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The Octet R8: a fast, sensitive, label-free protein analysis platform for characterising biomolecular interactions

Octet R8：快速、灵敏、无标记的蛋白质分析平台，用于表征生物分子相互作用

基本信息

批准号：
MR/X012646/1
负责人：
Jaswir Basran
金额：
$ 27.34万
依托单位：
University of Leicester
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2022
资助国家：
英国
起止时间：
2022 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=MR%2FX012646%2F1
关键词：
Octet R8 fast sensitive label

项目摘要

Interactions between molecules are central to all processes of life. These interactions are diverse in nature and include protein-protein interactions, protein-oligonucleotide interactions, protein-cofactor interactions and interactions involving complex bilayer assemblies. Because of their importance in terms of furthering our biological understanding and accelerating pharmaceutical development, all of these interactions are at the heart of innovative research programs across the globe. Life science research at the University of Leicester has an outstanding track record in conducting groundbreaking research in this important field. To allow the world-leading researchers to drive innovation and address the most important biological questions of our time requires highly sensitive and accurate quantitative analytical tools. The heavily used instrumentation at UoL is ageing and has limitations compared to the currently available techniques. We, therefore, seek to purchase the Sartorius Octet R8.The Octet R8 is a state-of-the-art in bio-layer interferometry (BLI), an optical technique for detecting biomolecular interactions by measuring the interference pattern of white light reflected from the surface of a biosensor. BLI compares the interference pattern of white light reflected from an internal reference layer with that reflected from the immobilised protein layer present on the biosensor tip. The binding of biomolecules (analytes) at the tip surface results in a shift in interference pattern allowing the quantification of binding events in real-time and with high precision. Only interactions of bound biomolecules are measured, making bio-layer interferometry a powerful analytical tool for a wide range of applications. The Octet R8 system measures binding events in small volumes of 200 microliters in a non-destructive manner to determine association rates (kon), dissociation rates (koff), and affinity constants (KD). Arguably one of the most powerful features of the Octet systems is the unique 'dip and read' biosensor format which permits the analysis of complex samples e.g. cell culture supernatants and lysates enabling biomolecular interactions to be studied in biologically relevant environments. Due to the multitude of different biosensors and surface chemistries BLI provides enormous flexibility for a range of multidisciplinary projects.The label-free Octet system negates the need for intrinsic/external chromophores or the addition of fluorescent labels/probes: a prerequisite of solution optical spectroscopies. Although a competing technology like isothermal titration calorimetry (ITC) is a label-free technology that is very useful for providing thermodynamic data, it does not allow for real-time kinetic analyses and is less sensitive than the Octet system. Surface Plasmon Resonance (SPR, a comparable label-free, surface-based technology) generates highly accurate, reproducible data but does not have the high throughput or flexibility offered by the Octet platform and poses significant challenges associated with its microfluidics-based nature.

分子之间的相互作用是所有生命过程的核心。这些相互作用在性质上是多样的，包括蛋白质-蛋白质相互作用、蛋白质-寡核苷酸相互作用、蛋白质-辅因子相互作用和涉及复杂双层组装的相互作用。由于它们在促进我们的生物学理解和加速药物开发方面的重要性，所有这些相互作用都是地球仪创新研究计划的核心。在生命科学研究在莱斯特大学在这一重要领域进行开创性的研究有着出色的记录。为了让世界领先的研究人员能够推动创新并解决我们这个时代最重要的生物学问题，需要高度灵敏和准确的定量分析工具。UoL大量使用的仪器正在老化，与目前可用的技术相比存在局限性。因此，我们寻求购买Sartorius Octet R8。Octet R8是生物层干涉测量（BLI）中最先进的技术，BLI是一种通过测量生物传感器表面反射的白色光的干涉图案来检测生物分子相互作用的光学技术。BLI将从内部参考层反射的白色光的干涉图案与从生物传感器尖端上存在的固定蛋白质层反射的干涉图案进行比较。生物分子（分析物）在尖端表面处的结合导致干涉图案的偏移，从而允许实时且高精度地定量结合事件。仅测量结合的生物分子的相互作用，使得生物层干涉测量成为广泛应用的强大分析工具。Octet R8系统以非破坏性方式测量200微升小体积中的结合事件，以确定结合速率（kon）、解离速率（koff）和亲和常数（KD）。可以说Octet系统最强大的功能之一是独特的“浸渍和读取”生物传感器格式，允许分析复杂样品，例如细胞培养上清液和裂解物，从而能够在生物相关环境中研究生物分子相互作用。由于BLI具有多种不同的生物传感器和表面化学性质，因此它为一系列多学科项目提供了巨大的灵活性。无标记Octet系统无需使用内部/外部发色团或添加荧光标记/探针：这是溶液光谱学的先决条件。虽然竞争技术如等温滴定量热法（ITC）是一种无标记技术，对于提供热力学数据非常有用，但它不允许实时动力学分析，并且不如Octet系统灵敏。表面等离子体共振（SPR，一种类似的无标记，基于表面的技术）产生高度准确，可重复的数据，但不具有Octet平台提供的高通量或灵活性，并提出了与其基于微流体的性质相关的重大挑战。