中文关键词:基质金属蛋白酶-2 金纳米棒荧光共振能量转移 分子探针荧光成像 英文关键词:Matrixmetalloprotease-2 (MMP-2), Goldnanorods, Fluorescenceresonance energytransfer (FRET), Molecularprobe, Fluorescentimaging 基金项目:

作者	单位	E-mail
尹 玲	苏州大学江苏省有机合成重点实验室 苏州大学材料与化学化工学部 苏州	yinling_1109@163.com
王安娜	苏州大学医学部放射医学与防护学院
赵梦	苏州大学医学部放射医学与防护学院
沙靖全	济宁学院化学与化工系
白永香	苏州大学医学部基础医学与生物科学学院
史海斌	苏州大学医学部放射医学与防护学院	hbshi@suda.edu.cn

摘要点击次数:1426 全文下载次数:0 中文摘要: 开发能特异性和灵敏性检测基质金属蛋白酶-2(MMP-2)的智能型荧光分子探针,对于癌症的早期精准诊断与治疗至关重要。本文基于MMP-2特异性识别的多肽底物,利用一价铜离子催化的“点击”化学反应将荧光素(FITC)和多肽底物GPLGVRGY相耦联,与SH-PEG-COOH修饰的金纳米棒在1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和N-羟基硫代琥珀酰亚胺钠盐(Sulfo-NHS)的作用下通过酰胺化反应,制备得到一种新的MMP-2特异性响应的荧光共振能量转移(FRET)金纳米棒探针GNR@FITC。首先,将探针与MMP-2在缓冲液中孵育,通过对溶液在520 nm处荧光信号变化的跟踪检测,对探针的特异性和检测灵敏度进行了评估。体外重组酶检测实验结果表明,探针对MMP-2具有较强的特异性和选择性,最低检测限可达1.28 ng.mL_-1。此外,基于小鼠乳腺癌细胞4T1的3-(4,5-二甲基-2-噻唑)-2,5-二苯基溴化四氮唑噻唑蓝(MTT)实验结果表明,探针在0-96 μg.mL_-1浓度范围内对4T1细胞存在较低的毒性。细胞共聚焦显微成像实验结果显示,相比于小鼠成纤维细胞NIH/3T3,4T1细胞在与探针孵育后,产生出较强的绿色荧光信号。如预先加入抑制剂,细胞的荧光信号则大大减弱。总之,该探针对MMP-2过表达小鼠乳腺癌细胞4T1表现出较好的特异性和检测灵敏度,具有进一步开发应用于乳腺癌早期诊断的极大潜力。 英文摘要: Developing a smart fluorescent probe which can specifically and sensitively detect matrix metalloproteinases-2 (MMP-2) is vital for the early accurate diagnosis and treatment of cancers. In this study, fluorescein isothiocyanate (FITC) was firstly conjugated to the peptide substrate GPLGVRGY by using copper (I)-catalyzed “click” reaction, and subsequently reacted with gold nanorods decorated with SH-PEG-COOH under the catalysis of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) andSN-hydroxysulfosuccinimide sodium salt (Sulfo-NHS) through amidation reaction to afford a novel fluorescence resonance energy transfer (FRET)-based probe GNR@FITC for specific detection of MMP-2 activity. To assess the specificity and sensitivity of the probe for activated MMP-2 in vitro, the enzymatic assays were firstly performed with recombinant MMP-2. Fluorescent signal change of the mixed solution of probe and MMP-2 with and without treatment of MMP-2 specific inhibitor was recorded at 520 nm with fluorescence spectrometer. In vitro enzyme detection experiments indicated that the probe showed good specificity and high sensitivity for MMP-2 in comparison with other proteins, and the minimum detection limit of MMP-2 was as low as 1.28 ng.mL_-1. In addition, the cytotoxicity of the probe was assessed through the widely used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. It exhibited very low cytotoxicity to mouse mammary tumour 4T1 cells in a range of 0-96 μg.mL_-1. The fluorescence imaging results showed that 4T1 cells had stronger green fluorescence signal comparing to the rat fibroblasts cells NIH/3T3. However, most of the fluorescence was readily competed away by pretreatment of the probe with MMP-2 inhibitor. In summary, this probe shows good specificity and high sensitivity for the detection of MMP-2-overexpressed 4T1 cells, which may have great potential for clinical application in early diagnosis of breast cancer. 查看全文查看/发表评论下载PDF阅读器 相关附件：附件1附件2附件3版权转让声明书附件1附件2 -->