Portable Bioassay System Based on Electronic p-Chips: Application to Detect Dengue Antibodies

摘要 本项目的目的是论证一种新型便携式分析仪的可行性 利用该平台技术进行多重免疫分析。使多路传输能力成为可能 通过使用小型电子p芯片。最初的概念验证应用将是免疫分析确定 血清或血浆样本中登革病毒(DENV)抗体的浓度。化验结果将是 用于快速确定患者以前是否感染过四种病毒中的一种 血清型。这一信息是至关重要的，因为先前存在的对DENV的免疫力会使疾病急剧恶化。 再次暴露于异源病毒血清型时的进展和最终的疾病结局。长期的 目标是为多重免疫分析提供一个便携式分析平台，用于分析关键的生物标记物 各种疾病。该系统的优点是分析速度快(从绘制 患者的血液样本到结果报告)，最少的样本处理和低成本的 分析器和消耗品。 电子芯片是激光激活的微应答器(“p芯片”)，体积很小(600微米x 600微米x 100微米)，化学惰性，高度稳定。P芯片，每个包含唯一的序列号，是 用探针衍生，在这种情况下是针对特定蛋白质生物标记物的抗体，然后与 样品和结合用荧光试剂检测(S)。P芯片的一个关键特征是它们是无源的。 仅当脉冲光直接照射时才会激活的设备，从而使它们能够 单独阅读，同时在一个封闭的旋转管中旋转，暴露在聚焦的激发激光束下。这个 读出依赖于荧光强度和芯片序列号的同时测定 (ID)，用于识别结合探针。多重分析是通过将不同的探针放置在 同一试管中的P-芯片，其方式很容易读取数十到数百个 在很短的时间内报告结果。该项目的主要目标是(1)建立一个 制作阅读器原型，开发控制设备和分析数据的软件；以及(2) 证明一种一管一步免清洗免疫检测登革热病毒的方法并对其进行表征。 建议的方法的结果将是一种新的系统，用于测试患者的暴露程度 最近或过去，登革热病毒。该系统将由便携式分析仪(“气旋”)组成。 该检测配置在p芯片上，用于定量血清中四种抗登革热抗体的血清型。系统 可以很容易地扩展以包括其他感兴趣的生物标志物。

Summary The purpose of the present project is to demonstrate a new type of portable analyzer and the feasibility of performing multiplex immunoassays utilizing this platform technology. The multiplex capability is made possible by using small, electronic p-Chips. The initial proof-of-concept application will be an immunoassay determining the concentration of antibodies to dengue virus (DENV) in serum or plasma samples. The assay will be designed to rapidly determine whether a patient has been previously infected with one of the four viral serotypes. This information is crucial as pre-existing immunity to DENV can drastically worsen disease progression and ultimately disease outcome upon re-exposure to heterologous viral serotypes. The long-term goal is to provide a portable assay platform for multiplex immunoassays that analyze key biomarkers for a variety of diseases. The advantages of this system are rapid assay speed (turnaround time from drawing a patient's blood sample to the reporting of the results), minimal sample handling and low cost for both the analyzer and consumables. The electronic chips are laser light-activated microtransponders ("p-Chips") that are small (600 µm x 600 µm x 100 µm), chemically inert and highly stable. p-Chips, each of which contains a unique serial number, are derivatized with probes, in this case antibodies to specific protein biomarkers, and then incubated with a sample and binding detected with fluorescent reagent(s). A key feature of p-Chips is that they are passive devices that become active only when directly illuminated with pulsed light, thus enabling them to be individually read while spinning in a closed, rotating tube exposed to a focused excitation laser beam. The readout relies on the simultaneous determination of the intensity of fluorescence and the chip serial number (ID), which identifies the binding probe. Multiplex assays are constructed by placing different probes on p-Chips within the same test tube in a manner that lends itself readily to reading dozens to hundreds of p-Chips and reporting results in a very short period of time. The main goals of this project are to (1) build a prototype reader and develop software that will both control the device and analyze the data; and (2) demonstrate and characterize a one-tube-one-step-no-wash immunoassay for detecting dengue virus. The results for the proposed approach will be a novel system for testing the exposure of a patient, either recently or in the past, to the dengue virus. The system will be composed of the portable analyzer ("Cyclone") and the assay configured on p-Chips quantifying four serotypes of the anti-dengue Abs in serum. The system can be expanded easily to include additional biomarkers of interest.