LLISA: ???Liposome-Linked Immunosorbant Assay??? for Detection of HIV Viral Load

描述(申请人提供)：艾滋病毒/艾滋病影响着全世界3300多万人，在撒哈拉以南非洲资源贫乏的地区尤其严重，那里居住着67%的艾滋病毒/艾滋病患者。抗逆转录病毒疗法(ART)提高了艾滋病毒患者的寿命和生活质量，2003至2008年间，全球努力使撒哈拉以南非洲地区获得这种治疗的机会增加了30倍。然而，缺乏客观的诊断测试来确定何时开始抗逆转录病毒治疗并监测其成功与否，阻碍了有效治疗的使用。除了对CD4+T淋巴细胞进行计数外，在护理点进行病毒载量计数也是非常必要的，因为两者 这些参数对于制定适当的治疗策略是必要的。HIV病毒可能发生在从10pfu/？L到数千pfu/？L的全血中，这给检测这些微量的颗粒物带来了挑战。目前的检测既包括基于抗体的检测，也包括基于聚合酶链式反应的检测，并且不能在护理地点使用，特别是在资源有限的情况下。还应该指出的是，这种设备对于发达国家来说当然也是非常有价值的，对于远程设置、床边或医生办公室中检测病毒的一系列应用来说。作为上述这些问题的解决方案，用于艾滋病毒/艾滋病分析的微型制造的护理点(POC)生物芯片具有巨大的前景。我们建议开发一种集成设备，用于在护理地点对艾滋病毒病毒载量进行电子检测。我们建议在我们广泛的前期工作的基础上，在微流控生物芯片内开发基于电子的传感方法，以极大地降低运营成本，增加便携性，并提供简单易用的诊断试剂盒，供医护人员在偏远设施或家访期间操作。我们的集成方法是创新的，我们将：(I)从全血中捕获特定的病毒，(Ii)使用标记有病毒颗粒抗体的脂质体标记这些病毒，(Iii)通过降低介质的电导率来裂解脂质体，并检测微流控捕捉室中介质的电阻抗变化。阻抗的变化预计与捕获的病毒颗粒的数量有关。我们计划在R21期间使用在全人类血液中添加的VSV-G病毒作为HIV的模型，以演示新检测方案的概念证明。这种跨学科协作汇聚了各合作伙伴的专门知识。我们将在Bashir Group(UIUC)和Lee Lab(UCI)在微流体方面的广泛工作的基础上开发基于电子的微流体护理点测试 以及大小和性质可控的脂质体的产生。随着这两套专业知识的整合，我们希望解决开发护理点式病毒载量分析的巨大挑战。

DESCRIPTION (provided by applicant): HIV/AIDS affects more than 33 million people throughout the world, and is especially a critical problem in resource-poor regions in sub-Saharan Africa, where 67% of all HIV/AIDS patients live. Antiretroviral therapy (ART) increases the longevity and quality of life for HIV patients, and global efforts have increased the accessibility of such treatment by 30-fold in sub-Saharan Africa between 2003 and 2008. However, the lack of objective diagnostic tests to determine when to start ART and to monitor its success hinders the effective use of treatment. In addition to the counting of CD4+ T Lymphocytes, it is also highly desirable to perform viral load counts at the point-of-care, as both these parameters are needed for the development of the appropriate treatment strategy. HIV viruses could occur at levels ranging from 10pfu/¿l to thousands of pfu/¿l of whole blood, making it challenging to detect these minute quantities of particles. Current tests include both antibody- based and PCR-based and are not available at point-of-care, especially for resource-limited settings. It should be also pointed out that such devices would of course be extremely valuable also for the developed world, for remote settings, at bedside, or at the doctor's office for a range of applications in detection of viruses. As a solution to these problems presented above, micro-fabricated point-of-care (POC) biochips for HIV/AIDS analysis hold tremendous promise. We propose to develop an integrated device for the electrical detection of HIV viral load at point-of-care. We propose to build on our extensive preliminary work to develop electrically- based sensing methods within micro-fluidic biochips to greatly reduce the operating costs, increase portability, and provide simple-to-use diagnostic kits that can be operated by healthcare workers in remote facilities or during home visits. Our integrated approach is innovative as we will; (i) capture the specific viruses from whole blood, (ii) label these viruses using liposomes tagged with antibodies for the viral particles, (iii) lyse the liposomes by lowerin the electrical conductivity of the medium, and detect the changes in the electrical impedance of the medium in the microfluidic capture chamber. The change in impedance is expected to correlate to the number of virus particles captured. We plan to use VSV-G virus spiked in whole human blood as a model for HIV during this R21 to demonstrate the proof of concept of the novel detection scheme. The interdisciplinary collaboration brings together the expertise of the Co-PIs. We will build on the extensive work in Bashir group (UIUC) on development of microfluidic point-of-care tests that are electrically based, and in Lee Lab (UCI) in microfluidics and generation of liposomes of controlled size and properties. With the integration of these two sets of expertise, we expect to address the grand challenge of developing point-of-care viral load assays.