CN109694396A - 一种双光子比率荧光探针在检测β-半乳糖苷酶中的应用 - Google Patents
一种双光子比率荧光探针在检测β-半乳糖苷酶中的应用 Download PDFInfo
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Abstract
本发明公开了一种双光子比率型荧光探针精确测量肿瘤细胞中的β‑半乳糖苷酶的制备方法和应用。该探针基于荧光共振能量转移的双光子萘衍生物骨架与对甲氨基酚荧光团连接，加上β‑半乳糖苷酶可活化单元构成。利用酶促反应切割保护基团以产生游离酚，导致荧光发射信号比例性的变化。优势在于，能量供体与受体之间的荧光光谱距离更大，光谱之间的干扰更小，灵敏度更高。为临床用于检测活体内原发性卵巢癌的重要标志酶提供了潜在工具。
Description
技术领域
本发明涉及小分子荧光探针鉴定异常的生物酶活性在癌细胞中的位置和表达水平，更具体地说是一种基于荧光共振能量转移的双光子萘衍生物荧光探针对原发性卵巢癌的重要标志物β-半乳糖苷酶（β-gal）的检测。
背景技术
当前，癌症一经发现基本都被判为晚期。在与癌症相关的死亡中，如果可以早期诊断，大约30％的人已经得救。癌症和癌症靶向治疗的早期准确诊断对于增加治愈的机会和提高癌症存活率具有重要意义。特别地，癌症相关生物标志物的临床测量对于癌症诊断，治疗和管理具有重要意义。在许多潜在的生物标志物中，酶在许多生理学、病理学和药理学过程中的重要作用受到了强烈关注。许多研究表明，某些酶的异常活性与各种癌症直接相关。典型的β-半乳糖苷酶（β-gal）在原发性卵巢癌中的酶活性明显增强。因此，鉴定生物标志酶在活癌细胞中的位置和表达水平成为早期癌症的诊断和监测治疗的关键，也是当前该研究领域亟需解决的问题之一。
目前已经应用了几种成像技术来检测或成像酶，例如磁共振成像（MRI），核成像（包括单光子发射计算机断层扫描（SPECT）和正电子发射断层扫描（PET））以及荧光成像。每一种技术都是不可替代的，具有自己的特征灵敏度，深度穿透和空间分辨率。例如，MRI对活癌细胞中低丰度的酶缺乏足够的特异性和敏感性。 SPECT和PET具有高灵敏度和断层扫描能力，但空间分辨率较差（1-2 mm）。其他一些检测方法，如使用标记抗体或蛋白质组学方法，可用于检测和跟踪固定细胞中的酶或在体外进行检测，但未能提供活细胞中酶的实时信息。为了克服这些缺点，基于小分子荧光探针的荧光成像已被广泛用于可视化和量化活细胞动态过程中，其具有高灵敏度，非破坏性快速分析和实时检测的优势。
大多数小分子酶荧光探针是基于与酶活性位点的特异性相互作用而设计的，包括催化位点和结合位点。酶具有区分各种底物之间微小差异和催化底物特异性反应的能力，通过将最佳底物基团连接到荧光团来产生基于底物的探针，荧光团在活性酶转化时其光谱性质相应改变。在基于FRET的探针中，荧光团和底物通过酶可切割的接头在一定距离内彼此连接，以产生比率型探针，其在底物通过特异性酶促反应释放后恢复荧光。总之，理想的小分子酶探针在靶酶催化的化学修饰后应经历可检测的荧光变化，本文中，设计并合成了一种新的双光子有效比率荧光化合物（TPR-βgal），用于检测生物系统中β-半乳糖苷酶（β-gal）的活性。
发明内容
本发明要解决的技术问题：一是提供一种原料廉价易得，合成步骤简单，反应条件温和的荧光探针合成方法。二是提供了一种具有大斯托克斯位移、检测速度快、选择性强、高特异性、灵敏度高、低细胞毒性，检测生物样品、活细胞和动物中的β-gal的新型比率双光子荧光探针。
为了解决上述技术问题，采取的技术方案如下，一种特异性识别β-gal的比率双光子荧光探针，具有如下分子结构式：
上述β-半乳糖苷酶荧光探针的制备方法如下：
1）将1 eq的Np-Rhod染料与1.25 eq碘化钠，溶解于 N, N-二甲基甲酰胺中，然后向混合液中加入0.6 eq的2,3,4,6-四乙酰氧基-alpha-D-吡喃糖溴化物，室温下搅拌4h, 后升温至80℃回流12h。用TCL板监测反应，反应完全后，用二氯甲烷萃取，无水硫酸镁干燥，减压蒸发，并用硅胶柱进行分离，得到化合物TPR-βgalAc，其结构式如下：
2）向1 eqTPR-βgalAc的无水甲醇溶液中滴加2 eq甲醇钠，使反应混合物在室温下搅拌5h。用TCL板监测反应，利用酸将pH调节至中性，反应完全后，用二氯甲烷萃取溶液。用无水硫酸镁干燥，有机相减压蒸发。通过硅胶色谱纯化残余物，得到目标探针化合物TPR-βgal。
所述步骤1）中硅胶柱分离洗脱剂配比为甲醇:二氯甲烷=1:100。
所述步骤1）中Np-Rhod染料与2,3,4,6-四乙酰氧基-alpha-D-吡喃糖溴化物的摩尔比值为1:0.5-0.7，2,3,4,6-四乙酰氧基-alpha-D-吡喃糖溴化物不能过量，会产生过多杂质点。
所述步骤2）中硅胶柱分离洗脱剂配比为甲醇:二氯甲烷=1:80。
所述步骤2）中中间产物TPR-βgalAc与甲醇钠的摩尔比值为1:2-2.5, 其中注意保护基团的活化应将pH调节至中性，有利于提高探针的产率。
本发明的优点:
本发明的荧光探针，用肉眼就可以观察到溶液颜色的变化，伴随着紫外灯下同样可以观察到荧光颜色变化，是一种具有生色传感功能的荧光探针。
本发明的荧光探针通过比率荧光成像可以用作区分正常细胞和卵巢癌细胞。主要用的活细胞为Hela细胞株。由lacZ基因编码大肠杆菌转染的卵巢癌细胞。
本发明的荧光探针，其本身不发射荧光，在与β-gal作用后，反应体系在542 nm处出现红移荧光峰，同时，448 nm处的荧光强度急剧下降，提供可视化的比率荧光信号（I541 nm / I448 nm），在与β-gal反应前后增加了54倍。
本发明的荧光探针对β-半乳糖苷酶的检测表现出很高的灵敏度，具有高光稳定性。荧光强度比（I541 nm / I448 nm）在反应前10分钟内迅速增加，在约70分钟内获得最大强度。探针溶液荧光强度与β-gal浓度有很好的线性关系。检出限为9.11×10-3 U / mL。
本发明pH和温度的适用范围较宽，有利于生命系统中β-gal的检测。在pH值从6.0到9.0的变化以及温度从25℃到45℃的范围内，都不影响探针分子对β-半乳糖苷酶的检测。
本发明的荧光探针对β-gal有很好的选择性和很强的抗干扰能力。可能干扰的离子Ba2+, Mg2+, Ca2+, Cr3+, Na+, Co2+; 活性硫物质S2O3 2-, SO3 2-, HSO3 -; 精氨酸、半胱氨酸; 活性氧物质H2O2, ClO-, NO3 -; 溶菌酶、纤维素酶的存在不影响探针分子对β-gal的响应。
本发明的荧光探针具有较好的肿瘤细胞靶向性、化学稳定性、生物兼容性和 β-gal选择性。双光子激光共聚焦成像实验表明该探针具有较好的细胞通透性，对细胞和生物体无毒副作用。
因此，本发明是一种简单、快速、灵敏的β-gal特异性检测试剂。在化学分析检测领域具有广阔的应用前景。
具体实施方式
下面结合具体实施例对本发明做进一步的详细说明。
实施例1
1）化合物TPR-βgalAc的合成：
将Np-Rhod染料（1.2 mmol，1eq），碘化钠 (1.5mmol ，1.25eq)溶解于20 mL N, N-二甲基甲酰胺中，然后向混合液中加入2,3,4,6-四乙酰氧基-alpha-D-吡喃糖溴化物（0.72mmol，0.6eq），室温下搅拌4 h, 后升温至80℃回流12 h。用TCL板监测反应，反应完全后，用二氯甲烷萃取，无水硫酸镁干燥，减压蒸发，并用硅胶柱进行分离，硅胶颗粒大小为200-300目，得到化合物TPR-βgalAc，为黄色固体，洗脱剂配比为甲醇:二氯甲烷=1:100，产率为75%。
2）化合物TPR-βgal的合成：
向10mL TPR-βgalAc（0.54mmol，1 eq）的无水甲醇溶液中滴加4mL甲醇钠（1.0mmol，2eq）的无水甲醇溶液，使反应混合物在室温下搅拌5h。用TCL板监测反应，利用盐酸将pH调节至7，反应完全后，用二氯甲烷萃取溶液。用无水硫酸镁干燥，有机相减压蒸发。通过硅胶色谱纯化残余物，硅胶颗粒大小为200-300目，得到目标探针化合物TPR-βgal，为粉红色固体，洗脱剂配比为甲醇:二氯甲烷=1:80，产率为50%。
实施例2
化合物TPR-βgal荧光探针随不同β-gal浓度（0-30 U）的I542 nm / I448 nm比率荧光发射的变化
取实施例1制备的TPR-βgal荧光探针溶于含有EtOH作为共溶剂（PBS / EtOH = 2：1，v/ v）的PBS（10mM，pH 7.4）溶液制备1mmol/L 储备溶液。从储备液中取出10 μL 加入到5 mL的离心管当中，加入不同量（0-30 U）的β-gal标准溶液，用PBS缓冲溶液（0.1 mol/L，pH=7.5）与EtOH体积比为2:1的溶液稀释至3 mL，测量其荧光性质。以385 nm 为激发光，随着β-gal加入量的增加，448 nm处的荧光强度急剧下降，并且在542 nm处出现红移荧光峰，提供可视化的比率荧光信号（I542 nm / I448 nm）。
实施例3
化合物TPR-βgal荧光探针的荧光线性范围测定
配制10份2 mL的TPR-βgal荧光探针溶液（10 μM），分别加入β-gal（0-25 U），进行荧光检测（λex = 385 nm），计算各体系中荧光强度，通过分析I542 nm / I448 nm处的荧光强度与β-gal浓度的关系，评估探针对β-gal的响应性能。表明探针在β-gal浓度0.1-2 U范围内呈线性关系，线性相关系数为R2=0.99592，而且探针对β-gal的检测限为9.11×10-3 U/mL。
实施例4
化合物TPR-βgal荧光探针对不同分子或离子的选择性
从实施例1 中荧光探针储备液中取出10 μL 加入到5 mL 的离心管当中，分别加入100eq的竞争分子标准溶液，其中一个加入等摩尔量的β-gal标准溶液，在37 °C下30 min 后以385 nm 为激发光检测溶液的荧光发射光谱变化，可以发现，其他干扰物质对化合物TPR-βgal的荧光几乎没有影响，而β-gal的加入使化合物TPR-βgal的荧光显著增强。
实施例5
化合物TPR-βgal荧光探针响应时间的变化
从实施例1 中荧光探针储备液中取出4份10 μL 溶液加入到5 mL 的离心管当中，分别加入0、2、5、10 U的β-gal，用荧光分光光度法测定荧光强度随时间的变化。荧光强度比（I542nm / I448 nm）在前10分钟内迅速增加，然后在约25分钟内获得最大强度。一旦达到平台，探针的荧光强度保持相对稳定，表明它具有光稳定性。此外，在没有β-gal的情况下，在系统中没有观察到明显的荧光强度比变化，证实在实验条件下探针TPR-βgal是稳定的。
实施例6
化合物TPR-βgal荧光探针对抑制剂的响应
我们将本发明探针与β-gal的特异性抑制剂D-半乳糖进行酶抑制反应实验。体系a：5 μM 化合物TPR-βgal荧光探针标准溶液；b：在反应体系a中加入1 U 的β-gal；c：在反应体系b中加入1 μM 的D-半乳糖。测定表明，在加入酶抑制剂后荧光强度比（I542 nm / I448 nm）会降低，说明探针确实与β-半乳糖苷酶发生了反应。对荧光探针TPR-βgal与荧光染料Np-Rhod对不同浓度抑制剂D-半乳糖的响应进行对比。体系中未加入β-半乳糖苷酶，探针和染料的荧光强度随着D-半乳糖浓度的增大并未发生明显变化，进一步说明了探针与酶的特异性反应。
上述仅为本发明的优选具体实施方式，但本发明的设计构思并不局限于此，反利用此构思对本发明进行非实质性的改动，均应属于侵犯本发明保护范围的行为。
Claims (1)
1.一种如式Ⅰ所示的荧光探针在β-半乳糖苷酶分子检测中的应用，其特征在于，所述荧光探针用于水环境和细胞中β-半乳糖苷酶的含量传感检测；所述的传感检测包含目视定性检测，荧光检测，细胞成像检测；
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