Microscale biosensors for HIV detection and viral load determination

The HIV/AIDS pandemic has killed 39 million people worldwide, and nearly as many people are living with HIV infection today. The global response to this disease has come a long way since the emergence of HIV in the early 1980s, including more than 64 billion USD in international spending between 2002 and 2013 alone [1]. Due to the worldwide effort, HIV infection has been transformed from a death sentence into a manageable, chronic illness that can have limited impact on lifespan when treated properly [2]. Antiretroviral therapy, public health campaigns, and other education and prevention efforts have facilitated an age in which no one, regardless of age, gender, sexual orientation, nationality, or socioeconomic status should face despair on account of this infection. However, barriers persist to bringing proper care to millions of people worldwide, including access to testing and the diagnostic tools necessary for proper administration of therapy. Following serological testing to establish HIV-positive status, the current standard of care requires monitoring of CD4+ T lymphocyte counts and plasma HIV viral load to guide administration of antiretroviral therapy. For many individuals living with HIV worldwide, the expensive and sophisticated laboratory instruments necessary for these measurements are extremely difficult to access due to poor healthcare infrastructure and lack of technical personnel. For those who are capable of bearing the expense and inconvenience of traveling to facilities that can provide one or both of these measurements, continuing care can be hindered by difficulties in patient follow-up. A point-of-care technology capable of performing these essential measurements to HIV therapy, therefore, is a critical need worldwide. Here we explore solutions rooted in micro- and nanotechnology principles to address this immense challenge in global health. Point-of-care diagnostics which meet the following criteria could improve the way that HIV/AIDS is treated, particularly in remote and resource-limited settings: low-cost assays (approximately $10 or less), small sample volumes (approximately 10 μL or less), rapid measurements (approximately 10 minutes or less), as well as technologies that are easy to use and portable. Our expertise in this area began with the development of a lab-on-a-chip micro-cytometer for CD4+ T lymphocyte enumeration from a drop of whole blood, which was tested on HIV-positive patients in the Champaign-Urbana, IL area and matched results from clinical flow cytometry at Carle Foundation Hospital in Urbana, IL [3]. This thesis describes work on the complementary measurement, viral load detection, aimed at meeting the ideal criteria described above for a point-of-care diagnostic technology. Our approaches to viral load measurements follow two broad themes. First, we describe an antigen-based approach which leverages immuno-affinity recognition for whole virus particle detection. In this method, the novel component of our sensing system is an ion-filled liposome which, upon stimulation (in this case, by heating), releases ions into low-conductivity media in a microchannel and can be quantitatively measured by a simple impedance measurement. We have termed this technique “ion-release impedance spectroscopy.” Employing the liposome in an immunoassay involving a primary capture antibody to HIV surface proteins and a secondary, identical antibody anchored to the exterior of the liposome, we are able to show qualitative detection of HIV virions in a microchannel [4]. We have improved aspects of this approach by performing ion-release impedance spectroscopy with liposomes exhibiting a higher melting temperature, and explored immuno-affinity capture of viruses on magnetic beads in an attempt to perform a concentration or separation step from a whole blood sample. Our second approach is detection of viral RNA in whole blood. In this technique, we employ loop-mediated isothermal amplification (LAMP) in the detection of viral RNA following a reverse-transcription test. One novel aspect of this approach is in performing the test from unprocessed whole blood, which we introduce into a microfluidic channel, mix with cell lysis buffer, add to RT-LAMP reagents, and distribute into nanoliter-scale droplets on a silicon microchip. Another novel step is to image this reaction with a consumer mobile smartphone device, which we integrate with the microchip setup using a 3-D printed platform. Results from our smartphone-imaged RT-LAMP technique show amplification in reactions containing as few as 3 virus particles per droplet, corresponding to 670 viruses per microliter of whole blood [5]. The true power of this approach, however, can be realized in a quantitative digital detection approach for which we describe a framework and preliminary designs, providing a basis for a highly-practical viral load test based on the proof-of-concept demonstrated in our lab. These micro- and nanotechnology approaches to HIV viral load measurements give hope for a portable diagnostics platform which could bring the standard of life-saving HIV/AIDS care to people in all parts of the world, no matter how remote or resource-limited