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Isothermal nucleic acid amplification tests (NAATs) are a vital tool for point-of-care (POC) diagnostics. These assays are well-suited for rapid, low-cost POC diagnostics for infectious diseases compared to traditional PCR tests conducted in central laboratories. There has been significant development of POC NAATs using paper-based diagnostic devices because they provide an affordable, user-friendly, and easy to store format; however, the difficulties in integrating separate liquid components, resuspending dried reagents, and achieving a low limit of detection hinder their use in commercial applications. Several studies report low assay efficiencies, poor detection output, and poorer limits of detection in porous membranes compared to traditional tube-based protocols. Recombinase polymerase amplification is a rapid, isothermal NAAT that is highly suited for POC applications, but requires viscous reaction conditions that has poor performance when amplifying in a porous paper membrane. In this work, we show that we can dramatically improve the performance of membrane-based recombinase polymerase amplification (RPA) of HIV-1 DNA and viral RNA by employing a coin cell-based vibration mixing platform. We achieve a limit of detection of 12 copies of DNA per reaction, nearly 50% reduction in time to threshold (from ∼10 minutes to ∼5 minutes), and an overall fluorescence output increase up to 16-fold when compared to unmixed experiments. This active mixing strategy enables reactions where the target and reaction cofactors are isolated from each other prior to the reaction. We also demonstrate amplification using a low-cost vibration motor for both temperature control and mixing, without the requirement of any additional heating components. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We present experimental measurements of electromigration current through a single cylindrical nanopore. A single cylindrical nanopore with 175 nm diameter was fabricated in silicon in series with two micropores with 2 and 100 [micro]m diameters. Thick electrical double layers (EDLs) (ka [approximately equal to] 1) exhibit current rectification due to asymmetric concentration polarization while thinner EDLs show nearly symmetric conductance. After the electric field is turned off, electrical current is measured and observed due to redistribution of ions in the concentration polarization layer.

A microfluidic method to rapidly measure the octanol--water partition coefficient in thousands of individual picoliter drops is described. AT-junction microfluidic chip is used to generate a segmented flow of monodisperse, fluorescein-laden water in octanol carder fluid. The partitioning of individual drops reaches equilibrium in less than 2 s. Epifluorescence microscopy is used measure the partition coefficient of fluorescein as a function of pH. Results compare well with previous measurements using traditional shake-flask methods. The methods presented here are rapid, provide detailed statistics, and can be run in parallel, enabling the simultaneous partitioning of thousands of compounds for various applications such as drug development, environmental testing, and combinatorial chemistry. Microfluidic partitioning and extraction in picoliter drops may be useful for studying molecules and particles away from their equilibrium state and in cases with limited samples.

This paper investigates a hydrodynamic particle separation technique that employs pinching of particles to a narrow microchannel. The particles are subject to a sudden expansion which results in a size-based particle separation transverse to the flow direction. The separation resolution and particle dispersion are measured using epifluorescence microscopy. The resolution and dispersion are predicted using a compact theoretical model. Devices are fabricated using conventional soft lithography of polydimethylsiloxane. The results show that the separation resolution is a function of the microchannel aspect ratio, particle size difference, and the microchannel sidewall roughness. A separation resolution as large as 3.8 is obtained in this work. This work shows that particles with diameters on the order of the sidewall roughness cannot be separated using pinched flow fractionation.

N-type porous silicon can be used to realize electroosmotic pumps with high flow rates per applied potential difference. The porosity and pore size of porous silicon membranes can be tuned, the pore geometry has near-unity tortuosity, and membranes can be made thin and with integrated support structures. The size of hexagonally packed pores is modified by low-pressure chemical vapor deposition (LPCVD) polysilicon deposition, followed by wet oxidation of the polysilicon layer, resulting in a pore radius varying from 1 to 3 [micro]m. Pumping performance of these devices is experimentally studied as a function of pore size and compared with theory. These 350-[micro]m-thick silicon membranes exhibit a maximum flow rate per applied field of 0.13 ml/min/ [cm.sup.2]/V. This figure of merit is five times larger than previously demonstrated porous glass EO pumps. [1494] Index Terms--Electroosmotic pump, porous silicon membrane, zeta potential.

Interferometry has been used for many years as a semi-quantitative image-based diagnostic for combustion research. In this paper, we use image-plane, double-pulse holographic interferograms of axisymmetric flames to infer their radial temperature distribution. An Abel inversion is performed on the fringe data to account for line-of-sight integration through the flame. The sensitivity of nonresonant refractive diagnostics decreases inversely with temperature, and the accuracy of the technique is discussed in this context. A small, nonpremixed capillary flame is investigated, and the temperatures inferred from interferometry are compared with those obtained with [N.sub.2] coherent anti-Stokes Raman spectroscopy thermometry. Additionally, the thermal field of a burning monodisperse methanol droplet stream is investigated interferometrically. Because of their small size, both of these flames challenge the performance limit of temperature interferometery. OCIS codes: 120.1740, 120.2880, 090.2880, 100.2650, 110.6960, 110.6980.

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. [ABSTRACT FROM AUTHOR]