Ultrabright Fluorescent Nanoconstruct Enabling Widely Accessible, High Performance Multiplexed Protein Assays

摘要 生物体液和组织中多种蛋白质的检测和定量具有基础性意义 在生物医学研究和临床诊断中的重要性，因为它是不可能理解的 复杂的、非线性的、生化系统，无法准确地询问 组件。同时询问多个蛋白质的需要在所有生物中普遍存在 生物医学研究领域，这是充分了解健康、老龄化、 疾病，以及对治疗干预措施的反应。尽管这种需求可能无处不在，但在 对于研究人员来说，这并不是一个广泛可用的解决方案来制造多路蛋白质 测量灵敏度高，动态范围大。 抗体微阵列有一个简单的、类似于ELISA的工作流程，可以用于测量 多达数千种蛋白质同时存在。不幸的是，他们的敏感度相当低，而且 需要使用专门的昂贵(&gt；$75K)阅读器。这些都是阻碍 为大多数生物医学研究人员所采用。我们已经开发出一种超亮荧光 纳米结构我们称之为等离子体氟，它比标准的&gt；亮5000倍 今天在微阵列中使用的荧光报告。简单地用等离子体氟取代了 现有的报告大大提高了抗体微阵列的灵敏度，而不需要 对工作流程的任何更改。重要的是，等离子体氟是如此明亮，以至于它允许荧光 使用更广泛的可获得和更多用途的读取器(例如荧光读取器)来读取微阵列 蛋白质印迹阅读器。此外，它还放宽了对读取器工具的要求 值得注意的是，这使我们能够创造出任何研究实验室都能做到的廉价阅读器 负担得起。为了更容易地采用等离子体荧光增强型微阵列，我们有 还开发了用于微阵列数据分析的软件。 在这个项目中，我们的目标是：将我们的等离子体氟的生产规模提高到早期 商业规模；更广泛地验证各种流行抗体的增强 微阵列；最终确定我们廉价阅读器的设计；并更全面地开发我们的用户- 友好的微阵列分析软件。 我们相信，等离子体氟将成为所有人的标准荧光报告分子 微阵列和等离子体荧光增强微阵列将成为一种广泛使用和强大的 用于阐明蛋白质网络在健康和疾病中的作用的工具。

Abstract Detection and quantification of multiple proteins in biological fluids and tissues is of fundamental importance in biomedical research and clinical diagnostics because it is impossible to understand complex, non-linear, biochemical systems without being able to accurately interrogate the components. The need to interrogate multiple proteins simultaneously is ubiquitous across all domains of biomedical research, and it is a major barrier to fully understanding health, ageing, disease, and response to therapeutic interventions. Though the need may be ubiquitous, there is not a widely accessible solution for researchers to make multiplexed protein measurement with high sensitivity and a large dynamic range. Antibody microarrays have a straightforward, ELISA-like workflow and can be used to measure up to thousands of proteins simultaneously. Unfortunately, they have rather poor sensitivity, and require a specialized and expensive (>$75k) reader to be used. These are significant barriers to adoption for the majority of biomedical researchers. We have developed an ultrabright fluorescent nanoconstruct we call the Plasmonic Fluor, which is >5,000X brighter than the standard fluorescent reporter used in microarrays today. Simply substituting the Plasmonic Fluor for the existing reporter significantly increases the sensitivity of antibody microarrays without requiring any change to the workflow. Importantly, the Plasmonic Fluor is so bright that it allows fluorescent microarrays to be read using more widely available and versatile readers such as a fluorescent Western blot reader. Additionally, it relaxes the requirements on the reader instrumentation significantly, which has enabled us to create an inexpensive reader that any research lab can afford. To allow even easier adoption of the Plasmonic Fluor enhanced microarrays, we have also created software for microarray data analysis. In this project, we aim to: increase the manufacturing scale of our Plasmonic Fluor to an early commercial scale; more extensively validate enhancement of a variety of popular antibody microarrays; finalize the design of our inexpensive reader; and more fully develop our user- friendly microarray analysis software. We believe the Plasmonic Fluor will become the standard fluorescent reporter molecule for all microarrays, and Plasmonic Fluor-enhanced microarrays will become a widely used and powerful tool for elucidating the role of protein networks in health and disease.