进行反应,反应结束后冷却析出,得到产物A,其中水杨醛或5-甲氧基水杨醛、苯并噻唑-2-乙腈、哌啶三者的摩尔比为1:1:0.1;
(2)向反应器中加入产物A,加入N,N-二甲基甲酰胺溶解,将盐酸羟胺和稀硫酸溶于甲醇,然后加入反应体系中进行反应,反应结束后冷却析出,得到所述的香豆素苯并噻唑衍生物,其中产物A与盐酸羟胺的摩尔比为1:1.5。
所述的香豆素吡啶盐衍生物的制备方法为:
(1)向反应器中加入水杨醛或5-甲氧基水杨醛和2-乙腈吡啶,加入乙醇溶解,再加入哌啶进行反应,反应结束后冷却析出,得到产物B,其中水杨醛或5-甲氧基水杨醛、2-乙腈吡啶、哌啶三者的摩尔比为1:1:0.1;
(2)向反应器中加入产物B,加入N,N-二甲基甲酰胺溶解,将盐酸羟胺和稀硫酸溶于甲醇中,然后加入反应体系中进行反应,反应结束后冷却析出,得到产物C,其中产物B与盐酸羟胺的摩尔比为1:1.5;
(3)向反应器中加入产物C,加入二氯甲烷溶解,将碘甲烷加入反应体系中进行反应,反应结束后冷却析出,得到所述的香豆素吡啶盐衍生物,其中产物C与碘甲烷的摩尔比为1:2。
所述的1,8-萘酰亚胺衍生物的制备方法为:
(1)向反应器中加入4-溴-1,8-萘二甲酸酐,加入乙二醇单甲醚溶解体系,加入吗啉进行反应,反应结束后冷却析出,得到产物D,其中4-溴-1,8-萘二甲酸酐与吗啉的摩尔比为1:2;
(2)向反应器中加入产物D,加入乙二醇单甲醚溶解体系,加入乙醇胺进行反应,反应结束后冷却析出,得到所述的1,8-萘酰亚胺衍生物,其中产物D与乙醇胺的摩尔比为1:2。
一种用于检测活细胞内次氯酸的比率型纳米荧光探针的制备方法,将1,8-萘酰亚胺衍生物和香豆素衍生物溶于N,N-二甲基甲酰胺中,-10℃~10℃下逐滴加入到两亲性共聚物水溶液中,超声分散,常温搅拌4~12 h,-10℃~10℃下过水系滤膜,得到所述的比率型纳米荧光探针。
优选的,1,8-萘酰亚胺衍生物和香豆素衍生物的质量比为(1~20):1。
优选的,1,8-萘酰亚胺衍生物和香豆素衍生物的质量比为10:1。
相比于现有技术,本发明的有益效果为:以F-127为基体制备自组装纳米颗粒,选择香豆素苯并噻唑衍生物或香豆素吡啶盐衍生物与1,8-萘酰亚胺衍生物作为荧光团制备纳米探针。利用肟基作为识别位点,与ClO‒发生氧化反应,形成羰基,释放出前体荧光信号,制备得到的比率型荧光探针具有优异的荧光性质,在PBS缓冲溶液中能够比率响应ClO‒信号,并且具有良好的灵敏性、特异的选择性及超快的响应速度,在较宽的pH范围内稳定且不受其它相关离子的干扰,是一种检测次氯酸的良好荧光分子探针,可应用于活细胞中外源性/内源性的ClO‒荧光成像检测。
附图说明
图1为Cou-NP1(a),Cou-NP2(b),Cou-NP3(c),Cou-NP4(d)在PBS缓冲溶液(10mM,pH 7.4)中的紫外-可见吸收光谱图;
图2为在室温下,Cou-NP1(a),Cou-NP2(b),Cou-NP3(c),Cou-NP4(d)以及加入ClO‒后在PBS缓冲溶液(10mM, pH7.4)中的荧光发射光谱图;
图3为Cou-NP1(a),Cou-NP2(b),Cou-NP3(c),Cou-NP4(d)在PBS缓冲溶液(10mM,pH 7.4)中与10当量的ClO‒和其他分析物反应的荧光发射比率,数据为平均值±S.E.M.,n= 3;
图4为Cou-NP1(a),Cou-NP2(b),Cou-NP3(c),Cou-NP4(d)在PBS缓冲溶液(10mM,pH7.4)中随着ClO‒的加入荧光发射光谱的变化;
图5为纳米探针的荧光发射比率与ClO‒浓度的线性相关性,数据为平均值±S.E.M.,n=3;
图6为Cou-NP1(a),Cou-NP2(b),Cou-NP3(c),Cou-NP4(d)在PBS缓冲溶液(10mM,pH7.4)中与不同浓度ClO‒反应后荧光发射比率随时间的变化;
图7为Cou-NP1(a),Cou-NP2(b),Cou-NP3(c),Cou-NP4(d)在不同pH的PBS缓冲溶液(10mM)中与ClO‒反应后的荧光发射强度变化;
图8为Cou-NP3在不同条件下对RAW264.7细胞的激光共聚焦荧光成像;
其中,(a)细胞与Cou-NP3(10μM,30min)孵育;
(b)将细胞用ClO‒清除剂ABH(250μM,4h)预处理,再与Cou-NP3(10μM,30min)孵育;
(c)用ABH(250μM,4h)预处理细胞,再用LPS(5µg/mL)和PMA(5µg/mL)处理细胞12h,再与Cou-NP3(10μM,30min)孵育;
(d)用ABH(250μM,4h)预处理细胞,再用脂多糖(LPS)(5µg/mL)和佛波酯(PMA)(5µg/mL)处理细胞12 h,用ClO‒清除剂ABH(250μM,4h)预处理,再与Cou-NP3(10μM,30min)孵育;
(e)将细胞用ClO‒清除剂ABH(250μM,4h)预处理,再用NaClO(20μM,4h)处理细胞,再与Cou-NP3(10μM,30min)孵育;
图a-e中的平均荧光强度,比例尺20μm,数据为平均值±S.E.M.,n=3;
图9为经图8中的条件处理后对RAW264.7细胞的双通道荧光成像比值(Iblue/Igreen)。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。
实施例中所用原料:
Pluronic F-127:(聚(乙二醇)-block-聚(丙二醇)-block-聚(乙二醇)),纯度AR级,购自西格玛奥德里奇(Sigma-Aldrich);
薄层层析:GF254,CP;
柱层析硅胶粉:200-300目,AR:
其余原料均为市售AR级;
1,8-萘酰亚胺衍生物的制备方法为:取烧瓶,加入3.6mmol 4-溴-1,8-萘二甲酸酐,加入乙二醇单甲醚溶解体系,加入7.2mmol吗啉,将反应体系的温度升至80℃,反应回流7h后,反应结束,冷却到室温,有固体析出,抽滤洗涤得到产物D。
取烧瓶,加入1.8mmol产物D,加入乙二醇单甲醚溶解体系,加入3.6mmol乙醇胺,将反应体系的温度升至80℃,反应回流1h后,反应结束,停止反应,冷却到室温,有固体析出,抽滤洗涤,得到N-羟乙基-4-吗啉-1,8-萘二甲酰亚胺,记为化合物E。
实施例中所用主要仪器:
紫外分光光度计:岛津UV-1750;
荧光光谱仪:HORIBA Fluoromax-4;
高效液相色谱:Shimadzu LC-20A;
核磁共振波谱仪:Bruker AVANCE III HD;
高分辨质谱:Bruker Daltonics micro TQF-Q II;
质谱采用ESI源正离子模式扫描;干燥气(N2)流速为10 mL/min,雾化气(N2)压力为30 Psi;毛细管电压为3.5 kV;毛细管温度设为350 ℃;
激光共聚焦荧光显微镜:Zeiss LSM880;
酶标仪:Synergy 2。
实施例1
(1)香豆素衍生物的制备:取烧瓶,加入8.19mmol水杨醛和8.19mmol苯并噻唑-2-乙腈,加入无水乙醇溶解,加入0.82mmol哌啶。将体系加热至80℃,搅拌回流1 h,反应结束,冷却至室温,有固体析出,抽滤洗涤得到固体产物A,产率为81%。
取烧瓶,加入1.8mmol 产物A,加入N,N-二甲基甲酰胺(DMF)溶解。将2.7mmol盐酸羟胺和10% H2SO4(1~2 d)溶于甲醇中,加入反应体系中。将反应体系加热至90℃,搅拌回流3h,停止反应,冷却至室温,有固体析出,抽滤洗涤得到香豆素衍生物,产率74%。
(2)比率型纳米荧光探针的制备:按照1,8-萘酰亚胺衍生物与香豆素衍生物的质量比为10:1,将二者溶于DMF中,4℃下逐滴加入到10% F-127水溶液中,超声分散,常温搅拌6h,4℃下过水系滤膜,得到比率型纳米荧光探针,其多分散性指数(PDI)为0.274±0.030,粒径分布均匀。
实施例2
(1)香豆素衍生物的制备:取烧瓶,加入13mmol 5-甲氧基水杨醛和13mmol苯并噻唑-2-乙腈,加入无水乙醇溶解,加入1.3mmol哌啶。将体系加热至80℃,搅拌回流1 h,反应结束。停止反应,冷却至室温,有固体析出,抽滤洗涤,得到产物A,产率为75%。
取烧瓶,加入3.2mmol 产物A,加入DMF溶解。将4.8mmol盐酸羟胺和10% H2SO4(1mL)溶于甲醇中,加入反应体系中。将反应体系加热至90℃,搅拌回流3 h。停止反应,冷却至室温,有固体析出,抽滤洗涤,得到0.59 g香豆素衍生物,产率57%。
(2)比率型纳米荧光探针的制备:按照1,8-萘酰亚胺衍生物与香豆素衍生物的质量比为10:1,将二者溶于DMF中,4℃下逐滴加入到10% F-127水溶液中,超声分散,常温搅拌6h,4℃下过水系滤膜,得到比率型纳米荧光探针,PDI为0.239±0.017,粒径分布均匀。
实施例3
(1)香豆素衍生物的制备:取烧瓶,加入8.2mmol水杨醛和8.2mmol 2-乙腈吡啶,加入无水乙醇溶解,加入0.82mmol哌啶。将体系加热至80℃,搅拌回流1h,反应结束。停止反应,冷却至室温,有固体析出,抽滤洗涤得到1.3 g产物B,产率为73%。
取烧瓶,加入11.2mmol 产物B,加入DMF溶解。将16.9mmol盐酸羟胺和10% H2SO4(100μL)溶于甲醇中,加入反应体系中。将反应体系加热至90℃,搅拌回流3 h。停止反应,冷却至室温,有固体析出,抽滤洗涤,得到1.9 g产物C,产率为72%。
取烧瓶,加入0.4mmol 产物C,加入二氯甲烷溶解。将0.8mmol碘甲烷加入反应体系中。将反应体系室温下搅拌24h。反应结束后,有固体析出,抽滤洗涤,得到120mg目标化合物即为香豆素衍生物,收率为81%。
(2)比率型纳米荧光探针的制备:按照1,8-萘酰亚胺衍生物与香豆素衍生物的质量比为10:1,将二者溶于DMF中,4℃下逐滴加入到10% F-127水溶液中,超声分散,常温搅拌6h,4℃下过水系滤膜,得到比率型纳米荧光探针,PDI为0.241±0.038,粒径分布均匀。
实施例4
(1)香豆素衍生物的制备:取烧瓶,加入3.3mmol 5-甲氧基水杨醛和3.3mmol 2-乙腈吡啶,加入无水乙醇溶解,加入0.3mmol哌啶。将体系加热至80℃,搅拌回流1h,反应结束。停止反应,冷却至室温,有固体析出,抽滤洗涤,得到0.7g产物B,产率为80%。
取烧瓶,加入2mmol产物B,加入DMF溶解。将3mmol盐酸羟胺和10% H2SO4(100μL)溶于甲醇中,加入反应体系中。将反应体系加热至90℃,搅拌回流3h。停止反应,冷却至室温,有固体析出,抽滤洗涤,得到0.33g产物C,产率为61%。
取烧瓶,加入0.75mmol产物C,加入二氯甲烷溶解。将1.5mmol碘甲烷加入反应体系中。将反应体系室温下搅拌24h。反应结束后,有固体析出,抽滤洗涤,得到200mg香豆素衍生物,收率为66%。
(2)比率型纳米荧光探针的制备:按照1,8-萘酰亚胺衍生物与香豆素衍生物的质量比为10:1,将二者溶于DMF中,4℃下逐滴加入到10% F-127水溶液中,超声分散,常温搅拌6h,4℃下过水系滤膜,得到比率型纳米荧光探针,PDI为0.256±0.009,粒径分布均匀。
实施例5
本实施例的制备方法与实施例1基本相同,不同之处仅在于,1,8-萘酰亚胺衍生物与香豆素衍生物的质量比为1:1,所得比率型纳米荧光探针的PDI为0.517±0.107,粒径分布较均匀。
实施例6
本实施例的制备方法与实施例2基本相同,不同之处仅在于,1,8-萘酰亚胺衍生物与香豆素衍生物的质量比为20:1,所得比率型纳米荧光探针的PDI为0.492±0.067,粒径分布较均匀。
实施例7
本实施例的制备方法与实施例3基本相同,不同之处仅在于,1,8-萘酰亚胺衍生物与香豆素衍生物的质量比为20:1,所得比率型纳米荧光探针的PDI为0.435±0.076,粒径分布较均匀。
实施例8
本实施例的制备方法与实施例4基本相同,不同之处仅在于,1,8-萘酰亚胺衍生物与香豆素衍生物的质量比为5:1,所得比率型纳米荧光探针的PDI为0.572±0.091,粒径分布较均匀。
实施例9
(1)香豆素衍生物的制备:取烧瓶,加入原料水杨醛和2-乙腈吡啶,加入无水乙醇溶解,加入哌啶。将体系加热至80℃,搅拌回流1h,反应结束。停止反应,冷却至室温,有固体析出,抽滤洗涤得到产物B。其中水杨醛、2-乙腈吡啶、哌啶三者的摩尔比为1:1:0.1。
取烧瓶,加入产物B,加入DMF溶解。将盐酸羟胺和10% H2SO4溶于甲醇中,加入反应体系。将反应体系加热至90℃,搅拌回流3h。停止反应,冷却至室温,有固体析出,抽滤洗涤,得到产物C。其中产物B与盐酸羟胺的摩尔比为1:1.5。
取烧瓶,加入产物C,加入二氯甲烷溶解。将碘甲烷加入反应体系中。将反应体系室温下搅拌24h。反应结束后,有固体析出,抽滤洗涤得到香豆素衍生物。其中产物C与碘甲烷的摩尔比为1:2。
(2)比率型纳米荧光探针的制备:按照1,8-萘酰亚胺衍生物与香豆素衍生物的质量比为10:1,将二者溶于DMF中,0℃下逐滴加入到10% F-127水溶液中,超声分散10min,常温搅拌8h,0℃下过水系滤膜,得到比率型纳米荧光探针。
将实施例1~4所得比率型纳米荧光探针分别记为Cou-NP1、Cou-NP2、Cou-NP3、Cou-NP4,进行相关测试,测试方法如下:
光物理学性质测试:
所有的光物理表征均在室温下表征。用Phosphate buffer缓冲液(PBS缓冲溶液)稀释原液(pH=7.4)。所有水溶液由去离子水制备。选择性响应测试中所用活性氧物种参考以下文献进行配制[Sun, Q. etc., Ultrafast Detection of Peroxynitrite inParkinson's Disease Models Using a Near-Infrared Fluorescent Probe [J]. Anal.Chem.,2020, 92(5): 4038-4045.]。
PBS缓冲溶液的配制:分别称取4.0g氯化钠、0.1g氯化钾、0.12g磷酸二氢钾、1.79g十二水合磷酸氢二钠,溶于450mL去离子水中,用盐酸调节pH至7.4,定容至500mL,得到浓度为10mM的PBS缓冲溶液。
测试溶液的配制:储备的纳米探针直接用PBS缓冲溶液稀释至10μM。
ROS及其他分析物溶液的配制:
a)过氧烷氧自由基(ROO•):2,2’-二异丁基脒二盐酸盐溶于PBS溶液中备用。
b)次氯酸根(ClO‒):将次氯酸钠溶于PBS中,用紫外分光光度计标定浓度,ClO‒在292nm处的摩尔消光系数为350 M-1cm-1。
c)过氧化氢(H2O2):将30%过氧化氢水溶液用PBS稀释制备。H2O2在240nm处的摩尔消光系数为43.6 M-1cm-1。
d)单线态氧(1O2):将等量的钼酸钠和过氧化氢溶液混合制备。
e)羟基自由基(HO•):根据Fenton反应,将10eq的过氧化氢溶液与硫酸亚铁溶液混合制备。
f)一氧化氮(NO•):先将去离子水用氩气鼓气泡30min,在氩气氛围下加入硝普钠,溶解之后,继续鼓30min。
g)过氧亚硝基阴离子(ONOO‒):在0℃下,将亚硝酸钠(0.6M)和H2O2(0.7M)的去离子水溶液混合,剧烈搅拌,加入盐酸(0.6M),随后迅速加入氢氧化钠溶液(1.5M),加入适量二氧化锰。继续在0℃下搅拌10~15min。用0.2μm的水系滤膜过滤除掉二氧化锰。ONOO‒在0.1M的氢氧化钠溶液中在302 nm处的摩尔消光系数为1670 M-1cm-1。
h)其他分析物:准确称取相应质量后,溶于PBS溶液(10mM,pH=7.4)中。
荧光量子产率:
以香豆素6为标准品(在乙醇中,φ standard= 0.8),测定量子产率。对探针和香豆素6在0.01至0.05的吸光度范围内进行吸收光谱的测量。根据以下公式计算量子产率:
其中,φ是量子产率,∑F是积分荧光强度,Abs是在λex=400nm处的吸光度,n表示溶剂的折射率。
生物细胞成像测试:
巨噬细胞(RAW264.7)在含有10%胎牛血清、100 units/mL青霉素以及100 μg/mL链霉素的Dulbecco改良Eagle培养基(DMEM)中培养。细胞在5%的二氧化碳、饱和湿度、37℃培养箱中培养。
细胞实验分为四组。第一组是将细胞与10μM探针一起孵育30min,PBS缓冲液洗涤三次,进行细胞成像。第二组细胞用ABH(250μM)预处理4h后洗去,再与10μM探针一起孵育30min,PBS缓冲液洗涤,进行细胞成像。第三组细胞用ABH(250μM)预处理4h后洗去,再用NaClO(20μM)孵育4h,与10μM探针一起孵育30min,PBS缓冲液洗涤,进行细胞成像。第四组细胞用ABH(250μM)预处理4h后洗去,再与LPS(5µg/mL)和PMA(5µg/mL)孵育12h,然后与10μM探针一起孵育30min,PBS缓冲液洗涤三次,进行细胞成像。激发波长405nm,波长收集范围为450~600nm。
从四个纳米探针Cou-NP1~4的紫外-可见吸收光谱图(图1)可以看出,四个纳米探针均表现出单峰,这是因为F-127包覆的两个化合物的紫外最大吸收波长位置相接近,且峰宽,未能分开。如图1所示,Cou-NP1~4的最大吸收波长分别在397nm、400nm、390nm、390nm,因此,Cou-NP1~4的荧光发射光谱以400nm为激发波长测试。
纳米探针Cou-NP1~4的荧光发射光谱如图2所示,Cou-NP1加入ClO‒后,在438nm处的荧光发射光谱明显增强(图2a),由于FRET效应,化合物E在565nm处的荧光强度也增加。Cou-NP2加入ClO‒后,在465nm处的荧光发射光谱明显增强(图2b),由于FRET效应,化合物E在565nm处的荧光强度也增加。Cou-NP3加入ClO‒后,在420nm处的荧光发射光谱明显增强(图2c),由于FRET效应,化合物E在565nm处的荧光强度也增加。Cou-NP4加入ClO‒后,在466nm处的荧光发射光谱明显增强(图2d),由于FRET效应,化合物E在565nm处的荧光强度也增加。结果表明,本申请的比率型荧光探针加入ClO‒后I420~466nm/I565nm比值增大,可用来检测ClO‒。
响应机理为:在比率型荧光探针内,香豆素衍生物在420~460nm处的荧光强度较弱,而化合物E在565nm处的荧光强度较强,荧光强度比值I420~460nm/I565nm很低。与ClO‒反应后,肟基被氧化水解,香豆素衍生物转化为具有良好发光性能的香豆素,荧光明显增强。化合物E在565nm处的荧光强度也稍有增强,从而增加了I420~460nm/I565nm,验证了该荧光纳米探针具有FRET效应,表现出比率型荧光探针的双发射特点,这对于检测ClO‒水平是可行的。值得注意的是,在565nm处始终保持强的荧光可以无创监测纳米探针在体内的传递,在450nm左右的激活荧光可以实时检测活细胞内的ClO‒。
为了验证纳米探针对ClO‒检测的特异性,本申请考察了探针对各种潜在的干扰物质(1: Blank, 2: Na+, 3: K+, 4: Mg2+, 5: Ca2+, 6: Cu2+, 7: Fe3+, 8: Fe2+, 9: Zn2+,10: CO3 2-, 11: SO4 2-, 12: SO3 2-, 13: HSO3 ‒, 14: S2-, 15: HS‒, 16: Hcy, 17: Cys,18: GSH, 19: ROO•, 20: H2O2, 21:1O2, 22: HO•, 23: NO, 24: ONOO‒, 25: ClO‒)存在下的荧光响应。如图3a所示,可以看出所有干扰物质对Cou-NP1没有明显的应响应,而在加入ClO‒后I438nm/I562nm荧光比值明显增强,并且加入ONOO‒后I438nm/I562nm荧光比值也有增强,对特异性检测ClO‒有干扰影响。如图3b所示,所有干扰物质对Cou-NP2没有明显的响应,而在加入ClO‒后引起I465nm/I565nm荧光比值明显增强,并且加入ONOO‒后的I465nm/I565nm荧光比值接近加入ClO‒后的I465nm/I565nm荧光比值响应,对特异性检测ClO‒干扰影响增大。因此可以看出,7位给电子基团的引入使得纳米探针对ONOO‒的响应增强。图3c和图3d得到同样的结论,7位给电子的存在使得纳米探针检测ClO‒的特异性干扰影响增大,但是吡啶盐比苯并噻唑的吸电子能力大,降低了纳米探针检测ClO‒特异性的干扰影响,Cou-NP3检测ClO‒的特异性优于其他三个纳米探针。结果表明,Cou-NP3可作为一种高选择性的荧光探针特异性检测ClO‒。
经过荧光发射光谱的测试,发现四个纳米探针均表现出两个发射峰。如图4,在420-466nm处的波长为香豆素衍生物探针,565nm处为化合物E的发射峰。加入ClO‒后,420-466nm处的荧光强度明显逐渐增强,565nm处的荧光强度随之缓慢逐渐增强。这种荧光强度变化,基于香豆素衍生物探针与ClO‒反应转化成具有良好发光性能的香豆素,420-466nm处的荧光强度增大,而香豆素和化合物E之间的FRET效应引起565nm处荧光强度的增加。
I420~460nm/I565nm的荧光强度比值如图5,在纳米探针中,香豆素衍生物探针在420-466nm处的荧光强度较弱,而化合物E在565nm处的荧光强度较强,荧光强度比值I420~460nm/I565nm很低。与ClO‒反应后,纳米探针内的香豆素衍生物探针的荧光释放,荧光强度明显增大。在565nm处的荧光强度缓慢增加,从而I420~460nm/I565nm荧光强度比值随着ClO‒的逐渐加入逐渐增加。如图5所示,Cou-NP1~4在ClO‒的浓度为0-20μM范围内荧光强度比值呈现线性相关,根据L=σ/k,计算出检测限分别为6.91 nM(Cou-NP1)、6.34 nM(Cou-NP2)、7.28 nM(Cou-NP3)、10.40 nM(Cou-NP1)。结果表明,该种纳米荧光探针对于检测ClO‒具有高的灵敏度。
Cou-NP1~4在PBS缓冲液中对ClO‒响应的反应动力学如图6所示,Cou-NP1~4在不同浓度ClO‒存在时,小分子探针的荧光发射强度逐渐快速增大,化合物E在565nm处的荧光强度逐渐缓慢增大。两个分子的荧光强度比值I420~460nm/I565nm在5s内快速增大,并且快速达到稳定,在100s内保持不变,表明Cou-NP1~4在PBS缓冲溶液中加入ClO‒后,可以在PBS缓冲溶液中保持稳定。进一步表明,Cou-NP1~4可以作为一种快速检测ClO‒的荧光探针。
在不同pH的PBS缓冲溶液中,Cou-NP1~4对ClO‒响应的荧光光谱如图7所示。在不同pH值中,随着ClO‒的加入,发射光谱发生了显著变化。如图7a中,香豆素苯并噻唑衍生物的荧光发射强度随着pH值的增大逐渐减小,化合物E的荧光发射强度随着pH的增大先增大后减小,考虑到比率型荧光探针双发射的特点,Cou-NP1在pH为7~9时表现出比率型荧光探针的特点。图7b中,香豆素苯并噻唑衍生物的荧光发射强度随着pH的增大逐渐减小,化合物E的荧光发射强度随着pH的增大而逐渐增大。Cou-NP2在pH为7时,比率型荧光探针双发射的特点较好,可以在检测ClO‒时出现双发射峰。图7c中,香豆素吡啶盐衍生物的荧光发射强度在过酸过碱时较小,中性时荧光强度最大,化合物E的荧光发射在中性增大。Cou-NP3在pH为6~9时,比率型荧光探针双发射的特点较好,可以在检测ClO‒时出现双发射峰。图7d中,香豆素吡啶盐衍生物的荧光发射强度随着pH的增大基本不变,化合物E的荧光发射强度在中性最大,比率型荧光探针双发射的特点较好。结果表明,Cou-NP1~4荧光探针在较宽的pH范围(4-11)内具有较高的稳定性,表明它可以在pH范围宽泛的活细胞中作用,可以在生理环境下对ClO‒进行比率检测。
利用激光共聚焦扫描显微镜研究Cou-NP3对活细胞中ClO‒比率荧光成像的能力,如图8所示。首先,将细胞与Cou-NP3(10μM)在25℃下孵育30min,然后通过激光共聚焦扫描显微镜双通道成像(λem= 400-480nm和λem= 500-680nm),如图8a所示,在蓝色通道中(λem=400-480nm)仅观察到微弱的荧光,同时,绿色通道显示强烈的荧光(λem=500-680nm)。双通道的荧光强度比值(Iblue/Igreen)为0.26(图9)。当ClO‒清除剂ABH(250µM)清除细胞内ClO‒浓度时(图8b),化合物E的荧光不变,但激活探针香豆素吡啶盐衍生物的荧光减小,因此,Iblue/Igreen仅降至0.13。当加入NaClO上调细胞内ClO‒的浓度时(图8c),Iblue/Igreen显著升高至1.11,这是因为香豆素吡啶盐衍生物的荧光明显增强,结果表明,Cou-NP3能够通过比率荧光成像监测外源性ClO‒的水平。
由于LPS/PMA是诱导细胞内ClO‒产生的生物试剂,因此利用Cou-NP3监测RAW264.7细胞内ClO‒的生成。经LPS(5µg/mL)和PMA(5µg/mL)刺激后,细胞内Cou-NP1的荧光变亮(图8d),Iblue/Igreen明显高于未刺激细胞5倍,这些成像结果表明,Cou-NP3能够通过比率荧光成像监测RAW264.7活细胞中的ClO‒水平。
Claims (8)
1. 用于检测活细胞内次氯酸的比率型纳米荧光探针,其特征在于,由两亲性共聚物F-127为外壳包裹有机染料分子构成,所述的有机染料分子为香豆素衍生物和1,8-萘酰亚胺衍生物,所述的香豆素衍生物为香豆素苯并噻唑衍生物或香豆素吡啶盐衍生物,
所述的1,8-萘酰亚胺衍生物的结构式为
;
所述的香豆素苯并噻唑衍生物的结构式为
,其中R为-H或-OCH3;
所述的香豆素吡啶盐衍生物的结构式为
,其中R为-H或-OCH3。
2.根据权利要求1所述的用于检测活细胞内次氯酸的比率型纳米荧光探针,其特征在于,所述的香豆素衍生物的结构式为
。
3.根据权利要求1所述的用于检测活细胞内次氯酸的比率型纳米荧光探针,其特征在于,所述的香豆素苯并噻唑衍生物的制备方法为:
(1)向反应器中加入水杨醛或5-甲氧基水杨醛和苯并噻唑-2-乙腈,加入乙醇溶解,再加入哌啶进行反应,反应结束后冷却析出,得到产物A,其中水杨醛或5-甲氧基水杨醛、苯并噻唑-2-乙腈、哌啶三者的摩尔比为1:1:0.1;
(2)向反应器中加入产物A,加入N,N-二甲基甲酰胺溶解,将盐酸羟胺和稀硫酸溶于甲醇,然后加入反应体系中进行反应,反应结束后冷却析出,得到所述的香豆素苯并噻唑衍生物,其中产物A与盐酸羟胺的摩尔比为1:1.5。
4.根据权利要求1所述的用于检测活细胞内次氯酸的比率型纳米荧光探针,其特征在于,所述的香豆素吡啶盐衍生物的制备方法为:
(1)向反应器中加入水杨醛或5-甲氧基水杨醛和2-乙腈吡啶,加入乙醇溶解,再加入哌啶进行反应,反应结束后冷却析出,得到产物B,其中水杨醛或5-甲氧基水杨醛、2-乙腈吡啶、哌啶三者的摩尔比为1:1:0.1;
(2)向反应器中加入产物B,加入N,N-二甲基甲酰胺溶解,将盐酸羟胺和稀硫酸溶于甲醇中,然后加入反应体系中进行反应,反应结束后冷却析出,得到产物C,其中产物B与盐酸羟胺的摩尔比为1:1.5;
(3)向反应器中加入产物C,加入二氯甲烷溶解,将碘甲烷加入反应体系中进行反应,反应结束后冷却析出,得到所述的香豆素吡啶盐衍生物,其中产物C与碘甲烷的摩尔比为1:2。
5.根据权利要求1所述的用于检测活细胞内次氯酸的比率型纳米荧光探针,其特征在于,所述的1,8-萘酰亚胺衍生物的制备方法为:
(1)向反应器中加入4-溴-1,8-萘二甲酸酐,加入乙二醇单甲醚溶解体系,加入吗啉进行反应,反应结束后冷却析出,得到产物D,其中4-溴-1,8-萘二甲酸酐与吗啉的摩尔比为1:2;
(2)向反应器中加入产物D,加入乙二醇单甲醚溶解体系,加入乙醇胺进行反应,反应结束后冷却析出,得到所述的1,8-萘酰亚胺衍生物,其中产物D与乙醇胺的摩尔比为1:2。
6. 一种根据权利要求1~5中任一项所述的用于检测活细胞内次氯酸的比率型纳米荧光探针的制备方法,其特征在于,将1,8-萘酰亚胺衍生物和香豆素衍生物溶于N,N-二甲基甲酰胺中,-10℃~10℃下逐滴加入到两亲性共聚物水溶液中,超声分散,常温搅拌4~12 h,-10℃~10℃下过水系滤膜,得到所述的比率型纳米荧光探针。
7.根据权利要求6所述的比率型纳米荧光探针的制备方法,其特征在于,1,8-萘酰亚胺衍生物和香豆素衍生物的质量比为(1~20):1。
8.根据权利要求6所述的比率型纳米荧光探针的制备方法,其特征在于,1,8-萘酰亚胺衍生物和香豆素衍生物的质量比为10:1。
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