CN106928263A - 一种用于快速检测过氧化氢的荧光探针的制备与应用 - Google Patents
一种用于快速检测过氧化氢的荧光探针的制备与应用 Download PDFInfo
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Abstract
本发明涉及一种过氧化氢开型荧光探针及其制备与应用,所述过氧化氢荧光探针化合物具有式I的结构。制备方法是:将邻羟基芘醛与4‑硼酯溴苄溶于丙酮并以无水碳酸钾为催化剂,回流制得产物Ⅱ。将9,10‑菲醌、乙酸铵、乙醇和化合物II回流得到最终的探针Ⅰ,该探针化合物对过氧化氢具有很好的选择性和灵敏性,检测限低,且对细胞没有毒性,不仅可应用于细胞内过氧化氢的检测和成像,而且还可应用于生物成像。
Description
技术背景
过氧化氢是人体内活性氧物种其中的一种,通过氧气的代谢及酶的催化而产生。适量水平的过氧化氢对免疫细胞的活化、生长和增殖具有促进作用,然而过量的过氧化氢可能会造成许多严重的疾病,例如,阿兹海默症、帕金森症、心脑血管疾病以及癌症等等。因此,识别以及实时监测细胞内过氧化氢的含量具有十分重要的意义。
目前,作为生物体内过氧化氢的检测方法主要有电化学方法、元素分析质谱等方法,这些方法的样品准备过程复杂,检测过程对样品的损伤较大,而且不能进行实时监测。与之对比,作为一种价格低廉的检测手段,荧光探针检测法由于其不仅具有灵敏度高、选择性好、响应速度快、能够进行实时可视化监测等优点,而且可在生物医学成像方面进行进一步的应用,近几年来备受研究人员的关注。
在现有的荧光探针技术中,过氧化氢探针种类较少,选择性低,检测不够灵敏等问题。比如DCFH是检测体内ROS的探针,但它容易被空气中的氧气氧化,容易对过氧化氢的检测产生干扰,降低灵敏度。另外,由于过氧化氢存在时间短,易与其它物质反应生成新的活性氧物种,因此对过氧化氢的检测本身就存在一定的难度,设计一种响应时间短,灵敏度高,选择性强的探针具有十分重要的意义。
发明内容
针对现有技术的不足,本发明提供一种反应型过氧化氢荧光探针化合物,既可以检测水中的过氧化氢,还可以在生物体内识别和检测过氧化氢并可应用于生物成像。
本发明还提供所述反应型过氧化氢荧光探针化合物的制备方法与应用。
本发明的技术方案如下:
一种反应型过氧化氢荧光探针化合物,具有式I所示的结构:
。
本发明所述的过氧化氢荧光探针化合物的制备方法,包括如下步骤:
1)将邻羟基芘醛,4-硼酯溴苄溶于丙酮,以无水碳酸钾为催化剂,在50℃下搅拌过夜反应,得到化合物Ⅱ;
(3)取9,10-菲醌和乙酸铵及乙醇,加热溶解后加入化合物II,回流得到最终的探针Ⅰ。
根据本发明,优选的,步骤(1)所述邻羟基芘醛、4-硼酯溴苄的摩尔比为1:1.1;
根据本发明,优选的,步骤(2)所述9,10-菲醌、化合物Ⅱ的摩尔比为1:1;
根据本发明,优选的,步骤(1)、(2)全程在氮气保护下进行;
根据本发明,优选的,步骤(1)反应温度为50℃;
根据本发明,优选的,步骤(2)反应温度为75℃;
更为详细的,本发明所述的反应型过氧化氢荧光探针化合物的制备方法,步骤如下:
(a)取邻羟基芘醛(109mg)和4-硼酯溴苄(130mg)溶于10ml丙酮,加入K2CO3(62mg),50℃下回流过夜,之后冷却到室温,除去溶剂,粗产品经柱层析提纯,得白色产物Ⅱ144mg,产率70%。
(b) 取9,10-菲醌(40mg),乙酸铵(0.16g)及8ml乙醇,加热至固体溶解,稍冷后,加入化合物Ⅱ(93mg),回流2小时。冷却后,抽滤得探针Ⅰ98mg,产率71%。
本发明所述的反应型过氧化氢荧光探针化合物,可用于测试细胞、组织和水中的过氧化氢含量。
进一步优选的,所述荧光探针用于在pH=7.4的DMSO和水的混合溶剂中过氧化氢的快速检测。
本发明通过实验验证,所述荧光探针在pH=7.4的DMSO和水的混合溶剂中,用波长427nm的光作为激发波长,可以发现其荧光很弱,加入过氧化氢后溶液在495nm处荧光迅速增强,而其他活性氧物种加入后溶液的荧光没有明显变化(如图1),因此该探针对过氧化氢具有很高的选择性。
将本发明的荧光探针化合物加入含有DMSO和水的混合溶剂中,配成荧光探针化合物浓度为10微摩尔每升的溶液,用PBS缓冲溶液调节pH=7.4,加入不同浓度的过氧化氢,用波长427nm的光激发,在波长495nm处的荧光依次增强,得出工作曲线如图2,据此通过测定待测样品的荧光强度,可定量计算出过氧化氢的浓度。
本发明所述的反应型过氧化氢荧光探针化合物不仅可以用于水中过氧化氢的检测,还可应用于组织细胞内过氧化氢的检测和成像。
将MCF-7细胞在含有10%牛胎儿血清的1毫升细胞培养基培养12小时,然后用100微摩尔每升的过氧化氢溶液处理10分钟,再用10微摩尔每升的本发明的荧光探针处理30分钟。细胞用激发波长为427nm的光源激发,在共聚焦显微镜下成像。
本发明的优良效果如下:
1、本发明的荧光探针化合物具有较好的水溶性、生物相容性以及较好的膜穿透性,可用于细胞内过氧化氢的探测及成像。
2、本发明的荧光探针化合物可用于水体中的定量过氧化氢检测且检测限较低;
3、本发明的荧光探针化合物具有良好的灵敏度和选择性,测试样品前处理简单;
4、本发明的荧光探针化合物为固体粉末,便于储存使用,并且合成方法简单、收率高、成本低、具有良好的推广前景;
5、与现有的技术相比,本发明的荧光探针在细胞成像研究中对组织细胞几乎无损伤。
附图说明
图1为本发明实施例1荧光离子探针在DMSO和水混合溶剂中(pH=7.4),与不同活性氧物种作用后的荧光强度。
图2为本发明的荧光探针与不同浓度的过氧化氢的荧光强度工作曲线图。
图3本发明的荧光探针的核磁图。
图4MCF-7细胞使用本发明的荧光探针和过氧化氢孵育后的共聚焦成像图。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明,但不限于此。实施例中的各种原料均来自于市场购买。
实施例1、探针化合物的合成
(a)化合物II的合成
取邻羟基芘醛(109mg)和4-硼酯溴苄(130mg)溶于10ml丙酮,然后加入K2CO3(62mg),50℃下回流过夜,旋干制样,柱层析提纯,得白色产物Ⅱ144mg,产率70%。
探针I的合成
取9,10-菲醌(40mg)和乙酸铵(160mg),混合溶于8ml乙醇中,加热回流至产物溶解,稍冷后加入化合物Ⅱ(93mg),回流2小时。冷却后,抽滤得固体产物Ⅰ98mg,产率71%,核磁如图3。
实施例2、荧光实验
取实施例1制备的荧光探针化合物,溶解到DMF和水的混合溶剂中,用PBS缓冲溶液调节pH=7.4;得荧光探针溶液,备用。
1、取荧光探针溶液,分10组,每组10毫升,其中1组不加活性氧物种,其它9组分别加入含有CH3COOOH, GSH, HOCl, O2•-, •OH , otBU, TBHF, Vc, H2O2溶液,使得每组溶液中含有探针化合物的浓度为10μΜ,活性氧物种浓度为300μM,使得活性氧物种与探针化合物的摩尔比为30:1;采用激发波长为427nm,荧光光度计测试其荧光强度,如图1所示,结果显示:本发明探针溶液本身没有荧光,一旦加入过氧化氢,溶液在495nm荧光迅速增强,而其他离子加入后溶液的荧光没有变化,因此探针对过氧化氢具有很高的选择性。
2、取荧光探针溶液,分16组,每组10毫升,分别加入不同浓度的过氧化氢溶液,调节到溶液中含有探针化合物的浓度为10μM,过氧化氢的浓度分别为探针化合物浓度的0、2、4、6、8、10、12、14、16、18、20、22、24、26、28、30倍,如图2所示:溶液在495nm处的荧光迅速增强,其荧光强度与浓度成线性关系。
实施例3、细胞成像实验
将MCF-7细胞在含有10%牛胎儿血清的1毫升细胞培养基培养12小时,之后在10微摩尔每升的本发明的荧光探针的溶液中孵育20分钟,然后加入100微摩尔每升的过氧化氢溶液处理10分钟。细胞用激发波长为405nm光源激发,在共聚焦显微镜下成像,如图4。
Claims (6)
1.一种开型过氧化氢荧光探针,具有式I所示的结构:
。
2.一种权利要求1所述的反应型过氧化氢荧光探针化合物的制备方法,包括如下步骤;
(1)将邻羟基芘醛和4-硼酯溴苄溶于丙酮中,加入无水碳酸钾为催化剂,在50℃下搅拌过夜反应,得到化合物Ⅱ;
(2)将9,10-菲醌、乙酸铵及乙醇混合,加热溶解,然后加入上步制得的化合物Ⅱ,回流2小时,冷却抽滤得最终的探针分子Ⅰ:
。
3.如权利要求2所述的开型过氧化氢荧光探针化合物的制备方法,其特征在于步骤(1)所述的邻羟基芘醛与4-硼酯溴苄的摩尔比为1:1.1;步骤(3)所述的Ⅱ、9,10-菲醌的摩尔比为1:1。
4.如权利要求2所述的开型过氧化氢荧光探针化合物的制备方法,其特征在于步骤(1)、(2)全程在氮气保护下进行。
5.如权利要求2所述的开型过氧化氢荧光探针化合物的制备方法,其特征在于步骤(1)反应温度为50℃,步骤(2)反应温度为75℃。
6.权利要求1所述的反应型过氧化氢荧光探针化合物的应用,可以用于检测水体中的过氧化氢含量以及细胞内成像研究。
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