UNS: Role of Reporter and Membrane Properties for the Sensitivity of Viral Nanoparticle Lateral Flow Assays

CBET - 1511789 Conrad，Jacinta研究人员已经证明，通过使用工程生物活性纳米颗粒可以显着提高灵敏度。所提出的方法开发的应用程序，如果成功的话，将简化病原体检测在各种情况下，包括食品，水和医药samples. Objective提出的研究是识别，理解，并最终工程的噬菌体报告和膜，在侧流检测（LFA）提供灵敏的响应的属性。 假设定制噬菌体纳米颗粒和LFA膜的性质将增强噬菌体转运和结合，并因此降低LFA检测限。具体目标是：（1）改变噬菌体形态以了解噬菌体作为LFA报道基因的性能;（2）鉴定增强噬菌体转运和粘附到膜上的膜性质;和（3）使用定向进化选择具有上级性能的噬菌体。当不同形状和大小的噬菌体报告分子扩散通过LFA膜并与LFA膜结合时，将用共聚焦显微镜成像。使用高通量图像处理算法，噬菌体取向和分散，结合将与膜性质和孔径相关。定向进化将用于从数十亿候选者的文库中选择具有改进的运输和特异性的噬菌体。 智力优势：该项目将PI在成像和受限运输方面的专业知识与co-PI在色谱相互作用和噬菌体工程方面的专业知识相结合，以开发灵敏度更高的LFA。噬菌体报告基因作为LFA报告基因具有潜在的变革性，能够以极低的非特异性结合进行快速、廉价和超灵敏的检测。拟议的研究将建立超灵敏的LFA作为一个广泛适用的平台技术。非球形纳米粒子在多孔介质中的输运研究尚处于起步阶段，本文将噬菌体作为一种新的模型体系，用于研究复杂受限介质中纳米粒子的分散行为。最后，该提案介绍了噬菌体进化与分离作为一种新的方法，工程噬菌体在新兴领域的纳米生物技术的应用。 更广泛的影响：病毒纳米颗粒LFA技术可以很容易地与微流体和基于智能手机的荧光成像集成，以产生一种廉价但超灵敏的使用点诊断工具。生物分离在现代生物制药生产的成本和工艺复杂性方面占主导地位;不需要经过培训的研究人员的过程分析（PAT）技术将立即影响这一领域。此外，现场便携式分析方法可能被证明对环境污染、食品安全监测和医疗诊断有用。休斯顿大学是美国第二大种族多元化的研究型大学，被指定为西班牙裔服务机构，为扩大代表性不足的群体的参与提供了肥沃的招生土壤。PI将继续针对当地科学界正在进行的外联工作，通过为学术和工业科学家组织德克萨斯州软物质会议，并在整个社会，通过编写和展示我们的学术分钟和引擎的科普广播节目。

CBET - 1511789 Conrad, Jacinta The investigators have shown that significantly improved sensitivity can be achieved by using engineered bio-active nanoparticles. The applications of the proposed method development, if successful, will simplify pathogen detection in a variety of contexts, including food, water and medical samples.The objective of the proposed research is to identify, understand, and ultimately engineer the properties of phage reporters and membranes that provide sensitive response in lateral flow assays (LFA). The hypothesis is tailoring the properties of phage nanoparticles and LFA membranes will enhance phage transport and binding and as a result reduce LFA limit of detection. The specific aims are to: (1) vary phage morphology to understand phage performance as LFA reporters; (2) identify membrane properties that enhance phage transport and adhesion onto the membrane; and (3) select phage for superior performance using directed evolution. Phage reporters of varied shape and size will be imaged with confocal microscopy as they diffuse through and bind to LFA membranes. Using high-throughput image-processing algorithms, phage orientation and dispersion, binding will be correlated to membrane properties and pore size. Directed evolution will be used to select phage with improved transport and specificity from libraries of billions of candidates. Intellectual Merit: This project integrates the expertise of the PI in imaging and confined transport with that of the co-PI in chromatographic interactions and phage engineering to develop LFAs of increased sensitivity. Phage reporters are potentially transformative as LFA reporters, enabling fast, inexpensive and ultrasensitive detection with extremely low non-specific binding. The proposed research will establish ultrasensitive LFAs as a broadly-applicable platform technology. The transport of nonspherical nanoparticles in porous media is under-investigated, and the proposed work will use phage as a new model system in for studing dispersion in complex and confined media. Finally, this proposal introduces phage evolution with separation as a novel method to engineer bacteriophage for applications in the emerging area of nanobiotechnology. Broader Impacts: Viral nanoparticle LFA technology can be readily integrated with microfluidics and smartphone-based fluorescence imaging to yield an inexpensive yet ultrasensitive point-of-use diagnostic tool. Bioseparations dominate the cost and process complexity of manufacturing of modern biopharmaceuticals; process analytical (PAT) technologies that do not require trained investigators will immediately impact this area. In addition, a field-portable analytical method may prove useful for environmental contamination, food safety monitoring, and medical diagnostics. The University of Houston is the second-most ethnically diverse research university in the US and is designated as a Hispanic-serving Institution, providing a fertile recruiting ground for broadening participation from underrepresented groups. The PIs will continue ongoing outreach efforts aimed at the local scientific community, by organizing the Texas Soft Matter Meeting for academic and industrial scientists, and at society at large, by writing and presenting popular science radio programs for The Academic Minute and Engines of our Ingenuity.