DNA microarray surface analysis to optimize detection

描述(由申请人提供)：这项生物工程研究小组R01竞争延续提案旨在延续上一资助时期在阐明商业和模型核酸微阵列诊断分析的关键表面特性方面的生产力和影响。尽管这些多重诊断格式具有巨大的临床和环境应用潜力，但只有两种微阵列分析被FDA批准用于临床诊断。阵列表面捕获问题阻碍了分析的可靠性、灵敏度和重复性，这些问题发生在所有商业平台上、单个平台中的批次内、不同临床实验室之间，甚至在单个载玻片上的微阵列元件内。这也是商业开发中相当一部分检测方法面临的最大问题：定制打印阵列，涉及不同的打印技术和检测试剂。因此，目前微阵列的临床影响是非常有限的。这项建议的意义来自于重点获得了解和解决分析可靠性、灵敏度和重现性问题所需的新信息。对于最近FDA、EPA和NIH的疾病、药物药理学和毒性筛选目标的关键路径倡议，广泛采用微阵列技术依赖于提高分析性能并对其局限性有明确的了解。这项提议直接回应了这一需求。工作假设是，使用最新可用的高分辨率仪器精确量化含有核酸的微阵列中的表面固定和捕获效率将识别当前的分析限制，并促进这些诊断方法临床使用所需的改进的表面捕获分析性能。这个为期3年的项目的具体目标包括：目标1.验证和关联用于核酸分子分析的新的表面分析技术方法，特别是量化微阵列表面上打印和捕获的核酸密度。目的2.比较DNA和PNA微阵列印刷斑点的均匀性与印刷底物条件和化学的关系，并解决高化学分辨率下的点对点变异性作为商业微阵列底物上观察到的微阵列分析偏差和不确定度的常见来源。目的3.通过直接定量和检测复杂介质中的少量目标DNA，提高标准微阵列分析的性能要求和捕获分析的检测限。这项建议的重点是提高目前核酸微阵列生物检测的能力。一套互补的最先进的表面分析方法将被用来评估影响DNA表面固定和目标杂交的变量，以模型和商业分析形式。拟议的战略解决了这些分析中的重要生物分析问题，以确定对监测公共卫生和疾病、药理学和个性化药物以及毒理学所涉及的高度优先的科学和技术产生影响的改进。开发可靠的生物检测方法，能够在阿托莫尔水平上检测来自真实世界样本的DNA杂交，将允许在当前研究用途之外的快速临床诊断应用。促进这些广泛使用的微阵列分析格式从当前的研究转化为未来真正的临床应用是本研究的主要目的。

DESCRIPTION (provided by applicant): This Bioengineering Research Group R01 competing continuation proposal aims to continue the productivity and impact of the preceding funded period in elucidating key surface properties of commercial and model nucleic acid microarray diagnostic assays. Despite immense potential clinical and environmental applications for these multiplexed diagnostic formats, only two microarray assays are FDA-approved for clinical diagnostic use. Array surface-capture problems preclude assay reliability, sensitivity, and reproducibility, occurring across all commercial platforms, within batches in a single platform, between different clinical labs, and even within microarray elements on a single slide. This is also the most significant problem for a substantial fraction of assays under commercial development: custom printed arrays involving different printing technologies and assay reagents. Hence, clinical impact of microarrays is currently very limiting. Significance is derived from this proposal's focus on obtaining new information necessary for understanding and resolving the issues of assay reliability, sensitivity and reproducibility. Wide adoption of microarray technology for recent FDA, EPA and NIH Critical Path initiatives for disease, drug pharmacology and toxicitiy screening goals relies on improving assay performance and developing clear understanding of their limitations. This proposal responds directly to that need. The working hypothesis is that precise quantitation of surface immobilization and capture efficiencies in microarrays comprising nucleic acids using newly available high-resolution instrumentation will identify current assay limitations, and facilitate improved surface-capture assay performance necessary for clinical use of these diagnostics. Specific aims for the 3-year project include: Aim 1. Validate and correlate new surface analytical technology methods for molecular analysis of nucleic acids, specifically, to quantify printed and captured nucleic acid density on microarrayed surfaces. Aim 2. Compare DNA and PNA microarray printed spot uniformity as it relates to printed substrate conditions and chemistry, and address spot-to-spot variability at high chemical resolution as common sources of microarray assay variance and uncertainty observed on commercial microarray substrates. Aim 3. Improve standard microarray assay performance requirements and capture assay detection limits by direct quantitation and detection of small amounts of target DNA in complex media without pre-purification. This proposal focuses on improving the current capabilities in nucleic acid microarray bioassay. A suite of complementary state-of-the-art surface analytical methods will be exploited to assess variables affecting DNA surface immobilization and target hybridization in model and commercial assay formats. The strategy proposed addresses important bio-analytical problems in these assays to identify improvements that impact high-priority science and technology involved in monitoring public health and disease, pharmacology and personalized medicine, and toxicology. Developing reliable bio- assays capable of detecting DNA hybridization from real-world samples at an attomole level would allow rapid clinical diagnostic applications beyond current research use. Facilitating translation of these widely used microarray assay formats from current research to true clinical future use is a major aim of this research.