This research employs pepsin-containing membranes to digest proteins online after a capillary electrophoresis (CE) separation and prior to tandem mass spectrometry. Proteolysis after the separation allows the peptides from a given protein to enter the mass spectrometer in a single plug. Thus, migration time can serve as an additional criterion for confirming the identification of a peptide. The membrane resides in a sheath-flow electrospray ionization (ESI) source to enable digestion immediately before spray into the mass spectrometer, thus limiting separation of the digested peptides. Using the same membrane, digestion occurred reproducibly during 20 consecutive CE analyses performed over a 10 h period. Additionally, after separating a mixture of six unreduced proteins with CE, online digestion facilitated protein identification with at least 2 identifiable peptides for all the proteins. Sequence coverages were >75% for myoglobin and carbonic anhydrase II but much lower for proteins containing disulfide bonds. Development of methods for efficient separation of reduced proteins or identification of cross-linked peptides should enhance sequence coverages for proteins with disulfide bonds. Migration times for the peptides identified from a specific protein differed by <∼30 s, which allows for rejection of some spurious peptide identifications.
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Charging YOYO-1 on Capillary Wall for Online DNA Intercalation and Integrating This Approach with Multiplex PCR and Bare Narrow Capillary–Hydrodynamic Chromatography for Online DNA Analysis
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Abstract. Heterogeneous ice nucleation is thought to be the primary pathway for the formation of ice in mixed-phase clouds, with the number of active ice-nucleating particles (INPs) increasing rapidly with decreasing temperature. Here, molecular-dynamics simulations of heterogeneous ice nucleation demonstrate that the ice nucleation rate is also sensitive to pressure and that negative pressure within supercooled water shifts freezing temperatures to higher temperatures. Negative pressure, or tension, occurs naturally in water capillary bridges and pores and can also result from water agitation. Capillary bridge simulations presented in this study confirm that negative Laplace pressure within the water increases heterogeneous-freezing temperatures. The increase in freezing temperatures with negative pressure is approximately linear within the atmospherically relevant range of 1 to −1000 atm. An equation describing the slope depends on the latent heat of freezing and the molar volume difference between liquid water and ice. Results indicate that negative pressures of −500 atm, which correspond to nanometer-scale water surface curvatures, lead to a roughly 4 K increase in heterogeneous-freezing temperatures. In mixed-phase clouds, this would result in an increase of approximately 1 order of magnitude in active INP concentrations. The findings presented here indicate that any process leading to negative pressure in supercooled water may play a role in ice formation, consistent with experimental evidence of enhanced ice nucleation due to surface geometry or mechanical agitation of water droplets. This points towards the potential for dynamic processes such as contact nucleation and droplet collision or breakup to increase ice nucleation rates through pressure perturbations.
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Madden, John D. ; Anderson, Iain A. ; Shea, Herbert R. (Ed.)Soft polymer actuators are in increasing demand due to their more fluid like motion and flexibility when actuated than compared with rigid actuators, which makes them valuable in diverse engineering applications. One of the main types of soft polymer actuators is the dielectric elastomer actuator, whose working principle is to apply a voltage potential difference between electrodes to reduce the thickness of the elastomeric material while expanding its area. This paper looks at manufacturing a micro soft polymer dielectric elastomer actuator utilizing two-photon polymerization 3D printing. The actuator contains micro channels that are filled with an electrode by using capillary action. A complex helical geometry is designed, printed, and tested for electrode filling capabilities. Quite a few obstacles are described in this paper including the use of a newly released two-photon polymerization resin which has limited supporting resources, as well as the complex helical geometry having a large compliance that vastly complicates its fabrication, post-processing, handling, electrode filling, electrode integration, and actuation testing. However, these challenges are overcome by using the standard printing recipes currently available for the resins, adding electrode isolation layers, and printing thicker elastomer zones for more structural support. The results found solidify the approach of filling microchannels with electrodes through capillary action and lead to further the focus and creation of multi-functional micro soft actuators.more » « less
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Small scale contact between a soft, liquid-coated layer and a stiff surface is common in many situations, from synovial fluid on articular cartilage to adhesives in humid environments. Moreover, many model studies on soft adhesive contacts are conducted with soft silicone elastomers, which possess uncrosslinked liquid molecules ( i.e. silicone oil) when the modulus is low. We investigate how the thickness of a silicone oil layer on a soft substrate relates to the indentation depth of glass microspheres in contact with crosslinked PDMS, which have a modulus of <10 kPa. The particles indent into the underlying substrate more as a function of decreasing oil layer thickness. This is due to the presence of the liquid layer at the surface that causes capillary forces to pull down on the particle. A simple model that balances the capillary force of the oil layer and the minimal particle–substrate adhesion with the elastic and surface tension forces from the substrate is proposed to predict the particle indentation depth.more » « less
