CN114560898A - 一种荧光探针及其在识别β-D-葡萄糖醛酸苷酶和/或内质网定位中的应用 - Google Patents
一种荧光探针及其在识别β-D-葡萄糖醛酸苷酶和/或内质网定位中的应用 Download PDFInfo
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Abstract
本发明提供一种基于萘酰亚胺和4‑甲苯磺酰胺的β‑葡萄糖醛酸苷酶的荧光探针ERNathG及其应用，其属于分子识别技术领域。它可用于测定β‑葡萄糖醛酸苷酶的酶活性，同时能够锚定酶促反应发生位点的内质网结构。以ERNathG作为特异性探针反应底物，借助β‑葡萄糖醛酸苷酶体内外反应体系，通过定量检测单位时间内酶促反应水解产物的生成量来测定各生物样品中β‑葡萄糖醛酸苷酶的活性。本发明可用于不同来源β‑葡萄糖醛酸苷酶活性的定性、定量测定。
Description
技术领域
本发明属于分子识别技术领域，尤其涉及一种荧光探针及其在识别β-D-葡萄糖醛酸苷酶和/或内质网定位中的应用。
背景技术
荧光探针能对某种特定分析物进行专一性识别检测，通过荧光信号变化来分析目标分子与荧光探针的反应过程。但是，荧光探针的荧光强度容易受到周围环境、自身浓度和检测仪器等因素的影响，导致检测结果的准确性降低。而同时具有两个荧光发射峰的比率型荧光探针在成像时，可以通过自身两个信号峰进行校正，避免以上因素的影响，提高检测的准确性。由于激发态分子经非辐射跃迁损失了部分能量，导致荧光发射光谱较相应的紫外吸收光谱有一定的红移，两种光谱之间的差值称为斯托克斯位移。荧光物质的斯托克斯位移越大，发生自吸收引起的荧光淬灭概率就越小，所以其值越大越好。
葡萄糖醛酸酶具有广泛应用，如β-D-葡萄糖醛酸酶(GUS)是检测大肠杆菌的标志性酶。现行国标法HJ1001-2018检测大肠杆菌，利用GUS分解荧光共轭物4-甲基伞形酮-β-D-葡萄糖醛酸(MUG)，释放4-甲基伞形酮(4-MU)作为信号分子。4-MU是羟基解离型荧光分子，pKa 8-9，而GUS活性最适pH 5.2-8。因此，国标采用手工法，以多孔板荧光有/无的概率估算大肠杆菌的最大可能数(MPN)。但是，目前多种行业需要自动在线监测方法，而自动程序需要记录荧光强度值算菌浓度，在测试pH区间荧光强度值不准。因此，需要pH适应中性条件的荧光信号分子，萘酰亚胺结构存在中性条件应用的可能性。
除了细菌以外，GUS广泛存在于哺乳动物和植物细胞。尤其是移植排异肾病，各种恶性肿瘤和风湿类关节炎等，都存在GUS的过量表达。所以，针对这些病变的原位成像有利于诊断。适当的锚定结构有利于实现原位成像，比如4-甲苯磺酰胺。
满足以上条件的分子，应当包含GUS识别结构即β-D-葡萄糖醛酸苷、萘酰亚胺结构和4-甲苯磺酰胺结构。但是，目前没有该分子报道。
发明内容
本发明的目的在于提供一种荧光探针及其在识别葡萄糖醛酸苷酶和/或内质网定位中的应用。本发明中的荧光探针同时具备葡萄糖醛酸酶识别功能和内质网锚定功能，可在产生葡萄糖醛酸酶的位点或细胞被水解断裂，产生荧光信号，荧光信号锚定于该位点的内质网结构。
本发明的目的在于提供一种具有式I所示结构的荧光探针，记为ERNathG，以及其在识别葡萄糖醛酸苷酶和/或内质网定位成像中的应用。本发明比率型双光子荧光探针以1,8-萘酰亚胺为双光子荧光基团，以β-D-葡萄糖醛酸苷为识别基团，糖苷键为酶切位点，以4-甲苯磺酰胺为内质网锚定基团。该探针通过与葡萄糖醛酸酶的酶促反应，使糖苷键断裂，连接着4-甲苯磺酰胺的1,8-萘酰亚胺基团整体离去，通过反应底物信号减少或反应产物的信号增加，以及二者荧光强度的比值变化，来检测葡萄糖醛酸酶的存在及其活性大小，同时，能够原位锚定内质网。
该特异性探针为一类比率型的双光子荧光探针，其不易受所在体系溶液背景及杂质的干扰，可用于各种葡萄糖醛酸酶活性的定量检测，包括但不限于大肠杆菌检测、肠道癌细胞成像等。
本发明还公开了上述化合物ERNathG作为GUS的特异性荧光探针底物的应用。采用ERNathG作为GUS的特异性底物，进行水解反应，通过检测单位时间内水解产物的荧光强度，测定不同生物体系中GUS的活性，同时，锚定酶促反应位点的内质网。所述生物体系包括但不限于各种产生GUS的细菌，例如大肠杆菌、粪肠球菌、无乳链球菌、产酸克雷伯氏菌、枯草芽孢杆菌，和各种重组表达等；多种病变细胞，例如肠道癌细胞、移植排异肾病、各种恶性肿瘤和风湿类关节炎等，尤其在具有能够被4-甲苯磺酰胺锚定的细胞器结构的细胞应用有优势。
具体应用如下：
以ERNathG作为特异性探针底物；底物浓度范围0～700μM，优选为10～100μM。在磷酸盐缓冲液中，反应温度：20℃至60℃，优选37℃。反应时间；反应体系pH 5.5至10.5之间，优选pH 7.0。反应时间：0至24小时。
本发明提供的ERNathG分子为底物，经GUS酶促反应，水解产物是ERNathOH分子。测定单位时间内底物减少量或水解产物生成量，或二者的比值作为GUS活性的评价指标。ERNathG分子和ERNathOH分子的最适激发波长/发射波长，分别为366/455nm和456/555nm。
本发明所述ERNathG分子作为GUS的特异性探针底物，具有以下突出优势：
(1)能够锚定GUS酶促反应发生部位的内质网。
(2)高特异性：ERNathG可被GUS高特异性识别并代谢。
(3)高灵敏度：ERNathG具有较长的荧光发射波长，可以较好的减弱生物背景荧光的干扰。
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，还可以根据提供的附图获得其他的附图。
图1为本发明中具有式I所示结构的荧光探针的HNMR图谱；
图2为ERNathG分子及其与GUS温育后的水解产物ERNathOH分子的激发/发射谱；
图3为ERNathG分子及其与GUS温育后的水解产物ERNathOH分子的激发与pH关系；
图4为ERNathG分子及其与GUS温育后的水解产物ERNathOH分子的发射与pH关系；
图5为溶液中，ERNathG分子及其与大肠杆菌温育后的水解产物ERNathOH分子的荧光强度变化；
图6为固体平板上，大肠杆菌菌落加入ERNathG和不加ERNathG对照的荧光照片。
具体实施方式
本发明对制备得到的荧光探针分子进行结构鉴定，结果如图1所示，图1为本发明荧光探针分子的核磁共振氢谱图，由图1可知，本发明成功得到了具有式I所示结构的荧光探针分子(ERNathG)，该分子可以通过多种合成路径获得。
实施例1ERNathG的体外GUS水解
本发明将荧光探针分子(ERNathG)作为底物，进行GUS水解实验操作，结果如图2所示，图2为ERNathG分子及其与GUS温育后的水解产物ERNathOH分子的激发/发射谱，其中，(a)为ERNathG分子，(b)为ERNathOH分子。由图2可以看出，ERNathG(a图)经过葡萄糖醛酸酶(大肠杆菌源)水解生成ERNathOH(b图)的激发发射谱，斯托克斯位移约为100nm。有益效果的原因是：1)大的斯托克斯位移是1,8-萘酰亚胺荧光分子的性质；2)ERNathG能够转化成ERNathOH，并且作为荧光探针，是因为葡萄糖醛酸酶识别位点的引入。
图3为ERNathG的体外GUS水解操作后，ERNathG分子及其与GUS温育后的水解产物ERNathOH分子的激发与pH关系，其中，(a)为ERNathG分子，(b)为ERNathOH分子。由图3可以看出，激发波长不受pH影响，稳定性好。
图4为ERNathG的体外GUS水解操作后，ERNathG分子及其与GUS温育后的水解产物ERNathOH分子的发射与pH关系其中，(a)为ERNathG分子，(b)为ERNathOH分子。由图4可以看出，ERNathG和ERNathOH的pH稳定性好。尤其ERNathOH为ERNathG分解(大肠杆菌源葡萄糖醛酸酶)后的产物分子，一般为各种应用中的检测目标荧光分子，在大于pH7，尤其是pH7.5之后，荧光强度不随pH改变。在葡萄糖醛酸酶相关应用中，尤其是中性水体大肠杆菌指标检测中，所合成分子适用于多种水体，且不受恒温增殖过程中，杂菌等对繁殖对溶液体系pH的改变的影响。
实施例2ERNathG液相检测大肠杆菌
本发明将荧光探针分子(ERNathG)作为反应底物，在液相中，进行大肠杆菌检测反应，具体步骤为配制LB液体培养基，接入大肠杆菌，37℃温育8小时，加入终浓度为20μM的ERNathG，继续温育1小时；结果如图5所示，图5为ERNathG分子及其与大肠杆菌温育后的水解产物ERNathOH分子的荧光强度变化。由图5可知，本发明的分子结构可用于大肠杆菌的液相检测。
实施例3ERNathG成像应用
本发明将荧光探针分子(ERNathG)的成像应用。具体步骤为配制LB固体培养基，接入大肠杆菌，37℃温育18小时，待长出肉眼可见菌落，加入终浓度为100μM的ERNathG，继续温育2小时；结果如图6所示，图6为固体平板上，大肠杆菌菌落加入ERNathG和不加ERNathG对照的荧光照片。由图6可知，本发明的分子结构经GUS水解后，可锚定于反应发生位点的内质网，用于成像研究。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (7)
1.一种荧光探针，具有式I所示结构，
2.如权利要求1所述的荧光探针在识别葡萄糖醛酸苷酶和/或内质网定位中的应用。
3.根据权利要求2所述的应用，其特征在于，所述葡萄糖醛酸苷酶为细菌来源葡萄糖醛酸苷酶、或者哺乳动物来源葡萄糖醛酸苷酶、或者植物来源葡萄糖醛酸苷酶。
4.根据权利要求3所述的应用，其特征在于，所述细菌包括大肠杆菌、粪肠球菌、无乳链球菌、产酸克雷伯氏菌和枯草芽孢杆菌中的一种或几种。
5.根据权利要求2所述的应用，其特征在于，所述内质网定位为具有能够被4-甲苯磺酰胺锚定的细胞器结构。
6.根据权利要求2所述的应用，其特征在于，所述内质网定位包括哺乳动物病变细胞定位成像。
7.根据权利要求6所述的应用，其特征在于，所述病变包括肠道癌、移植排异肾病、恶性肿瘤、风湿类关节炎及GUS特异表达相关疾病。
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