Qualitative and semiquantitative isothermal detection of nucleic acids for point-of-care testing applications

Nucleic acid amplification tests (NAATs) are widely implemented as infectious disease diagnostics due to their high sensitivity and specificity. Polymerase chain reaction (PCR) tests are the most commonly diagnostic NAAT. PCR requires the use of equipment for temperature cycling and instrumentation to monitor reaction progress and often require upstream sample preparation to isolate nucleic acids from a raw clinical samples. As such, PCR-based tests are most commonly used large and well-resourced healthcare systems where clinical samples are collected from patients and transported to a centralized testing laboratory. Point-of-care diagnostic testing (POCT) is a complementary paradigm to centralized testing where testing infrastructure and consumables are disseminated throughout a healthcare system such that diagnostic testing can be performed on-site with the patient nearby. POCT methods enable test-and-treat strategies commonly implemented in STI screening programs and obviate issues associated with patient loss to follow up. When developing NAATs for POCT, it is often necessary to simplify assay protocols and reduce required overhead especially in low resourced settings where access to laboratory resources may be limited. Recently, isothermal NAA methods, which eliminate the need for costly thermocycling equipment, have garnered use in POCT applications often coupled with visual readout methods to eliminate instrumentation requirements. In this work, I expand upon these existing isothermal NAA methods to develop POCT with both qualitative and semiquantitative detection capabilities. First, I developed a thermophilic helicase dependent amplification (tHDA) assay for the detection of the STI Trichomonas vaginalis (TV) and a competitive internal control. This assay is compatible with visual readout via a low cost lateral flow immunoassay (LFIA) device. I also developed a simple yet effective strategy for rapid nucleic acid extraction and enrichment from urine. When coupled, the sample preparation strategy and tHDA assay were able to detect TV at a concentration of 0.25 TV genomes per mL. I observed 96.6% sensitivity and 100% specificity when testing clinical urine samples. Second, I developed a novel test for semiquantitative detection of hepatitis B virus (HBV) DNA at two tunable thresholds with LFIA readout. Chronic HBV patients require serial viral load monitoring to inform initiation of antiretroviral therapy (ART) and ensure continued treatment efficacy for patients receiving ART. I demonstrated the tunability of the semiquantitative ligation and amplification assay by controllably setting two independent quantification thresholds to specific HBV viral load concentrations used in chronic HBV case management. This assay can be performed in under one hour and is compatible with isothermal tHDA

A paperfluidic platform to detect Neisseria gonorrhoeae in clinical samples

Globally, the microbe Neisseria gonorrhoeae (NG) causes 106 million newly documented sexually transmitted infections each year. Once appropriately diagnosed, NG infections can be readily treated with antibiotics, but high-risk patients often do not return to the clinic for treatment if results are not provided at the point of care. A rapid, sensitive molecular diagnostic would help increase NG treatment and reduce the prevalence of this sexually transmitted disease. Here, we report on the design and development of a rapid, highly sensitive, paperfluidic device for point-of-care diagnosis of NG. The device integrates patient swab sample lysis, nucleic acid extraction, thermophilic helicase-dependent amplification (tHDA), an internal amplification control (NGIC), and visual lateral flow detection within an 80 min run time. Limits of NG detection for the NG/NGIC multiplex tHDA assay were determined within the device, and clinical performance was validated retroactively against qPCR-quantified patient samples in a proof-of-concept study. This paperfluidic diagnostic has a clinically relevant limit of detection of 500 NG cells per device with analytical sensitivity down to 10 NG cells per device. In triplicate testing of 40 total urethral and vaginal swab samples, the device had 95% overall sensitivity and 100% specificity, approaching current laboratory-based molecular NG diagnostics. This diagnostic platform could increase access to accurate NG diagnoses to those most in need.This work was funded by the National Institute of Health National Institute of Allergy and Infectious Diseases award number R01 AI113927 to Boston University and the NIH National Institute of Biomedical and Bioengineering award number U54 EB007958 to Johns Hopkins University. (R01 AI113927 - National Institute of Health National Institute of Allergy and Infectious Diseases; U54 EB007958 - NIH National Institute of Biomedical and Bioengineering)Accepted manuscrip

Microfabricated platforms to investigate cell mechanical properties

Mechanical stimulation has been imposed on living cells using several approaches. Most early investigations were conducted on groups of cells, utilizing techniques such as substrate deformation and flow-induced shear. To investigate the properties of cells individually, many conventional techniques were utilized, such as AFM, optical traps/optical tweezers, magnetic beads, and micropipette aspiration. In specific mechanical interrogations, microelectro- mechanical systems (MEMS) have been designed to probe single cells in different interrogation modes. To exert loads on the cells, these devices often comprise piezo-electric driven actuators that attach directly to the cell or move a structure on which cells are attached. Uniaxial and biaxial pullers, micropillars, and cantilever beams are examples of MEMS devices. In this review, the methodologies to analyze single cell activity under external loads using microfabricated devices will be examined. We will focus on the mechanical interrogation in three different regimes: compression, traction, and tension, and discuss different microfabricated platforms designed for these purposes