Nanostructured Optical Photonic Crystal Biosensor for HIV Viral Load Measurement

Detecting and quantifying biomarkers and viruses in biological samples have broad applications in early disease diagnosis and treatment monitoring. We have demonstrated a label-free optical sensing mechanism using nanostructured photonic crystals (PC) to capture and quantify intact viruses (HIV-1) from biologically relevant samples. The nanostructured surface of the PC biosensor resonantly reflects a narrow wavelength band during illumination with a broadband light source. Surface-adsorbed biotarget induces a shift in the resonant Peak Wavelength Value (PWV) that is detectable with <10 pm wavelength resolution, enabling detection of both biomolecular layers and small number of viruses that sparsely populate the transducer surface. We have successfully captured and detected HIV-1 in serum and phosphate buffered saline (PBS) samples with viral loads ranging from 104 to 108 copies/mL. The surface density of immobilized biomolecular layers used in the sensor functionalization process, including 3-mercaptopropyltrimethoxysilane (3-MPS), N-gamma-Maleimidobutyryl-oxysuccinimide ester (GMBS), NeutrAvidin, anti-gp120, and bovine serum albumin (BSA) were also quantified by the PC biosensor

Human Herpes Virus 8 in HIV-1 infected individuals receiving cancer chemotherapy and stem cell transplantation

Background: Human Herpes Virus 8 (HHV8) can cause Kaposi’s Sarcoma (KS) in immunosuppressed individuals. However, little is known about the association between chemotherapy or hematopoietic stem cell transplantation (HSCT), circulating HHV8 DNA levels, and clinical KS in HIV-1-infected individuals with various malignancies. Therefore, we examined the associations between various malignancies, systemic cancer chemotherapy, T cell phenotypes, and circulating HHV8 DNA in 29 HIV-1-infected participants with concomitant KS or other cancer diagnoses. Methods: We quantified HHV8 plasma viral loads and cell-associated HHV8 DNA and determined the relationship between circulating HHV8 DNA and lymphocyte counts, and markers of early and late lymphocyte activation, proliferation and exhaustion. Results: There were no significant differences in plasma HHV8 DNA levels between baseline and post-chemotherapy time points or with the presence or absence of clinical KS. However, in two participants circulating HHV8 DNA increased following treatment for KS or HSCT for lymphoma,. We observed an approximately 2-log10 reduction in plasma HHV8 DNA in an individual with KS and multicentric Castleman disease following rituximab monotherapy. Although individuals with clinical KS had lower mean CD4+ T cell counts and percentages as expected, there were no significant associations with these factors and plasma HHV8 levels. We identified increased proportions of CD8+ and CD4+ T cells expressing CD69 (P = 0.01 & P = 0.04 respectively), and increased CD57 expression on CD4+ T cells (P = 0.003) in participants with detectable HHV8. Conclusion: These results suggest there is a complex relationship between circulating HHV8 DNA and tissue-based disease in HIV-1 and HHV8 co-infected individuals with various malignancies

Increased HIV-1 transcriptional activity and infectious burden in peripheral blood and gut-associated CD4+ T cells expressing CD30

HIV-1-infected cells persist indefinitely despite the use of combination antiretroviral therapy (ART), and novel therapeutic strategies to target and purge residual infected cells in individuals on ART are urgently needed. Here, we demonstrate that CD4+ T cell-associated HIV-1 RNA is often highly enriched in cells expressing CD30, and that cells expressing this marker considerably contribute to the total pool of transcriptionally active CD4+ lymphocytes in individuals on suppressive ART. Using in situ RNA hybridization studies, we show co-localization of CD30 with HIV-1 transcriptional activity in gut-associated lymphoid tissues. We also demonstrate that ex vivo treatment with brentuximab vedotin, an antibody-drug conjugate (ADC) that targets CD30, significantly reduces the total amount of HIV-1 DNA in peripheral blood mononuclear cells obtained from infected, ART-suppressed individuals. Finally, we observed that an HIV-1-infected individual, who received repeated brentuximab vedotin infusions for lymphoma, had no detectable virus in peripheral blood mononuclear cells. Overall, CD30 may be a marker of residual, transcriptionally active HIV-1 infected cells in the setting of suppressive ART. Given that CD30 is only expressed on a small number of total mononuclear cells, it is a potential therapeutic target of persistent HIV-1 infection

Miniature bioanalytical assays utilizing mechanical actuation of microspheres

An estimated 70% of medical decisions rely on diagnostic results, yet half of the global population lacks access to diagnostic testing. Geography contributes to this gap, with decreased access found in decentralized settings, which can lack the physical and labor infrastructure necessary for gold standard bioanalytical assays. In such settings, lateral flow affinity assays remain the technique of choice, due to their fast time-to-result, ease-of-use and deliverable geometry. However, these assays suffer in sensitivity, with limits of detection 10 - 1000x higher than their gold standard counterparts. Capitalizing on transport advantages at small scales, many microfluidic devices enable high sensitivity assays in a compact form, but their translation to the field is often challenging due to their complexity and the required supporting instrumentation. As a result, gaps in diagnostics, suitable for the realities of operating in decentralized settings, remain. Inspired by challenges demonstrated during the height of the COVID-19 pandemic, this thesis introduces an affinity assay platform that utilizes simple mechanical, nonfluidic actuation of microspheres on a sensing surface to quantify bioanalytes in liquid samples. The assay integrates two processes, capture of analytes during bead sedimentation, and analysis based on bead interactions with a sensing surface using optical microscopy and image processing. The assay can be performed in a standalone device using minimal manual steps and in less than 30 minutes. Combined with single molecule sensitivity demonstrated by previous bead-based assays, this platform has the potential to enable high sensitivity bioanalysis with the ease-of-use profile required for decentralized settings. In the first part of this thesis, we describe a framework for predicting bead-analyte capture over a range of bioanalytes to aid in design across applications. We apply this method to design and analyze our proposed capture method, and to examine and predict transport advantages that arise from settling microspheres. In the second part, we investigate multiple simple actuation mechanisms that lead to bioanalyte quantification based on bead-surface interactions. While nearly all mechanisms show initial efficacy, thermal diffusion was selected for further development, with demonstrated picomolar limits of detection using a model assay, and bead sliding is also explored for its potential versatility and simplicity of imaging. As nonspecific binding of beads to the surface limits sensitivity, we also theoretically model and experimentally investigate surface coating techniques to minimize this effect and demonstrate preliminary enhanced assay sensitivity using a zwitterionic coating. Finally, using a simple device prototype, we demonstrate the ability of our assay to quantify cardiac troponin I (cTnI), an established biomarker for cardiac injury. Because elevated levels can indicate heart attack or other critical conditions, measuring cTnI accurately and rapidly is essential. cTnI is quantified via immunoaffinity techniques, with current generation high sensitivity lab assays reaching 0.001 ng/mL limit of detection (LOD) in 30 minutes to one hour and bedside point-of-care (POC) devices having 20x higher LOD but in 15 minutes. The instrumentation requirements of these assays make them expensive and challenging to perform in decentralized settings. As the global leading causes of death continue to transition from infectious to chronic diseases, increasing access to cTnI diagnostics will be crucial to improving health outcomes. Our results show that our integrated assay can detect cTnI at 0.01 ng/mL in buffer and at 0.1 - 1 ng/mL in 10x diluted serum in a 1% BSA buffer in under 30 minutes. This is a clinically-useful result, with our assay showing LODs 5-25x higher than current bedside POC assays and on par with previous generation high sensitivity lab assays (0.1 – 1 ng/mL), but in a format that requires a single manual transfer step and no specialized or dedicated instrumentation. The performance of the current assay is limited by sensing surface variability, which could be improved by optimization of surface chemistry and blocking reagents. Overall, the platform presented in this thesis could enable quantification of bioanalytes at sensitivities approaching current standard methods but in a user-friendly, high-throughput, distributable and rapid format, an important step forward towards filling the gap in technology for decentralized diagnostics and for other monitoring applications, such as those in water and food safety.Ph.D

Dry preservation of heavy metal contaminants using cation exchange resins for improved water quality monitoring

Thesis: S.M., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2017.Cataloged from PDF version of thesis.Includes bibliographical references (pages 141-148).Water quality monitoring is crucial to identifying and sustaining safe drinking water sources, tracking contamination events and remediating polluted sources. Monitoring of water sources is usually performed using a combination of field test kits and centralized laboratory analysis. While field test kits are easy and rapid to use, they are often inaccurate and cannot test for all parameters of interest. On the other hand, centralized laboratory testing can quantify parameters at trace levels using high throughput instrumentation, but preserving and transporting large volume water samples to these labs is time consuming and labor intensive. These challenges are especially problematic for rural or resource-limited settings, such that monitoring of drinking water sources in these areas is limited. Inspired by dried blood spotting, which revolutionized centralized testing for clinical trials, this thesis seeks to explore and develop a dry preservation technology for improved water quality monitoring. The technology includes a dry preservation device incorporating a sorbent, which collects and preserves contaminants from a water sample in a format that is easily transported or shippable. When the device arrives at a centralized lab, the contaminants can be removed from the device using a simple solvent elution step, yielding contaminants in a ready to test format, or the sorbent from the device can itself be directly analyzed using testing methods that accept solid samples. Such a paradigm has the potential to increase the ease and reach of water quality monitoring. This thesis describes field work, dry sampling sorbent identification and selection, kinetic parameter testing of the dry sampling sorbent, lab contaminant recovery protocol development and initial dry sampling device design for the dry storage of heavy metal cation contaminants. Heavy metals, such as lead, nickel and copper, area selected because they cause chronic health issues at very small concentrations and are increasingly found in drinking water sources due to anthropogenic contamination. The technology is developed in the context of India; however, dry sampling technology would likely find use in rural and resource limited communities around the world.by Emily B. Hanhauser.S.M

Iron oxide xerogels for improved water quality monitoring of arsenic(<scp>iii</scp>) in resource-limited environments<i>via</i>solid-phase extraction, preservation, storage, transportation, and analysis of trace contaminants (SEPSTAT)

We show that iron oxide xerogels can quantitatively adsorb, store, and release aqueous arsenic(iii), enabling a new arsenic monitoring paradigm where sample contamination is adsorbed onto solid sorbents for transportation to laboratories for analysis.</p

Sexual dimorphism in locus coeruleus dendritic morphology: A structural basis for sex differences in emotional arousal

Stress-related psychiatric disorders, such as depression and anxiety, affect a disproportionate number of women. We previously demonstrated that the major brain norepinephrine (NE)-containing nucleus, locus coeruleus (LC) is more sensitive to stressors and to the stress-related neuropeptide, corticotropin-releasing factor (CRF) in female compared to male rats. Because the LC-NE system is a stress-responsive system that is thought to be dysregulated in affective disorders, sex differences in LC structure or function could play a role in female vulnerability to these diseases. The present study used different approaches to compare LC dendritic characteristics between male and female rats. Immunofluorescence labeling of tyrosine hydroxylase, the norepinephrine synthetic enzyme, revealed that LC dendrites of female rats extend further into the peri-LC region, covering a significantly greater area than those of males. Optical density measurements of dendrites in the peri-LC revealed increased dendritic density in females compared to their male counterparts. Additionally, immunoreactivity for synaptophysin, a synaptic vesicle protein, was significantly greater in the LC in female rats, suggesting an increased number of synaptic contacts onto LC processes. Individual LC neurons were juxtacellularly labeled with neurobiotin in vivo for morphological analysis. LC dendritic trees of females were longer and had more branch points and ends. Consistent with this, Sholl analysis determined that, compared to males, LC dendrites of females had a more complex pattern of branching. The greater dendritic extension and complexity seen in females predicts a higher probability of communication with diverse afferents that terminate in the peri-LC. This may be a structural basis for heightened arousal in females, an effect which may, in part, account for the sex bias in incidence of stress-related psychiatric disorders

Iron oxide xerogels for improved water quality monitoring of arsenic( iii ) in resource-limited environments via solid-phase extraction, preservation, storage, transportation, and analysis of trace contaminants (SEPSTAT)

We show that iron oxide xerogels can quantitatively adsorb, store, and release aqueous arsenic(iii), enabling a new arsenic monitoring paradigm where sample contamination is adsorbed onto solid sorbents for transportation to laboratories for analysis.</p