Habit Reversal Therapy: An Underutilized Treatment for Atopic Dermatitis.

Atopic dermatitis (AD) is a common inflammatory skin condition characterized by a history of recurring pruritic lesions that are worsened by scratching. Therapeutic outcomes may be optimized by minimizing the scratching of pruritic AD lesions, which is often particularly challenging for pediatric populations. Alongside topical and systemic therapies, research supports the use of habit reversal therapy for AD to mitigate the urge to scratch.

A reinforced PDMS mold for hot embossing of cyclic olefin polymer in the fabrication of microfluidic chips.

Hot embossing is a cost-effective and flexible fabrication technology with high replication accuracy for feature sizes as small as 50 nm. Here we develop a reinforced polydimethylsiloxane (PDMS) mold for hot embossing of cyclic olefin polymer (COP) sheets in the fabrication of microfluidic chips and demonstrate the method by fabricating chips for automated sample digitization in digital nucleic acid assays. The PDMS is hardened by adding an investment powder as a dopant and is constrained with an aluminum frame to prevent lateral expansion during hot pressing.

Adapting Child Health Knowledge Translation Tools for Use by Indigenous Communities: Qualitative Study Exploring Health Care Providers' Perspectives.

Background: Our research groups have developed a number of parental knowledge translation (KT) tools to help families understand common childhood illnesses and make informed decisions regarding when to seek urgent care. We have developed a series of videos to help parents understand how to manage common acute childhood illnesses at home and when to contact emergency health care services. It is unclear whether the videos in their current form and language are useful for a wider range of populations, including Indigenous groups.

Digital Quantification of Human Viral RNA and DNA Using a Self-Digitization Chip.

Digital nucleic acid quantitation methods show excellent sensitivity and specificity for pathogen detection. Droplet digital PCR (ddPCR) is the most advanced digital nucleic acid quantitation method and has been commercialized, but is not suitable for many point-of-care applications due to its complex instrumentation. Here we describe a simple microfluidics-based self-digitization (SD) chip for quantifying nucleic acids at the point of care with minimal instrumentation.

Detection of 14 High-Risk Human Papillomaviruses Using Digital LAMP Assays on a Self-Digitization Chip.

Cervical cancer is the fourth-leading cause of cancer deaths among women worldwide and most cases occur in developing countries. Detection of high-risk (HR) HPV, the etiologic agent of cervical cancer, is a primary screening method for cervical cancer. However, the current gold standard for HPV detection, real-time PCR, is expensive, time-consuming, and instrumentation-intensive.

Self-digitization chip for quantitative detection of human papillomavirus gene using digital LAMP.

Digital nucleic acid amplification and detection methods provide excellent sensitivity and specificity and allow absolute quantification of target nucleic acids. Isothermal methods such as digital loop-mediated isothermal amplification (digital LAMP) have potential for use in rapid disease diagnosis in low-resource settings due to their speed and lack of thermal cycling. We previously developed a self-digitization (SD) chip, a simple microfluidics device that automatically digitizes a sample into an array of nanoliter wells, for use in digital LAMP.

Statistical Analysis of Nonuniform Volume Distributions for Droplet-Based Digital PCR Assays.

We present a method to determine the concentration of nucleic acids in a sample by partitioning it into droplets with a nonuniform volume distribution. This digital PCR method requires no special equipment for partitioning, unlike other methods that require nearly identical volumes. Droplets are generated by vortexing a sample in an immiscible oil to create an emulsion.

SD-chip enabled quantitative detection of HIV RNA using digital nucleic acid sequence-based amplification (dNASBA).

Quantitative detection of RNA is important in molecular biology and clinical diagnostics. Nucleic acid sequence-based amplification (NASBA), a single-step method to amplify single-stranded RNA, is attractive for use in point-of-care (POC) diagnostics because it is an isothermal technique that is as sensitive as RT-PCR with a shorter reaction time. However, NASBA is limited in its ability to provide accurate quantitative information, such as viral load or RNA copy number.

Self-digitization chip for single-cell genotyping of cancer-related mutations.

Cancer is a heterogeneous disease, and patient-level genetic assessments can guide therapy choice and impact prognosis. However, little is known about the impact of genetic variability within a tumor, intratumoral heterogeneity (ITH), on disease progression or outcome. Current approaches using bulk tumor specimens can suggest the presence of ITH, but only single-cell genetic methods have the resolution to describe the underlying clonal structures themselves.

Self-digitization microfluidic chip for absolute quantification of mRNA in single cells.

Quantification of mRNA in single cells provides direct insight into how intercellular heterogeneity plays a role in disease progression and outcomes. Quantitative polymerase chain reaction (qPCR), the current gold standard for evaluating gene expression, is insufficient for providing absolute measurement of single-cell mRNA transcript abundance. Challenges include difficulties in handling small sample volumes and the high variability in measurements.

The potential impact of droplet microfluidics in biology.

Droplet microfluidics, which involves micrometer-sized emulsion droplets on a microfabricated platform, is an active research endeavor that evolved out of the larger field of microfluidics. Recently, this subfield of microfluidics has started to attract greater interest because researchers have been able to demonstrate applications of droplets as miniaturized laboratories for biological measurements. This perspective explores the recent developments and the potential future biological applications of droplet microfluidics.

Mechanistic evaluation of the pros and cons of digital RT-LAMP for HIV-1 viral load quantification on a microfluidic device and improved efficiency via a two-step digital protocol.

Here we used a SlipChip microfluidic device to evaluate the performance of digital reverse transcription-loop-mediated isothermal amplification (dRT-LAMP) for quantification of HIV viral RNA. Tests are needed for monitoring HIV viral load to control the emergence of drug resistance and to diagnose acute HIV infections. In resource-limited settings, in vitro measurement of HIV viral load in a simple format is especially needed, and single-molecule counting using a digital format could provide a potential solution.

Identification of a new gene required for the biosynthesis of rhodoquinone in Rhodospirillum rubrum.

Rhodoquinone (RQ) is a required cofactor for anaerobic respiration in Rhodospirillum rubrum, and it is also found in several helminth parasites that utilize a fumarate reductase pathway. RQ is an aminoquinone that is structurally similar to ubiquinone (Q), a polyprenylated benzoquinone used in the aerobic respiratory chain. RQ is not found in humans or other mammals, and therefore, the inhibition of its biosynthesis may provide a novel antiparasitic drug target.

Multiplexed quantification of nucleic acids with large dynamic range using multivolume digital RT-PCR on a rotational SlipChip tested with HIV and hepatitis C viral load.

In this paper, we are working toward a problem of great importance to global health: determination of viral HIV and hepatitis C (HCV) loads under point-of-care and resource limited settings. While antiretroviral treatments are becoming widely available, viral load must be evaluated at regular intervals to prevent the spread of drug resistance and requires a quantitative measurement of RNA concentration over a wide dynamic range (from 50 up to 10(6) molecules/mL for HIV and up to 10(8) molecules/mL for HCV). "Digital" single molecule measurements are attractive for quantification, but the dynamic range of such systems is typically limited or requires excessive numbers of compartments.

Theoretical design and analysis of multivolume digital assays with wide dynamic range validated experimentally with microfluidic digital PCR.

This paper presents a protocol using theoretical methods and free software to design and analyze multivolume digital PCR (MV digital PCR) devices; the theory and software are also applicable to design and analysis of dilution series in digital PCR. MV digital PCR minimizes the total number of wells required for "digital" (single molecule) measurements while maintaining high dynamic range and high resolution. In some examples, multivolume designs with fewer than 200 total wells are predicted to provide dynamic range with 5-fold resolution similar to that of single-volume designs requiring 12,000 wells.

Digital isothermal quantification of nucleic acids via simultaneous chemical initiation of recombinase polymerase amplification reactions on SlipChip.

In this paper, digital quantitative detection of nucleic acids was achieved at the single-molecule level by chemical initiation of over one thousand sequence-specific, nanoliter isothermal amplification reactions in parallel. Digital polymerase chain reaction (digital PCR), a method used for quantification of nucleic acids, counts the presence or absence of amplification of individual molecules. However, it still requires temperature cycling, which is undesirable under resource-limited conditions.

Digital PCR on a SlipChip.

This paper describes a SlipChip to perform digital PCR in a very simple and inexpensive format. The fluidic path for introducing the sample combined with the PCR mixture was formed using elongated wells in the two plates of the SlipChip designed to overlap during sample loading. This fluidic path was broken up by simple slipping of the two plates that removed the overlap among wells and brought each well in contact with a reservoir preloaded with oil to generate 1280 reaction compartments (2.

Evolution of catalysts directed by genetic algorithms in a plug-based microfluidic device tested with oxidation of methane by oxygen.

This paper uses microfluidics to implement genetic algorithms (GA) to discover new homogeneous catalysts using the oxidation of methane by molecular oxygen as a model system. The parameters of the GA were the catalyst, a cocatalyst capable of using molecular oxygen as the terminal oxidant, and ligands that could tune the catalytic system. The GA required running hundreds of reactions to discover and optimize catalyst systems of high fitness, and microfluidics enabled these numerous reactions to be run in parallel.

Laterally mobile, functionalized self-assembled monolayers at the fluorous-aqueous interface in a plug-based microfluidic system: characterization and testing with membrane protein crystallization.

This paper describes a method to generate functionalizable, mobile self-assembled monolayers (SAMs) in plug-based microfluidics. Control of interfaces is advancing studies of biological interfaces, heterogeneous reactions, and nanotechnology. SAMs have been useful for such studies, but they are not laterally mobile.