CN112574252B - 基于试卤灵染料专一响应onoo-的荧光探针、制备方法及应用 - Google Patents
基于试卤灵染料专一响应onoo-的荧光探针、制备方法及应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及荧光探针领域,具体涉及一种基于试卤灵染料专一响应ONOO‒的荧光探针、制备及应用。
背景技术
作为一种寿命短且反应活性高的活性氮物种(RNS),过氧亚硝酸盐(ONOO‒)是由一氧化氮(NO)和超氧阴离子(O2˙‒)通过自由扩散反应在线粒体中产生。由于其具有极强的跨膜弥散性,可通过阴离子通道穿透各种生物膜作用于酶、蛋白质以及DNA等大分子物质,参与机体的各种生理和病理过程。到目前为止,越来越多的证据表明,过量的ONOO‒可能会引起多种疾病,例如炎症,神经退行性疾病,阿尔茨海默氏病,帕金森氏病,心血管,缺血性再灌注损伤和癌症。因此,开发一种检测细胞ONOO‒的方法并探讨其在疾病诊断和各种病理生理特性中的作用至关重要。
迄今为止,已经探索出了多种方法用于ONOO‒的检测,其中包括电化学分析、免疫组织化学、光致发光和化学发光等方法。在这些方法中荧光探针法具有响应速度快,灵敏度高,可视化能力强和生物创伤性低等优点,在化学分析、生物分析以及医学研究中得到了广泛的应用。近年来,这些化学反应型的荧光探针主要基于以下几种机理:硼酸酯的氧化,膦酰/次膦酸酯的氧化,酮的氧化,硒的氧化还原反应,氧化性N-脱芳基化和其他反应策略等。尽管已报道的荧光探针能够实现了对ONOO‒的检测,但是仍然存在一些局限性,例如选择性低(某些ONOO‒探针也可能对H2O2和NaClO产生响应),荧光发射波长较短(在紫外可见光区)且在生物体内成像应用匮乏等,所以构建对ONOO‒具有高特异性的新型长波长荧光探针并实现对细胞和活体成像的研究仍迫在眉睫。试卤灵染料因其具有荧光量子产率高,发射波长长,生物相容性好等优点而被广泛关注。因此,基于试卤灵荧光染料专一性响应生物细胞内ONOO‒的长波长荧光探针,并实现在活体层面的应用具有重要意义。
发明内容
本发明提出了一种基于试卤灵染料专一响应ONOO‒的荧光探针、制备方法及应用,该探针制备方法操作方便,原料易得,能够实现细胞以及小鼠活体内ONOO‒的专一性检测。
实现本发明的技术方案是:
一种基于试卤灵染料专一响应ONOO‒的荧光探针,化学分子式为:C31H12NO5P,命名为RFP,探针的结构式为:
所述的荧光探针的制备方法,步骤如下:
(1)N2保护、冰水浴下,将对羟基苯甲醇溶于混合溶剂中,再加入二苯基次膦酰氯,室温反应,冰水淬灭,二氯甲烷萃取,柱层析分离得到化合物1;
(2)步骤(1)制得的化合物1溶于二氯甲烷中,加入四溴化碳和三苯基膦,室温搅拌,然后减压去除溶剂,柱层析分离得到化合物2;
(3)先将试卤灵和碳酸钾溶于N,N-二甲基甲酰胺中,再加入步骤(2)所得化合物2,室温反应,冰水淬灭,二氯甲烷萃取,柱层析分离得到荧光探针。
所述步骤(1)中混合溶剂为三乙胺加入到四氢呋喃溶剂中混合得到,对羟基苯甲醇和二苯基次磷酰氯按照当量为1:(1~2)的比例溶于混合溶剂中。
所述步骤(2)中化合物1、四溴化碳和三苯基膦按照当量为1:1:(1~2)比例添加,室温搅拌1~2h。
所述步骤(3)中试卤灵、化合物2和碳酸钾按照当量为1:1:(1~2)比例添加,室温搅拌1~2h。
制备的荧光探针在制备细胞/活体中在专一检测ONOO‒试剂中的应用。
荧光探针的合成路线如下:
本发明基于试卤灵染料用于专一检测ONOO‒的荧光探针,该探针在溶液测试中,利用紫外和荧光光谱可判断出探针与ONOO‒反应时间,浓度依赖性关系;在通过选择性和抗干扰能力测试观察出,探针可专一性检测ONOO‒,不与活性氧化物反应且抗干扰能力强;并且探针的pH稳定性强,细胞毒性小。还可通过荧光成像技术达到对RAW264.7细胞和小鼠活体中的ONOO‒检测的目的。
本发明的有益效果是:
(1)本发明一种利用试卤灵染料专一性检测ONOO‒的荧光探针,合成方法简单,操作方便;
(2)本发明的检测方法可以实现ONOO‒专一性检测,而且不受其他活性氮和活性氧化物类的干扰;
(3)本发明检测信号明显,为长波长增强型荧光探针。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为荧光探针RFP的核磁氢谱图;
图2为荧光探针RFP的核磁碳谱图;
图3为荧光探针RFP与ONOO‒作用的时间紫外变化;
图4为荧光探针RFP与ONOO‒作用的时间荧光变化;
图5为荧光探针RFP测定ONOO‒浓度滴定实验荧光变化;
图6为荧光最强发射波长590 nm与ONOO‒的浓度线性拟合图;
图7为常见氨基酸对探针RFP检测ONOO‒的荧光选择性;
图8为常见氨基酸对探针RFP检测ONOO‒的荧光干扰性;
图9为荧光探针RFP和探针加ONOO‒在不同pH缓冲溶液中最大荧光强度变化图;
图10为探针RFP检测ONOO‒的细胞毒性;
图11为荧光探针RFP检测ONOO‒RAW264.7细胞成像图;
图12为荧光探针RFP检测ONOO‒小鼠活体成像。
具体实施方式
下面将结合本发明实施例,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有付出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
探针的制备步骤如下:
(1)化合物1的制备
N2保护冰水浴下,将对羟基苯甲醇溶于混合溶剂中,再加入二苯基次膦酰氯,室温反应1~2h,冰水淬灭,二氯甲烷萃取,柱层析分离得到化合物1;
(2)化合物2的制备
步骤(1)制得的化合物1溶于二氯甲烷中,再将四溴化碳和三苯基膦按照当量1:1~2的比例加入上述体系中,室温搅拌反应1~2小时,然后减压去除溶剂,柱层析分离得到化合物2;
(3)探针RFP的制备
先将试卤灵和碳酸钾按照当量1:2的比例溶于N,N-二甲基甲酰胺中,再将步骤(2)所得化合物2按照1~1.5当量溶于N,N-二甲基甲酰胺中加入上述体系,室温搅拌反应1~2h,冰水淬灭,二氯甲烷萃取,柱层析分离得到探针RFP;
1H NMR (400 MHz, CDCl3) δ 7.90 (dd, J = 12.6, 7.0 Hz, 4H), 7.70 (d, J= 8.9 Hz, 1H), 7.55 (q, J = 6.9, 6.4 Hz, 2H), 7.49 (dd, J = 7.6, 3.7 Hz, 4H),7.42 (d, J = 9.8 Hz, 1H), 7.32 (d, J = 8.6 Hz, 2H), 7.00 – 6.94 (m, 1H), 6.84– 6.82 (m, 1H), 6.32 (d, J = 1.9 Hz, 1H), 5.08 (s, 1H). 13C NMR (101 MHz,CDCl3) δ 186.31, 162.48, 149.80, 145.59, 134.71, 134.28, 132.59, 131.86,131.73, 129.01, 128.61, 121.11, 114.19, 106.78, 101.08, 77.03, 76.71, 70.25,64.60.
实施例2
探针的制备步骤如下:
(1)化合物1的制备
N2保护冰水浴下,三乙胺加入到四氢呋喃溶剂中混合得到混合溶剂,将对羟基苯甲醇酸肟酯与二苯基次膦酰氯按照当量为1:1.5的比例溶于混合溶剂中,室温反应2小时,冰水淬灭,二氯甲烷萃取,柱层析分离得到化合物1;
(2)化合物2的制备
将化合物1溶于二氯甲烷中,将四溴化碳和三苯基膦按照当量为1:1.5的比例加入,25℃搅拌1-1.5h,然后减压去除溶剂,柱层析分离得到化合物2;
(3)化合物3的制备
先将试卤灵和碳酸钾按照当量1:2的比例溶于N,N-二甲基甲酰胺中,再将步骤(2)所得化合物2按照1当量溶于N,N-二甲基甲酰胺中加入上述体系中,室温搅拌反应1~2h,冰水淬灭,二氯甲烷萃取,柱层析分离得到探针RFP。
实施例3
探针的制备步骤如下:
(1)化合物1的制备
N2保护冰水浴下,三乙胺加入到四氢呋喃溶剂中混合得到混合溶剂,将对羟基苯甲醇与二苯基次膦酰氯按照当量为1:2的比例溶于混合溶剂中,室温反应2小时,冰水淬灭,二氯甲烷萃取,柱层析分离得到化合物1;
(2)化合物2的制备
将化合物1溶于二氯甲烷中,将四溴化碳和三苯基膦按照当量为1:2的比例加入,25℃搅拌1-1.5h,然后减压去除溶剂,柱层析分离得到化合物2;
(3)化合物3的制备
先将试卤灵和碳酸钾按照当量1:2的比例溶于N,N-二甲基甲酰胺中,再将步骤(2)所得化合物2按照1.5当量溶于N,N-二甲基甲酰胺中加入上述体系中,室温搅拌反应1~2h,冰水淬灭,二氯甲烷萃取,柱层析分离得到探针RFP。
利用实施例1中的探针进行性能测试:
(1)溶液中检测ONOO‒的荧光探针RFP的反应时间测试
用二甲基亚砜(DMSO)配制1 mM RFP的荧光探针储备液;探针RFP(10 µM)与ONOO‒(100 µM)在溶液体系为PBS缓冲液(3Mm CTAB,pH=7.4)中进行反应,如图3所示,从紫外吸收图谱观察RFP在469 nm处的吸收峰伴随着时间的推移逐渐下降,并在20 min内下降到最低;而新生成的585 nm处紫外吸收峰逐渐增大至最高值,反应前后的吸收峰发生了116 nm的红移。同时,在荧光图谱上可观察到,在用525 nm的激发下,590 nm处出现一个新的荧光发射峰并增强约25倍(图4所示)。
(2)溶液中检测ONOO‒的荧光探针RFP的浓度滴定测试以及浓度线性关系
在浓度滴定实验中,发现随着ONOO‒的浓度逐渐增加,590 nm处的荧光峰也逐渐增强,其荧光强度在ONOO‒浓度至100 µM时达到最大。(见图5所示)。
以ONOO‒浓度为横坐标,探针RFP在590 nm处的荧光强度为纵坐标,绘制图并进行线性拟合,得到该探针的线性回归方程为:Y = 26.05 X + 0.445,线性相关系数R2 =0.992并计算出检测限为238 nM。(见图6所示)。
(3)干扰性和抗干扰性离子实验
在不同的荧光比色皿中,分别加入4 mL PBS缓冲液(3Mm CTAB,pH=7.4)和40 μL荧光探针储备液,如图7所示,当探针RFP分别加入上述选择的活性氮以及活性氧化物分析物种(100 µM)后(0: probe blank, 1:˙O2, 2: 1O2, 3:˙OH, 4:˙OtBu, 5: H2O2, 6: OCl‒,7: H2S, 8: HSO3 ‒, 9: SO4 2‒, 10: SO3 2‒, 11: CO3 2‒, 12: HCO3 ‒, 13: NO2 ‒, 14: NO, 15:K+, 16: Na+, 17: Ca+,18: Cys, 19: Hcy, 20: GSH, 21: ONOO‒),探针RFP可以对ONOO‒进行专一性识别,并在590 nm处出现明亮的红色荧光,而跟其它种类分析物反应后没有荧光出现,因此,探针可以实现专一性响应ONOO‒。当RFP(10 µM)在分别加入上述分析物(0:O2 .‒,1:1O2, 2:˙OH, 3:˙OtBu, 4:H2O2, 5:OCl‒, 6: H2S, 7:HSO3 ‒, 8:SO4 2‒, 9:SO3 2‒, 10:CO3 2‒,11:HCO3 ‒, 12:NO2 ‒, 13:NO, 14:K+, 15:Na+, 16:Ca+, 17:Cys, 18:Hcy, 19:GSH, and20:ONOO‒)后再加入100 µM ONOO‒反应60分钟后,可以看出ONOO‒在与各种分析物共存情况下,RFP在复杂的溶液体系内依然能够专一性检测到ONOO‒,且荧光程度较强。实验证明,RFP能够在响应ONOO‒时不受其它物质的干扰(见图8所示)。
(4)pH值的响应性实验
将探针RFP溶于二甲基亚砜中得到10mM探针母液,配置pH为6.0、6.4、6.8、7.2、7.6、8.0的溶液,对探针以及探针和ONOO‒反应后荧光强度的变化进行了研究。
结果如图9所示,探针荧光强度在pH值为6.0‒8.0的溶液中基本保持不变;在加入ONOO‒后,随着pH值的不断升高,其位于590 nm处荧光逐渐增强,且在生理范围7.2‒8.0的pH值下,荧光强度最强。实验证明探针RFP能够适应生物体内的pH环境。
(5)MTT细胞毒性实验
利用探针RFP对RAW264.7细胞的MTT细胞毒性实验,结果如图10所示。RAW264.7细胞用含有不同浓度的探针(1.25, 2.5, 5.0, 10, 20 μM)培养液孵育后,计算细胞的存活百分比。结果如图10所示,低浓度下细胞存活率可高达90%,探针几乎没有细胞毒性。
(6)检测ONOO‒的荧光探针对RAW264.7细胞内ONOO‒的检测性能测试
检测ONOO‒的荧光探针RFP对小鼠单核巨噬细胞(RAW264.7)细胞中ONOO‒的荧光共聚焦成像。结果如图11所示,(a)明场和(b)RFP(10 μM)和RAW264.7细胞一起孵育30分钟后,再用PBS缓冲液洗涤三次后的显微镜下荧光成像;(d)明场和(e)RAW264.7细胞先用脂多糖(LPS)和γ-干扰素(INF-γ)(LPS和INF-γ联用可刺激小鼠单核巨噬细胞产生内源性的ONOO‒)孵育30分钟,用PBS缓冲液洗涤三次,RFP(10 μM)再与细胞共孵育30分钟;(c)和(f)为共聚焦荧光与明场的重叠。通过共聚焦红色通道观察,检测ONOO‒的荧光探针在RAW264.7细胞中产生明亮的红色荧光;对照组实验中,细胞与RFP共同孵育,由成像实验结果可以观察红色通道荧光响应微弱。由此说明探针可以检测细胞内源性ONOO‒。该结果也表明,本发明的荧光探针具有检测RAW264.7细胞内ONOO‒的良好应用前景。
(7)检测ONOO‒的荧光探针对小鼠活体内ONOO‒的检测性能测试
通过小鼠胫跗关节皮下注射方式将LPS(200 μL,1 mg/mL,生理盐水)注射到裸鼠左侧胫跗关节(左脚踝)孵育12小时作为实验组;将等量生理盐水注射到裸鼠右侧胫跗关节(右脚踝)孵育12小时作为对照组,将探针RFP(50 μL,500 μM,DMSO/生理盐水=1/9)原位注射到裸鼠左、右侧胫跗关节(左脚踝和右脚踝)。如图12所示,注射前/注射后进行成像系统内荧光监测,通过共聚焦红色通道观察,检测ONOO‒的荧光探针在LPS刺激的小鼠发炎模型左腿中产生明亮的红色荧光,并在15分钟时即可达到最大荧光值;对照组实验中,仅用生理盐水和探针孵育的右腿成像实验结果中可以观察红色通道荧光响应微弱。由此说明探针可以快速地原位监测小鼠活体内源性ONOO‒的产生。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (7)
2.权利要求1所述的荧光探针的制备方法,其特征在于,步骤如下:
(1)N2保护、冰水浴下,将对羟基苯甲醇溶于混合溶剂中,再加入二苯基次膦酰氯,室温反应,冰水淬灭,二氯甲烷萃取,柱层析分离得到化合物1;
(2)步骤(1)制得的化合物1溶于二氯甲烷中,加入四溴化碳和三苯基膦,室温搅拌,然后减压去除溶剂,柱层析分离得到化合物2;
(3)先将试卤灵和碳酸钾溶于N,N-二甲基甲酰胺中,再加入步骤(2)所得化合物2,室温反应,冰水淬灭,二氯甲烷萃取,柱层析分离得到荧光探针。
3.根据权利要求2所述的荧光探针的制备方法,其特征在于:所述步骤(1)中混合溶剂为三乙胺加入到四氢呋喃溶剂中混合得到,对羟基苯甲醇和二苯基次磷酰氯按照当量为1:(1~2)的比例溶于混合溶剂中。
4.根据权利要求2所述的荧光探针的制备方法,其特征在于:所述步骤(2)中化合物1、四溴化碳和三苯基膦按照当量为1:1:(1~2)比例添加,室温搅拌1~2h。
5.根据权利要求2所述的荧光探针的制备方法,其特征在于:所述步骤(3)中试卤灵、化合物2和碳酸钾按照当量为1:1:(1~2)比例添加,室温下搅拌1~2h。
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