CN110563708B - 一种快速检测亚硫酸(氢)盐的turn-on型荧光探针及合成方法和应用 - Google Patents
一种快速检测亚硫酸(氢)盐的turn-on型荧光探针及合成方法和应用 Download PDFInfo
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Abstract
Description
技术领域
本发明属于化学分析检测技术领域,尤其是一种快速检测亚硫酸(氢)盐的turn-on型荧光探针及合成方法和应用。
背景技术
二氧化硫(SO2)长期以来一直被认为是一种对人体有害的环境污染物,是造成全球范围内“酸雨”的主要原因。其主要来源为火山喷发,硫磺类化石燃料的燃烧等。SO2吸入人体后主要以SO2衍生物——亚硫酸(氢)盐的形式存在。亚硫酸(氢)盐也可作为食品添加剂,但是过量摄入将导致不良反应如低血压、腹泻、荨麻疹等。毒理学和流行病学研究证明,内源性的亚硫酸(氢)盐浓度异常与多种疾病密切相关,如呼吸系统疾病、神经系统疾病、心血管系统疾病和癌症等。因此,有必要开发一种实时检测亚硫酸(氢)盐浓度的方法。
目前为止,有很多关于亚硫酸(氢)盐的检测方法,如色谱法,分光光度法,电化学法,毛细管电泳法,荧光分析法等。在这些方法中,荧光分析法因其具有预处理简单,灵敏度高,检测限低,选择性好等优点,在最近几年得到快速发展。香豆素类化合物具有良好的荧光量子产率和光稳定性,溶解性与细胞渗透性好、易于合成与修饰等特点,常用于构建有机小分子的荧光探针。
通过检索,尚未发现与本发明专利申请相关的专利公开文献。
发明内容
本发明目的在于克服现有技术中的不足之处,提供一种快速检测亚硫酸(氢)盐的turn-on 型荧光探针及合成方法和应用,该该探针能够对细胞、食品以及中药材中的亚硫酸(氢)盐进行快速、灵敏及专一性的检测。
本发明解决其技术问题所采用的技术方案是:
一种快速检测亚硫酸(氢)盐的turn-on型荧光探针,所述荧光探针的分子式为:C21+nH15+mN1+xO3+y,其中n,m,x,y均为1-20的整数,且分子具有以下结构:
其中取代基R1为H、烷基、芳基、硝基、酯基、醚基和二乙胺基中的一种,3-吲哚甲醛衍生物中的n为1-20的整数。
一种如上所述的快速检测亚硫酸(氢)盐的turn-on型荧光探针的合成方法,步骤如下:
将带有取代基R1的香豆素溶解在有机溶剂中,在有催化剂条件下加入3-吲哚甲醛衍生物,在反应温度90℃下反应2-8小时,然后过滤,利用硅胶柱层析得到目标化合物turn-on型荧光探针。
而且,所述有机溶剂为甲醇、乙醇、二甲基亚砜、乙腈、二氧六环、二氯甲烷、苯和甲苯中的一种或两种以上任意比例的混合物。
而且,所述催化剂为吡啶、三乙胺、碳酸钾或氢氧化钠。
如上所述的快速检测亚硫酸(氢)盐的turn-on型荧光探针在检测亚硫酸(氢)盐方面中的应用。
如上所述的快速检测亚硫酸(氢)盐的turn-on型荧光探针在食品或中药方面中的应用。
如上所述的快速检测亚硫酸(氢)盐的turn-on型荧光探针在细胞成像方面中的应用。
利用如上所述的快速检测亚硫酸(氢)盐的turn-on型荧光探针检测亚硫酸(氢)盐的方法,步骤如下:
将探针分子溶解在生理盐水或磷酸盐的缓冲溶液中,或者将探针分子溶解在甲醇、乙醇、乙腈或二甲基亚砜有机溶剂中,或者将探针分子溶解在水和甲醇、乙醇、乙腈或二甲基亚砜有机溶剂中的一种或两种以上任意比例的混合溶剂中,最终配置成浓度为1×10-5摩尔/升的探针溶液;然后在溶解有探针分子的上述探针溶液中加入浓度为含亚硫酸(氢)盐的样品溶液,即可检测亚硫酸(氢)盐。
本发明取得的优点和积极效果为:
1、本发明荧光探针对亚硫酸(氢)盐有很好的选择性,该荧光探针溶液的荧光强度与亚硫酸(氢)盐的浓度在一定的浓度范围内(0~8×10-5摩尔/升)具有良好的线性关系,表现出良好的实际应用性。本发明荧光探针响应时间短(10秒),测定灵敏度高,分子结构简单,合成方法简便,可用于细胞成像,检测食品及中药材中亚硫酸(氢)盐,使得其极易实际推广应用。
2、本发明探针能够对细胞、食品以及中药材中的亚硫酸(氢)盐进行快速、灵敏及专一性的检测。
3、本发明方法通过以香豆素为骨架,设计合成一种荧光探针,以实现对细胞、食品以及中药材中亚硫酸(氢)盐定性及定量检测。
附图说明
图1为本发明中的荧光探针的一维氢核磁谱图,横坐标为化学位移,纵坐标为信号强度图;
图2为本发明中的荧光探针对亚硫酸(氢)盐的选择性,横坐标为不同的离子,纵坐标为荧光强度图;
图3为本发明中的荧光探针对不同分子或离子的选择性检测图;
图4为本发明中的荧光探针的荧光强度和亚硫酸(氢)盐浓度的线性关系,横坐标为亚硫酸(氢)盐的浓度,纵坐标为荧光强度图;
图5为本发明中的荧光探针在细胞内对亚硫酸(氢)盐的响应图。
具体实施方式
下面详细叙述本发明的实施例,需要说明的是,本实施例是叙述性的,不是限定性的,不能以此限定本发明的保护范围。
本发明中所使用的原料,如无特殊说明,均为常规的市售产品;本发明中所使用的方法,如无特殊说明,均为本领域的常规方法。
一种快速检测亚硫酸(氢)盐的turn-on型荧光探针CISD,所述荧光探针的分子式为: C21+nH15+mN1+xO3+y,其中n,m,x,y均为1-20的整数,且分子具有以下结构:
其中取代基R1为H、烷基、芳基、硝基、酯基、醚基和二乙胺基中的一种,3-吲哚甲醛衍生物中的n为1-20的整数。
一种如上所述的快速检测亚硫酸(氢)盐的turn-on型荧光探针的合成方法,步骤如下:
将带有取代基R1的香豆素溶解在有机溶剂中,在有催化剂条件下加入3-吲哚甲醛衍生物,在反应温度90℃下反应2-8小时,然后过滤,利用硅胶柱层析得到目标化合物turn-on型荧光探针。
而且,所述有机溶剂为甲醇、乙醇、二甲基亚砜、乙腈、二氧六环、二氯甲烷、苯和甲苯中的一种或两种以上任意比例的混合物。
而且,所述催化剂为吡啶、三乙胺、碳酸钾或氢氧化钠。
如上所述的快速检测亚硫酸(氢)盐的turn-on型荧光探针在检测亚硫酸(氢)盐方面中的应用。
如上所述的快速检测亚硫酸(氢)盐的turn-on型荧光探针在食品或中药方面中的应用。
如上所述的快速检测亚硫酸(氢)盐的turn-on型荧光探针在细胞成像方面中的应用。
利用如上所述的快速检测亚硫酸(氢)盐的turn-on型荧光探针检测亚硫酸(氢)盐的方法,步骤如下:
将探针分子溶解在生理盐水或磷酸盐的缓冲溶液(PBS)中,或者将探针分子溶解在甲醇、乙醇、乙腈或二甲基亚砜有机溶剂中,或者将探针分子溶解在水和甲醇、乙醇、乙腈或二甲基亚砜有机溶剂中的一种或两种以上任意比例的混合溶剂中,最终配置成浓度为1×10-5摩尔/升的探针溶液;然后在溶解有探针分子的上述探针溶液中加入浓度为含亚硫酸(氢)盐的样品溶液,即可检测亚硫酸(氢)盐。
本发明的相关制备及检测:
一、一种快速检测亚硫酸(氢)盐的turn-on型荧光探针的合成:
将3-乙酰香豆素0.564g(3mmol)溶解在50mL的无水乙醇中,然后加入2-甲基吲哚3- 甲醛0.477g(3mmol),加入0.1mL的吡啶作为催化剂,反应温度90℃,在氮气保护下反应8小时,冷却至室温,过滤,经过硅胶柱层析得到红色固体化合物荧光探针CISD。
1H NMR(400MHz,DMSO-d6)δ(ppm)11.95(s,1H),8.68(s,1H),8.04(d,J=15.6Hz,1H), 7.97(dd,J=7.5,1.2Hz,1H),7.89–7.92(m,1H),7.73(td,J=7.8,1.2Hz,1H),7.71(d,J=15.6 Hz,1H),7.49(d,J=8.4Hz,1H),7.39–7.45(m,2H),7.18–7.21(m,2H),2.58(s,3H).
结果如图1所示,图1为本发明中的荧光探针的一维氢核磁谱图,横坐标为化学位移,纵坐标为信号强度。
二、检测荧光探针对亚硫酸(氢)盐的光谱响应:
结果如图2所示,本发明中的荧光探针CISD(浓度1×10-5摩尔/升)在PBS缓冲溶液中 (pH=7.4),没有亚硫酸(氢)盐时,溶液没有荧光。当加入荧光探针0~52倍摩尔量亚硫酸(氢)盐时溶液荧光在510纳米处逐渐增强。说明该荧光探针对亚硫酸(氢)盐有响应。
三、检测荧光探针对不同分子或离子的选择性:
结果如图3所示,检测本发明中的荧光探针CISD(浓度1×10-5摩尔/升)在PBS缓冲溶液中(pH=7.4),当加入各种常见离子和氨基酸时溶液荧光强度的变化,比如荧光探针52倍摩尔量的F-、Cl-、Br-、I-、NO3 -、AcO-、Ca2+、Zn2+、Fe3+、Al3+、H2PO4 -、HPO4 2-、 S2O3 2-、HS-、SO4 2-、HCO3 -、Hcy、H2O2、ClO–、Cys、GSH。荧光探针激发波长440纳米,发射波长510纳米。当在荧光探针溶液中加入的是荧光探针52倍摩尔量的亚硫酸(氢)盐时,该荧光探针溶液的荧光强度增强。而加入各种常见干扰物种时,该荧光探针溶液的荧光强度几乎没有明显变化,说明该荧光探针对亚硫酸(氢)盐有良好的选择性识别和实际应用性。
四、荧光探针的荧光强度和亚硫酸(氢)盐浓度的线性关系:
结果如图4所示,使用本发明中的荧光探针来评价该荧光探针的荧光强度与亚硫酸(氢) 盐浓度的线性关系。图4是在发射波长为510纳米处的荧光强度随亚硫酸氢钠浓度的变化曲线,图4的结果表明:探针溶液(浓度为1×10-5摩尔/升)荧光强度与亚硫酸(氢)盐的浓度 0~8×10-5摩尔/升范围内线性关系良好,具有优异的定量检测亚硫酸(氢)盐的能力,因此表现出良好的实际应用性。
五、细胞培养和荧光成像:
在含有10%(v/v)热杀菌的胎牛血清、1%(v/v)青霉素/链霉素的高糖培养基(DMEM) 中培养Hela细胞,在37℃含有5%CO2的潮湿培养箱中培养。将细胞孵育24小时之后,将探针加入到20mm直径玻璃底细胞培养皿中。然后用磷酸盐缓冲盐水(PBS)溶液冲洗细胞,用含有该荧光探针的无血清DMEM在37℃孵育1小时,然后用PBS洗涤三次以除去多余的探针。在共聚焦激光扫描显微镜上获取荧光图像。
荧光探针在细胞内对亚硫酸(氢)盐的响应结果见图5,如图5所示,使用本发明中的荧光探针来评价该荧光探针在细胞内对亚硫酸(氢)盐的响应。图5上、下两列表示加入亚硫酸(氢)盐前后荧光变化对比。未加亚硫酸(氢)盐细胞就没有荧光,加入亚硫酸(氢) 盐,荧光增强。结果表明:该荧光探针可以识别细胞中的亚硫酸(氢)盐。
尽管为说明目的公开了本发明的实施例,但是本领域的技术人员可以理解:在不脱离本发明及所附权利要求的精神和范围内,各种替换、变化和修改都是可能的,因此,本发明的范围不局限于实施例和附图所公开的内容。
Claims (7)
2.根据权利要求1所述的应用,其特征在于:所述快速检测亚硫酸(氢)盐的turn-on型荧光探针的合成方法,步骤如下:
将带有取代基R1的香豆素溶解在有机溶剂中,在有催化剂条件下加入3-吲哚甲醛衍生物,在反应温度90℃下反应2-8小时,然后过滤,利用硅胶柱层析得到目标化合物turn-on型荧光探针。
3.根据权利要求2所述的应用,其特征在于:所述有机溶剂为甲醇、乙醇、二甲基亚砜、乙腈、二氧六环、二氯甲烷、苯和甲苯中的一种或两种以上任意比例的混合物。
4.根据权利要求2或3所述的应用,其特征在于:所述催化剂为吡啶、三乙胺、碳酸钾或氢氧化钠。
7.利用快速检测亚硫酸(氢)盐的turn-on型荧光探针检测亚硫酸(氢)盐的方法,其特征在于:步骤如下:
将探针分子溶解在生理盐水或磷酸盐的缓冲溶液中,或者将探针分子溶解在甲醇、乙醇、乙腈或二甲基亚砜有机溶剂中,或者将探针分子溶解在水和甲醇、乙醇、乙腈或二甲基亚砜有机溶剂中的一种或两种以上任意比例的混合溶剂中,最终配置成浓度为1×10-5摩尔/升的探针溶液;然后在溶解有探针分子的上述探针溶液中加入浓度为含亚硫酸(氢)盐的样品溶液,即可检测亚硫酸(氢)盐;
所述快速检测亚硫酸(氢)盐的turn-on型荧光探针的分子式为:C21+nH15+mN1+xO3+y,其中n,m,x,y均为1-20的整数,且分子具有以下结构:
其中取代基R1为H、烷基、芳基、硝基、酯基、醚基和二乙胺基中的一种,3-吲哚甲醛衍生物中的n为1-20的整数。
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