Single-photon counting APD arrays for high-through-put single-molecule assays

用于高通量单分子检测的单光子计数 APD 阵列

• 批准号: 8297138
• 负责人: Massimo Antonio Ghioni
• 金额: $ 40.51万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8297138
• 关键词: Algorithms; Area; Biological Assay; Biotechnology; Color; Communities; Custom; Data Analyses; Detection; Development; Diagnostic; Drug Industry; Energy Transfer; Event; Fluorescence; Fluorescence Spectroscopy; Foundations; Goals; Holoenzymes; Lasers; Life; Mainstreaming; Measurement; Methods; Molecular Conformation; Optics; Outcome; Photons; Preclinical Drug Evaluation; Prevention; Process; Research; Research Personnel; Screening procedure; Spectrum Analysis; Spottings; Techniques; Technology; Transcription Elongation; Transcription Initiation; analytical tool; biological research; data reduction; detector; disease diagnosis; drug development; drug discovery; point-of-care diagnostics; single molecule; tool development

The long term goal of this proposal is the development of tools enabling high throughput single-molecule fluorescence spectroscopy. The specific aims of this proposal are: (1) Develop an optical setup for 3 color, 48 spot excitation and detection. (2) Develop a 48 pixel custom single-photon avalanche photodiode array (48p-SPADA). (3) Develop field-programmable-gate-array (FPGA) data reduction algorithms for up to 144 channels single-molecule burst analysis. (4) Develop high-throughput fluorescence (cross)-correlation spectroscopy data analysis algorithms in graphical processing unit (GPU). (5) Study fast and transient events in transcription initiation and elongation, focusing on the conformation and interactions of ¿70 with the bacterial RNAP holoenzyme. The expected outcome of this proposal is an increase in throughput for single molecule fluorescence techniques of a factor 48. This will include most single-molecule fluorescence techniques such as single-molecule fluorescence resonant energy transfer (smFRET) and microsecond alternating laser excitation (¿s-ALEX) as well as fluorescence correlation spectroscopy (FCS). The impact of these developments will be twofold. First, single-molecule fluorescence spectroscopy methods will become attractive to a larger research community, which will be able to use is as an efficient discovery and analytical tool. Additionally, it will give access to new temporal regimes that are unattainable with the current technology. Second, the leap in throughput (by a factor 48) will render this technology attractive for point-of-care diagnostics and large scale screening assays used by the biotechnology and pharmaceutical industry. For these reasons, this proposal is very likely to significantly impact the progresses of many areas of biological research including drug discovery and development as well as diagnostics, thus helping increase our understanding of life processes and lays the foundation for advances in disease diagnosis, treatment, and prevention.

该提案的长期目标是开发能够实现高吞吐量的工具 单分子荧光光谱。这项建议的具体目标是： (1)研制了一套3色、48点激发和探测的光学装置。 (2)研制了48像素定制单光子雪崩光电二极管阵列(48P-SPADA)。 (3)开发现场可编程门阵列(现场可编程门阵列)数据简化算法，最高可用于 144道单分子爆发分析。 (4)开展高通量荧光(交叉)相关光谱数据分析 图形处理器(GPU)中的算法。 (5)研究转录起始和延伸过程中的快速和瞬时事件，重点是 ？70与细菌RNAP全酶的构象及相互作用。 这一提议的预期结果是增加单分子的产量 因子48的荧光技术。这将包括大多数单分子 单分子荧光共振能量转移等荧光技术 (SmFRET)和微秒交变激光激发(S-亚历克斯)以及 荧光相关光谱(FCS)。 这些发展的影响将是双重的。第一，单分子荧光 光谱学方法将对更大的研究社区具有吸引力，这将 能够将其作为一种有效的发现和分析工具。 此外，它将提供访问新的时间制度的途径，而这些制度是通过 当前的技术。其次，吞吐量的跃升(提高了48倍)将导致这一点 适用于点式诊断和大规模筛查分析的技术 被生物技术和制药行业使用。出于这些原因，这项提议 极有可能对许多生物学研究领域的进展产生重大影响 包括药物发现和开发以及诊断，从而帮助增加 我们对生命过程的理解，为疾病的进展奠定了基础 诊断、治疗和预防。