Methods for Detecting Nucleic Acid Hybridization Using Liquid Crystals

这个项目的总体目标包括开发使用液晶（LC）检测核酸杂交的方法。此外，为了更好地了解核酸的界面相互作用影响LC取向的基本分子水平机制。这项研究的最终长期目标是在医学诊断应用中实现基于LC的检测，例如基于LC对DNA杂交的定向响应的多路复用方法（即DNA微阵列），该方法使用偏振光的传输来检测LC响应。两种截然不同的方法将被开发出来，因此这个项目的具体目标和研究策略分为两组。第一种方法涉及在存在单层阳离子表面活性剂的情况下LC/水界面上的LC极性倾角的动态变化，与该方法相关的具体目标包括开发多路复用平台，定量研究灵敏度和目标普遍性，开发可用于研究LC/水界面的荧光显微镜方法。以及应用这些方法来了解LC对DNA杂交反应的分子机制。最后，该方法将直接验证的实验，将区分密切相关的细菌病原体。第二种方法涉及当LC与延伸的dsDNA接触时，平面锚定LC的方位角方向的特征手性旋转。相关的具体目标包括宏观光学响应的定量研究，将该方法扩展到单杂交分子的检测，开发使用共价固定探针进行多重检测的方法，以及旨在理解对ssDNA和dsDNA的独特LC响应的机制研究。将对该方法进行与第一种方法类似的验证实验（即相关细菌之间的区分）。更广泛的影响这里开发的方法具有长期潜力，可用于适用于使用点应用（包括资源匮乏环境中的应用）的新型医疗诊断技术。此外，对lc -生物分子相互作用的基本理解将为研究基于lc的生物传感方法提供广泛的帮助。更广泛地说，所开发的光学表征方法将用于研究lc -液界面和其他液-液界面。参与该项目的研究人员将获得在未来几十年预计处于科学技术前沿的多学科领域的专业知识，包括液晶设备，生物检测，单分子显微镜和自组装分子系统。此外，通过参与各种教育和推广项目（例如REU，RET， GAANN， CU- discovery学习中心，CU K-12材料科学推广，科罗拉多高中荣誉学院），研究人员将继续与学生，教师和其他社区成员分享这个项目。正如提案中所总结的那样，PI和之前参与该项目的学生和博士后已经表现出真诚和持续的承诺，即在追求教育和推广的过程中创造性地使用他们的研究；在这个项目中，基本发现和尖端显微镜方法的整合将提供一个理想的平台，从这个平台上向公众传达研究的兴奋。

CBET-1160202SchwartzThe overall objectives of this project involve the development of methods where liquid crystals(LC) are used to detect nucleic acid hybridization. Also, to obtain an improved understanding ofthe fundamental molecular-level mechanisms by which the interfacial interactions of nucleicacids affect LC orientation. The ultimate long-term goal of this research is to enable LC-baseddetection in medical diagnostic applications, e.g. multiplexed methods (i.e. DNA microarrays)based on the orientational response of LCs to DNA hybridization, that use the transmission ofpolarized light to detect the LC response.Intellectual MeritTwo distinct approaches will be developed, and so the specific aims and research strategies ofthis project fall into two groups. The first approach involves a dynamic change of the LC polar tiltangle at the LC/aqueous interface in the presence of a monolayer of cationic surfactant, and thespecific aims associated with this approach include the development of a multiplexed platform,quantitative studies of sensitivity and target generality, the development of fluorescencemicroscopy methods that can be used to study the LC/aqueous interface, and the application ofthese methods to understand the molecular mechanisms of LC response to DNA hybridization.Finally, this method will be directly validated by experiments that will discriminate betweenclosely-related bacterial pathogens.The second approach involves a characteristic chiral rotation of the azimuthal orientation ofplanar-anchored LC when the LC is in contact with extended dsDNA. The associated specificaims include quantitative studies of the macroscopic optical response, extension of the methodto the detection of single hybridized molecules, development of methods that enable multiplexeddetection using covalently immobilized probes, and mechanistic studies aimed at understandingthe distinctive LC response to ssDNA and dsDNA. Similar validation experiments (i.e.discrimination between related bacteria) will be pursued for this method as for the first method.Broader ImpactThe methods developed here have the long-term potential to be used in new medical diagnostictechnologies that are appropriate for point-of-use applications including those in low resourceenvironments. Furthermore, the fundamental understandings obtained about LC-biomoleculeinteractions will be broadly useful for researchers pursuing LC-based biosensing approaches.More broadly, the optical characterization methods developed will be of use for studies of theLC-liquid interface and for other liquid-liquid interfaces.The researchers involved in this project will gain expertise in multidisciplinary areas projected tobe at the forefront of science and technology in the coming decades, including liquid crystaldevices, biodetection, single-molecule microscopy, and self-assembled molecular systems.Also, through their participation in a variety of educational and outreach programs (e.g. REU,RET, GAANN, CU-Discovery Learning Center, Materials Science from CU K-12 outreach,Colorado High School Honors Institute), the researchers will continue to share this project withstudents, teachers, and other community members. As summarized in the proposal the PI andprevious students and post-docs involved in this project have demonstrated a sincere andcontinuing commitment to the creative use of their research in the pursuit of education andoutreach; the integration of basic discovery and cutting-edge microscopy methods in this projectwill provide an ideal platform from which to communicate the excitement of research to thegeneral public.