Pilot evaluation of an enzymatic assay for rapid measurement of antiretroviral drug concentrations.

OBJECTIVE: Maintaining adequate drug adherence is crucial to ensure the HIV prevention benefits of pre-exposure prophylaxis (PrEP). We developed an enzymatic assay for rapidly measuring tenofovir-diphosphate (TFV-DP) concentrations-a metabolite that indicates long-term PrEP adherence. SETTING: The study was conducted at the Madison HIV Clinic at Harborview Medical Center in Seattle. METHODS: We enrolled adults receiving standard oral PrEP, and individuals not receiving any antiretrovirals. We measured TFV-DP concentrations in diluted whole blood using our novel REverSe TRanscrIptase Chain Termination (RESTRICT) assay, based on inhibition of HIV reverse transcriptase (RT) enzyme. Blood samples were diluted in water, DNA templates, nucleotides, RT, and intercalating dye added, and results measured with a fluorescence reader-stronger fluorescence indicated higher RT activity. We compared RESTRICT assay results to TFV-DP concentrations from matched dried blood spot samples measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) using ≥ 700 fmol/punch TFV-DP as a threshold for adequate adherence (≥ 4 doses/week). RESULTS: Among 18 adults enrolled, 4 of 7 participants receiving PrEP had TFV-DP levels ≥ 700 fmol/punch by LC-MS/MS. RESTRICT fluorescence correlated with LC-MS/MS measurements (r = - 0.845, p < 0.0001). Median fluorescence was 93.3 (95% confidence interval [CI] 90.9 to 114) for samples < 700 fmol/punch and 54.4 (CI 38.0 to 72.0) for samples ≥ 700 fmol/punch. When calibrated to an a priori defined threshold of 82.7, RESTRICT distinguished both groups with 100% sensitivity and 92.9% specificity. CONCLUSIONS: This novel enzymatic assay for measuring HIV reverse transcriptase activity may be suitable for distinguishing TFV-DP concentrations in blood that correspond to protective PrEP adherence.

Current state of commercial point-of-care nucleic acid tests for infectious diseases.

The COVID-19 pandemic has put the spotlight on the urgent need for integrated nucleic acid tests (NATs) for infectious diseases, especially those that can be used near patient ("point-of-care", POC), with rapid results and low cost, but without sacrificing sensitivity or specificity of gold standard PCR tests. In the US, the Clinical Laboratory Improvement Amendments Certificate of Waiver (CLIA-waiver) is mandated by the Food and Drug Administration (FDA) and designated to any laboratory testing with high simplicity and low risk for error, suitable for application in the POC. Since the first issuance of CLIA-waiver to Abbot's ID NOW Influenza A&B in 2015, many more NAT systems have been developed, received the CLIA-waiver in the US or World Health Organization (WHO)'s pre-qualification, and deployed to the front line of infectious disease detection. This review highlights the regulatory process for FDA and WHO in evaluating these NATs and the technology innovation of existing CLIA-waived systems. Understanding the technical advancement and challenges, unmet needs, and the trends of commercialization facilitated through the regulatory processes will help pave the foundation for future development and technology transfer from research to the market place.

HIV detection from human serum with paper-based isotachophoretic RNA extraction and reverse transcription recombinase polymerase amplification.

The number of people living with HIV continues to increase with the current total near 38 million, of which about 26 million are receiving antiretroviral therapy (ART). These treatment regimens are highly effective when properly managed, requiring routine viral load monitoring to assess successful viral suppression. Efforts to expand access by decentralizing HIV nucleic acid testing in low- and middle-income countries (LMICs) has been hampered by the cost and complexity of current tests. Sample preparation of blood samples has traditionally relied on cumbersome RNA extraction methods, and it continues to be a key bottleneck for developing low-cost POC nucleic acid tests. We present a microfluidic paper-based analytical device (µPAD) for extracting RNA and detecting HIV in serum, leveraging low-cost materials, simple buffers, and an electric field. We detect HIV virions and MS2 bacteriophage internal control in human serum using a novel lysis and RNase inactivation method, paper-based isotachophoresis (ITP) for RNA extraction, and duplexed reverse transcription recombinase polymerase amplification (RT-RPA) for nucleic acid amplification. We design a specialized ITP system to extract and concentrate RNA, while excluding harsh reagents used for lysis and RNase inactivation. We found the ITP µPAD can extract and purify 5000 HIV RNA copies per mL of serum. We then demonstrate detection of HIV virions and MS2 bacteriophage in human serum within 45-minutes.

Source Apportionment of Environmental Combustion Sources using Excitation Emission Matrix Fluorescence Spectroscopy and Machine Learning.

The link between particulate matter (PM) air pollution and negative health effects is well-established. Air pollution was estimated to cause 4.9 million deaths in 2017 and PM was responsible for 94% of these deaths. In order to inform effective mitigation strategies in the future, further study of PM and its health effects is important. Here, we present a method for identifying sources of combustion generated PM using excitation-emission matrix (EEM) fluorescence spectroscopy and machine learning (ML) algorithms. PM samples were collected during a health effects exposure assessment panel study in Seattle. We use archived field samples from the exposure study and the associated positive matrix factorization (PMF) source apportionment based on X-ray fluorescence and light absorbing carbon measurements to train convolutional neural network and principal component regression algorithms. We show EEM spectra from cyclohexane extracts of the archived filter samples can be used to accurately apportion mobile and vegetative burning sources but were unable to detect crustal dust, Cl-rich, secondary sulfate and fuel oil sources. The use of this EEM-ML approach may be used to conduct PM exposure studies that include source apportionment of combustion sources.

Point-of-Care HIV Viral Load Testing: an Essential Tool for a Sustainable Global HIV/AIDS Response.

The global public health community has set ambitious treatment targets to end the HIV/AIDS pandemic. With the notable absence of a cure, the goal of HIV treatment is to achieve sustained suppression of an HIV viral load, which allows for immunological recovery and reduces the risk of onward HIV transmission. Monitoring HIV viral load in people living with HIV is therefore central to maintaining effective individual antiretroviral therapy as well as monitoring progress toward achieving population targets for viral suppression. The capacity for laboratory-based HIV viral load testing has increased rapidly in low- and middle-income countries, but implementation of universal viral load monitoring is still hindered by several barriers and delays. New devices for point-of-care HIV viral load testing may be used near patients to improve HIV management by reducing the turnaround time for clinical test results. The implementation of near-patient testing using these new and emerging technologies may be an essential tool for ensuring a sustainable response that will ultimately enable an end to the HIV/AIDS pandemic. In this report, we review the current and emerging technology, the evidence for decentralized viral load monitoring by non-laboratory health care workers, and the additional considerations for expanding point-of-care HIV viral load testing.

Excitation-Emission Matrix Spectroscopy for Analysis of Chemical Composition of Combustion Generated Particulate Matter.

Analysis of particulate matter (PM) is important for the assessment of human exposures to potentially harmful agents, notably combustion-generated PM. Specifically, polycyclic aromatic hydrocarbons (PAHs) found in ultrafine PM have been linked to cardiovascular diseases and carcinogenic and mutagenic effects. In this study, we quantify the presence and concentrations of PAHs with lower molecular weight (LMW, 126 < MW < 202) and higher molecular weight (HMW, 226 < MW < 302), i.e., smaller and larger than Pyrene, in combustion-generated PM using excitation-emission matrix (EEM) fluorescence spectroscopy. Laboratory combustion PM samples were generated in a laminar diffusion inverted gravity flame reactor (IGFR) operated on ethylene and ethane. Fuel dilution by Ar in 0% to 90% range controlled the flame temperature. The colder flames result in lower PM yields however, the PM PAH content increases significantly. Temperature thresholds for PM transition from low to high organic carbon content were characterized based on the maximum flame temperature (Tmax,c ∼ 1791 to 1857 K) and the highest soot luminosity region temperature (T*c ∼ 1600 to 1650K). Principal component regression (PCR) analysis of the EEM spectra of IGFR samples correlates to GCMS data with R2 = 0.988 for LMW and 0.998 for HMW PAHs. PCR-EEM analysis trained on the IGFR samples was applied to PM samples from woodsmoke and diesel exhaust, the model accurately predicts HMW PAH concentrations with R2 = 0.976 and overestimates LMW PAHs.

Excitation Emission Matrix Fluorescence Spectroscopy for Combustion Generated Particulate Matter Source Identification.

The inhalation of particulate matter (PM) is a significant health risk associated with reduced life expectancy due to increased cardio-pulmonary disease and exacerbation of respiratory diseases such as asthma and pneumonia. PM originates from natural and anthropogenic sources including combustion engines, cigarettes, agricultural burning, and forest fires. Identifying the source of PM can inform effective mitigation strategies and policies, but this is difficult to do using current techniques. Here we present a method for identifying PM source using excitation emission matrix (EEM) fluorescence spectroscopy and a machine learning algorithm. We collected combustion generated PM2.5 from wood burning, diesel exhaust, and cigarettes using filters. Filters were weighted to determine mass concentration followed by extraction into cyclohexane and analysis by EEM fluorescence spectroscopy. Spectra obtained from each source served as training data for a convolutional neural network (CNN) used for source identification in mixed samples. This method can predict the presence or absence of the three laboratory sources with an overall accuracy of 89% when the threshold for classifying a source as present is 1.1 µg/m3 in air over a 24-hour sampling time. The limit of detection for cigarette, diesel and wood are 0.7, 2.6, 0.9 µg/m3, respectively, in air assuming a 24-hour sampling time at an air sampling rate of 1.8 liters per minute. We applied the CNN algorithm developed using the laboratory training data to a small set of field samples and found the algorithm was effective in some cases but would require a training data set containing more samples to be more broadly applicable.

Semiquantitative Nucleic Acid Test with Simultaneous Isotachophoretic Extraction and Amplification.

Nucleic acid amplification tests (NAATs) provide high diagnostic accuracy for infectious diseases and quantitative results for monitoring viral infections. The majority of NAATs require complex equipment, cold chain dependent reagents, and skilled technicians to perform the tests. This largely confines NAATs to centralized laboratories and can significantly delay appropriate patient care. Low-cost, point-of-care (POC) NAATs are especially needed in low-resource settings to provide patients with diagnosis and treatment planning in a single visit to improve patient care. In this work, we present a rapid POC NAAT with integrated sample preparation and amplification using electrokinetics and paper substrates. We use simultaneous isotachophoresis (ITP) and recombinase polymerase amplification (RPA) to rapidly extract, amplify, and detect target nucleic acids from serum and whole blood in a paper-based format. We demonstrate simultaneous ITP and RPA can consistently detect 5 copies per reaction in buffer and 10 000 copies per milliliter of human serum with no intermediate user steps. We also show preliminary extraction and amplification of DNA from whole blood samples. Our test is rapid (results in less than 20 min) and made from low-cost materials, indicating its potential for detecting infectious diseases and monitoring viral infections at the POC in low resource settings.

Shape-Dependent Surface Reactivity and Antimicrobial Activity of Nano-Cupric Oxide.

Shape of engineered nanomaterials (ENMs) can be used as a design handle to achieve controlled manipulation of physicochemical properties. This tailored material property approach necessitates the establishment of relationships between specific ENM properties that result from such manipulations (e.g., surface area, reactivity, or charge) and the observed trend in behavior, from both a functional performance and hazard perspective. In this study, these structure-property-function (SPF) and structure-property-hazard (SPH) relationships are established for nano-cupric oxide (n-CuO) as a function of shape, including nanospheres and nanosheets. In addition to comparing these shapes at the nanoscale, bulk CuO is studied to compare across length scales. The results from comprehensive material characterization revealed correlations between CuO surface reactivity and bacterial toxicity with CuO nanosheets having the highest surface reactivity, electrochemical activity, and antimicrobial activity. While less active than the nanosheets, CuO nanoparticles (sphere-like shape) demonstrated enhanced reactivity compared to the bulk CuO. This is in agreement with previous studies investigating differences across length-scales. To elucidate the underlying mechanisms of action to further explain the shape-dependent behavior, kinetic models applied to the toxicity data. In addition to revealing different CuO material kinetics, trends in observed response cannot be explained by surface area alone. The compiled results contribute to further elucidate pathways toward controlled design of ENMs.

Two orders of magnitude improvement in detection limit of lateral flow assays using isotachophoresis.

Lateral flow immunoassays (LFA) are one of the most prevalent point-of-care (POC) diagnostics due to their simplicity, low cost, and robust operation. A common criticism of LFA tests is that they have poor detection limits compared to those of analytical techniques, like ELISA, which confines their application as a diagnostic tool. The low detection limit of LFA and associated long equilibration times are due to kinetically limited surface reactions that result from low target concentration. Here, we use isotachophoresis (ITP), a powerful electrokinetic preconcentration and separation technique, to focus target analytes into a thin band and transport them to the LFA capture line, resulting in a dramatic increase in the surface reaction rate and equilibrium binding. We show that ITP is able to improve the limit of detection (LoD) of LFA by 400-fold for 90 s assay time and by 160-fold for a longer 5 min time scale. ITP-enhanced LFA (ITP-LF) also shows up to 30% target extraction from 100 µL of the sample, whereas conventional LFA captures less than 1% of the target. ITP improves the LoD of LFA to the level of some lab-based immunoassays, such as ELISA, and may provide sufficient analytical sensitivity for application to a broader range of analytes and diseases that require higher sensitivity and lower detection limits.

Colorimetric detection of catalytic reactivity of nanoparticles in complex matrices.

There is a need for new methodologies to quickly assess the presence and reactivity of nanoparticles (NPs) in commercial, environmental, and biological samples since current detection techniques require expensive and complex analytical instrumentation. Here, we investigate a simple and portable colorimetric detection assay that assesses the surface reactivity of NPs, which can be used to detect the presence of NPs, in complex matrices (e.g., environmental waters, serum, urine, and in dissolved organic matter) at as low as part per billion (ppb) or ng/mL concentration levels. Surface redox reactivity is a key emerging property related to potential toxicity of NPs with living cells, and is used in our assays as a key surrogate for the presence of NPs and a first tier analytical strategy toward assessing NP exposures. We detect a wide range of metal (e.g., Ag and Au) and oxide (e.g., CeO2, SiO2, VO2) NPs with a diameter range of 5 to 400 nm and multiple capping agents (tannic acid (TA), polyvinylpyrrolidone (PVP), branched polyethylenimine (BPEI), polyethylene glycol (PEG)). This method is sufficiently sensitive (ppb levels) to measure concentrations typically used in toxicological studies, and uses inexpensive, commercially available reagents.

Electric fields yield chaos in microflows.

We present an investigation of chaotic dynamics of a low Reynolds number electrokinetic flow. Electrokinetic flows arise due to couplings of electric fields and electric double layers. In these flows, applied (steady) electric fields can couple with ionic conductivity gradients outside electric double layers to produce flow instabilities. The threshold of these instabilities is controlled by an electric Rayleigh number, Ra(e). As Ra(e) increases monotonically, we show here flow dynamics can transition from steady state to a time-dependent periodic state and then to an aperiodic, chaotic state. Interestingly, further monotonic increase of Ra(e) shows a transition back to a well-ordered state, followed by a second transition to a chaotic state. Temporal power spectra and time-delay phase maps of low dimensional attractors graphically depict the sequence between periodic and chaotic states. To our knowledge, this is a unique report of a low Reynolds number flow with such a sequence of periodic-to-aperiodic transitions. Also unique is a report of strange attractors triggered and sustained through electric fluid body forces.

Isotachophoretic preconcenetration on paper-based microfluidic devices.

Paper substrates have been widely used to construct point-of-care lateral flow immunoassay (LFIA) diagnostic devices. Paper based microfluidic devices are robust and relatively simple to operate, compared to channel microfluidic devices, which is perhaps their greatest advantage and the reason they have reached a high level of commercial success. However, paper devices may not be well suited for integrated sample preparation, such as sample extraction and preconcentration, which is required in complex samples with low analyte concentrations. In this study, we investigate integration of isotachophoresis (ITP), an electrokinetic preconcentration and extraction technique, onto nitrocellulose-based paper microfluidic devices with the goal to improve the limit of detection of LFIA. ITP has been largely used in traditional capillary based microfluidic devices as a pretreatment method to preconcentrate and separate a variety of ionic compounds. Our findings show that ITP on nitrocellulose is capable of up to a 900 fold increase in initial sample concentration and up to 60% extraction from 100 µL samples and more than 80% extraction from smaller sample volumes. Paper based ITP is challenged by Joule heating and evaporation because it is open to the environment. We achieved high preconcentration by mitigating evaporation induced dispersion using novel cross-shaped device structures that keep the paper hydrated. We show that ITP on the nitrocellulose membrane can be powered and run several times by a small button battery suggesting that it could be integrated to a portable point-of-care diagnostic device. These results highlight the potential of ITP to increase the sensitivity of paper based LFIA under conditions where small analyte concentrations are present in complex biological samples.

SARS-CoV-2 recombinase polymerase amplification assay with lateral flow readout and duplexed full process internal control.

Nucleic acid amplification tests for the detection of SARS-CoV-2 have been an important testing mechanism for the COVID-19 pandemic. While these traditional nucleic acid diagnostic methods are highly sensitive and selective, they are not suited to home or clinic-based uses. Comparatively, rapid antigen tests are cost-effective and user friendly but lack in sensitivity and specificity. Here we report on the development of a one-pot, duplexed reverse transcriptase recombinase polymerase amplification SARS-CoV-2 assay with MS2 bacteriophage as a full process control. Detection is carried out with either real-time fluorescence or lateral flow readout with an analytical sensitivity of 50 copies per reaction. Unlike previously published assays, the RNA-based MS2 bacteriophage control reports on successful operation of lysis, reverse transcription, and amplification. This SARS-CoV-2 assay features highly sensitive detection, visual readout through an LFA strip, results in less than 25 minutes, minimal instrumentation, and a useful process internal control to rule out false negative test results.

Simple, low-cost styrene-ethylene/butylene-styrene microdevices for electrokinetic applications.

Styrene-ethylene/butylene-styrene (SEBS) copolymers combine thermoplastic and elastomeric properties to provide microdevices with the advantageous properties of hard thermoplastics and ease of fabrication similar to PDMS. This work describes the electrical surface properties of SEBS block copolymers using current monitoring experiments to determine zeta potential. We show that SEBS exhibits a stable and relatively high zeta potential magnitude compared to similar polymers. The zeta potential of SEBS is stable when stored in air over time, and no significant differences are observed between different batches and devices, demonstrating reproducibility of results. We show zeta potential trends for varying pH and counterion concentration and demonstrate that SEBS has a repeatable surface potential comparable to glass. Oxygen plasma treatment greatly increases the zeta potential magnitude immediately following treatment before undergoing a moderate hydrophobic recovery to a stable zeta potential. SEBS copolymers also offer simple rapid prototyping fabrication and mass production potential. The presented electrokinetic properties combined with simple, low-cost fabrication of microdevices make SEBS a quality material for electrokinetic research and application development.

Disruption of Model Cell Membranes by Carbon Nanotubes.

Carbon nanotubes (CNTs) have one of the highest production volumes among carbonaceous engineered nanoparticles (ENPs) worldwide and are have potential uses in applications including biomedicine, nanocomposites, and energy conversion. However, CNTs possible widespread usage and associated likelihood for biological exposures have driven concerns regarding their nanotoxicity and ecological impact. In this work, we probe the responses of planar suspended lipid bilayer membranes, used as model cell membranes, to functionalized multi-walled carbon nanotubes (MWCNT), CdSe/ZnS quantum dots, and a control organic compound, melittin, using an electrophysiological measurement platform. The electrophysiological measurements show that MWCNTs in a concentration range of 1.6 to 12 ppm disrupt lipid membranes by inducing significant transmembrane current fluxes, which suggest that MWCNTs insert and traverse the lipid bilayer membrane, forming transmembrane carbon nanotubes channels that allow the transport of ions. This paper demonstrates a direct measurement of ion migration across lipid bilayers induced by CNTs. Electrophysiological measurements can provide unique insights into the lipid bilayer-ENPs interactions and have the potential to serve as a preliminary screening tool for nanotoxicity.

REverSe TRanscrIptase chain termination (RESTRICT) for selective measurement of nucleotide analogs used in HIV care and prevention.

Sufficient drug concentrations are required for efficacy of antiretroviral drugs used in HIV care and prevention. Measurement of nucleotide analogs, included in most HIV medication regimens, enables monitoring of short- and long-term adherence and the risk of treatment failure. The REverSe TRanscrIptase Chain Termination (RESTRICT) assay rapidly infers the concentration of intracellular nucleotide analogs based on the inhibition of DNA synthesis by HIV reverse transcriptase enzyme. Here, we introduce a probabilistic model for RESTRICT and demonstrate selective measurement of multiple nucleotide analogs using DNA templates designed according to the chemical structure of each drug. We measure clinically relevant concentrations of tenofovir diphosphate, emtricitabine triphosphate, lamivudine triphosphate, and azidothymidine triphosphate with agreement between experiment and theory. RESTRICT represents a new class of activity-based assays for therapeutic drug monitoring in HIV care and could be extended to other diseases treated with nucleotide analogs.

Role of nanoparticle surface functionality in the disruption of model cell membranes.

Lipid bilayers are biomembranes common to cellular life and constitute a continuous barrier between cells and their environment. Understanding the interaction of engineered nanomaterials (ENMs) with lipid bilayers is an important step toward predicting subsequent biological effects. In this study, we assess the effect of varying the surface functionality and concentration of 10-nm-diameter gold (Au) and titanium dioxide (TiO(2)) ENMs on the disruption of negatively charged lipid bilayer vesicles (liposomes) using a dye-leakage assay. Our findings show that Au ENMs having both positive and negative surface charge induce leakage that reaches a steady state after several hours. Positively charged particles with identical surface functionality and different core compositions show similar leakage effects and result in faster and greater leakage than negatively charged particles, which suggests that surface functionality, not particle core composition, is a critical factor in determining the interaction between ENMs and lipid bilayers. The results suggest that particles permanently adsorb to bilayers and that only one positively charged particle is required to disrupt a liposome and trigger the leakage of its entire contents in contrast to mellitin molecules, the most widely studied membrane lytic peptide, which requires hundred of molecules to generate leakage.

Distribution of functionalized gold nanoparticles between water and lipid bilayers as model cell membranes.

Lipid bilayers are biomembranes common to cellular life and constitute a continuous barrier between cells and their environment. Understanding the interaction of nanoparticles with lipid bilayers is an important step toward predicting subsequent biological effects. In this study, we assessed the affinity of functionalized gold nanoparticles (Au NPs) with sizes from 5 to 100 nm to lipid bilayers by determining the Au NP distribution between aqueous electrolytes and lipid bilayers. The Au NP distribution to lipid bilayers reached an apparent steady state in 24 h with smaller Au NPs distributing onto lipid bilayers more rapidly than larger ones. Au NPs distributed to lipid bilayers to a larger extent at lower pH. Tannic acid-functionalized Au NPs exhibited greater distribution to lipid bilayers than polyvinylpyrrolidone-functionalized Au NPs of the same size. Across the various Au NP sizes, we measure the lipid bilayer-water distribution coefficient (K(lipw) = C(lip)/C(w)) as 450 L/kg lipid, which is independent of dosimetric units. This work suggests that the nanoparticle-cell membrane interaction is dependent on solution chemistry and nanoparticle surface functionality. The K(lipw) value may be used to predict the affinity of spherical Au NPs across a certain size range toward lipid membranes.

Quantitative isothermal amplification on paper membranes using amplification nucleation site analysis.

Quantitative nucleic acid amplification tests (qNAATs) are critical in treating infectious diseases, such as in HIV viral load monitoring or SARS-CoV-2 testing, in which viral load indicates viral suppression or infectivity. Quantitative PCR is the gold standard tool for qNAATs; however, there is a need to develop point-of-care (POC) qNAATs to manage infectious diseases in outpatient clinics, low- and middle-income countries, and the home. Isothermal amplification methods are an emerging tool for POC NAATs as an alternative to traditional PCR-based workflows. Previous works have focused on relating isothermal amplification bulk fluorescence signals to input copies of target nucleic acids for sample quantification with limited success. In this work, we show that recombinase polymerase amplification (RPA) reactions on paper membranes exhibit discrete fluorescent amplification nucleation sites. We demonstrate that the number of nucleation sites can be used to quantify HIV-1 DNA and viral RNA in less than 20 minutes. An image-analysis algorithm quantifies nucleation sites and determines the input nucleic acid copies in the range of 67-3000 copies per reaction. We demonstrate a mobile phone-based system for image capture and onboard processing, illustrating that this method may be used at the point-of-care for qNAATs with minimal instrumentation.