CN105017271B - 一种次氯酸荧光比率探针及其应用 - Google Patents

一种次氯酸荧光比率探针及其应用 Download PDF

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CN105017271B
CN105017271B CN201510432403.8A CN201510432403A CN105017271B CN 105017271 B CN105017271 B CN 105017271B CN 201510432403 A CN201510432403 A CN 201510432403A CN 105017271 B CN105017271 B CN 105017271B
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赵宝祥
苗俊英
张延如
孟宁
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Abstract

本发明公开了一种基于TBET机理的次氯酸荧光比率探针，所述探针是由罗丹明B硫代酰肼与6‑二甲氨基‑2‑萘甲酰基相连接，形成单硫代双酰肼核心结构的有机小分子化合物，其化学结构式如式(I)所示。本发明的探针可以选择性地与次氯酸作用，随着次氯酸浓度的增加，其荧光发射强度在585nm处逐渐增强，在440nm处逐渐减弱；二者比值(I585/I440)与次氯酸浓度在一定范围内呈线性关系。实现了细胞内的比率成像，有望在工业生产和临床医学中发挥作用，具有广阔的应用前景。

Description

一种次氯酸荧光比率探针及其应用
技术领域
本发明涉及一种荧光比率探针及其应用，尤其涉及一种基于TBET(through bondenergy transfer)机理的次氯酸荧光比率探针及其应用；属于有机小分子荧光探针领域。
背景技术
次氯酸是生物体内最重要的活性氧之一，在生物体内是由双氧水和氯离子经髓过氧化酶催化而成【A.Hammer et al,Lab.Invest.,2001,81,543】。一方面，次氯酸作为内源性杀菌剂在抵抗细菌入侵起着重要的作用[Z.M.Prokopowicz et al,Immunol.,2010,184,824]；另一方面，次氯酸生成量的失控与某些疾病有关，比如：关节炎、肾脏疾病、肺损伤、动脉硬化以及肿瘤等疾病[R.Rao et al,Front.Biosci.,2008,13,7210；D.I.Pattison etal,Biochemistry,2006,45,8152]。因此发展生物体内次氯酸成像技术对研究其在生理活动的作用具有重要的意义。
次氯酸荧光探针作为一种生物成像技术，因其具有高选择、超灵敏及快速响应等优点吸引了科学家的更多关注[H.Zhu et al,J.Am.Chem.Soc.,2014,136,12820；Q.A.Bestet al,J.Am.Chem.Soc.,2013,135,13365]；荧光比率探针因其能够有效消除环境、探针浓度及激发光强度的干扰而备受青睐[L.Yuan et al,Acc.Chem.Res.,2013,46,1462]。在众多类荧光比率探针中，TBET机理的荧光比率探针是通过共轭键将能量供体荧光团与能量受体荧光团连接而成。因其能量传递是经过共轭键来完成并不需要供体发射峰与受体吸收峰的大量重叠，所以这类探针具有较高的能量传递效率，较大的pseudo-Stokes位移，进而提高了该类探针在成像中的分辨率。
尽管次氯酸荧光比率探针的报道很多，但是到目前为止基于TBET机理的次氯酸荧光比率探针尚属空白。因此开发基于TBET机理的次氯酸荧光比率探针成为目前亟待解决的课题。
发明内容
针对现有技术的不足，本发明要解决的问题是提供一种基于TBET机理的次氯酸荧光比率探针及其应用。
本发明所述的基于TBET机理的次氯酸荧光比率探针，其特征在于所述荧光探针由罗丹明B硫代酰肼与6-二甲氨基-2-萘甲酰基相连接，形成单硫代双酰肼核心结构，其化学结构式如式(I)所示，
上述的基于TBET机理的次氯酸荧光比率探针的制备方法是：用已知方法合成6-二甲氨基-2-萘甲酰氯和罗丹明B硫代酰肼，然后二者反应得到上述次氯酸荧光比率探针。
上述的次氯酸荧光比率探针在检测含次氯酸样品中的应用。
其中：所述含次氯酸样品优选是生物细胞、含次氯酸的溶液。
本发明所述的探针的核心结构单硫代双酰肼与次氯酸发生反应生成噁二唑(见图1)，从而将两个荧光团用共轭键相连接，同时罗丹明B螺环打开，能量传递体系建立，导致萘蓝色荧光强度减弱罗丹明红色荧光增强；从而达到荧光比率检测次氯酸的效果。
具体的：配制上述氯酸荧光比率探针的乙醇与磷酸盐(0.05M)缓冲液(v/v＝3：7，pH＝6，)的溶液，分别加入定量的活性氧或活性氮的水溶液，如：t-BuOOH,t-BuOO·,H2O2,HO·,NO,NOOO-,1O2,-O2,及HOCl。然后对上述溶液进行荧光测试，结果表明上述探针对次氯酸有很好的选择性，其在585nm与440nm处的荧光强度比值(I585/I440)变化明显，见图2，3。
在加入上述探针的RAW264.7活细胞中，对照不加LPS和加入LPS(LPS：细胞产生次氯酸的刺激剂)的细胞染色荧光显微成像变化。可以看出对照组A细胞蓝色通道荧光较强，红色通道荧光较弱；对照组B蓝色通道荧光较A组减弱，红色通道荧光较A组增强；其红色通道荧光与蓝色通道荧光强度统计值的比值变化非常明显，见图4。
本发明所述的荧光探针随次氯酸浓度的增加，585nm处荧光强度逐渐增强，440nm处荧光强度逐渐减弱；二者比值相对次氯酸浓度在一定范围内呈线性关系。所以该探针能够定量检测低浓度的HOCl。见图5。
综上，本发明所述的基于TBET机理的次氯酸荧光比率探针不仅可以定量检测低浓度次氯酸，而且能够用于细胞内的比率成像；所以有望在工业生产和临床医学中发挥作用，具有广阔的应用前景。
附图说明
图1为该次氯酸探针与次氯酸生成产物的高分辨质谱。
图2为该次氯酸探针对各种活性氧响应荧光发射谱图。
图3为探针与各种活性氧作用时，荧光发射在585nm与440nm处的荧光强度比值(I585/I440)图。
图4为该次氯酸探针在RAW细胞内荧光显微成像图。
其中：细胞用10μM该次氯酸探针溶液孵化4小时后的(a)蓝色和(b)红色通道成像；细胞先用1μg mL-1LPS孵化12h后，再用10μM该次氯酸探针溶液孵化4小时后的(c)蓝色和(d)红色通道成像；两组成像中，红色通道荧光与蓝色通道荧光强度统计值的比值对比图(e)。
图5为该次氯酸探针在585nm与440nm处的荧光强度变化，及其比值与次氯酸浓度间的线性关系图。
具体实施方式
实施例1
将6-二甲氨基-2-萘甲酰氯(0.25mmol)和罗丹明B硫代酰肼(0.25mmol)溶于5ml干燥的吡啶中，在氮气保护下，室温搅拌2h，然后回流状态下搅拌6小时。减压蒸馏除去反应溶剂，粗产品经柱层析纯化得到淡黄色固体，50.6mg，产率：30.2％。
结构确认谱图数据，1H NMR(300MHz,DMSO)δ10.60(s,1H),8.09(s,1H),8.01(d,J＝6.6Hz,1H),7.74(d,J＝9.1Hz,1H),7.68–7.48(m,4H),7.24(dd,J＝9.1,2.3Hz,1H),7.13(d,J＝6.5Hz,1H),6.91(d,J＝1.9Hz,1H),6.49(d,J＝8.7Hz,2H),6.34(m,4H),3.33(s,8H),3.03(s,6H),1.07(t,J＝6.9Hz,12H).13C NMR(75MHz,DMSO)δ164.53,153.15,149.51,148.55,136.31,135.86,132.92,129.73,129.65,128.74,128.20,125.34,125.11,124.74,124.35,123.74,116.50,107.60,104.81,103.33,96.99,63.45,59.71,43.60,12.40.HRMS:m/z[M+H]+calcd for C41H44N5O2S:670.3216,found:670.3261；m/z[M+2H]2+calcd forC41H45N5O2S:335.6647,found:335.6663。
上述荧光探针的制备及检测次氯酸的机理如下式所示：
实施例2
向装有5μM该次氯酸探针的10ml容量瓶中，用微量进样器分别加入10当量的t-BuOOH,t-BuOO·,H2O2,HO·,NO,NOOO-,1O2,-O2,和1当量的HOCl。作用1h后进行荧光测试。
结果表明，该探针只对HOCl有较好的响应，对次氯酸比其它活性氧有较高的选择性。见图2，3。
实施例3
细胞内荧光成像测试：
将RAW264.7细胞转移到小的玻璃瓶中孵化24h后，一组用10μM该探针孵化4h，然后用PBS洗涤三次进行共聚焦细胞成像检测。另一组用1μg mL-1的LPS孵化12h，然后用PBS洗涤三次，再用10μM该探针孵化4h，接着用PBS洗涤三次进行共聚焦细胞成像检测。所用激发波长为405nm，蓝色通道收集波长为405-550nm，红色通道收集波长为550-650nm。见图4。
实施例4
向装有5μM该次氯酸探针的10ml容量瓶中，用微量进样器分别加入不同当量的HOCl，作用1h后进行荧光测试。
结果表明，585nm处荧光强度与440nm处荧光强度的比值相对次氯酸浓度在一定范围内呈线性关系。见图5。

Claims (2)

1.一种次氯酸荧光比率探针，其特征在于：所述荧光探针的化学结构式如式(I)所示，
2.权利要求1所述的次氯酸荧光比率探针在检测含次氯酸的溶液中的应用。
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CN102827176B (zh) * 2012-09-14 2015-02-11 天津市亚马逊科技发展有限公司 一种荧光素分子探针材料的制备方法与应用
CN102994072A (zh) * 2012-12-12 2013-03-27 东华大学 一种检测次氯酸根的荧光探针及其制备方法
CN103880853B (zh) * 2014-03-12 2016-06-29 西安交通大学 罗丹明6g酰肼衍生物、其制备方法、应用及作为荧光探针对次氯酸进行荧光分析的方法

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