Sensitive and quantitative point-of-care viral load testing on paper

 描述(申请人提供)：人类免疫缺陷病毒(HIV)已造成3900万多人死亡，每年夺走150多万人的生命。抗逆转录病毒疗法在降低死亡率方面非常有效，在发展中国家扩大获得抗逆转录病毒疗法的机会，避免了500多万与艾滋病毒有关的死亡。扩大获得抗逆转录病毒治疗的机会，在实验室基础设施和训练有素的工作人员有限的情况下，在早期诊断、及时启动抗逆转录病毒治疗和及时诊断抗逆转录病毒治疗失败的治疗监测方面提出了紧迫的挑战。定期病毒载量检测是抗逆转录病毒治疗监测的最准确和最受欢迎的方法，世界卫生组织(世卫组织)在资源有限的情况下管理艾滋病毒的指导方针承认并推荐这种方法。在发达国家，艾滋病毒载量检测采用基于核酸的检测方法。然而，这些检测方法昂贵，以实验室为基础，技术复杂，在资源有限的情况下不容易获得。因此，为了增加低收入和中等收入国家通过定期抗逆转录病毒治疗监测获得艾滋病毒护理的机会，迫切需要廉价、快速、敏感和特定的艾滋病毒载量监测工具。在这份提案中，我们将基于我们先前的专业知识，使用纳米和微米级方法来开发用于护理点(POC)病毒负载测试的可靠微芯片平台。我们建议的平台技术依赖于三项与工程和生物学相关的技术进步：(I)使用高效和特异性的抗gp120/gp41抗体在芯片上捕获多种HIV亚型；(Ii)使用便携式系统对病毒裂解物进行灵敏的无标签电子检测；以及(Iii)使用印刷柔性石墨烯修饰电极的纸基微流控技术。我们的微芯片制造简单、廉价，而且可以批量生产，因为我们使用导电油墨在纸衬底上打印微电极，每个芯片只需几便士。我们之前发表的工作已经证明了概念证明，可以有选择地从指尖体积的血液(&lt；100µL)中捕获和检测多种艾滋病毒亚型(A、B、C、D、E、G和Panel)，并使用芯片上病毒裂解物的电子传感进行ART监测。该诊断平台技术广泛适用于其他传染病，如肝炎、疟疾、水痘、结核病和流感。我们已经证明了所提出的微芯片技术能够检测生物样本中的KSHV、EBV和大肠杆菌。这项拟议研究的主要目的是开发一种价格低廉(1美元)、一次性、可批量生产的纸基微流控设备，它可以自动处理全血样本(&lt；100微米L)，并快速(&lt；30分钟)检测和计数艾滋病毒。

 DESCRIPTION (provided by applicant): Human immunodeficiency virus (HIV) has caused more than 39 million deaths and takes the lives of more than 1.5 million people per year. Antiretroviral therapy (ART) has been highly effective in reducing mortality and expanding access to ART in developing countries has averted more than 5 million HIV-related deaths. The expansion of access to ART has given rise to urgent challenges in early diagnosis, timely ART initiation, and treatment monitoring for timely diagnosis of ART failure in resource-limited settings, where there are limited laboratory infrastructure and trained staff. Regular viral load testing is the most accurate and preferred approach for ART monitoring and is recognized and recommended by the World Health Organization (WHO) guidelines for HIV management in resource-limited settings. In developed countries, nucleic acid-based assays are used for HIV load testing. However, these assays are expensive, laboratory-based, and technically complex, and cannot be easily accessed in resource-limited settings. Thus, to increase access to HIV care with regular ART monitoring in low- and middle-income countries, there is an urgent need for inexpensive, rapid, sensitive, and specific HIV load monitoring tools. In this proposal, building upon our prior expertise, we will use nano- and micro-scale approaches to develop a reliable microchip platform for point-of-care (POC) viral load testing. Our proposed platform technology relies on three engineering and biology related technological advances: (i) on-chip capture of multiple HIV subtypes using anti-gp120/gp41 antibodies with high efficiency and specificity, (ii) sensitive label-free electrical detection of viral lysate using a portable system and (iii) paper- based microfluidics with printed flexible graphene-modified electrodes. Our microchip fabrication is simple, inexpensive, and mass-producible as we print microelectrodes on paper substrates using conductive inks for only a few pennies per chip. Our prior published work has shown the proof-of-concept that multiple HIV subtypes (A, B, C, D, E, G, and panel) can be selectively captured and detected from a fingerprick volume of blood (<100 µL) with clinically relevant virus concentrations for ART monitoring using electrical sensing of viral lysate on-chip. This diagnostic platform technology is broadly applicable to other infectious diseases such as hepatitis, malaria, pox, tuberculosis, and influenza. We have shown the ability of the proposed microchip technology to detect KSHV, EBV, and E. coli in biological samples. The main aim of the proposed study is developing an inexpensive (<$1), disposable, and mass-producible paper-based microfluidic device that is automated to handle whole blood samples (<100 µL) and to rapidly (<30 minutes) detect and count HIV.