CN111848671A - 一种过氧亚硝酰阴离子比率远红外荧光探针、制备方法和应用 - Google Patents
一种过氧亚硝酰阴离子比率远红外荧光探针、制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种过氧亚硝酰阴离子比率远红外荧光探针、制备方法和应用，过氧亚硝酰阴离子比率远红外荧光探针的结构式如下：
本申请的荧光探针，是一种高选择性、低检测下线、较大的伪斯托克斯位移以及比率远红外ONOO‑荧光探针，能满足医学能对ONOO‑引起的病变及时诊断的需求。
Description
技术领域
本发明属于离子检测技术领域，具体是涉及到一种过氧亚硝酰阴离子比率远红外荧光探针、制备方法和应用。
背景技术
过氧亚硝酸酰阴离子(ONOO-)是体内最重要的活性氧之一，在生命活动中起着至关重要的作用。正常浓度的过氧亚硝酸酰阴离子在生理和病理过程中起到信号转导和抗菌作用。然而，由于ONOO-较强的氧化氧化和亲核能力，高浓度的ONOO-对许多生物分子造成损伤，如蛋白质、脂类、DNA等，从而可能影响细胞生长过程以及导致细胞坏死和凋亡。因此发展一种高选择、灵敏检测的方法用于ONOO-的检测具有重要意义。近几年来，发展了许多用于ONOO-检测的方法比如电子顺磁共振法、电化学法、色谱法、化学发光和紫外-可见吸收光谱法荧光探针法等，但是上述检测方法需要大型仪器，耗时耗力，不适用于生物体内ONOO-的原位、实时快速定量分析。
荧光探针法由于具有高灵敏性、实时性、较高的时空分辨性以及可以直接用于生物体中活性物质的检测等优势，广泛用于生物体中ONOO-的检测。然而，目前用于ONOO-检测的荧光探针大多以开-关型以及短发射波长为主，其中开-关型荧光探针易环境条件的改变、光漂白以及样品浓度的变化的影响；短发射波长荧光探针不能对生物体中深层组织检测、对光有较大的损伤以及易受激发光的干扰。因此，上述荧光探针不利于ONOO-的精准检测以及对深层组织ONOO-的检测。另外，较小的伪斯托克斯位移的荧光探针易受到生物体荧光的干扰。
用于细胞内H2O2和ONOO-检测的荧光探针的设计合成与生物分析应用，杨秀娥，2016年山东师范大学硕士论文，第一章2.3节公开了过氧亚硝基阴离子荧光探针的研究进展，有荧光光谱法、紫外可见光谱法、化学发光法、电化学方法、酶学法和免疫组织化学法等，包括如下探针化合物，如：
苯基酰肼类
苯并噻唑(BT)类
以花菁为荧光母体，二价硒为响应基团的探针
以香豆素和半花菁为荧光骨架的比率型探针CHCN
以派洛宁为荧光母体
中国专利申请201810684964.0公开了对称的化合物
发明内容
本发明要解决的技术问题是提供一种过氧亚硝酰阴离子比率远红外荧光探针、制备方法和应用，具有高选择性，低检测下线以及较大的伪斯托克斯位移。
本发明的内容包括一种过氧亚硝酰阴离子比率远红外荧光探针，结构式如下：
一种过氧亚硝酰阴离子比率远红外荧光探针的制备方法，步骤为：
将化合物ANI和碘乙烷溶于有机溶剂中，搅拌回流，待反应完成后，过滤，得到过氧亚硝酰阴离子比率远红外荧光探针，所述化合物ANI的结构为：
所述化合物ANI和碘乙烷的摩尔比为1:2；所述有机溶剂为无水乙腈；所述化合物ANI和无水乙腈的重量比为10-15:1。
具体的反应过程为：
所述化合物ANI的制备方法为，将化合物AN和二苯基次磷酰氯溶于三乙胺和有机溶剂(优选为无水四氢呋喃)中，搅拌，反应得到化合物ANI；化合物AN的结构为：
所述化合物AN、二苯基次磷酰氯和三乙胺的摩尔比为1:1.5:1.5。
本申请还提供一种过氧亚硝酰阴离子比率远红外荧光探针在检测过氧亚硝酰阴离子中的应用。
本发明的有益效果是，本申请的荧光探针，是一种高选择性、低检测下线、较大的伪斯托克斯位移以及比率远红外ONOO-荧光探针，能满足医学能对ONOO-引起的病变及时诊断的需求。
本申请以异佛尔酮吡啶盐为荧光基团，二苯基次磷酰酯为识别基团，探针ANI-DP自身由于羟基被保护，在535nm处有荧光发射。当加入过氧亚硝酰阴离子时，过氧亚硝酰阴离子与二苯基次磷酰酯发生亲核加成反应，二苯基次磷酰酯脱去，生成酚羟基，荧光红移至628nm。
相比于本发明人先前申请的荧光探针，即：
其中，R基团为-H、-OH、-Cl或-OCH3；本申请的反应机理不同，本申请属于分子内质子转移(ICT)机理，属于比率远红外荧光探针，伪斯托克斯位移较大，不易受环境条件的改变、光漂白以及样品浓度变化的影响。
本申请的荧光探针对过氧亚硝酰阴离子检测下线比较低，为13.3nM，说明该探针比较灵敏。
附图说明
图1为探针化合物ANI-DP的1H NMR图谱。
图2为探针化合物ANI-DP的13C NMR图谱。
图3为探针化合物ANI-DP加入(带竖直线的线条)和不加ONOO-的紫外可见光吸收光谱图。
图4为探针化合物ANI-DP与ONOO-作用前后的荧光强度变化图。
图5为本发明荧光探针的荧光强度(I628/I535)比值和ONOO-浓度线性关系图。
图6为探针化合物ANI-DP与ONOO-作用前后荧光强度(I628/I535)比值随pH的变化图。
图7为探针化合物ANI-DP与ONOO-作用前后荧光强度(I628/I535)比值随时间的变化图。
图8为探针化合物ANI-DP与不同的生物活性物种作用后的荧光强度(I628/I535)比值变化图。
(1)ANI-DP，(2)Zn2+，(3)Fe3+，(4)K+，(5)Al3+，(6)Cu2+，(7)NO2 -，(8)NO3 -，(9)F-，(10)Cl-，(11)Br-，(12)CO3 2-，(13)H2PO4 -，(14)AcO-，(15)Cys，(16)Hcy，(17)GSH，(18)H2O2，(19)HClO，(20)HO·-，(21)tBuHO·-，(22)ROO·-，(23)O2·-，(24)1O2 -，(25)ONOO-。
具体实施方式
实施例1
将化合物AN(400mg，1mmol)、二苯基次磷酰氯(0.356mg，1.5mmol)和三乙胺(0.21mL)溶于10mL无水四氢呋喃中，溶液搅拌室温反应12小时，待反应完成后，旋干柱层析的产品ANI(0.288mg，产率48％)。
1H NMR(400MHz,DMSO-d6)δ(ppm):8.60(d,J＝6.4Hz,2H),7.94-7.85(m,5H),7.65-7.62(m,4H),7.58-7.53(m,4H)),7.30(d,J＝8.0Hz,2H),7.12(d,J＝8.0Hz,2H),5.53(s,2H),2.51(s,2H),1.01(s,6H)；13C NMR(100MHz,DMSO-d6)δ(ppm):166.5,162.0,154.7,154.1,151.8,150.3,145.3,135.5,133.3,132.0,131.7,130.6,130.4,129.7,129.5,129.4,125.9,124.0,122.1,121.5,121.4,117.3,116.5,98.2,97.5。
将化合物ANI(600mg，1mmol)和碘乙烷(940mg，2mmol)溶于5mL无水乙腈中，溶液搅拌回流反应8小时，待反应完成后，过滤，收集黄色产品470mg，产率62％)。
1H NMR(400MHz，DMSO-d6)δ(ppm):9.12(d，J＝6.8Hz,2H)，8.13(d,J＝6.8Hz,2H)，7.94-7.85(m,5H)，7.66-7.62(m,4H)，7.57-7.54(m,4H))，7.30(d,J＝8.0Hz,2H)，7.14(d,J＝6.0Hz,2H)，5.59(s,2H)，4.62(q,2H)，2.66(s,2H)，2.49(s,2H)，1.55(t,3H)，1.02(s,6H)；13C NMR(100MHz,DMSO-d6)δ(ppm):167.0,164.0,161.6,155.8,154.6,152.1,151.8,144.9,135.8,133.4,132.0,131.9,131.7,130.3,129.5,127.8,125.5,123.9,121.4,99.7,64.5,56.3,44.8,31.9,28.1,16.7。具体如图1-2所示。
总反应式为：
实施例2
一、探针ANI-DP对ONOO-响应的吸收光谱和荧光光谱
将探针化合物ANI-DP溶于DMSO中，配置成1mmol/L的溶液。向移取的探针ANI-DP溶液中加入DMSO和PBS缓冲液(pH＝7.4)，配置成10μM(DMSO：PBS＝10:90，V/V)的待测溶液。向待测溶液加入ONOO-，用紫外光谱和荧光光谱仪测试其荧光发射光谱的变化情况。由图3可知，探针ANI-DP在388nm处有强的吸收峰，加入ONOO-之后，探针ANI-DP在451nm处有出现了新的吸收峰。由图4可知，探针ANI-DP自身在535nm处有发射峰，加入ONOO-之后，探针在628nm处出现了强荧光发射峰，并且该发射峰强度随着ONOO-浓度的增加而增强，而535nm处发射峰则逐渐减弱。
该探针ANI-DP可以对ONOO-实现比率远红外检测，其伪斯托克斯位移可达到177nm。由图5通过计算可得测下线为13.3nM(计算公式为3σ/k，其中σ为空白样品连续测定多次的标准偏差，k为标准曲线斜率)。
二、pH值变化对探针ANI-DP与ONOO-作用的荧光强度(I554/I473)比值影响
在不同的pH条件下，测试了探针ANI-DP对ONOO-的荧光强度比值(I628/I535)变化。由图6可知，pH为2-4时，探针ANI-DP的荧光强度比值(I628/I535)逐渐增强，pH为4-10时，探针ANI-DP的荧光强度比值(I628/I535)达到最佳，并保持不变。因此，探针ANI-DP可在生理pH条件下实现对ONOO-的检测。
三、探针ANI-DP对ONOO-的时间研究
为了考察探针ANI-DP对ONOO-响应的时间，测试了探针ANI-DP对ONOO-的荧光强度比值(I628/I535)随时间变化情况。由图7可知，在探针ANI-DP溶液加入ONOO-后，荧光强度比值(I628/I535)随时间的变化而逐渐增强，荧光强度比值(I628/I535)在20分钟内达到最大，说明探针ANI-DP可实时检测ONOO-。
四、探针ANI-DP对ONOO-的选择性研究
在探针ANI-DP溶液中分别加入不同的生物活性物种，分别测试了探针ANI-DP的荧光强度比值(I628/I535)变化情况。由图8可知，在探针ANI-DP溶液中加入ONOO-后，荧光强度比值(I628/I535)发生了明显的改变，而加入其它生物活性物种，荧光强度比值(I628/I535)几乎没有发生变化，说明探针ANI-DP能够选择性的实现ONOO-检测。
Claims (6)
1.一种过氧亚硝酰阴离子比率远红外荧光探针，其特征是，结构式如下：
2.一种如权利要求1所述的过氧亚硝酰阴离子比率远红外荧光探针的制备方法，其特征是，步骤为：
将化合物ANI和碘乙烷溶于有机溶剂中，搅拌回流，待反应完成后，过滤，得到过氧亚硝酰阴离子比率远红外荧光探针，所述化合物ANI的结构为：
3.如权利要求2所述的制备方法，其特征是，所述化合物ANI和碘乙烷的摩尔比为1:2。
4.如权利要求2或3所述的制备方法，其特征是，所述有机溶剂为无水乙腈。
5.如权利要求3所述的制备方法，其特征是，所述化合物ANI和无水乙腈的重量比为10-15:1。
6.一种如权利要求1所述的过氧亚硝酰阴离子比率远红外荧光探针在检测过氧亚硝酰阴离子中的应用。
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