Tza-Huei Wang, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Detection of point mutations in tissues and body fluid DNA have wide-spread implications in studying molecular etiology of cancer as well as in developing new technologies for future clinical applications. In this application we propose to develop a clinically relevant genetic analysis technology that enables multiplex detection of point mutations in unamplified genomic DNA using limited amounts of clinical samples. This amplification-free detection technology will be developed using a combination of two innovative technologies, single-molecule detection (SMD) and quantum dot (QD)-mediated fluorescence resonance energy transfer (qFRET). Preliminary studies have yielded promising results indicating that this integrative SMD-qFRET technology is able to detect DNA targets at extremely low concentrations (~ 5 fM), obviating the need for target amplification. When incorporated with allele-specific oligonucleotide ligation, this technology can enable detection of low- abundance point mutations in unamplified genomic DNA. This project consists of three Specific Aims. First, we will develop an amplification-free point mutation detection method and evaluate it by analyzing four representative point mutations in the KRAS gene (at codon 12 and codon 13) and one commonly occurring mutation in the BRAF gene (at codon 599) in unamplified genomic DNA from ovarian serous tumors. Second, we will enhance the sensitivity and resolution of this new method to 0.5 fM and 0.5 % (mutant/wild-type ratio of 1:200) respectively by optimizing both the design of the QD-mediated fluorescence energy transfer system and the ligation reaction conditions. Third, we will increase the analysis throughput and mass detection efficiency of the assays by implementing this new detection method in a multiplex, microfluidic format. We will design and fabricate a microfluidic array device and use it to dispense and guide micro-volumes of genomic DNA samples for multiplex analysis using SMD spectroscopy for seven mutation assays simultaneously. It is expected that, as compared to conventional PCR-based mutational analysis, this new technology will provide a more rapid and reliable measure in detecting point mutation using a 5 ?l or less assay volume. If successfully established, it could provide a relatively straightforward molecular diagnostic platform for cancer detection and can potentially be performed in many laboratories and clinical settings. [unreadable] [unreadable] [unreadable]

The most studied epigenetic abnormality in cancer is gene silencing associated with DNA hypermethylation. Recent studies demonstrate that gene inactivation by promoter hypermethylation can occur at early stages of cancer progression, perhaps even before mutations can be detected. Using DNA methylation as a cancer biomarker shows great promise for early diagnosis, assessments in high risk individuals, and post-therapy monitoring. While a noninvasive test with bodily fluids to detect cancer is seen as a holy grail by clinicians, the ability to detect specific DNA methylation changes in bodily fluids, including blood, sputum or stool represents a greater challenge, due to the small amounts of DNA available in these samples and the limited tumor content of such samples. Therefore, a clinically useful technology that allows for detection of DNA methylation in bodily fluids will have a substantial impact in both cancer diagnosis and management. We propose to develop a new methylation detection platform which integrates improved methods for each of the critical processes involved: DNA isolation, bisulfite treatment, and detection of methylation. All steps will ultimately be integrated and performed on a microfiuidic chip, utilizing superparamagnetic particles as a common carrier for fluidic and molecular manipulations and the quantum dots-mediated fluorescence resonance energy transfer technology (QD-FRET) for biosensing. This approach will facilitate highly efficient sample preparation and sensitive detection of DNA methylation in bodily fluids. In addition, the proposed technology can perform integrated and automatic analysis, minimizing manual labor and time while providing more reproducible results and being useful for a large scale screening. The potential use in pre-cUnical applications will be determined by testing bodily samples from patients with early stage cancers and controls, including serum/plasma, sputum and stool. In addition, the detection of cancer specific methylation in the blood/plasma will be used to monitor therapeutic response in Projects 2, 3 and 4, facihtating development prior to examination of cancer patients.

DESCRIPTION (provided by applicant): Circulating cell-free DNA have been found to freely exist in human body fluids such as blood, urine, and stool. These rare nucleic acids contain a bevy of genetic and epigenetic biomarkers that can be used to reveal hidden pathologies. In cancer, cell-free DNA can be used to determine the status of remote tumors through detecting tumor-associated molecular alterations, such as point mutations and DNA methylation, with great promise for non-invasive monitoring of tumor dynamics, therapeutic response, and disease progression. Since cell-free DNA are present at very low physiological concentrations, PCR-based methodologies, such as mutation allele specific amplification (MASA) and methylation-specific PCR (MSP), are current mainstream methods for their analysis. Unfortunately, the throughput and multiplexing of these methods have been limited. Given that substantial heterogeneity in molecular alterations exists among cancers, analysis of a panel of biomarkers is needed to determine the tissue type and malignant transformation. However, efforts to multiplex PCR have been hampered by issues including mispriming and limited availability of colored florescent dyes. Employing a large number of separate PCRs for each sample is costly and problematic as it requires large amounts of DNA where DNA is the limiting factor for cell-free DNA analysis. We seek to develop a microfluidic single-molecule detection platform to address the unmet need for multiplexed and high-throughput analysis of cell-free DNA biomarkers. Our platform takes advantage of the high sensitivity of single-molecule spectroscopy to enable direct analysis of low-concentration DNA without reliance on PCR. It aims to achieve highly multiplexed detection of e 48 biomarkers in a single measurement via the use of a molecular coding approach to generate biomarker-specific fluoro-codes that are subsequently decoded by multi-color, single-molecule spectroscopy. In addition, the platform incorporates a microfluidic device for parallel target concentration and arrayed detection, facilitating high-throughput measurements of 50 samples on a chip. The capability of the platform is exemplified by detection of both genetic and epigenetic cancer biomarkers, including point mutations and promoter DNA methylation in serum, sputum and stool samples, collected from patients with ovarian, lung and colon cancer.

DESCRIPTION (provided by applicant): Micro RNAs (miRNAs) are short nucleotides (~ 20 nt) that act as regulators of gene expression in nearly all cellular processes including differentiatio, proliferation, and apoptosis. In tumors, miRNAs have been shown to play key roles in cancer processes such as metastasis and tumorigenesis. Since a single miRNA can regulate many mRNAs, dysregulation of one miRNA can have far-reaching biological consequences and that a small panel of miRNAs may suffice for diagnostic purposes. Recent discovery of the existence of circulating miRNAs in the blood stream further raises the potential of miRNAs as noninvasive biomarkers for remote cancer detection. In addition, due to their small size and protection within an exosomal shell, miRNAs robustly resist RNA degradation in tissue and blood. These features make miRNAs exciting targets for cancer diagnosis and prognosis. Since cell-free, circulating miRNAs exist at very low physiological concentrations, current methods to detect these targets predominantly rely on highly sensitive RT-qPCR. However, RT-qPCR is generally limited to single-plex analyses while clinical assessment of miRNAs requires that a panel (e.g.10-100) miRNAs to be quantified in a rapid and inexpensive manner. Employing a large number of separate PCRs for each sample is costly and requires large amounts of miRNA, which is difficult to obtain from blood samples. On the other hand, existing multiplexed technologies such as miRNA microarrays are woefully lacking in the requisite sensitivity to detect these circulating miRNA panels in body fluids. In this project, we propose to develop a single molecule length coding platform to address the unmet clinical need for highly sensitive and multiplexed detection of circulating miRNA. The platform employs a ligation- based molecular length coding scheme to generate miRNA-specific length-encoded strands that are deciphered by size separation to facilitate multiplexed detection. It utilizes cylindrical illumination confocal spectroscopy to quantify low concentration targets through single molecule counting, achieving high sensitivity and quantitative accuracy. In addition, a microfluidic device will be developed to simultaneously concentrate multiple microliter-sized samples into picoliter-sized plugs for arrayed separation in sub-micron channels to enhance both the resolution of separation and the throughput of analysis. Finally, we will validate the proposed platform by determining the analytical sensitivity and specificity using control serum samples spiked with synthetic miRNA sequences. We aim to achieve PCR-equivalent sensitivity of <10-22 mole, specificity of >1000:1 for unrelated miRNA and > 100:1 for related miRNA. Validation with clinical samples will be performed by analyzing a panel of 20 miRNAs in the serum of patients with advanced esophageal adenocarcinoma (20 samples) and healthy controls (20 samples) using d 200 mL of serum in a single reaction. The result will be compared to that obtained by RT-qPCR using 4 mL of serum split into 20 separate single-plex reactions.

DESCRIPTION (provided by applicant): Aberrantly methylated DNA is found abundantly in all forms of cancer and the tumors that produce them. In fact, it has been estimated that within the cells of every tumor are several hundreds of aberrantly methylated CpG islands, many of which are promoters of tumor suppressor genes. The ability to detect and quantify promoter methylation will allow much more refined diagnostic and prognostic stratification. Recently, several groups including ours have reported the detection of tumor-associated methylated DNA circulating in serum/plasma. The use of circulating methylated DNA is a particularly attractive for screening and companion diagnostics for cancer, as serum/plasma is obtained through a simple, relatively noninvasive procedure. Currently, detection of circulating methylated DNA is mainly performed using bisulfite-based methods such as methylation specific PCR (MSP) due to their high sensitivity and specificity. However, clinical applications of these tests have been encumbered by a number of hurdles, resulting in highly variable success. For any bisulfite based methods, the process of DNA extraction and bisulfite conversion involves several disconnected steps on different platforms, resulting in substantial sample loss. Furthermore, MSP is designed to detect a specific methylation pattern; however, the promoter methylation patterns may be highly variable in tumors, compromising its clinical sensitivity. While sequence-based methods including bisulfite sequencing and pyrosequencing can be used to analyze methylation heterogeneity, these methods do not have the requisite sensitivity to detect the extremely low ratios (<0.1%) of methylated epialleles present in the bloodstream. In order to address these issues, we propose a streamlined methylation detection platform combining a silica paramagnetic bead (SSB)-based method for processing circulating DNA from large volume plasma samples to maximize assay fidelity and a microfluidic digital high resolution melt (HRM) approach for detecting and quantifying heterogeneous promoter epialleles at the single molecule level. A microfluidic device will be developed to facilitate high fidelity digital PCR and HRM in 1.6x106 microfluidic reaction chambers. A melt curve analysis program will be developed for analyzing the specific methylation allele in each reaction chamber according to the respective melt profile. The platform will be validated using both synthetic control samples and clinical samples from lung cancer patients undergoing epigenetic therapy. The proposed technology will enable detection and quantification of heterogeneous methylation with a low LOD (<10 copies of methylated DNA in ? 2 ml plasma sample), high sensitivity (1/100,000; methylated/unmethylated alleles) and a wide dynamic range (7 orders of magnitude), a level of performance unattainable by any other existing technologies (MSP, real- time qMSP and sequencing).

? DESCRIPTION (provided by applicant): The ability for clinicians to provide effective treatments to bacterial infections with targeted antibiotics hinges on molecular diagnostics capable of identifying the pathogen and determining its susceptibility to antibiotics in a timely manner. Urinary tract infection (UTI) is a particularly representative infection because it one of the most common bacterial infections that affect all ages but is currently only diagnosed in centralized laboratories via bacterial culture, which typically takes 2-3 days for definitive diagnosis. The significant time delay between sample collection and result reporting contributes to widespread empiric use of broad-spectrum antibiotics, which has contributed towards emergence of multidrug-resistant pathogen. Toward addressing this important unmet need, our overall goal is to develop and validate an integrated diagnostic platform for bacterial pathogen identification (ID) and antibiotic susceptibility testing (AST) in a sample-to-answer manner in under 3 hours. Such rapid molecular diagnostics will transform the clinicians' ability to provide evidence-based diagnosis of bacterial infections, expedite treatments based on objective data, promote effective utilization of antibiotics. Specifically, we propose to develop an innovative droplet microfluidic network capable of combinatorially generating millions of picoliter (pL)-sized droplets of different compositions, i.e. mixtures of samples and probes or antibiotics at varying concentration levels, as the backbone technology. The microfluidic chip enables a streamlined approach for sample-reagent mixing, compartmentalization of mixtures into a massive number of droplets, and serial dilutions to simultaneously carry out pathogen ID and AST. In the pathogen ID module, single bacterial cells are encapsulated in droplets, achieving an effective concentration equivalent to 108-109 cfu/ml and thereby enabling rapid identification via the hybridization of molecular beacon probes in an amplification-free approach. In the AST module, individual bacterial cells are encapsulated and cultured in droplets that enhance local culture condition for bacterial growth and enable direct measurements of single bacterial doublings, thereby facilitating direct phenotypic AST from urine samples. We have assembled an academic-industry partnership consisted of Johns Hopkins University (droplet microfluidics and diagnostics), Stanford University (UTI, molecular probes, validation studies), University of Arizona (microfluidic AST), and GE Global Research (manufacturing and system integration). We propose the following aims: 1) to achieve single cell, amplification-free pathogen ID in a droplet-based microfluidic chip using a panel of peptide nucleic acid molecular beacons that target bacterial 16S rRNA; 2) to develop a droplet-based single cell AST capable of determining the minimum inhibitory concentration (MIC) for commonly used antibiotics for UTI; 3) to perform system integration and instrument development through partnership with our industry partner; and 4) to perform analytical and clinical validation with the integrated device. To facilitate technology translation, a Product Development Plan for future clinical deployment is proposed.

? DESCRIPTION: Micellar nanoparticles that mimic the size and shape of viral particles are attractive as a DNA delivery vehicle because of their improved colloidal stability and transport properties, ability to evade immune clearance, and high payload packaging capacity. Moreover, nanoparticle shape has explicitly been identified as an important factor determining their transport properties and delivery efficiency. However, there is no available nanoparticle synthesis method for packaging plasmid DNA payloads while allowing sufficient control over particle size and shape. Recently, we have shown that distinct shape control and tuning for DNA micelles can be achieved through complexation of plasmid DNA with engineered block or graft copolymers of polycation and poly (ethylene glycol) under controlled assembly conditions. In this proposed study, we will develop a synergistic research program comprising parallel and integrated experimental and computational strategies to (1) develop and understand new methods for DNA micelle assembly that permit scalable, high-uniformity synthesis with shape control and high stability; (2) reveal shape-dependent nanoparticle diffusion and transport properties in physiologically media in vitro and in vivo; and (3) demonstrate the delivery efficiency of a theranostic vector by shape-controlled DNA micelles and their imaging and therapeutic efficacy using mouse models of human metastatic cancers. The proposed study brings together a unique combination of expertise in DNA nanoparticle assembly, microfluidics-based single-particle analysis/fluorescence correlation spectroscopy, in vivo imaging, cancer theranostics, and computer simulations to address a crucial knowledge gap in the engineering and delivery of DNA nano-therapeutics. It will not only offer a new, generalizable method for synthesizing shape-controlled DNA micelles, but also provide a mechanistic understanding of shape- dependent transport properties of nanoparticles. Moreover, the integrated nature of our experimental and computational approach establishes a new paradigm that will greatly accelerate the discovery and development of new DNA nanoparticle systems for efficient gene medicine delivery.

TITLE  Ultrasensitive Detection of Tumor Specific DNA Methylation Changes for the Early Detection of Lung Cancer  ABSTRACT  This proposal seeks to improve upon the management of lung cancer through detection of tumor specific abnormal DNA methylation. Despite highly publicized advances in genomics and proteomics, the promise of non-invasive diagnostics and personalized medicine remains largely unrealized. Recently, comprehensive determination of genetic and epigenetic aberrations has become a major activity in cancer research since it is well understood that these aberrations provide clues to the process of tumorigenesis. The applicants have developed extremely sensitive assays for the detection of hypermethylated DNA sequences. They have also optimized the isolation and processing of circulating cell free DNA from tumors for these sensitive methods. The comprehensive genome wide analysis of molecular changes in cancer completed by The Cancer Genome Atlas (TCGA) has been used to identify many highly frequent cancer specific methylation events in lung cancer that will be combined with an integrated approach to sample processing and preparation and novel sensitive detection strategies to provide utrasensitive detection of tumor specific changes in DNA methylation in blood and sputum samples. With a large population based screening cohort, the Pittsburgh Lung Screening Study, they will develop and characterize the performance of sensitive methods for detecting cancer specific changes in DNA methylation. This molecular detection will compliment CT screening to address the important issue of early detection of lung cancer.

Esophageal squamous cell carcinoma (ESCC) ranks sixth among all cancers worldwide, with 450,000 new cases diagnosed per year and a very poor prognosis. Low-cost, minimally invasive point-of-care population screening for ESCC is badly needed, particularly in LMICs, where 5-year ESCC survival is less than 10%. Altered methylation occurs frequently in human malignancies, including EC, constituting an early event that can serve as an early cancer detection biomarker. However, for DNA methylation to be used in this manner, we need cost-effective, user-friendly and robust tests that permit clinical translation in LMICs. We propose an early ESCC diagnostic strategy comprising a single-use, swallowable sponge to collect esophageal specimens coupled with a smartphone-manipulated microfluidic chip for automated sample processing and methylation detection. This strategy does not require endoscopy (EGD), can be administered by healthcare workers without medical degrees, and uses an on-phone analytic app. The sponge is cheaper (approximately $30 each), less invasive, and easier to perform than EGD ($1500 total cost, including facility fees); there are no room charges, unlike EGD. The microchip integrates DNA extraction, bisulfite DNA conversion, and methylation analysis into a single device. In addition, the microchip interfaces with a cellphone, for both device control and methylation detection and analysis. The integrated device enables detection of DNA methylation from crude samples in a ?sample-to-answer? manner, without the need for sending data back to an analytic center off-site. Thus, the proposed platform promises a cost-effective and user-friendly POC strategy for early ESCC detection that is implementable in LMICs. We have also assembled a talented interdisciplinary, intercontinental team to execute this strategy. Our task will be achieved in 2 phases via the following Aims: UG3 PHASE: Aim 1: To assess a streamlined protocol for sample collection, processing and methylation detection. Aim 2: To implement DNA sample processing and methylation detection into a mobile phone- manipulated microfluidic chip system. Aim 3: To test a prototype ESCC diagnostic strategy integrating the DNA methylation detection system with the swallowable sponge for sample collection. UH3 PHASE: Aim 1: To improve the cost-effectiveness and robustness of the methylation diagnostic system for use in LMICs. Aim 2: To develop a method for ambient chip storage and perform on-chip QC tests to verify assay functionality. Aim 3: To conduct an ESCC diagnostic trial in Uganda using our point-of-care strategy.

PROJECT SUMMARY The ability for clinicians to effectively treat bacterial infections with targeted antibacterials in the acute-care settings hinges on diagnostics capable of identifying the pathogen broadly and determining its susceptibility to antibacterials in a timely manner. Bloodstream infection (BSI) is a particularly representative disease because it is the leading cause of death due to infections with rapid disease progression. Unfortunately, the inconvenient delay of blood culture for definitive diagnosis contributes to widespread empiric use of broad- spectrum antibacterials and emergence of multi-drug-resistant pathogens. Toward addressing this critical unmet need, we propose to develop a new molecular diagnostic platform that integrates bacterial detection, species identification (ID), and antibacterial susceptibility testing (AST) from blood samples in a streamlined test. The expected sample-to-answer turnaround time is 90 min for ID and as early as 2-3 hr for AST. Such integrated diagnostic solution within the proposed timeframe will transform acute-care clinicians? ability to establish diagnosis of bacterial infections, need for infection control, and antibacterial treatment based on objective data to improve clinical outcome. Using an innovative microfluidic digital array chip for assaying single cells as a backbone technology, we propose to develop a new molecular diagnostic platform which promises rapid ID and AST and allows customizable workflow and assay tailored to the clinical scenario while adjustable based on real-time results. The array chip seamlessly integrates digitization of cells, brief incubation (under various drug conditions), single-cell PCR (scPCR) or reverse transcriptase PCR (scRT-PCR) and single-cell high-resolution melt (scHRM). Thereby, bacterial pathogen can be detected at the level of single-cells, identified based on species- specific melt curves, and their antibacterial susceptibility profile subsequently assessed by measuring changes in rRNA level as a biosynthetic marker of cell viability. ScPCR/scRT-PCR enables sensitive detection and absolute quantification of rRNA of individual cells critical to rapid and reliable differentiation between viable and no-viable cells; while scHRM overcomes a key limitation of bulk HRM to resolve multiple species for diagnosing polymicrobial infections or discarding contaminations. Since both ID and AST do not rely on culture, they reduce total turnaround time from days to minutes/hours. We have assembled a superb team of multi-disciplinary investigators and industry advisors with complementary expertise and strong track record of team science. We propose the following aims:1) to develop a streamlined BSI diagnostic protocol for integrated ID and AST; 2) to develop a microfluidic array chip that enables ID and AST with single-cell resolution; 3) to develop instrument and analysis programs for single- cell ID and AST; and 4) to demonstrate the single-cell diagnostic platform, we will perform analytical and pilot clinical validation studies.

PROJECT SUMMARY Antimicrobial resistance (AMR) in Neisseria gonorrhoeae (NG) is in the top tier of AMR l threats as defined by WHO. Over the past decades, NG has developed resistance to all antimicrobials previously recommended for treatment of gonorrhea, leaving dual therapy of ceftriaxone plus azithromycin as currently the only appropriate option for empirical first-line therapy in most countries world-wide. Now NG strains have been reported resistant to both ceftriaxone and azithromycin. Conversely, although ciprofloxacin is no longer recommended by the CDC for the treatment of NG, recent studies suggest that a large percentage of GC infections could be potentially treated with ciprofloxacin. With the continued evolution of AMR, there is an urgent need for personalized treatment approaches that target an individual infection, in contrast to the current ?globally uniform? empiric approach. However, this requires clinicians to know drug resistance or susceptibility quickly enough to inform prescription decisions. To mitigate the emergence and spread of AMR in NG, CDC periodically publishes STD treatment guidelines to assist clinicians. These guidelines are informed by susceptibility data generated by the national CDC Gonococcal Isolate Surveillance Project (GISP). GISP?s impact, however, is being jeopardized by technological evolution. Determining AMR requires prolonged (24-48 hours) microbiological cultivation in sophisticated laboratory facilities. But the advent of nucleic acid amplification tests (NAAT) with enhanced speed and accuracy has supplanted culture-based diagnosis of NG infections, leading to limited specimen collection for culture and loss of capability to perform culture of NG in most testing clinics. Consequently, the success of widespread NAAT has inadvertently created a critical void in AMR testing. We propose to develop a complete diagnostic solution capable of performing identification (ID) of NG infection and phenotypic antimicrobial susceptibility testing (AST). Specifically, ID is achieved using PCR to detect NG- specific DNA markers; while AST is carried out using quantitative PCR to measure the difference in nucleic acid markers (bacterial DNA or RNA) which correlate with the physiologic state of pathogen between drug- treated samples and no-drug controls. Our combined ID-AST platform, which capitalizes on innovative advances in NAAT and microfluidics, has the potential to deliver all essential NG diagnostic information specific to each suspected patient at the POC to tailor personalized treatment; its practical design is also well- suited to resolve the technical challenges confronting GISP for routine surveillance of NG AMR. We propose the following aims: 1) to develop a streamlined diagnostic protocol for integrated ID and AST of NG; 2) to develop a droplet microfluidic cartridge implementing the integrated ID-AST assay; 3) system integration and instrument development; and 4) analytical and clinical validation of the integrated system. To facilitate technology translation, a Product Development Plan for future clinical deployment is proposed.

The revolutionary advances in human immunodeficiency virus (HIV) antiretroviral therapy (ART) which could reach and maintain viral suppression have gradually transitioned HIV/acquired immunodeficiency syndrome (AIDS) to a manageable, chronic disease in the past 2 decades. However, it is still one of the leading public threats in the world with approximately 37.9 million people living with HIV (PLWHA), 1.7 million new infections, and 770,000 HIV-related deaths in 2018. As a comprehensive strategy for improving the clinical management of HIV, the World Health Organization (WHO) specifically calls for improved accessibility of not only ART, but also HIV testing to reach untested high-risk populations and to help PLWHA ascertain their viral load (VL) and potential for transmission. In response, this project seeks to develop a diagnostic technology for rapid, affordable and easy-to-use HIV VL detection that is amenable for self-testing and even in-home testing. Unlike conventional diagnostic technologies that are impractical for self-testing due to requirements of sophisticated devices and instruments, our platform employs magnetofluidic technology to replace bulk fluid transport with magnetic particle manipulation, enabling the integration of sample processing and PCR without the need for complex fluidic cartridges and supporting instrumentation. Miniaturization of instrumentation and assay facilitated by magnetofluidics minimizes reagent consumption and the thermal mass, leading to >10-fold reduction in cost and assay time. The integrated device facilitates HIV RNA detection from finger-stick blood in a facile ?sample-to- answer? manner, promising a rapid, inexpensive and user-friendly strategy for VL self-testing. We have assembled a multi-disciplinary team with complementary expertise and strong track record and history of collaboration in team science. In order to achieve the above goal, we propose (i) to develop a streamlined assay protocol for sample processing and PCR detection of HIV in whole blood, (ii) to develop an inexpensive magnetofluidic cartridge implementing the HIV detection assay as a simple blood-to-result test, (iii) to develop a portable instrument to enable facile HIV self-testing, (iv) to develop a method for ambient cartridge storage, and finally (v) to evaluate performance and patient acceptability of the HIV self-testing platform in both acute care and resource-limited settings. The development of this HIV VL self-testing technology has the potential for dramatically increasing the accessibility of detecting acute infections and monitoring viral suppression in PLWHA, thus presenting a game changer in the global HIV prevention strategy.