Isotachophoresis for Rapid Nucleic Acid Hybridization and Sensitive Detection of Urinary Tract Infections

用于快速核酸杂交和灵敏检测尿路感染的等速电泳

• 批准号: 1159092
• 负责人: Juan Santiago
• 金额: $ 30万
• 依托单位: Stanford University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1159092&HistoricalAwards=false
• 关键词: Isotachophoresis; Rapid; Nucleic; Acid; Hybridization

1159092SantiagoProposed is a study of the theory and practice of a novel assay which integrates on-chip ITP with sequence detection capability of molecular beacons (MBs). MB are synthetic oligonucleotide DNA which fluoresce strongly upon hybridization with their complimentary nucleic acid sequence. ITP is a non-linear electrokinetic technique which can focus target analytes with high selectivity in a sharp electric field gradient. The Santiago group proposes the use of on-chip ITP to achieve 100,000-fold preconcentration and mixing of MBs and rRNA into the same 10 picoliter virtual reaction chamber at an ITP focal zone. This focusing causes ~100,000-fold increase of hybridization rates. The Santiago group has demonstrated the principle of the ITP/MB assay, and here propose a transformative new method which can attain a dramatic, 1000-fold increase in sensitivity over their preliminary work combining ITP and MBs. The proposed 100 fM sensitivity would cover the full clinically relevant range, including presymptomatic UTI infections. They propose a novel two-step assay which initially preconcentrates, mixes, and hybridizes MBs, and subsequently separates un-reacted MBs from hybrids immediately prior to detection to minimize non-specific background fluorescence. They will minimize sample dispersion using geometric modifications to on-chip channels. To improve their design and optimization capabilities, they will, in parallel, develop a novel and unique computational tool for coupling finite rate reactions with non-linear electrokinetic flows. They will validate this computational tool using a set of well-controlled experiments. These computational tools will be incorporated into a free, open-source code with a user-friendly interface, and be portable across major platforms (Windows, MAC, and Unix). This unique code will be made available through downloads, a blog website for updates and commentary, tutorial videos, relevant data bases, documentation (including peer-reviewed journal papers describing the solution methods), and example input and output files. In summary, the proposed study will (1) explore the basic limits and potential of leveraging ITP to create widely applicable on-chip virtual reaction chambers for fast hybridization; (2) create a novel react-then-separate assay which speeds up hybridization rates by 100,000 fold and maximizes sensitivity; (3) demonstrate prototype miniaturized systems for UTI diagnosis from human urine in 10 min; and (4) develop new, free, open-source computational tools for the electrokinetics community. Infectious diseases caused by bacterial pathogens remain one of the most common causes of mortality worldwide. This proposal focuses on urinary tract infections (UTI), the second most common infection in the US. UTI affects all patient demographics; and causes $3.4 billion total medical expenditures, approximately 8 million clinic or emergency department visits, and over 100,000 hospitalizations per year. Similar to most other bacterial infections, diagnosis of UTI requires a centralized clinical microbiology laboratory and trained professionals to perform bacterial culture and phenotyping, which typically takes 1-3 days. Molecular amplification and diagnostic methods exist, but are rarely used for UTI and still require trained personnel in centralized laboratories. This delay time in diagnosis is at least partially responsible for overuse of antibiotics and the associated emergence of drug resistant bacterial strains. To address this need, the Santiago group proposes to develop a prototype demonstration of a low-cost microfluidic device with potential to reduce bacterial diagnostic time from ~3 days to ~10 min including sample loading, on-chip sample preparation, and sequence specific detection. This system will be designed with practical implementation in mind, including ease of use and minimal fluid handling. This work has application to both conventional clinical microbiology laboratories and point-of-care settings, and can potentially eliminate dependence on trained operators. Santiago will use isotachophoresis (ITP) to extract and concentrate nucleic acid bacterial signatures, including 16S ribosomal RNA (rRNA), directly from untreated urine lysate, and detect and quantify its presence using sequence specific molecular beacon probes on a miniaturized device. Websites will be developed to describe the work to the general community and to share models and methods with the scientific community; host a science high school teacher in their laboratory each summer; incorporate assignments based on this research into a Fluids Engineering undergraduate course; host 1-2 undergraduates in the lab; and incorporate research-level material into an advance graduate course.

1159092提出了一种新型检测方法的理论和实践研究，该方法将芯片上ITP与分子信标（MB）的序列检测能力相结合。MB是合成的寡核苷酸DNA，其在与它们的互补核酸序列杂交时发出强烈荧光。ITP是一种非线性电动技术，可以在尖锐的电场梯度中以高选择性聚焦目标分析物。圣地亚哥小组提出使用芯片上ITP来实现100，000倍的预浓缩，并将MB和rRNA混合到ITP焦点区的相同10皮升虚拟反应室中。这种聚焦导致杂交率增加约100，000倍。圣地亚哥小组已经证明了ITP/MB检测的原理，并在此提出了一种变革性的新方法，该方法的灵敏度比他们结合ITP和MB的初步工作高出1000倍。拟定的100 fM灵敏度将涵盖全部临床相关范围，包括症状前UTI感染。他们提出了一种新的两步测定法，该测定法首先预浓缩、混合和杂交MB，随后在检测之前立即将未反应的MB与杂交体分离，以最大限度地减少非特异性背景荧光。他们将通过对片上通道进行几何修改来最大限度地减少样品分散。为了提高他们的设计和优化能力，他们将同时开发一种新颖独特的计算工具，用于将有限速率反应与非线性电动流动相耦合。他们将使用一组控制良好的实验来验证这种计算工具。这些计算工具将被纳入一个免费的开放源代码，具有用户友好的界面，并可跨主要平台（Windows，MAC和Unix）移植。这个独特的代码将通过下载、更新和评论的博客网站、教程视频、相关数据库、文档（包括描述解决方案方法的同行评审期刊论文）以及示例输入和输出文件提供。总之，拟议的研究将（1）探索利用ITP创建用于快速杂交的广泛适用的芯片上虚拟反应室的基本限制和潜力;（2）创建一种新型的反应然后分离的测定，其将杂交速率加快100，000倍并使灵敏度最大化;（3）展示用于在10分钟内从人尿液中诊断UTI的原型小型化系统;以及（4）为电动力学社区开发新的、免费的、开源的计算工具。由细菌病原体引起的传染病仍然是全世界最常见的死亡原因之一。该提案的重点是尿路感染（UTI），这是美国第二大常见感染。UTI影响所有患者的人口统计数据;每年造成34亿美元的总医疗支出，约800万次门诊或急诊就诊，以及超过10万次住院治疗。与大多数其他细菌感染类似，UTI的诊断需要集中的临床微生物学实验室和训练有素的专业人员进行细菌培养和表型分析，通常需要1-3天。存在分子扩增和诊断方法，但很少用于UTI，并且仍然需要在集中实验室中训练有素的人员。这种诊断上的延迟时间至少部分地导致了抗生素的过度使用和相关的耐药菌株的出现。为了满足这一需求，圣地亚哥小组提出开发一种低成本微流体装置的原型演示，该装置有可能将细菌诊断时间从约3天减少到约10分钟，包括样品加载、芯片上样品制备和序列特异性检测。该系统的设计将考虑到实际实施，包括易用性和最小的流体处理。这项工作适用于传统的临床微生物实验室和护理点设置，并可能消除对训练有素的操作人员的依赖。圣地亚哥将使用等速电泳法（ITP）直接从未处理的尿液裂解物中提取和浓缩核酸细菌特征，包括16 S核糖体RNA（rRNA），并使用小型化设备上的序列特异性分子信标探针检测和定量其存在。将开发网站来向一般社区描述这项工作，并与科学界分享模型和方法;每年夏天在他们的实验室里接待一名科学高中教师;将基于这项研究的作业纳入流体工程本科课程;在实验室里接待1-2名本科生;并将研究水平的材料纳入高级研究生课程。