CN113563279B - 一种检测硝基还原酶的双光子荧光探针及其制备方法和用途 - Google Patents
一种检测硝基还原酶的双光子荧光探针及其制备方法和用途 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及一种检测硝基还原酶的双光子荧光探针及其制备方法和用途,以实现双光子共聚焦成像检测缺氧和常氧下细胞线粒体内硝基还原酶的变化,具有高选择性、高灵敏度、低生物毒性的优点。
背景技术
缺氧是一种组织供氧不足的情况,大量的实验表明,缺氧可以由肿瘤造成,一是肿瘤通过减少血液的供应从而减少了氧气的运输,二是肿瘤细胞的耗氧量较大,从而导致缺氧。临床研究表明,肿瘤的缺氧情况与其生长进展有着十分密切的关系,缺氧会使肿瘤更加容易发生转移和恶化,因此开发一种检测细胞缺氧的新方法对临床研究十分有意义。缺氧会使得生物体内发生很多还原反应,导致大量还原型酶的积累包括醛氧化酶、酯酰脱氢酶和硝基还原酶(nitroreductase,NTR)。硝基还原酶在近些年被大量的研究,是理想的缺氧标记物,具有很高的选择性和代表性。在缺氧的情况下,硝基还原酶可以在烟酰胺腺嘌呤二核苷酸(NADH)的存在下,将NADH作为电子供体还原含有硝基的化合物,并且硝基还原酶含量会异常地增加,所以硝基还原酶与生物体内缺氧的水平有着密切的关系。
荧光探针具有反应迅速,高灵敏性,高特异性和优良的生物相容性在硝基还原酶的检测中得到了广泛的应用。近些年来,许多探针在检测硝基还原酶方面取得了重大进展。但是这类探针在细胞成像方面的应用较少,或者无法定位线粒体,且不具有双光子性质。双光子共聚焦成像由于能够提供更深的穿透力和高分辨成像等优势已成为一种在亚细胞水平上对分析物进行可视化监测的有效方法。因此设计出一种双光子荧光探针,实现活细胞线粒体内硝基还原酶的灵敏检测显得十分重要。
发明内容
本发明旨在提供一种检测硝基还原酶的双光子荧光探针及其制备方法和用途,所要解决的技术问题是通过分子设计得到一种可以特异性识别硝基还原酶的有机小分子结构,以实现通过双光子共聚焦荧光成像监测活细胞线粒体内硝基还原酶含量的变化,具有选择性高、灵敏度高、光稳定性好等优点,细胞毒性测试表明本发明荧光探针的细胞相容性良好。
本发明双光子荧光探针,简记为NO2-1,其结构式如下所示:
本发明双光子荧光探针的制备方法,包括如下步骤:
步骤1:将Compound1(0.5g,1.17mmol)和对羟基苯甲醛(1.25g,10.23mmol)放入史莱克瓶中,在无氧的条件下加入乙醇(10mL)和哌啶(2滴),60℃加热12h,等到反应完全后,冷却至室温,用石油醚醚抽滤后,真空干燥,得到灰红色固体OH-1,0.4g,产率59.25%。
步骤2:在45℃下,将OH-1(1g,2.53mmol),Cs2CO3(1.5g,3.06mmol)和4-硝基苄基溴苯(0.82g,3.8mmol)放入到史莱克瓶中,并在N2氛围下,滴加10ml的DMF,在45℃下搅拌4h,冷却蒸掉溶剂,用石油醚抽滤,得到的固体通过色谱柱(二氯甲烷:石油醚=2:1)的方法提纯,减压蒸馏后真空干燥,得到淡黄色粉末NO2-1,0.6g,产率64%。
本发明双光子荧光探针的合成路线如下所示:
本发明双光子荧光探针的用途,是用于制备检测活细胞线粒体内的硝基还原酶变化时的检测试剂。检测方法包括如下:
将本发明NO2-1溶于DMSO(5mL)中制得2mM的母液,取15μL NO2-1母液加入到3mL不同浓度的硝基还原酶的测试液中。NO2-1的单光子激发波长为500nm,检测520-640nm范围内的荧光光谱变化,可以明显观察到从硝基还原酶含量0μg/mL到7μg/mL,在558nm左右处荧光强度增强了80倍,硝基还原酶浓度和荧光强度呈现良好的线性关系(R2=0.98)。
为了测试NO2-1对于硝基还原酶的反应时间,取15μLNO2-1的母液加入到3mL不同浓度的硝基还原酶溶液中,得到最终的测试液。激发波长同上,检测520-640nm范围内的荧光光谱变化,可以观察到当时间在30min时,NO2-1的荧光强度基本达到稳定。探针对硝基还原酶的特异性响应能力是评价荧光探针的重要指标,采用相同浓度的测试液测试了NO2-1在不同阴阳离子及生物成分的荧光强度,发现NO2-1只在硝基还原酶和NADH共同作用下才能表现出较强的荧光变化。还探究了NO2-1在缺氧的HeLa细胞和常氧的HeLa细胞中的检测硝基还原酶,因为在细胞缺氧的过程中,细胞内硝基还原酶的含量会发生明显变化,这会影响探针的荧光强度变化。
附图说明
图1是探针NO2-1响应硝基还原酶的反应机理示意图。
图2是探针NO2-1响应硝基还原酶的紫外吸收光谱验证图。
图3是探针NO2-1响应硝基还原酶的荧光发射光谱验证图。
图4是探针NO2-1在不同浓度的硝基还原酶溶液的(a)荧光光谱图,(b)和(c)荧光强度与NTR浓度的关系图和线性关系图。
图5是探针NO2-1在(a)不同浓度(0μg/mL、0.5μg/mL、1μg/mL和3μg/mL)的硝基还原酶测试液中的反应时间图,(b)探针NO2-1与不同阴阳离子及生物成分的荧光光谱图。
图6是探针NO2-1与硝基还原酶反应前后溶液中的双光子吸收截面。
图7是探针NO2-1的细胞毒性测试图。
图8是探针NO2-1(10μM)和0.5μM商用线粒体探针(Mito-Tracker Green FM)同时共染HeLa细胞的共聚焦荧光成像图。探究NO2-1的线粒体靶向能力。
图9是探针NO2-1染色HeLa细胞的共聚焦成像。(a)图为NO2-1在常氧下染色HeLa细胞,(b)图为NO2-1在缺氧条件下染色HeLa细胞
具体实施方式
下面通过实施例对本发明做进一步说明。
实施例1:NO2-1的合成
在45℃下,将OH-1(1g,2.53mmol),Cs2CO3(1.5g,3.06mmol)和4-硝基苄基溴苯(0.82g,3.8mmol)放入到史莱克瓶中,并在N2氛围下,滴加10ml的DMF。在45℃下搅拌4h,冷却蒸掉溶剂,用石油醚抽滤,得到的固体通过柱层析(二氯甲烷:石油醚=2:1)的方法提纯,减压蒸馏后真空干燥,得到淡黄色粉末NO2-1 0.6g,产率64%。1H NMR(400MHz,DMSO-d6)δ8.26(d,2H)8.23(d,2H),8.13(d,2H),8.03(d,2H),7.86(m,2H),7.71(t,2H),7.21(d,2H),5.39(s,2H),4.03(s,3H),13C NMR(100MHz,CDCl3)δ171.92,161.34,148.31,147.01,144.2,141.89,132.03,129.18,128.32,127.50,127.23,124.09,123.56,116.63,115.54,111.54,68.31,36.24.ESI-MS:m/z 403.1099([M-I]-calcd 403.3842)。
实施例2:NO2-1的光谱测试
将0.0053g的NO2-1加入到5ml的DMSO配成浓度为2×10-3mol/L的测试母液,并用移液枪移取15μL的母液到不同浓度的硝基还原酶溶液中(0.125μg/ml,0.25μg/ml,0.5μg/ml,1μg/ml,2μg/ml,3μg/ml,4μg/ml,5μg/ml,7μg/ml)。随后,对上述测试也进行荧光测试,随着硝基还原酶的浓度从0.125μg/mL增加到7μg/mL,探针的荧光强度也在不断增加(图4a),并在3μg/mL之后,探针的荧光强度增长幅度较小,并且如图4b和4c所示硝基还原酶浓度与探针的最大荧光强度有着良好的线性关系(R2=0.98)。
实施例3:NO2-1的反应机理测试
为了研究NO2-1的反应机理(图1),随后对NO2-1进行紫外测试。如图2所示,NO2-1在400nm左右处出现紫外吸收峰,在加入硝基还原酶和NADH后发现在500nm左右处出现新的紫外吸收峰,并且与OH-1的紫外吸收峰位置相同,从紫外吸收光谱可以看出,NO2-1在硝基还原酶和NADH的作用下被还原成OH-1。随后从荧光光谱来验证,NO2-1的响应机理。分别测试了在硝基还原酶存在下、在NAHD存在下和同时存在硝基还原酶和NADH的条件下反应后探针NO2-1的荧光强度的变化。如图3所示,NO2-1与硝基还原酶反应后出现了微弱的荧光信号,而探针NO2-1与NADH作用后几乎没有荧光,但是在NADH作用下,探针NO2-1与硝基还原酶反应后在558nm左右处出现了很强的荧光信号并且相比于没有NADH存在情况下探针的荧光强度增加了80多倍,这是由于NADH在该反应过程中提供能量,由此也可以看出探针NO2-1与硝基还原酶的反应对NADH的依赖性。
实施例4:细胞毒性测试
在探针NO2-1进行细胞成像的应用之前,需要对探针的毒性进行测试,采用MTT法进行实验。分别加入0μM、5μM、10μM、20μM的探针NO2-1培养HeLa细胞24h,发现细胞存活率始终保持在85%以上(图7)。因而探针NO2-1对于HeLa细胞的毒性较低,可以进行生物应用。
实施例5:NO2-1的灵敏性测试和干扰性测试
为了研究NO2-1对响应硝基还原酶的时间,我们分别在浓度为0.5μg/mL、1μg/mL、3μg/mL的响应硝基还原酶溶液中对探针进行实时荧光测试。如图5a所示,随着时间的延长,探针的荧光强度逐渐增强,并且硝基还原酶与NO2-1的反应体系约在30min就达到了平稳并且NO2-1可以稳定存在反应的体系当中。随后为了验证NO2-1对硝基还原酶的选择性,防止其他物质对NO2-1的干扰,我们测试了在PH=7.4的PBS缓冲溶液中加入不同的阴阳离子及生物成分(Ca2+、Cl-、Mg2+、Na+、Cl-、SO4 2-、ClO-、SO3 2-、K+、HSO3 -、Cys、GSH、H2O2、Glucose、S2-、NADH),如图5b所示这些干扰物质并没有引起NO2-1的荧光强度的变化。
实施例6:NO2-1的双光子性能测试
探针NO2-1在PBS溶剂中时,没有明显的双光子吸收。当探针NO2-1与硝基还原酶和NADH反应后,其有效双光子吸收截面(Φδ)在820nm出现最大,为82GM(图6)。证明探针NO2-1有潜力用于细胞线粒体内硝基还原酶的双光子共聚焦荧光成像。
实施例7:细胞定位测试
为了研究NO2-1的线粒体定位性能,这里使用线粒体的商业染料(Mito-TrackerGreen FM,0.5μM)与NO2-1在HeLa细胞中进行共定位研究。结果表明NO2-1的红色通道(λem=500nm,λex=560nm)和Mito-Tracker Green FM(λem=510±20nm,λex=480nm)的荧光图像重叠良好,并且NO2-1与线粒体商业染料(Mito-Tracker Green FM)的Pearson共定位系数计算为0.91(图8)。这些结果表明,NO2-1可以很好地定位于活细胞的线粒体中。
实施例8:NO2-1检测硝基还原酶来检测细胞缺氧
为了研究NO2-1在细胞中检测硝基还原酶的能力,我们把NO2-1(10μM)分别加入2组HeLa细胞中培养0.5h,一组为缺氧环境下与NO2-1培养,另一组为常氧下与NO2-1培养。之后用500nm的激发光源来进行共聚焦成像,检测荧光强度。如图9所示,NO2-1在常氧条件下与HeLa细胞孵育会发出微弱的红色荧光,而在缺氧条件下与HeLa细胞孵育后会发出很强红色荧光。表明随着氧气含量的下降,HeLa细胞的硝基还原酶水平升高,探针荧光强度增强,说明了探针NO2-1可以检测HeLa细胞中的硝基还原酶,并且NO2-1具有通过检测硝基还原酶来检验细胞是否缺氧的潜力。
Claims (3)
3.一种权利要求1所述的双光子荧光探针的用途,其特征在于:
所述双光子荧光探针用于制备监测活细胞线粒体内的硝基还原酶的检测试剂;所述检测试剂在细胞缺氧过程中对于线粒体内硝基还原酶的变化具有荧光响应能力。
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