| Resolving Structural Changes of Photoreceptors in Living Escherichia coli via In-cell Infrared Difference Spectroscopy
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Author:
Date:
2021-02-05
[Abstract] Several in-cell spectroscopic techniques have been developed recently to investigate the structure and mechanism of proteins in their native environment. Conditions in vivo differ dramatically from those selected for in vitro experiments. Accordingly, the cellular environment can affect the protein mechanism for example by molecular crowding or binding of small molecules. Fourier transform infrared (FTIR) difference spectroscopy is a well-suited method to study the light-induced structural responses of photoreceptors including changes in cofactor, side chains and secondary structure. Here, we describe a protocol to study the response of cofactor and protein in living E. coli cells via in-cell infrared difference (ICIRD) spectroscopy using the attenuated total reflection (ATR) ...
[摘要] [摘要]最近开发了几种细胞内光谱技术，以研究蛋白质在其天然环境中的结构和机理。体内条件与为体外实验选择的条件大不相同。因此，细胞环境可例如通过分子拥挤或小分子结合来影响蛋白质机制。傅立叶TRANSF ORM红外（FTIR）光谱差异是非常适合的方法来研究的光诱导结构响应小号光感受器，包括辅因子的变化，侧链和二级结构。在这里，我们描述了一种协议，用于研究活大肠杆菌中辅因子和蛋白质的响应 通过使用衰减全反射（ATR）配置的细胞内红外差（ICIRD）光谱分析细胞。蛋白质在大肠杆菌中过表达，将细胞转移到盐溶液中，并使用荧光光谱法确定每个细胞的拷贝数。将悬浮液离心，并将浓缩的细胞转移到FTIR光谱仪内的ATR池中。该恒温细胞密封并从与LED的顶部照明。在照射之前和之后记录强度光谱，以产生活细胞内部受体的差异光谱。利用ICIRD光谱，可在近乎自然的环境中解决可溶性感光体的结构变化。该方法在环境条件下可在H 2 O中运行，无标记，对蛋白质大小没有任何限制，并且不需要任何纯化步骤。
图形摘要：
使用衰减全反射对活大肠杆菌中的感光细胞进行细胞内红外差异光谱分析。
[背景]光感受器在从细菌，真菌，藻类和植物到动物的各种生物体的光感测和光适应中起着至关重要的作用（Möglich等，2010 ; ...
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| RETRACTED: Paper Lateral Flow Biosensor for Nodavirus Reverse Transcribed RNA Detection
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Author:
Date:
2020-08-05
[Abstract] Paper nanobiosensors have been established as an excellent platform for analysis of veterinary and human pathogens causing various diseases. Especially, lateral flow assays or biosensors ideal for sensitive, rapid, robust and accurate analysis in laboratory setups and on-site analysis. Viral RNA detection is of great importance for public health as well as animal health protection. In that aspect, the present protocol focuses on the development of functionalized gold nanoparticle-based lateral flow biosensor for fish nervous necrosis virus (Nodavirus) nucleic acids detection. Total viral RNA, isolated from fish samples was subjected to reverse transcription PCR amplification and the amplification products were mixed with specific oligonucleotide probe. A red test line was formed when ...
[摘要] [摘要] 纸纳米生物传感器已经成为分析导致各种疾病的兽医和人类病原体的一个极好的平台。尤其是侧向流分析或生物传感器是实验室设备和现场分析中灵敏、快速、可靠和准确分析的理想选择。病毒RNA检测对公共卫生和动物健康保护具有重要意义。在这一方面，本协议的重点是开发功能化金纳米粒子侧流生物传感器，用于鱼类神经坏死病毒（Nodavirus）核酸检测。从鱼类标本中分离出的总病毒RNA进行逆转录PCR扩增，扩增产物与特异性寡核苷酸探针混合。当有nodavirus产物存在时，形成红色检测线。这种方法对基础研究有很大的意义，因为它消除了耗时、繁琐的电泳程序的需要，并且可以在养鱼场对养鱼户进行调整。利用这种生物分析平台进行病害监测，无需耗费大量时间和成本，对水产养殖和环境安全有很大影响。
[背景] 关注点和/或现场生物分析一直是关注人类和动物福祉的研究工作的最终目标。基于纸基的传感平台具有功能化简单、重现性好、制造成本低等优点，是一种极具吸引力的分析平台。纸基分析设备已应用于小分子、蛋白质和各种核酸的分析（Parolo和Merkoçi，2013；Bahadir和Sezgintürk，2016；Jiang等人，2019）。侧流生物传感器（LFB）是一种带有干试剂的预制材料条带，通过流体样品激活。它们专为一次性一次性使用而设计，只要有足够的开/关信号（Posthuma ...
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