An Advanced Biosensor for Molecular Interaction Studies.

用于分子相互作用研究的先进生物传感器。

• 批准号: 8516144
• 负责人: DARRYL J. BORNHOP
• 金额: $ 60.46万
• 依托单位: MOLECULAR SENSING, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8516144
• 关键词: Address; Adoption; Affinity; Algorithms; Back; Baclofen; Benchmarking; Benign; Binding; Biological; Biological Assay; Biological Markers; Biological Process; Biological Sciences; Biology; Biosensor; Buffers; CXCR4 gene; Calibration; Calorimetry; Carbonic Anhydrase II; Catalysis; Cell Wall; Cell physiology; Cells; Chemicals; Chemistry; Commercial Sectors; Communities; Complex; Computers; Data; Data Collection; Detection; Detergents; Devices; Diagnostic; Diagnostic Procedure; Dimethyl Sulfoxide; Edetic Acid; Electronics; Environment; Event; Excision; Experimental Models; Feedback; Flowers; Fluorescence; Foundations; G Protein-Coupled Receptor Genes; Glass; Glycerol; Grant; HIV Protease; Hydrogen Bonding; Immobilization; Injection of therapeutic agent; Interferometry; Kinetics; Label; Laboratories; Lasers; Lead; Life; Light; Liquid substance; Literature; Malignant Neoplasms; Measurement; Measures; Medical; Membrane; Membrane Proteins; Methodology; Methods; Microfluidics; Molecular; Monitor; Noise; Optics; Organic solvent product; PTGS2 gene; Pattern; Performance; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Physiological; Plastics; Positioning Attribute; Process; Protocols documentation; Radioactive; Reaction; Refractive Indices; Reproducibility; Research; Research Personnel; Running; Sampling; Sampling Studies; Scheme; Science; Serum; Signal Transduction; Silicon Dioxide; Site; Small Business Technology Transfer Research; Solutions; Speed; Stromal Cell-Derived Factor 1; Sucrose; Sulfanilamide; Surface; System; Techniques; Technology; Technology Transfer; Testing; Thermodynamics; Time; Titrations; Training; Translations; Treatment Efficacy; Validation; Vesicle; Water; base; catalyst; commercialization; cost; design; drug discovery; evaluation/testing; gamma-Aminobutyric Acid; graphical user interface; improved; innovation; instrument; interest; meetings; multidisciplinary; operation; prototype; public health relevance; research and development; sensor; small molecule; tool; user-friendly

DESCRIPTION (provided by applicant): Molecular interactions form the foundation of biology and chemistry. They are central to life itself and determine catalytic activity, cellular function, and therapeutic efficacy. The vast majority of diagnostic procedures depend on some type of specific molecular interaction. Therefore, the ability to perform pure liquid-phase molecular binding analysis at high sensitivity, without modifying the interacting species, and at physiological concentrations would be revolutionary. Yet, the tools available to quantify these interactions have limitations. Traditional methods such as the sucrose gradient technique or isothermal titrimetric calorimetry are laborious and require substantial quantities of sample to perform an assay. Fluorescence and radioactive methods are sensitive, but rely on the incorporation of signaling labels to enable detection, slowing the assay and increasing its cost. Numerous techniques, particularly the label-free methods, require surface immobilization of one of the interacting moieties putting the species in a non-native environment. Labels and tethers can be benign, but often alter the interacting molecules and can lead to a biased result. Recently my group and others demonstrated that back-scattering interferometry (BSI) can be used in the academic laboratory to perform molecular interaction determinations label-free and in free-solution, with sensitivity that allows assays on small quantities of sample, at physiologically relevant concentrations. BSI is a universal sensing method that only requires the product of a reaction to refract or interact with light differently than the participating species, therefore has the potential to be widely applicable for general use as a Molecular Interaction Photometer (MIP). BSI is a prime candidate to become an MIP because it has a simple and inexpensive optical train comprised of a He-Ne laser, a microfluidic channel, and a position sensor allowing minute refractive index changes to be monitored. Measurements are made within a microfluidic channel formed in glass, fused silica, or plastic and at physiologically relevant concentrations in complex matrices such as serum or with native membrane- proteins. Yet the current academic embodiment of BSI is not commercially viable. Tedious alignment methods, immature transduction schemes, poorly refined sample handling and introduction methods, and performance limitations due to environmental noise sensitivity all impede the wide dissemination and adoption of BSI in the life science and drug discovery communities. Under Phase I of this STTR grant we met our milestones demonstrating a two-channel BSI instrument with a pipette-driven injection method and a fringe detection algorithm that simplified fringe selection and alignment. Here we propose to build on these results, while capitalizing on two new innovations to transform our academic laboratory BSI into the MIP instrument we call NanoBIND. Under this STTR Phase II, we propose the completion of four aims to refine BSI through research development and technology transfer, facilitate commercialization by Molecular Sensing Inc. and allow the subsequent broad dissemination in the research community. In Aim 1 we further simplify the optical train, while retaining the advantages of performing a simultaneous sample-reference determination. Aim 2 implements a sample introduction method that is user friendly, minimizes the potential for contamination and constrains volumes to <1?L. An improved algorithm enhances sensitivity, enables electronic fringe selection and alignment and addresses non-specific interactions (at cell wall) to improve assay reproducibility in Aim 3. And in Aim 4 ?- prototype version BSI systems will be constructed and used, external to Vanderbilt and MSI, to demonstrate that BSI gives meaningful and quantitative binding affinity values (from ?M to pM) and that it can be used to screen for molecular interactions in complex matrices such as serum and cell-free media, as well as DMSO. Upon completion of these Aims, three identical prototypes will have been constructed and evaluated for field utility. Feedback from these laboratories and users will provide the formal framework for refining the design under Phase III commercial deployment.

描述（由申请人提供）：分子相互作用构成了生物学和化学的基础。它们是生命本身的核心，并确定催化活性，细胞功能， 和治疗功效。绝大多数诊断程序取决于某种类型的特定分子相互作用。因此，在高灵敏度上执行纯液相分子结合分析的能力，而无需修改相互作用的物种，并以生理浓度是革命性的。但是，可用于量化这些交互的工具有局限性。传统方法，例如蔗糖梯度技术或等温滴定量热法是艰苦的，需要大量样品才能执行测定。荧光和放射性方法是敏感的，但依靠信号标签的掺入来实现检测，减慢测定并增加其成本。许多技术，尤其是无标签方法，都需要将一种相互作用的部分固定在非本地环境中。标签和系数可以是良性的，但通常会改变相互作用的分子，并可能导致偏见。最近，我的小组和其他人表明，可以在学术实验室中使用反散射干涉法（BSI）来执行分子相互作用确定的标签和自由溶液，并具有敏感性，可以在生理相关浓度下对少量样品进行测定。 BSI是一种通用感应方法，仅需要反应的乘积以折射或与光相互作用的方式与参与物种不同， 因此，有可能广泛用于一般用作分子相互作用光度计（MIP）的潜力。 BSI是成为MIP的主要候选者，因为它具有由He-Ne激光器，微流体通道和位置传感器组成的简单且廉价的光学序列，并且可以监视微小的折射率变化。测量是在形成的微流体通道中进行的，该通道在玻璃，融合二氧化硅或塑料中形成，并在复杂矩阵（例如血清或天然膜蛋白）的生理相关浓度下进行。然而，BSI的当前学术体现在商业上并不可行。乏味的对准方法，不成熟的转导方案，不成熟的样品处理和引入方法以及由于环境噪声敏感性而引起的绩效限制都阻碍了生命科学和药物发现社区中BSI的广泛传播和采用。在此STTR授予的第一阶段，我们遇到了我们的里程碑，该里程碑展示了具有移液管驱动的注入方法的两通道BSI仪器，以及简化条纹选择和对齐的条纹检测算法。在这里，我们建议以这些结果为基础，同时利用两项新的创新，将我们的学术实验室BSI转变为我们称为Nanobind的MIP工具。在此STTR II阶段，我们提出了四个旨在通过研究开发和技术转移来完善BSI的目标，从而促进了Molecular Sensing Inc.的商业化，并允许随后的研究界进行广泛的传播。在AIM 1中，我们进一步简化了光列，同时保留了同时执行样品引用确定的优势。 AIM 2实现了一种样本简介方法，该方法是用户友好的，可以最大程度地减少污染的可能性，并将量限制为<1？l。改进的算法可以提高灵敏度，使电子条纹选择和对齐方式解决非特异性相互作用（在细胞壁上），以提高目标3的测定可重复性。复杂基质中的分子相互作用，例如血清和无细胞培养基以及DMSO。这些目标完成后，将构建和评估三个相同的原型。这些实验室和用户的反馈将提供正式 在第三阶段商业部署下完善设计的框架。