"LLISA:'Liposome-Linked Immunosorbant Assay' for Detection of HIV Viral Load at Point-of-Care"

艾滋病毒/艾滋病影响着全世界3300多万人，尤其是撒哈拉以南非洲资源贫乏地区的一个严重问题，那里居住着67%的艾滋病毒/艾滋病患者。抗逆转录病毒疗法延长了艾滋病毒患者的寿命，提高了他们的生活质量，2003年至2008年期间，全球努力使撒哈拉以南非洲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疗法并监测其成功与否，这阻碍了治疗的有效利用。除了CD 4 + T淋巴细胞的计数之外，还非常希望在护理点进行病毒载量计数，因为这两种方法都可以检测到病毒载量。 这些参数是制定适当治疗策略所必需的。HIV病毒在全血中的含量范围从10 pfu/L到数千pfu/L，这使得检测这些微量颗粒具有挑战性。目前的测试包括基于抗体和基于PCR的测试，并且在护理点不可用，特别是对于资源有限的环境。还应当指出的是，这样的设备对于发达国家、对于远程设置、在床边或在医生办公室对于检测病毒的一系列应用当然也是极其有价值的。作为上述问题的解决方案，用于HIV/AIDS分析的微制造护理点（POC）生物芯片具有巨大的前景。我们建议开发一种集成设备，用于在护理点进行HIV病毒载量的电检测。我们建议在我们广泛的初步工作的基础上，在微流控生物芯片内开发基于电学的传感方法，以大大降低运营成本，增加便携性，并提供简单易用的诊断试剂盒，可由医疗保健工作者在远程设施或家访期间操作。我们的综合方法是创新的，因为我们将：（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，并检测微流体捕获室中介质的电阻抗变化。预计阻抗的变化与捕获的病毒颗粒数量相关。我们计划在R21期间使用加标在人全血中的VSV-G病毒作为HIV模型，以证明新检测方案的概念验证。跨学科的合作汇集了Co-PI的专业知识。我们将在Bashir小组（UIUC）开发基于电的微流体即时检测和Lee Lab（UCI）开发微流体的广泛工作的基础上继续发展 以及产生尺寸和性质可控的脂质体。通过整合这两套专业知识，我们有望解决开发即时病毒载量测定的巨大挑战。

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.