CN112920081B - 一种选择性识别次氯酸的荧光探针及其制备方法和应用 - Google Patents
一种选择性识别次氯酸的荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
Description
技术领域
本发明属于有机合成技术领域,具体涉及一种选择性识别次氯酸的荧光探针及其制备方法和应用。
背景技术
活性氧分子是许多生理和病理过程的标志物,与人体健康密切相关,在生命体系中起着至关重要的作用,而次氯酸作为一种活性氧,在机体内正常的浓度范围内可以对病原体起到杀灭作用参与免疫反应。但是次氯酸的含量超出正常范围,就会对机体造成氧化损伤,甚至引发许多疾病,如动脉粥样硬化、心血管疾病、类风湿性关节炎、癌症、肾脏和肝脏疾病等,因此,实时监测次氯酸的动态分布及浓度变化非常重要。
针对活性氧的分析检测方法有很多,包括质谱、电子自旋共振和化学发光等。己报导的识别HClO/ClO-的方法主要有电化学分析法、碘量法、色谱法和荧光标记法。而传统的检测手段与活性氧之间缺乏选择性、灵敏度差、耗时长、操作繁琐等局限性,很难对活性氧物质进行很好的检测。
荧光分析法,与其他分析方法相比,具有操作方便、响应快速、选择性好、灵敏度高,并且在对目标分析物的识别过程中经常伴随着紫外可见吸收的变化,可实现肉眼检测,近几年来,荧光分析法受到了科研工作者的关注,实现其在化学环境下或者生物体中对分析物的实时、动态、原位检测。
发明内容
为了克服现有技术中的问题,本发明提供了一种选择性识别次氯酸的荧光探针。该荧光探针对次氯酸根离子的响应具有良好的线性关系,对次氯酸根离子的特异性识别强,展现出明显的“裸眼”比色识别能力,检测限低。
本发明还提供了上述荧光探针的制备方法和应用。
为实现上述目的,本发明的技术方案如下:
一种选择性识别次氯酸的荧光探针,所述荧光探针的分子式为C24H20N6O,结构式如下:
上述选择性识别次氯酸的荧光探针的合成路线如下:
具体包括以下步骤:
(1)将丙二腈溶于N,N-二甲基甲酰胺中,然后于室温下依次加入异佛尔酮、哌啶、冰醋酸并搅拌,接着在氮气保护下,升温回流,反应4~6h,得到的产物经水洗、乙酸乙酯萃取、干燥、过滤,得到粗品化合物1;将粗品化合物1用柱色谱分离,经干燥得到化合物1;
(2)将步骤(1)制备的化合物1溶于无水乙醇中,然后加入对羟基苯甲醛和哌啶,搅拌条件下回流3~5h,得到反应液;除去反应液中的有机溶剂,得到粗品化合物2;将粗品化合物2用柱色谱分离,经干燥得到化合物2;
(3)将步骤(2)制备的化合物2溶于三氟乙酸中,然后加入六次甲基四胺,于搅拌条件下回流5~7h,得到反应液;将反应液冷却,析出固体,经抽滤、干燥得到粗品化合物3;将粗品化合物3用柱色谱分离,经干燥得到化合物3;
(4)将步骤(3)制备的化合物3和二氨基马来腈溶于无水乙醇,于搅拌条件下回流1~3h,然后经抽滤、干燥即得所述荧光探针。
优选的,步骤(1)中丙二腈与异佛尔酮的摩尔比为1:(1~1.5);其中,其中丙二腈的投加量为1.2~1.4mol/L,柱色谱分离时洗脱剂为石油醚:乙酸乙酯=(18~22):1(体积比,下同)。
优选的,步骤(2)中化合物1与对羟基苯甲醛的摩尔比为1:(1~1.2);对羟基苯甲醛的投加量为1.0~1.2mol/L,柱色谱分离时洗脱剂为石油醚:乙酸乙酯=(6~10):1。
优选的,步骤(3)中化合物2与六次甲基四胺的摩尔比为1:(2~2.5);六次甲基四胺的投加量为0.4~0.5mol/L,柱色谱分离时洗脱剂为二氯甲烷:甲醇=(110~130):1。
优选的,步骤(4)中化合物3与二氨基马来腈的摩尔比为1:(1~1.2);二氨基马来腈的投加量为1.0~1.2mol/L。
上述荧光探针在次氯酸荧光检测中的应用。
和现有技术相比,本发明的有益效果是:
1.本发明基于本文基于反应型传感机制,利用异佛尔酮和丙二腈为原料,合成中间体,然后中间体与二氨基马来腈反应,得到以C=N键为响应基团的反应型探针,荧光强度显著性增强并伴随明显的颜色变化,选取的干扰离等对检测效果几乎无影响,因而实现了对次氯酸的特异性识别响应,并展现出明显的“裸眼”比色识别能力;
2.该探针具有良好的选择性和较高的灵敏度,检测限可达0.058μmol·L-1。
附图说明
图1为实施例1制备的荧光探针的核磁氢谱;
图2为实施例1制备的荧光探针的核磁碳谱;
图3为实施例1制备的荧光探针与不同离子反应后的紫外吸收光谱;
图4为实施例1制备的荧光探针与不同离子反应后的荧光光谱;
图5为实施例1制备的荧光探针与不同离子反应后的荧光强度图;
图6为实施例1制备的荧光探针在日光灯下对不同离子反应后的荧光颜色变化;
图7为实施例1制备的荧光探针与次氯酸离子反应随时间变化的紫外吸收光谱;
图8中(a)为实施例1制备的荧光探针与次氯酸离子反应随时间变化的荧光光谱;(b)为实施例1制备的荧光探针与次氯酸离子反应前后的颜色变化;
图9为实施例1制备的荧光探针与不同浓度ClO-离子响应后的紫外吸收光谱;
图10中(a)为实施例1制备的荧光探针与不同浓度ClO-离子响应后的荧光光谱;(b)为溶液的荧光强度与ClO-离子浓度之间的线性关系;
图11为实施例1制备的荧光探针在不同pH体系溶液中与ClO-离子响应后的紫外吸收光谱;
图12为实施例1制备的荧光探针在不同pH体系溶液中与ClO-离子响应后的荧光光谱。
具体实施方式
下面结合实施例和附图对本发明进行进一步说明,但并不是对本发明的限制。
实施例1
本实施例荧光探针的制备方法包括以下步骤:
(1)化合物1的制备
向50mL圆底烧瓶中加入1.67g(25mmol)丙二腈,用14mLN,N-二甲基甲酰胺溶解,依次向烧瓶中加入4.0mL异佛尔酮(26.7mmol)、0.5mL哌啶、0.1mL冰醋酸,并在室温下搅拌12h;然后在氮气保护下,升温至140℃回流4h,反应得到的产物依次经静置、冷却、50mL去离子水水洗、30mL乙酸乙酯萃取三次、无水硫酸钠干燥、过滤,得到粗品化合物1。将粗品化合物1进一步用硅胶色谱柱分离,洗脱剂为石油醚:乙酸乙酯=20:1(体积比,下同),经真空干燥得到3.09g化合物1,其收率为65%。
合成路线如下:
(2)化合物2的制备
向25mL二口瓶中加入0.20g化合物1(1.08mmol),用8mL无水乙醇溶解,然后加入0.15g对羟基苯甲醛(1.23mmol)和30uL哌啶,于85℃下搅拌回流3h,得到反应液。将反应液进行旋蒸,除去有机溶剂,得到粗品化合物2。将粗品化合物2进一步用柱色谱分离,洗脱剂为石油醚:乙酸乙酯=8:1,经真空干燥得到化合物2,其产率为85%。
合成路线如下:
(3)化合物3的制备
向25mL二口烧瓶中加入0.5g的化合物2(1.72mmol),再加入8.5mL三氟乙酸,磁力搅拌下加入0.5g的六次甲基四胺(3.57mmol),于90℃下搅拌回流5h,得到暗红色反应液,然后将其缓慢倒入冰水中,搅拌、析出固体,经抽滤、干燥得到粗品化合物3。将粗品化合物3进一步用柱色谱分离,洗脱剂为二氯甲烷:甲醇=120:1,经真空干燥得到化合物3,其收率为20%。
合成路线如下:
(4)探针的合成
将0.1g化合物3(0.31mmol)和0.04g二氨基马来腈(0.37mmol)溶于6mL无水乙醇中,于90℃下搅拌回流2h,有固体析出,经抽滤、洗涤、真空干燥,不需要进一步纯化得到0.11g探针化合物,收率为85%。
合成路线如下:
对本实施例制备的荧光探针进行核磁氢谱和核磁碳谱分析,结果分别如图1、图2所示。
1HNMR(400MHz,DMSO)δ10.95(s,1H),8.56(s,1H),8.42(s,1H),8.00(s,2H),7.68(d,J=8.5Hz,1H),7.31(d,J=16.1Hz,1H),7.21(d,J=16.2Hz,1H),6.98(d,J=8.5Hz,1H),6.83(s,1H),2.61(s,2H),2.53(s,2H),1.02(s,6H).13C NMR(101MHz,DMSO)δ170.68(s),159.94(s),156.58(s),151.67(s),133.52(s),128.18(d,J=8.5Hz),127.88(s),126.88(s),122.37(s),117.53(s),114.96(s),114.38(d,J=11.0Hz),113.70(s),103.81(s),76.00(s),56.50(s),42.74(s),40.52(d,J=21.0Hz),40.24(s),40.20(s),40.03(s),39.99(s),39.68(d,J=21.0Hz),39.37(s),32.15(s),27.91(s),19.03(s).
本实施例制备的荧光探针的应用试验如下:
1)检测用探针储备液的配置
a.选择性识别次氯酸的探针样品溶液(1.00×10-3mol·L-1)的配制:取0.0022g(M=446)反应型巯基化合物探针溶于5mL二甲亚砜中,配成浓度为1.00×10-3mol·L-1的溶液。
b.离子储备液的配置:(1)探针空白对照、(2)硫酸氢钠(HSO3 -)、(3)硫氢化钠(H2S)、(4)碳酸氢钠(HCO3 -)、(5)氯化钠(Cl-)、(6)硫酸氢钠(HSO4 -)、(7)氰化钠(CN-)、(8)磷酸钠(PO4 3-)、(9)醋酸钠(Ac-)、(10)硫酸钠(SO4 2-)、(11)碳酸钠(CO3 2-)、(12)硫氰化钠(SCN-)、(13)过氧化氢(H2O2)、(14)氟化铵(F-)、(15)碘化钾(I-)、(16)溴化钾(Br-)、(17)甲酸铵(HCOO-)、(18)亚硝酸钠(NO2 -)、(19)硝酸钠(NO3 -)和(20)次氯酸钠(ClO-)均用去离子水配置成为浓度为1.00×10-3mol·L-1或1.00×10-2mol·L-1(本实施例使用的浓度为1.00×10-3mol·L-1)的溶液。
c.PBS缓冲溶液(pH=7.4)的配制:
母液配置:0.2mol·L-1K2HPO4:称取71.6g Na2HPO4·12H2O溶于1000mL水中配置成0.2mol·L-1Na2HPO4:称取31.2g NaH2PO4·2H2O,溶于1000mL水中配置成0.2mol·L-1K2HPO4。
0.2mol·L-1PBS母液(pH=7.4):取19mL的0.2mol·L-1Na2HPO4,81mL 0.2mol·L- 1NaH2PO4,加水稀释至1000mL即可。
下述检测中使用的缓冲溶液均为PBS(pH=7.4),实验用水均为去离子水。
2)检测分析
a、反应体系
表1 不用溶剂配比的测试体系(体积比)
说明:“-”表示不响应或者响应差,“√”表示响应较好。
取3mL配置体系溶液(表1中各体系),加入30μL探针储备液(1.00×10-3mol·L-1),再加30μL次氯酸根离子(ClO-)储备液,换不同比例的体系溶液进行紫外检测,结果如表1显示,在PBS:DMSO=2:8的反应体系中,探针和次氯酸根(ClO-)离子反应现象最为明显,紫外光谱中,探针的最大吸光度为0.533,加入次氯酸根(ClO-)离子后变为0.244,差值为0.289,在PBS:乙腈和PBS:EtOH体系中不反应或差值小于0.289,最终确定PBS:DMSO=2:8作为反应体系。
b、对探针的检测
取30μL浓度为1.0×10-3mol·L-1的探针储备液,用3mLPBS:DMSO=2:8(体积比)的反应溶液配置成1.0×10-5mol·L-1,加入10当量的上述各种离子储备液,检测其紫外吸收和荧光光谱,结果分别如图3、图4所示;其中,紫外吸收检测范围:800~300nm,荧光参数设置:激发狭缝宽度2.5nm,发射狭缝宽度5nm,激发波长Ex=425nm,发射波长扫描范围Em=435~830nm。由图3可以看出,在加入这些离子后,紫外-可见吸收光谱显示探针在625nm处的最强吸收峰无明显变化,加入ClO-离子后,最强吸收峰发生50nm的蓝移,吸光度明显降低,而在425nm处的吸光度显著增高。由图4可以看出,在加入ClO-离子后(Ex=425nm,Em=435~830nm),荧光光谱显示探针的荧光发射波长发生迁移,且荧光强度增强5倍,而加入其他离子后荧光发射波长及强度无明显变化,因此探针对ClO-离子存在良好的选择性,而对其他离子无特异选择性。
荧光探针与上述编号1-20的不同离子反应后的荧光强度如图5所示,由图5可以看出,荧光探针与ClO-反应后荧光强度明显高于空白样和其余干扰离子,由此我们认定该探针可以作为一种高选择性检测次氯酸的荧光增强型探针。
同时还考察了本实施例探针在待测离子溶液中的颜色变化,结果如图6所示,图6中为在日光灯下的荧光颜色变化,从左往右依次为空白样、编号2-20的干扰离子。通过图6对比可以看出,反应后溶液在日光下由蓝色(空白样和除ClO-外的其余干扰离子)变为绿色(ClO-),即ClO-具有“裸眼”识别能力。
c、探针的时间效应检测
取30μL浓度为1.0×10-3mol·L-1的探针储备液,用PBS:DMSO=2:8的反应溶液(3mL)配置成1.0×10-5mol·L-1,加入10Equiv的ClO-离子,不间断扫描8min,进行紫外-可见吸收光谱及荧光光谱扫描,结果分别如图7、图8中(a)所示。由图7可以看出,在紫外光谱中,ClO-离子的加入会立即引起625nm处吸收峰的降低,425nm处吸收峰的升高,吸光度能在3min内达到稳定;由图8中(a)可以看出,在荧光光谱中,荧光发射波长立即向短波方向发生迁移,且强度增强,荧光强度能在3min内达到稳定,溶液的颜色也立即由蓝色变成绿色,如图图8中(b)所示。说明探针对ClO-离子的检测具有较高的灵敏性,整个检测过程能在3min内快速完成。
d、探针浓度梯度的检测
取3mL缓冲溶液,加入30μL的1.00×10-3mol·L-1探针储备液,加入不同当量的ClO-溶液,摇匀,检测紫外-吸收光谱及荧光光谱的变化,结果分别如图9、图10中(a)所示。由图9可以看出,在紫外光谱中,625nm处的吸收峰随ClO-离子浓度增大而逐渐降低,并发生小范围蓝移,425nm处的吸收峰随ClO-离子浓度增大而逐渐升高;由图10中(a)可以看出,在荧光光谱中,750nm处荧光强度随ClO-离子浓度增大而增强,并向短波方向发生迁移。当ClO-离子浓度在0~1.48×10-4mol·L-1范围内,溶液的荧光强度与ClO-离子浓度之间具有良好的线性关系,结果如图10中(b)所示,经线性拟合,一元一次方程的表达式为:y=4.02644x+48.1985,线性相关系数R2=0.9931,说明拟合程度好。根据检出限计算公式D=3σ/k(其中σ为空白标准偏差,k为拟合曲线斜率)得出检测限为0.058μmol·L-1。
e、pH范围的检测
取30μL浓度为1.0×10-3mol·L-1的探针储备液,将PBS:DMSO=2:8的反应溶液中的PBS组分,换成相应的pH为1~14的反应溶液,用3mLpH反应溶液配置成1.0×10-5mol·L-1。加入10Equiv的ClO-离子,检测其紫外吸收及荧光的光谱性质,结果分别如图11、图12所示。由图11-12可以看出,紫外吸收光谱及荧光光谱都表明在pH=3~9之间探针对ClO-离子的响应具有良好的稳定性,因此探针可以在生理环境下检测ClO-离子。
Claims (7)
2.权利要求1所述选择性识别次氯酸的荧光探针的制备方法,其特征在于,合成路线如下:
具体包括以下步骤:
(1)将丙二腈溶于 N,N-二甲基甲酰胺中,然后于室温下依次加入异佛尔酮、哌啶、冰醋酸并搅拌,接着在氮气保护下,升温回流,反应4~6 h,得到的产物经水洗、乙酸乙酯萃取、干燥、过滤,得到粗品化合物1;将粗品化合物1用柱色谱分离,经干燥得到化合物1;
(2)将步骤(1)制备的化合物 1溶于无水乙醇中,然后加入对羟基苯甲醛和哌啶,搅拌条件下回流3 ~5h,得到反应液;除去反应液中的有机溶剂,得到粗品化合物2;将粗品化合物2用柱色谱分离,经干燥得到化合物2;
(3)将步骤(2)制备的化合物2 溶于三氟乙酸中,然后加入六次甲基四胺,于搅拌条件下回流5~7 h,得到反应液;将反应液冷却,析出固体,经抽滤、干燥得到粗品化合物3;将粗品化合物3用柱色谱分离,经干燥得到化合物3;
(4)将步骤(3)制备的化合物3和二氨基马来腈溶于无水乙醇,于搅拌条件下回流1~3h,然后经抽滤、干燥即得所述荧光探针。
3.根据权利要求2所述选择性识别次氯酸的荧光探针的制备方法,其特征在于,步骤(1)中丙二腈与异佛尔酮的摩尔比为1:(1~1.5)。
4.根据权利要求2所述选择性识别次氯酸的荧光探针的制备方法,其特征在于,步骤(2)中化合物1与对羟基苯甲醛的摩尔比为1:(1~1.2)。
5.根据权利要求2所述选择性识别次氯酸的荧光探针的制备方法,其特征在于,步骤(3)中化合物2与六次甲基四胺的摩尔比为1:(2~2.5)。
6.根据权利要求2所述选择性识别次氯酸的荧光探针的制备方法,其特征在于,步骤(4)中化合物3与二氨基马来腈的摩尔比为1:(1~1.2)。
7.权利要求1所述荧光探针以非疾病的诊断为目的在次氯酸荧光检测中的应用。
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