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

研究人员已经证明，使用工程生物活性纳米颗粒可以显著提高灵敏度。所提议的方法开发的应用如果成功，将简化在各种情况下的病原体检测，包括食品、水和医疗样品。该研究的目的是鉴定、理解并最终设计噬菌体报告体和膜的特性，这些特性在横向流动试验（LFA）中提供敏感反应。假设是调整噬菌体纳米颗粒和LFA膜的特性将增强噬菌体的运输和结合，从而降低LFA的检测极限。具体目的是：(1)改变噬菌体形态，了解噬菌体作为LFA报告者的表现；(2)识别增强噬菌体运输和粘附在膜上的膜特性；(3)利用定向进化技术选择具有优良性能的噬菌体。不同形状和大小的噬菌体报告体将用共聚焦显微镜成像，因为它们通过LFA膜扩散并结合。利用高通量图像处理算法，噬菌体的方向和分散，结合将与膜性质和孔径有关。定向进化将用于从数十亿候选噬菌体库中选择具有改进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副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。噬菌体报告基因与LFA报告基因一样具有潜在的变动性，能够以极低的非特异性结合实现快速、廉价和超灵敏的检测。提出的研究将建立超灵敏lfa作为广泛应用的平台技术。非球形纳米颗粒在多孔介质中的输运研究正在进行中，本研究将利用噬菌体作为研究复杂和受限介质中分散的新模型系统。最后，本提案介绍了噬菌体进化与分离作为一种新的方法来设计噬菌体应用于新兴的纳米生物技术领域。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相结合，从而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制造的成本和工艺复杂性方面占主导地位；不需要训练有素的调查人员的过程分析（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多种族的研究型大学，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聘土壤。pi将通过组织德克萨斯软物质学术和工业科学家会议，以及在整个社会中，通过为学术分钟和我们的创造力引擎编写和展示科普广播节目，继续针对当地科学界进行持续的推广工作。

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.