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（分钟），灵敏，廉价的基于纳米弹簧（NS）的生物平台，由于在小的二维足迹中具有高度活性的生物表面积，因此不需要昂贵，复杂的仪器来获得结果。目前的多重ELISA分析限于抗原或抗体包被的珠粒，其需要微流体通道中的激光检测系统，或基于电致发光的测定，其具有高设备成本和复杂的电子芯片抗体捕获平台。本项目的长期具体目标是：1）确定NS结合抗体的负载能力和生物活性。（第一阶段）; 2）根据第一阶段开发的方案评估抗原下降和捕获（夹心）形式; 3）开发能够对多个样本进行快速、同时分析的多重ELISA形式; 4）评估纳米弹簧平台的潜力“微型化”的分析; 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测定的能力具有相关性。除了测定用于疾病诊断、监测疾病进展或人类治疗程序的几种不同生物标志物的能力之外，多重测定在兽医学中的细菌和病毒疾病检测和诊断以及药物和毒素筛选中具有已证明的需求。将其他生物活性分子附着到NS上的能力在开发用于药物和药物生产、废水处理和细胞培养中的组织生长的更小和更有效的生物反应器中具有实用性。对于阶段I，我们提出使用二氧化硅纳米弹簧作为固相用于包被捕获抗体或抗原，并且确定抗体和抗原结合的最大能力，以及与标准ELISA测定中使用的当前可用材料（即微量滴定板）相比这些分子的最大活性。对于这些研究，NS将沉积在玻璃微珠的支持介质上。我们将研究纳米弹簧沉积的密度作为包被蛋白质活性的函数。由于可用于生物材料的表面积的显著增加、可用于涂覆的材料的种类繁多以及这些材料可被涂覆的容易性、使用NS的低成本，NS提供了优于这些其他材料的明显优势。由于可用于结合的大表面积，我们预期可以增加测定的灵敏度，同时将测定的足迹从标准96孔格式减少到384孔或1536孔或阵列格式。这种“小型化”将有效地减少所需的样品和试剂的体积，从而将测定时间从数小时减少到数分钟。 公共卫生相关性：纳米弹簧提供的更大可用表面积（250倍）将促进多种捕获蛋白和/或抗体的结合，允许单个测定测试孔用于检测多种化合物（多重免疫测定）。测试可以在更短的时间内完成在一个较小的测试平台上和检查现场，它将在各种与健康相关的应用中发挥不可估量的作用：病毒检测和疫苗开发;多种症状疾病中促炎细胞因子的多重检测（例如反射性交感神经营养不良）;检测人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B型和C型肝炎以及HIV-1型病毒（常见的输血传播病原体）;衣原体的联合检测。例如，这些活动的目的包括：检测沙眼和人乳头瘤病毒（两种无症状性传播疾病）;评估特定生物标志物的变化，作为化疗阳性结果的预测指标;或在分发受污染产品之前检测食品病原体和毒素。

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.