Monitoring botulinum neurotoxin a activity with peptide-functionalized quantum dot resonance energy transfer sensors.
Monitoring botulinum neurotoxin a activity with peptide-functionalized quantum dot resonance energy transfer sensors.
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DOI：
10.1021/nn102997b
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发表时间：
2011-02
期刊：
影响因子：
17.1
通讯作者：
K. Sapsford;Jessica Granek;J. Deschamps;K. Boeneman;J. Blanco-Canosa;P. Dawson;K. Susumu;M. Stewart;Igor L. Medintz
中科院分区：
文献类型：
--
作者：
K. Sapsford;Jessica Granek;J. Deschamps;K. Boeneman;J. Blanco-Canosa;P. Dawson;K. Susumu;M. Stewart;Igor L. Medintz
肉毒神经毒素（BoNTs）是一种极其强效的细菌毒素，沿着污染食品供应，并具有很高的开发潜力作为生物恐怖主义制剂。目前仍然需要迅速和灵敏地检测这些毒素的暴露情况，并核实其活性状态，因为后者直接影响诊断并有助于提供有效的治疗。我们研究了使用半导体量子点（QD）-肽福斯特共振能量转移（FRET）组件来监测BoNT血清型A轻链蛋白酶（LcA）的活性。设计并优化模块化LcA肽底物，以包含中心LcA识别/切割区、允许用Cy3受体染料标记的独特残基、延伸的接头-间隔序列和允许最终比率肽-QD-自组装的末端寡聚组氨酸。通过监测FRET相互作用，评价了显示带电或PEG化表面涂层的许多不同QD材料自组装染料标记的LcA肽底物的能力。利用直接肽-QD形式或备选地在QD组装之前将肽间接预暴露于LcA进行蛋白水解测定。根据QD材料和使用的形式获得可变活性，最灵敏的暴露前测定结果表明检测限为350 pM LcA。对各种QD-肽传感器构建的建模提供了对所得组装结构如何影响LcA识别相互作用和随后活性的深入了解。这些结果还突出了肽设计和QD特征，特别是表面封端剂，对整体传感器活性的独特作用。
Botulinum neurotoxins (BoNTs) are extremely potent bacterial toxins that contaminate food supplies along with having a high potential for exploitation as bioterrorism agents. There is a continuing need to rapidly and sensitively detect exposure to these toxins and to verify their active state, as the latter directly affects diagnosis and helps provide effective treatments. We investigate the use of semiconductor quantum dot (QD)-peptide Förster resonance energy transfer (FRET) assemblies to monitor the activity of the BoNT serotype A light chain protease (LcA). A modular LcA peptide substrate was designed and optimized to contain a central LcA recognition/cleavage region, a unique residue to allow labeling with a Cy3 acceptor dye, an extended linker-spacer sequence, and a terminal oligohistidine that allows for final ratiometric peptide-QD-self-assembly. A number of different QD materials displaying charged or PEGylated surface-coatings were evaluated for their ability to self-assemble dye-labeled LcA peptide substrates by monitoring FRET interactions. Proteolytic assays were performed utilizing either a direct peptide-on-QD format or alternatively an indirect pre-exposure of peptide to LcA prior to QD assembly. Variable activities were obtained depending on QD materials and formats used with the most sensitive pre-exposure assay result demonstrating a 350 pM LcA limit of detection. Modeling the various QD-peptide sensor constructs provided insight into how the resulting assembly architecture influenced LcA recognition interactions and subsequent activity. These results also highlight the unique roles that both peptide design and QD features, especially surface-capping agents, contribute to overall sensor activity.