RESUMO
Particle removal from the surface of a substrate has been an issue in numerous fields for a long time. In semiconductor processes, for instance, the formation of clean surfaces by removing photoresist (PR) must be followed in order to create neat patterns. Although PR removal has been intensively investigated recently, little is known about how ultraviolet (UV) and developer solutions alter the PR resin (and in what manner) near the surface. While varying the exposure times of UV and developer solution, we investigated the topographic changes on the surfaces of PR resin films and particles. The measured surface properties were then correlated with the detachment force determined using films, and eventually with the residual PR particle removal percentages obtained in a microchannel. Using a positive PR and a base developer solution, we demonstrated that UV causes the surface of PR resin to become hydrophilic and wavy, whereas the developer solution produces a surface with a larger degree of roughness by swelling and partially dissolving the resin. Ultimately, the increased roughness decreased the effective contact area between PR resins, hence decreasing the detachment force and increasing the particle removal percentages. We anticipate that our findings will help understand residual particle issues, particularly on the removal mechanism of PR resins based on surface topography.
RESUMO
Cell membranes actively change their local compositions, serving essential biological processes such as cellular signaling and endocytosis. Although membrane dynamics is vital in the cellular functions, the complexity of natural membranes has made its fundamental understanding and systematic assessment difficult. Here, a powerful artificial membrane system is developed for real-time visualization of the spatiotemporal dynamics of membrane remodeling. Through well-defined air/oil/water interfaces on grid holes, tens of planar lipid bilayer membranes are easily created, and their reproducibility, controllability, and generality are highlighted. The freestanding membranes are large but also highly stable, facilitating direct long-term monitoring of dynamic membrane reconstitution caused by external stimuli. As an example to demonstrate the superiority of this membrane system, the effect of cholesterol trafficking, which significantly affects biophysical properties of cell membranes, is investigated at different membrane compositions. Cholesterol transport into and out of the membranes at different rates causes anomalous lipid arrangements through cholesterol-mediated phase transitions and decomposition, which have never been witnessed before. Furthermore, enzyme-induced membrane dynamics is successfully shown in this platform; sphingomyelinases locally generate asymmetry between two membrane leaflets. This technique is broadly applicable for exploring the membrane heterogeneity under various membrane-based reactions, providing valuable insight into the membrane dynamics.
Assuntos
Colesterol , Bicamadas Lipídicas , Transporte Biológico , Membrana Celular/metabolismo , Colesterol/metabolismo , Bicamadas Lipídicas/metabolismo , Reprodutibilidade dos TestesRESUMO
Because numerous drugs are administered through an oral route and primarily absorbed at the intestine, the prediction of drug permeability across an intestinal epithelial cell membrane has been a crucial issue in drug discovery. Thus, various in vitro permeability assays have been developed such as the Caco-2 assay, the parallel artificial membrane permeability assay (PAMPA), the phospholipid vesicle-based permeation assays (PVPA) and Permeapad. However, because of the time-consuming and quite expensive process for culturing cells in the Caco-2 assay and the unknown microscopic membrane structures of the other assays, a simpler yet more accurate and versatile technique is still required. Accordingly, we developed a new platform to measure the permeability of small molecules across a planar freestanding lipid bilayer with a well-defined area and structure. The lipid bilayer was constructed within a conventional UV spectrometer cell, and the transport of drug molecules across the bilayer was recorded by UV absorbance over time. We then computed the permeability from the time-dependent diffusion equation. We tested this assay for five exemplary hydrophilic drugs and compared their values with previously reported ones. We found that our assay has a much higher permeability compared to the other techniques, and this higher permeability is related to the thickness of the lipid bilayer. Also we were able to measure the dynamic permeability upon the addition of a membrane-disrupting surfactant demonstrating that our assay has the capability to detect real-time changes in permeability across the lipid bilayer.
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Living cells efflux intracellular ions for maintaining cellular life, so intravital measurements of specific ion signals are of significant importance for studying cellular functions and pharmacokinetics. In this work, de novo synthesis of artificial K+ -selective membrane and its integration with polyelectrolyte hydrogel-based open-junction ionic diode (OJID) is demonstrated, achieving a real-time K+ -selective ion-to-ion current amplification in complex bioenvironments. By mimicking biological K+ channels and nerve impulse transmitters, in-line K+ -binding G-quartets are introduced across freestanding lipid bilayers by G-specific hexylation of monolithic G-quadruplex, and the pre-filtered K+ flow is directly converted to amplified ionic currents by the OJID with a fast response time at 100 ms intervals. By the synergistic combination of charge repulsion, sieving, and ion recognition, the synthetic membrane allows K+ transport exclusively without water leakage; it is 250× and 17× more permeable toward K+ than monovalent anion, Cl- , and polyatomic cation, N-methyl-d-glucamine+ , respectively. The molecular recognition-mediated ion channeling provides a 500% larger signal for K+ as compared to Li+ (0.6× smaller than K+ ) despite the same valence. Using the miniaturized device, non-invasive, direct, and real-time K+ efflux monitoring from living cell spheroids is achieved with minimal crosstalk, specifically in identifying osmotic shock-induced necrosis and drug-antidote dynamics.
Assuntos
Quadruplex G , Canais Iônicos , Canais Iônicos/metabolismo , Transporte Biológico , Cátions/química , Fenômenos Fisiológicos Celulares , PotássioRESUMO
Micrometer-sized aqueous droplets serve as a unique reactor that drives various chemical reactions not seen in bulk solutions. However, their utilization has been limited to the synthesis of low molecular weight products at low reactant concentrations (nM to µM). Moreover, the nature of chemical reactions occurring outside the droplet remains unknown. This study demonstrated that oil-confined aqueous microdroplets continuously generated hydroxyl radicals near the interface and enabled the synthesis of polymers at high reactant concentrations (mM to M), thus successfully converting the interfacial energy into the synthesis of polymeric materials. The polymerized products maintained the properties of controlled radical polymerization, and a triblock copolymer with tapered interfaces was prepared by the sequential addition of different monomers into the aqueous microdroplets. Furthermore, a polymerization reaction in the continuous oil phase was effectively achieved by the transport of the hydroxyl radicals through the oil/water interface. This interfacial phenomenon is also successfully applied to the chain extension of a hydrophilic polymer with an oil-soluble monomer across the microdroplet interface. Our comprehensive study of radical polymerization using compartmentalization in microdroplets is expected to have important implications for the emerging field of microdroplet chemistry and polymerization in cellular biochemistry without any invasive chemical initiators.
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Several signaling processes in the plasma membrane are intensified by ceramides that are formed by sphingomyelinase-mediated hydrolysis of sphingomyelin. These ceramides trigger clustering of signaling-related biomolecules, but how they concentrate such biomolecules remains unclear. Here, the spatiotemporal localization of ganglioside GM1, a glycolipid receptor involved in signaling, during sphingomyelinase-mediated hydrolysis is described. Real-time visualization of the dynamic remodeling of the heterogeneous lipid membrane that occurs due to sphingomyelinase action is used to examine GM1 clustering, and unexpectedly, it is found that it is more complex than previously thought. Specifically, lipid membranes generate two distinct types of condensed GM1: 1) rapidly formed but short-lived GM1 clusters that are formed in ceramide-rich domains nucleated from the liquid-disordered phase; and 2) late-onset yet long-lasting, high-density GM1 clusters that are formed in the liquid-ordered phase. These findings suggest that multiple pathways exist in a plasma membrane to synergistically facilitate the rapid amplification and persistence of signals.
Assuntos
Ceramidas/genética , Gangliosídeo G(M1)/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielinas/genética , Bacillus cereus/enzimologia , Membrana Celular/genética , Membrana Celular/metabolismo , Ceramidas/biossíntese , Ceramidas/química , Análise por Conglomerados , Gangliosídeo G(M1)/genética , Hidrólise , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipídeos/química , Lipídeos/genética , Lipídeos de Membrana/química , Lipídeos de Membrana/genética , Transdução de Sinais/genética , Esfingomielina Fosfodiesterase/química , Esfingomielinas/química , Esfingomielinas/metabolismoRESUMO
Serum proteins are believed to trigger a sudden failure of lung function, but to date the mechanism remains elusive. Most studies have focused on the transport of the proteins from the subphase to the lung surfactant interface, although the opposite direction of transport, i.e., from air-to-interface, could be equally important. Here, we report that physiological concentrations of serum droplets can rapidly form a film upon exposure to air, and the entire film can be transferred to the lung surfactant interface upon coalescence, displacing it. This film was mechanically stable and remains intact even for multiple biaxial compression/expansion cycles. Our findings provide a mechanism of lung surfactant replacement by serum proteins that is fundamentally different from the subphase-to-interface transport and demonstrate that it is nearly impossible to remove the film from the interface where the lung surfactant should be, thus impairing the lung in a permanent way.
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Despite the enormous potential of cellulose nanofibrils (CNFs) as a reinforcing filler in various fields, the use of them has been limited by high-energy mechanical treatments that require a lot of energy and time consumption. To reduce the demands of energy and time required for mechanical treatments, microalgae, in particular, Nannochloropsis oceanica, which has small size, rapid growth rate, and high productivity was used as a CNFs source. This study obtains the CNFs by lipid/protein extraction, purification, and TEMPO-mediated oxidation processes under gentle mixing without high-energy mechanical treatments. Furthermore, to evaluate the applicability of microalgal CNFs as a reinforcing filler, this study estimated the mechanical strength of the fibrils by the sonication-induced scission method. To achieve a precise estimation, an effective method to distinguish straight fibrils from buckled fibrils was also developed, and subsequently, only straight fibrils were used to calculate the mechanical strength in the sonication-induced scission method. Consequently, the tensile strength of the N. oceanica CNFs is around 3-4GPa on average which is comparable with the mechanical strength of general reinforcing fillers and even higher than that of wood CNFs. Thus, this study has shown that the newly proposed simplified method using N. oceanica is very successful in producing CNFs with great mechanical strength which could be used in various reinforcement fields.