【作者】 高健；

【导师】 丁彩凤； 张鹏；

【作者基本信息】 青岛科技大学 ， 化学， 2023， 硕士

【摘要】 大多数疾病的发生都会伴随着某些生物活性分子的异常表达,因此众多具有标志性的生物活性分子在疾病的诊断和治疗过程中发挥着极其重要的作用。随着人们对超高灵敏度、实时成像检测需求的增长,具有高选择性、良好生物相容性、反应迅速、操作简便、灵敏度高、无创等优势的荧光探针,在疾病早期诊断和治疗以及生物体实时成像中显示出巨大的潜力。本文构建了两种新型小分子荧光探针,能够在对生物系统产生最小不良影响的情况下实现对某些生物活性分子的高灵敏度和高选择性检测。具体研究内容如下:(1)γ-谷氨酰转移酶(GGT)广泛存在于活细胞中,并在许多肿瘤组织中过表达。因此,它通常被认为是检测肿瘤,特别是肝癌的重要生物标志物。准确测定其活性有助于相关疾病的早期诊断和治疗。本工作以丹磺酰胺为荧光团,设计合成了一种基于谷氨酰胺键的“开启”型荧光探针NSA-GGT用于检测GGT活性。该探针具有良好的水溶性,能很好地分散在水溶液中。在磷酸盐缓冲液中与GGT孵育后,探针NSA-GGT在～523 nm处的荧光增加了25倍以上。这种传感模式对GGT活性检测显示出较好的灵敏度,并且在细胞内GGT荧光成像方面有良好的表现。后续的生物学实验表明,探针NSA-GGT还可用于活细胞和动物组织中GGT活性的荧光成像。(2)黑色素瘤原发于表皮和真皮交界处,多出现于皮肤、眼部等部位,其恶性程度高、易转移,肝、肺、骨、腹膜后为转移多发部位。肝脏黑色素瘤是一种极难诊断、致死率极高的恶性肿瘤,目前并没有一种系统性的诊疗标准,基于此我们首次开发了一种基于酶级联反应检测生物体内BChE与TYR活性的近红外小分子荧光探针MB-BChE-TYR,用于肝脏黑色素瘤的早期诊断和术后评估。选用亚甲基蓝(Methylene blue)作为近红外荧光基团,引入对羟基苯乙胺与环丙基甲酸酯,分别作为酪氨酸酶(TYR)与丁酰胆碱酯酶(BChE)的特异性反应位点。而当TYR与BChE存在时,BChE催化酯键断裂,释放出MB-TYR,再经TYR的氧化成为醌,然后经历快速分子内环化导致脲键的自发水解,近红外荧光被激活。实验证实,探针MB-BChE-TYR可以通过荧光强度的变化实现对BChE与TYR的检测,进而实现对肝脏黑色素瘤的监测,有望成为肝脏黑色素瘤的早期诊断与术后评估的辅助治疗手段,提高患者的生存率。更多还原

【Abstract】 The occurrence of most diseases is accompanied by abnormal expression of certain bioactive molecules,and thus numerous landmark bioactive molecules play an extremely important role in the diagnosis and treatment of diseases.As the demand for ultra-high-sensitivity,real-time imaging assays grows,fluorescent probes with the advantages of high selectivity,good biocompatibility,rapid response,ease of operation,high sensitivity,and non-invasiveness show great potential for early diagnosis and treatment of diseases as well as real-time imaging of organisms.In this paper,two novel small-molecule fluorescent probes were constructed that can achieve highly sensitive and selective detection of certain bioactive molecules with minimal adverse effects on biological systems.The specific studies are as follows:(1)γ-glutamyltransferase(GGT)is widely present in living cells and is overexpressed in many tumor tissues.Therefore,it is often considered an important biomarker for the detection of tumors,especially hepatocellular carcinoma.Accurate determination of its activity can help in the early diagnosis and treatment of related diseases.In this work,a glutamine bond-based "turn-on" fluorescent probe,NSAGGT,was designed and synthesized for the detection of GGT activity using dansylamide as the fluorophore.The probe has good water solubility and can be well dispersed in aqueous solution.After incubation with GGT in phosphate buffer,the fluorescence of the probe NSA-GGT at ～523 nm increased more than 25-fold.This sensing mode showed good sensitivity for GGT activity detection and good performance in intracellular GGT fluorescence imaging.Follow-up biological experiments showed that the probe NSA-GGT can also be used for fluorescence imaging of GGT activity in live cells and animal tissues.(2)Melanoma originates at the junction of epidermis and dermis,and is mostly found in the skin and eye.It is highly malignant and metastatic,with liver,lung,bone,and retroperitoneum as the most frequent sites of metastasis.Based on this,we developed the first near-infrared small molecule fluorescent probe MB-BChE-TYR based on enzyme cascade reaction to detect BChE and TYR activity in organisms for early diagnosis and postoperative evaluation of hepatic melanoma.Methylene blue(METE blue)was selected as the NIR fluorescent moiety,and phydroxyphenethylamine and cyclopropyl formate were introduced as specific reaction sites for tyrosinase(TYR)and butyrylcholinesterase(BChE),respectively.And when TYR is present with BChE,BChE catalyzes the ester bond breakage to release MB-TYR,which then becomes quinone by the oxidation of TYR and then undergoes rapid intramolecular cyclization leading to spontaneous hydrolysis of the uranyl bond and activation of NIR fluorescence.It was confirmed that the probe MB-BChE-TYR can achieve the detection of BChE and TYR by the change of fluorescence intensity,which in turn enables the monitoring of hepatic melanoma,and is expected to be an adjuvant therapy for the early diagnosis and postoperative evaluation of hepatic melanoma and improve the survival rate of patients.更多还原