Enhanced surface-bound biological activity using a nanospring platform

描述(由申请人提供)：该项目的长期商业目标是开发一种快速(几分钟)、灵敏、廉价的基于纳米弹簧(NS)的生物平台，该平台不需要昂贵、复杂的仪器来获得结果，因为在小的二维足迹中具有高活性的生物表面积。目前的多重ELISA分析仅限于需要微流控通道中的激光检测系统的抗原或抗体包被的小球，或设备成本高且复杂的电子芯片抗体捕获平台的基于电化学扩散的分析。本项目的长期目标是：1)确定NS结合抗体的载量和生物学活性。第一阶段)；2)根据第一阶段制定的方案评估抗原下降和捕获(夹心)两种形式；3)开发一种能够对多个样本进行快速、同时分析的多重ELISA法；4)评估纳米弹簧平台用于“小型化”检测的潜力；5)确定纳米弹簧增强多种其他生物制剂活性的能力。从几个不同的角度来看，快速进行多重ELISA检测的能力具有相关性。除了能够检测用于疾病诊断、监测人类疾病进展或治疗程序的几种不同生物标志物外，多重检测在兽医中的细菌和病毒疾病检测和诊断以及药物和毒素筛选方面也有明显的需求。将其他生物活性分子连接到NS上的能力在开发更小和更高效的生物反应器方面具有实用价值，用于药物和制药生产、废水处理和细胞培养中的组织生长。对于第一阶段，我们建议使用二氧化硅纳米弹簧作为包被捕获抗体或抗原的固相，并与目前标准ELISA法中使用的材料(即微滴定板)相比，确定抗体和抗原结合的最大容量，以及这些分子的最大活性。在这些研究中，NS将被沉积在玻璃微珠的支撑介质上。我们将研究纳米弹簧沉积密度与包被蛋白活性之间的关系。与其他材料相比，NS具有明显的优势，这是因为生物材料的表面积显著增加，可用于涂层的材料种类繁多，这些材料易于涂层，使用NS的成本较低。由于可用于结合的大表面积，我们预计该分析的灵敏度可以提高，同时将分析的足迹从标准的96孔形式减少到384孔或1536孔或阵列形式。这种“小型化”将有效地减少所需的样品和试剂的体积，从而将分析时间从几个小时减少到几分钟。 与公共卫生相关：纳米弹簧提供更大的可用表面积(250倍)将有助于结合多个捕获蛋白和/或抗体，从而允许使用单一测试井检测多种化合物(多重免疫分析)。检测可能在较短的时间内(几分钟而不是几小时)、在较小的测试平台上和在检查现场完成，并且将在各种与健康有关的应用中具有不可估量的价值：病毒检测和疫苗开发；在多症状疾病(如反射性交感神经营养不良)中多项检测促炎细胞因子；检测人类肿瘤组织中的血管生成细胞因子(允许选择特定的化疗药物)；检测和区分乙肝、丙型肝炎和艾滋病毒-1病毒(常见的经输血传播的病原体)；衣原体的联合检测。沙眼衣原体和人类乳头瘤病毒(两种无症状的性传播疾病)；评估特定生物标志物的变化，作为阳性结果化疗的预测指标；或在分发受污染的产品之前检测食品病原体和毒素，仅举几例。

DESCRIPTION (provided by applicant): The long-term commercial objective of this project is to develop a rapid (minutes), sensitive, inexpensive nanospring-based (NS) biological platform that does not require expensive, complex instrumentation for obtaining results due to the highly active biological surface area in a small two-dimensional footprint. Current multiplex ELISA analysis is limited to antigen- or antibody-coated beads which require laser detection systems in microfluidic channels, or electrochemilumiscent-based assays with high equipment costs and complex electronic chip antibody-capture platforms. The long-term specific aims of this project are to: 1) to determine loading capacity and biological activity of NS bound antibodies. (Phase I); 2) evaluate both antigen-down and capture (sandwich) formats based on the protocols developed in Phase I; 3) develop a multiplex ELISA format that is capable of performing rapid, simultaneous analyses on multiple samples; 4) evaluate the potential of the nanospring platform for "miniaturization" of the assay; 5) determine the ability of nanosprings to enhance the activity of a wide variety of other biological agents. The ability to rapidly perform multiplex ELISA assays has relevance from several different perspectives. Beyond the ability to assay for several different biomarkers which are used in disease diagnosis, monitoring disease progression or treatment procedures in humans, multiplex assays have a demonstrated need in bacterial and viral disease detection and diagnosis in veterinary medicine, and drug and toxin screening. The ability to attach other bioreactive molecules onto NS has utility in the development of smaller and more efficient bioreactors for drug and pharmaceutical production, wasterwater treatment, and tissue growth in cell culture. For Phase I we propose to use silica nanosprings as the solid- phase for the coating of capture antibodies or antigens, and to determine the maximum capacity of antibody and antigen binding, and maximum activity of these molecules as compared to currently available materials used in standard ELISA assays (i.e microtiter plates). For these studies, NS will be deposited on a support medium of glass microbeads. We will investigate both the density of nanospring deposition as a function of activity of the coated proteins. NS offer a distinct advantage over these other materials due to the dramatic increase in the surface area available for biological materials, the wide variety of materials available for coating and the ease at which these materials can be coated, the low cost of using NS. As a result of the large surface area available for binding, we anticipate that the sensitivity of the assay can be increased while reducing the footprint of the assay from a standard 96-well format to a 384- or 1536-well or array format. This "miniaturization" will effectively reduce the volume of sample and reagents needed thus reducing the time of the assay from hours to minutes. PUBLIC HEALTH RELEVANCE: A greater available surface area afforded by nanosprings (250 fold) will facilitate the binding of multiple capture proteins and/or antibodies, allowing for a single assay test-well to be used in the detection of multiple chemical compounds (multiplex immunoassay). Tests may be completed in a shorter time (minutes rather than hours), on a smaller test platform and on the site of examination and will be invaluable in a variety of health-related applications: viral detection and vaccine development; multiple detection of pro-inflamatory cytokines in multi- symptomatic diseases (e.g. reflex sympathetic dystrophy); the detection of angiogenic cytokines in human tumor tissue (allowing for the selection of specific chemotherapeutic drugs); detection and discrimination of hepatitis B and C, and HIV type-1 viruses (common transfusion-transmitted pathogens); combined detection of Chlamydia. trachomatis and human papillomaviruses (two asymptomatic, sexually transmitted diseases); to evaluate the changes in specific biomarkers as a predictive indicator of positive-outcome chemotherapy; or to detect food pathogens and toxins prior to distribution of contaminated products, to name a few.