The generation of multi-laminar reagent streams for rapid, sequential (bio)chemical reactions on magnetic particles in a continuous flow microreactor

This paper was presented at the 2nd Micro and Nano Flows Conference (MNF2009), which was held at Brunel University, West London, UK. The conference was organised by Brunel University and supported by the Institution of Mechanical Engineers, IPEM, the Italian Union of Thermofluid dynamics, the Process Intensification Network, HEXAG - the Heat Exchange Action Group and the Institute of Mathematics and its Applications.We demonstrate a versatile microfluidic system for performing rapid, consecutive (bio)chemical reactions in continuous flow. Surface-functionalised magnetic microparticles are introduced into a chamber and pulled, via a magnet, across a series of laminar flow streams containing different reagents, thus performing multiple sequential reactions on the particles’ surface. Such a continuous flow method eliminates many of the inefficiencies associated with batch techniques, such as the time-consuming, laborious sequential reaction and washing steps, to yield a system that can perform analyses far more rapidly and with less reagent volume than conventional methods. This innovative device has been applied to a two-reaction step mouse IgG sandwich immunoassay and one- and two-reaction step DNA hybridisation assays, all of which were completed within one minute. These results pave the way for a multi-purpose microreactor that can perform a variety of analytical and synthetic processes.This study is funded by the Engineering and Physical Sciences Research Council (EPSRC)

Temperature-based tuning of magnetic particle separation by on-chip free-flow magnetophoresis

This paper was presented at the 2nd Micro and Nano Flows Conference (MNF2009), which was held at Brunel University, West London, UK. The conference was organised by Brunel University and supported by the Institution of Mechanical Engineers, IPEM, the Italian Union of Thermofluid dynamics, the Process Intensification Network, HEXAG - the Heat Exchange Action Group and the Institute of Mathematics and its Applications.Free-flow magnetophoresis provides a fast and efficient means of continuous flow magnetic separation for the detection of biological analytes, due to the wide variety of magnetic particle surface properties available for binding specific targets. Here, we investigate the effect of temperature changes on the deflection behaviour of magnetic particles in a microfluidic magnetophoresis separation chamber. It was found that the extent of deflection was greatly increased at higher temperatures due to decreased solution viscosity and thus reduced resistance against particle motion. This concept was used to improve the resolution of the separation of 2.8 μm and 1 μm diameter magnetic particles. Hence, controlling the temperature of the separation system provides a simple but highly effective means of enhancing magnetic separation efficiency. This concept could also be applied to the temperature-based tuning of microparticle trajectories in many others types of continuous flow processes, such as those using optical, electrical or acoustic forces.This study is funded by the Engineering and Physical Sciences Research Council (EPSRC)

Physician Gender Impact on Obesity Care in the Academic Ambulatory Setting

BACKGROUND & OBJECTIVE: The purpose of this study was to assess a nationally representative sample of academic family physicians to determine whether personal physician characteristics are associated with attitudes towards and care of overweight and/or obese patients. METHODS: Questions pertaining to physician’s interactions with overweight and obese patients was administered as the 2012 Council of Academic Family Medicine Educational Research Alliance (CERA) survey to academic family physicians. We analyzed self-reported demographic responses physicians gave with the main outcome the association between self-reported likelihood of engaging in weight loss discussions with overweight or obese patients and physicians’ personal characteristics (gender and physician BMI). RESULTS: 36% of the 1099 physicians surveyed were overweight and 14% were obese (Table 1). We found no differences in the self-reported likelihood of discussing weight loss strategies with either their overweight patients or obese patients by physician BMI category. 77% female vs. 64% male physicians reported being very/extremely likely to discuss weight loss with their obese patients at their most recent visit (p=0.002). Female physicians reported discussing weight loss strategies often or at every visit more than males (females 79%, males 69%; p=0.02). Finally, female physicians self-reported more minutes spent counseling overweight and obese patients (p = 0.0001) (Table 3). CONCLUSION: Female family medicine physicians are more likely than male counterparts to discuss weight loss strategies with obese patients and they report spending more time discussing weight loss strategies with their patients. However, self-reported physician BMI was not associated with these behaviors

An evaluation of the heat test for the ice-nucleating ability of minerals and biological material

Ice-nucleating particles (INPs) are atmospheric aerosol particles that can strongly influence the radiative properties and precipitation onset in mixed-phase clouds by triggering ice formation in supercooled cloud water droplets. The ability to distinguish between INPs of mineral and biological origin in samples collected from the environment is needed to better understand their distribution and sources. A common method for assessing the relative contributions of mineral and biogenic INPs in samples collected from the environment (e.g. aerosol, rainwater, soil) is to determine the ice-nucleating ability (INA) before and after heating, where heat is expected to denature proteins associated with some biological ice nucleants. The key assumption is that the ice nucleation sites of biological origin are denatured by heat, while those associated with mineral surfaces remain unaffected; we test this assumption here. We exposed atmospherically relevant mineral samples to wet heat (INP suspensions warmed to above 90 ∘C) or dry heat (dry samples heated up to 250 ∘C) and assessed the effects on their immersion mode INA using a droplet freezing assay. K-feldspar, thought to be the dominant mineral-based atmospheric INP type where present, was not significantly affected by wet heating, while quartz, plagioclase feldspars and Arizona Test Dust (ATD) lost INA when heated in this mode. We argue that these reductions in INA in the aqueous phase result from direct alteration of the mineral particle surfaces by heat treatment rather than from biological or organic contamination. We hypothesise that degradation of active sites by dissolution of mineral surfaces is the mechanism in all cases due to the correlation between mineral INA deactivation magnitudes and their dissolution rates. Dry heating produced minor but repeatable deactivations in K-feldspar particles but was generally less likely to deactivate minerals compared to wet heating. We also heat-tested biogenic INP proxy materials and found that cellulose and pollen washings were relatively resistant to wet heat. In contrast, bacterially and fungally derived ice-nucleating samples were highly sensitive to wet heat as expected, although their activity remained non-negligible after wet heating. Dry heating at 250 ∘C leads to deactivation of all biogenic INPs. However, the use of dry heat at 250 ∘C for the detection of biological INPs is limited since K-feldspar's activity is also reduced under these conditions. Future work should focus on finding a set of dry heat conditions where all biological material is deactivated, but key mineral types are not. We conclude that, while wet INP heat tests at (>90 ∘C) have the potential to produce false positives, i.e. deactivation of a mineral INA that could be misconstrued as the presence of biogenic INPs, they are still a valid method for qualitatively detecting very heat-sensitive biogenic INPs in ambient samples if the mineral-based INA is controlled by K-feldspar

Resolving the size of ice-nucleating particles with a balloon deployable aerosol sampler: the SHARK

Ice-nucleating particles (INPs) affect cloud development, lifetime, and radiative properties, hence it is important to know the abundance of INPs throughout the atmosphere. A critical factor in determining the lifetime and transport of INPs is their size; however very little size-resolved atmospheric INP concentration information exists. Here we present the development and application of a radio-controlled payload capable of collecting size-resolved aerosol from a tethered balloon for the primary purpose of offline INP analysis. This payload, known as the SHARK (Selective Height Aerosol Research Kit), consists of two complementary cascade impactors for aerosol size-segregation from 0.25 to 10 µm, with an after-filter and top stage to collect particles below and above this range at flow rates of up to 100 L min−1. The SHARK also contains an optical particle counter to quantify aerosol size distribution between 0.38 and 10 µm, and a radiosonde for the measurement of temperature, pressure, GPS altitude, and relative humidity. This is all housed within a weatherproof box, can be run from batteries for up to 11 h, and has a total weight of 9 kg. The radio control and live data link with the radiosonde allow the user to start and stop sampling depending on meteorological conditions and height, which can, for example, allow the user to avoid sampling in very humid or cloudy air, even when the SHARK is out of sight. While the collected aerosol could, in principle, be studied with an array of analytical techniques, this study demonstrates that the collected aerosol can be analysed with an offline droplet freezing instrument to determine size-resolved INP concentrations, activated fractions, and active site densities, producing similar results to those obtained using a standard PM10 aerosol sampler when summed over the appropriate size range. Test data, where the SHARK was sampling near ground level or suspended from a tethered balloon at 20 m altitude, are presented from four contrasting locations having very different size-resolved INP spectra: Hyytiälä (southern Finland), Leeds (northern England), Longyearbyen (Svalbard), and Cardington (southern England)

Homogeneous Freezing of Water Using Microfluidics

The homogeneous freezing of water is important in the formation of ice in clouds, but there remains a great deal of variability in the representation of the homogeneous freezing of water in the literature. The development of new instrumentation, such as droplet microfluidic platforms, may help to constrain our understanding of the kinetics of homogeneous freezing via the analysis of monodisperse, size-selected water droplets in temporally and spatially controlled environments. Here, we evaluate droplet freezing data obtained using the Lab-on-a-Chip Nucleation by Immersed Particle Instrument (LOC-NIPI), in which droplets are generated and frozen in continuous flow. This high-throughput method was used to analyse over 16,000 water droplets (86 μm diameter) across three experimental runs, generating data with high precision and reproducibility that has largely been unrepresented in the microfluidic literature. Using this data, a new LOC-NIPI parameterisation of the volume nucleation rate coefficient (JV(T)) was determined in the temperature region of −35.1 to −36.9 °C, covering a greater JV(T) compared to most other microfluidic techniques thanks to the number of droplets analysed. Comparison to recent theory suggests inconsistencies in the theoretical representation, further implying that microfluidics could be used to inform on changes to parameterisations. By applying classical nucleation theory (CNT) to our JV(T) data, we have gone a step further than other microfluidic homogeneous freezing examples by calculating the stacking-disordered ice–supercooled water interfacial energy, estimated to be 22.5 ± 0.7 mJ m−2, again finding inconsistencies when compared to theoretical predictions. Further, we briefly review and compile all available microfluidic homogeneous freezing data in the literature, finding that the LOC-NIPI and other microfluidically generated data compare well with commonly used non-microfluidic datasets, but have generally been obtained with greater ease and with higher numbers of monodisperse droplets

On-chip analysis of atmospheric ice-nucleating particles in continuous flow

Ice-nucleating particles (INPs) are of atmospheric importance because they catalyse the freezing of supercooled cloud droplets, strongly affecting the lifetime and radiative properties of clouds. There is a need to improve our knowledge of the global distribution of INPs, their seasonal cycles and long-term trends, but our capability to make these measurements is limited. Atmospheric INP concentrations are often determined using assays involving arrays of droplets on a cold stage, but such assays are frequently limited by the number of droplets that can be analysed per experiment, often involve manual processing (e.g. pipetting of droplets), and can be susceptible to contamination. Here, we present a microfluidic platform, the LOC-NIPI (Lab-on-a-Chip Nucleation by Immersed Particle Instrument), for the generation of water-in-oil droplets and their freezing in continuous flow as they pass over a cold plate for atmospheric INP analysis. LOC-NIPI allows the user to define the number of droplets analysed by simply running the platform for as long as required. The use of small (∼100 μm diameter) droplets minimises the probability of contamination in any one droplet and therefore allows supercooling all the way down to homogeneous freezing (around −36 °C), while a temperature probe in a proxy channel provides an accurate measure of temperature without the need for temperature modelling. The platform was validated using samples of pollen extract and Snomax®, with hundreds of droplets analysed per temperature step and thousands of droplets being measured per experiment. Homogeneous freezing of purified water was studied using >10 000 droplets with temperature increments of 0.1 °C. The results were reproducible, independent of flow rate in the ranges tested, and the data compared well to conventional instrumentation and literature data. The LOC-NIPI was further benchmarked in a field campaign in the Eastern Mediterranean against other well-characterised instrumentation. The continuous flow nature of the system provides a route, with future development, to the automated monitoring of atmospheric INP at field sites around the globe

On-chip density-based sorting of supercooled droplets and frozen droplets in continuous flow

The freezing of supercooled water to ice and the materials which catalyse this process are of fundamental interest to a wide range of fields. At present, our ability to control, predict or monitor ice formation processes is poor. The isolation and characterisation of frozen droplets from supercooled liquid droplets would provide a means of improving our understanding and control of these processes. Here, we have developed a microfluidic platform for the continuous flow separation of frozen from unfrozen picolitre droplets based on differences in their density, thus allowing the sorting of ice crystals and supercooled water droplets into different outlet channels with 94 ± 2% efficiency. This will, in future, facilitate downstream or off-chip processing of the frozen and unfrozen populations, which could include the analysis and characterisation of ice-active materials or the selection of droplets with a particular ice-nucleating activity

Two Prp19-Like U-Box Proteins in the MOS4-Associated Complex Play Redundant Roles in Plant Innate Immunity

Plant Resistance (R) proteins play an integral role in defense against pathogen infection. A unique gain-of-function mutation in the R gene SNC1, snc1, results in constitutive activation of plant immune pathways and enhanced resistance against pathogen infection. We previously found that mutations in MOS4 suppress the autoimmune phenotypes of snc1, and that MOS4 is part of a nuclear complex called the MOS4-Associated Complex (MAC) along with the transcription factor AtCDC5 and the WD-40 protein PRL1. Here we report the immuno-affinity purification of the MAC using HA-tagged MOS4 followed by protein sequence analysis by mass spectrometry. A total of 24 MAC proteins were identified, 19 of which have predicted roles in RNA processing based on their homology to proteins in the Prp19-Complex, an evolutionarily conserved spliceosome-associated complex containing homologs of MOS4, AtCDC5, and PRL1. Among these were two highly similar U-box proteins with homology to the yeast and human E3 ubiquitin ligase Prp19, which we named MAC3A and MAC3B. MAC3B was recently shown to exhibit E3 ligase activity in vitro. Through reverse genetics analysis we show that MAC3A and MAC3B are functionally redundant and are required for basal and R protein–mediated resistance in Arabidopsis. Like mos4-1 and Atcdc5-1, mac3a mac3b suppresses snc1-mediated autoimmunity. MAC3 localizes to the nucleus and interacts with AtCDC5 in planta. Our results suggest that MAC3A and MAC3B are members of the MAC that function redundantly in the regulation of plant innate immunity

Systematic Two-Hybrid and Comparative Proteomic Analyses Reveal Novel Yeast Pre-mRNA Splicing Factors Connected to Prp19

Prp19 is the founding member of the NineTeen Complex, or NTC, which is a spliceosomal subcomplex essential for spliceosome activation. To define Prp19 connectivity and dynamic protein interactions within the spliceosome, we systematically queried the Saccharomyces cerevisiae proteome for Prp19 WD40 domain interaction partners by two-hybrid analysis. We report that in addition to S. cerevisiae Cwc2, the splicing factor Prp17 binds directly to the Prp19 WD40 domain in a 1∶1 ratio. Prp17 binds simultaneously with Cwc2 indicating that it is part of the core NTC complex. We also find that the previously uncharacterized protein Urn1 (Dre4 in Schizosaccharomyces pombe) directly interacts with Prp19, and that Dre4 is conditionally required for pre-mRNA splicing in S. pombe. S. pombe Dre4 and S. cerevisiae Urn1 co-purify U2, U5, and U6 snRNAs and multiple splicing factors, and dre4Δ and urn1Δ strains display numerous negative genetic interactions with known splicing mutants. The S. pombe Prp19-containing Dre4 complex co-purifies three previously uncharacterized proteins that participate in pre-mRNA splicing, likely before spliceosome activation. Our multi-faceted approach has revealed new low abundance splicing factors connected to NTC function, provides evidence for distinct Prp19 containing complexes, and underscores the role of the Prp19 WD40 domain as a splicing scaffold