Micropipette Technique Research Articles

Microplastics destabilize lipid membranes by mechanical stretching

Estimated millions of tons of plastic are dumped annually into oceans. Plastic has been produced only for 70 y, but the exponential rise of mass production leads to its widespread proliferation in all environments. As a consequence of their large abundance globally, microplastics are also found in many living organisms including humans. While the health impact of digested microplastics on living organisms is debatable, we reveal a physical mechanism of mechanical stretching of model cell lipid membranes induced by adsorbed micrometer-sized microplastic particles most commonly found in oceans. Combining experimental and theoretical approaches, we demonstrate that microplastic particles adsorbed on lipid membranes considerably increase membrane tension even at low particle concentrations. Each particle adsorbed at the membrane consumes surface area that is proportional to the contact area between particle and the membrane. Although lipid membranes are liquid and able to accommodate mechanical stress, the relaxation time is much slower than the rate of adsorption; thus, the cumulative effect from arriving microplastic particles to the membrane leads to the global reduction of the membrane area and increase of membrane tension. This, in turn, leads to a strong reduction of membrane lifetime. The effect of mechanical stretching of microplastics on living cells membranes was demonstrated by using the aspiration micropipette technique on red blood cells. The described mechanical stretching mechanism on lipid bilayers may provide better understanding of the impact of microplastic particles in living systems.

An intervention to increase accuracy and understanding in undergraduate micropipette use

Abstract Micropipetting is an essential skill for the budding immunologist and is widely used across biological and physical science disciplines, yet few undergraduates are adequately introduced to this skill. The mentors of undergraduates seeking research experience point to micropipette inexperience as a common barrier to success. As part of our SEA-PHAGES authentic research laboratory, we designed a hands-on intervention to give thorough training in micropipetting to first-year undergraduates. The intervention included assignment of a pre-class video on micropipetting, an activity to orient students to the micropipette parts and functions, and a TA-led demonstration of proper micropipette use and volume adjustment. Success of the intervention was analyzed using data collected from a practical test following the lab activity. In addition, we compared the students’ end-of-semester experimental success to that of students from previous semesters lacking the micropipetting activity, using archived electronic laboratory notebooks. We found that students progressed through experiments faster and with more success after experiencing the micropipette intervention. Our analysis showed that some aspects of proper micropipette use need to be emphasized, such as proper depth of tip entry and the correct tactile discrimination of the first/second stop on the micropipette. In addition, we found that students have misconceptions and gaps in knowledge about the metric system that are an additional barrier to proper micropipette use. This project presents a successful platform for teaching micropipette technique, and demonstrates that this leads to increased likelihood of success for the students in this real-world laboratory setting.

Micro-Surface and -Interfacial Tensions Measured Using the Micropipette Technique: Applications in Ultrasound-Microbubbles, Oil-Recovery, Lung-Surfactants, Nanoprecipitation, and Microfluidics.

This review presents a series of measurements of the surface and interfacial tensions we have been able to make using the micropipette technique. These include: equilibrium tensions at the air-water surface and oil-water interface, as well as equilibrium and dynamic adsorption of water-soluble surfactants and water-insoluble and lipids. At its essence, the micropipette technique is one of capillary-action, glass-wetting, and applied pressure. A micropipette, as a parallel or tapered shaft, is mounted horizontally in a microchamber and viewed in an inverted microscope. When filled with air or oil, and inserted into an aqueous-filled chamber, the position of the surface or interface meniscus is controlled by applied micropipette pressure. The position and hence radius of curvature of the meniscus can be moved in a controlled fashion from dimensions associated with the capillary tip (~5–10 μm), to back down the micropipette that can taper out to 450 μm. All measurements are therefore actually made at the microscale. Following the Young–Laplace equation and geometry of the capillary, the surface or interfacial tension value is simply obtained from the radius of the meniscus in the tapered pipette and the applied pressure to keep it there. Motivated by Franklin’s early experiments that demonstrated molecularity and monolayer formation, we also give a brief potted-historical perspective that includes fundamental surfactancy driven by margarine, the first use of a micropipette to circuitously measure bilayer membrane tensions and free energies of formation, and its basis for revolutionising the study and applications of membrane ion-channels in Droplet Interface Bilayers. Finally, we give five examples of where our measurements have had an impact on applications in micro-surfaces and microfluidics, including gas microbubbles for ultrasound contrast; interfacial tensions for micro-oil droplets in oil recovery; surface tensions and tensions-in-the surface for natural and synthetic lung surfactants; interfacial tension in nanoprecipitation; and micro-surface tensions in microfluidics.

Self-Coacervation of a Silk-Like Protein and Its Use As an Adhesive for Cellulosic Materials.

Liquid–liquid phase separation of biomacromolecules plays a critical role in many of their functions, both as cellular components and in structural assembly. Phase separation is also a key mechanism in the assembly of engineered recombinant proteins for the general aim to build new materials with unique structures and properties. Here the phase separation process of an engineered protein with a block-architecture was studied. As a central block, we used a modified spider silk sequence, predicted to be unstructured. In each terminus, folded globular blocks were used. We studied the kinetics and mechanisms of phase formation and analyzed the evolving structures and their viscoelastic properties. Individual droplets were studied with a micropipette technique, showing both how properties vary between individual drops and explaining overall bulk rheological properties. A very low surface energy allowed easy deformation of droplets and led to efficient infiltration into cellulosic fiber networks. Based on these findings, we demonstrated an efficient use of the phase-separated material as an adhesive for cellulose. We also conclude that the condensed state is metastable, showing an ensemble of properties in individual droplets and that an understanding of protein phase behavior will lead to developing a wider use of proteins as structural polymers.

Manipulating Single Microdroplets of NaCl Solutions: Solvent Dissolution, Microcrystallization, and Crystal Morphology.

A new "three-micropipette manipulation technique" for forming, dehydrating, crystallizing, and resolvating nanograms of salt material has been developed to study supersaturated single microdroplets and microcrystals. This is the first report of studies that have measured in situ both supersaturation (as homogeneous nucleation) and saturation (as microcrystal redissolution) for single microdroplets of NaCl solution using the micropipette technique. This work reports a measure of the critical supersaturation concentration for homogeneous nucleation of NaCl (10.3 ± 0.3 M) at a supersaturation fraction of S = 1.9, the saturation concentration of NaCl in aqueous solution as measured with nanograms of material (5.5 ± 0.1 M), the diffusion coefficient for water in octanol, D = (1.96 ± 0.10) × 10-6 cm2/s, and the effect of the solvent's activity on dissolution kinetics. It is further shown that the same Epstein-Plesset (EP) model, which was originally developed for diffusion-controlled dissolution and uptake of gas, and successfully applied to liquid-in-liquid dissolution, can now also be applied to describe the diffusion-controlled uptake of water from a water-saturated environment using the extended activity-based model of Bitterfield et al. This aspect of the EP model has not previously been tested using single microdroplets. Finally, it is also reported how the water dissolution rate, rate of NaCl concentration change, resulting crystal structure, and the time frame of initial crystal growth are affected by changing the bathing medium from octanol to decane. A much slower loss of water-solvent and concomitant slower up-concentration of the NaCl solute resulted in a lower tendency to nucleate and slower crystal growth because much less excess material was available at the onset of nucleation in the decane system as compared to the octanol system. Thus, the crystal structure is reported to be dendritic for NaCl solution microdroplets dissolving rapidly and nucleating violently in octanol, while they are formed as single cubic crystals in a gentler way for solution-dissolution in decane. These new techniques and analyses can now also be used for any other system where all relevant parameters are known. An example of this is control of drug/hydrogel/emulsion particle size change due to solvent uptake.

From Single Microparticles to Microfluidic Emulsification: Fundamental Properties (Solubility, Density, Phase Separation) from Micropipette Manipulation of Solvent, Drug and Polymer Microspheres

The micropipette manipulation technique is capable of making fundamental single particle measurements and analyses. This information is critical for establishing processing parameters in systems such as microfluidics and homogenization. To demonstrate what can be achieved at the single particle level, the micropipette technique was used to form and characterize the encapsulation of Ibuprofen (Ibp) into poly(lactic-co-glycolic acid) (PLGA) microspheres from dichloromethane (DCM) solutions, measuring the loading capacity and solubility limits of Ibp in typical PLGA microspheres. Formed in phosphate buffered saline (PBS), pH 7.4, Ibp/PLGA/DCM microdroplets were uniformly solidified into Ibp/PLGA microparticles up to drug loadings (DL) of 41%. However, at DL 50 wt% and above, microparticles showed a phase separated pattern. Working with single microparticles, we also estimated the dissolution time of pure Ibp microspheres in the buffer or in detergent micelle solutions, as a function of the microsphere size and compare that to calculated dissolution times using the Epstein-Plesset (EP) model. Single, pure Ibp microparticles precipitated as liquid phase microdroplets that then gradually dissolved into the surrounding PBS medium. Analyzing the dissolution profiles of Ibp over time, a diffusion coefficient of 5.5 ± 0.2 × 10−6 cm2/s was obtained by using the EP model, which was in excellent agreement with the literature. Finally, solubilization of Ibp into sodium dodecyl sulfate (SDS) micelles was directly visualized microscopically for the first time by the micropipette technique, showing that such micellization could increase the solubility of Ibp from 4 to 80 mM at 100 mM SDS. We also introduce a particular microfluidic device that has recently been used to make PLGA microspheres, showing the importance of optimizing the flow parameters. Using this device, perfectly smooth and size-homogeneous microparticles were formed for flow rates of 0.167 mL/h for the dispersed phase (Qd) and 1.67 mL/h for the water phase (Qc), i.e., a flow rate ratio Qd/Qc of 10, based on parameters such as interfacial tension, dissolution rates and final concentrations. Thus, using the micropipette technique to observe the formation, and quantify solvent dissolution, solidification or precipitation of an active pharmaceutical ingredient (API) or excipient for single and individual microparticles, represents a very useful tool for understanding microsphere-processes and hence can help to establish process conditions without resorting to expensive and material-consuming bulk particle runs.

Micropipette Technique Study of Natural and Synthetic Lung Surfactants at the Air-Water Interface: Presence of a SP-B Analog Peptide Promotes Membrane Aggregation, Formation of Tightly Stacked Lamellae, and Growth of Myelin Figures.

The present study is a microscopic interfacial characterization of a series of lung surfactant materials performed with the micropipette technique. The advantages of this technique include the measurement of equilibrium and dynamic surface tensions while acquiring structural and dynamic information at microscopic air-water interfaces in real time and upon compression. Here, we characterized a series of animal-derived and synthetic lung surfactant formulations, including native surfactant obtained from porcine lungs (NS); the commercial Curosurf, Infasurf, and Survanta; and a synthetic Super Mini-B (SMB)-containing formulation. It was observed that the presence of the natural hydrophobic proteins and, more strikingly, the peptide SMB, promoted vesicle condensation as thick membrane stacks beneath the interface. Only in the presence of SMB, these stacks underwent spontaneous structural transformations, consisting of the nucleation and growth of microtubes and in some cases their subsequent coiling into helices. The dimensions of these tubes (2-15 μm diameter) and their linear (2-3 μm/s) and volumetric growth rates (20-30 μm3/s) were quantified, and no specific effects were found on them for increasing SMB concentrations from 0.1 to 4%. Nevertheless, a direct correlation between the number of tubes and SMB contents was found, suggesting that SMB molecules are the promoters of tube nucleation in these membranes. A detailed analysis of the tube formation process was performed following previous models for the growth of myelin figures, proposing a combined mechanism between dehydration-rehydration of the lipid bilayers and induction of mechanical defects by SMB that would act as nucleation sites for the tubes. The formation of tubes was also observed in Infasurf, and in NS only after subsequent expansion and compression but neither in the other clinical surfactants nor in protein-free preparations. Finally, the connection between this data and the observations from the lung surfactant literature concerning the widely reported "near-zero surface tension" for lung surfactant films and intact alveolar surfaces is also discussed.

Single Microgels in Core-Shell Equilibrium: A Novel Method for Limited Volume Studies.

The interactions of cationic surfactant dodecyltrimethylammonium bromide and cationic protein cytochrome c with anionic polyacrylate microgels have been investigated in microscopic liquid droplets by means of a micropipette technique at ionic strength 0.01 M and pH 8. Experiments on single microgels in solutions of limited amounts of the surfactant provide the first evidence of microgels in a stable biphasic core-shell state with the surfactant partitioned to the shell. Under the same conditions, the protein is found to distribute uniformly in the microgels. Quantitative data in the form of swelling and binding isotherms are presented and compared with literature data for macrogels and with predictions of a recent gel theory. Theory is found to be in semiquantitative agreement with the experiments. The importance of polyion-mediated attractions between the protein molecules is analyzed theoretically and proposed to explain the continuous but highly cooperative binding isotherms.

P-Selectin Glycoprotein Ligand-1 Forms Dimeric Interactions with E-Selectin but Monomeric Interactions with L-Selectin on Cell Surfaces

Interactions of selectins with cell surface glycoconjugates mediate the first step of the adhesion and signaling cascade that recruits circulating leukocytes to sites of infection or injury. P-selectin dimerizes on the surface of endothelial cells and forms dimeric bonds with P-selectin glycoprotein ligand-1 (PSGL-1), a homodimeric sialomucin on leukocytes. It is not known whether leukocyte L-selectin or endothelial cell E-selectin are monomeric or oligomeric. Here we used the micropipette technique to analyze two-dimensional binding of monomeric or dimeric L- and E-selectin with monomeric or dimeric PSGL-1. Adhesion frequency analysis demonstrated that E-selectin on human aortic endothelial cells supported dimeric interactions with dimeric PSGL-1 and monomeric interactions with monomeric PSGL-1. In contrast, L-selectin on human neutrophils supported monomeric interactions with dimeric or monomeric PSGL-1. Our work provides a new method to analyze oligomeric cross-junctional molecular binding at the interface of two interacting cells.

Micropipette aspiration of an inflated fluid-filled spherical membrane

The micropipette technique has been widely used to determine the mechanical response of living cells. Cell is a complex structure and has highly nonlinear behavior when aspirated by the micropipette. Different models exist in the literature to determine the physical properties of the cells from the micropipette aspiration. In this work, we model the cell’s response during micropipette aspiration using continuum nonlinear membrane theory. The peripheral surface of the cell is modeled as a nonlinear membrane. The membrane is homogenous, isotropic, and of hyper-elastic material. The cytoplasm of the cell is modeled as an incompressible fluid. Our results show good agreement with the experiments reported in the literature.

Considerations when determining low interfacial tensions

Surfactants are often used to create low or “ultralow” oil–water interfacial tensions (IFTs). These molecules, in some cases, may also lead to the inadvertent formation of microemulsion phases which can obscure IFT measurements. Here, we present a case study of such an issue. We also introduce a little-known micropipette technique which has many advantages over the more common spinning drop method for determining low/ultralow IFTs.

Shear stress and force required for tether formation of neonatal and adult erythrocytes

Red blood cells (RBC) of neonates have a shorter survival time and they are more susceptible to mechanical alterations than RBC of adults. Irreversible alteration of the membrane of RBC of preterm neonates, term neonates and adults due to tether formation was studied by means of a micropipette technique. Shear stress and forces were applied with this technique in an axisymmetric configuration and were calculated with an approximation method. The applied shear stress and forces that induced tether formation were lowest for RBC of preterm neonates (1.5 Pa, 1.8 pN), in between for RBC of neonates (1.7 Pa, 2.1 pN) and highest for RBC in adults (1.9 Pa, 2.6 pN). Decreased mechanical stability of the membrane of neonatal RBC may in part cause the shorter life span, the greater tendency to endocytosis and the higher amount of irregularly shaped cells of neonatal RBC compared to RBC in adults.

Comparison of three different techniques for application of water solutions to Finn Chambers®

With regard to contact allergy, the dose of a sensitizer per unit skin area is an important factor for both sensitization and elicitation, and therefore a known amount/volume of test preparation should be applied at patch testing. To compare three different techniques for the application of aqueous solutions to Finn Chambers, in order to determine the precision and accuracy of each technique when the recommended 15 µl volume is applied. Four technicians applied formaldehyde 1.0% aq. (wt/vol) and methylchloroisothiazolinone/methylisothiazolinone 200 ppm (wt/vol) in sets of 10 onto Finn Chambers, with three different techniques: (i) micro-pipetting; (ii) dripping the solutions; and (iii) dripping the solutions followed by removal of excess solution with a soft tissue. Assessment of the variations was performed with the use of descriptive data. The ability to apply the exact amount was assessed by Fisher's exact test by categorizing each application as in or out of the range 12-18 µl. The micro-pipette technique had the best accuracy and precision, as well as the lowest inter-individual variation. The technique in which excess solution was removed had good precision, but failed in the application of the defined amount, i.e. 15 µl.

Hydration Potential of Lysozyme: Protein Dehydration Using a Single Microparticle Technique

For biological molecules in aqueous solution, the hydration pressure as a function of distance from the molecular surface represents a very short-range repulsive pressure that limits atom-atom contact, opposing the attractive van der Waals pressure. Whereas the separation distance for molecules that easily arrange into ordered arrays (e.g., lipids, DNA, collagen fibers) can be determined from x-ray diffraction, many globular proteins are not as easily structured. Using a new micropipette technique, spherical, glassified protein microbeads can be made that allow determination of protein hydration as a function of the water activity ( a w) in a surrounding medium (decanol). By adjusting a w of the dehydration medium, the final protein concentration of the solid microbead is controlled, and ranges from 700 to 1150 mg/mL. By controlling a w (and thus the osmotic pressure) around lysozyme, the repulsive pressure was determined as a function of distance between each globular, ellipsoid protein. For separation distances, d, between 2.5 and 9 Å, the repulsive decay length was 1.7 Å and the pressure extrapolated to d = 0 was 2.2 × 10 8 N/m 2, indicating that the hydration pressure for lysozyme is similar to other biological interfaces such as phospholipid bilayers.

The effect of hydrogen bonding on the diffusion of water in n-alkanes and n-alcohols measured with a novel single microdroplet method

While the Stokes-Einstein (SE) equation predicts that the diffusion coefficient of a solute will be inversely proportional to the viscosity of the solvent, this relation is commonly known to fail for solutes, which are the same size or smaller than the solvent. Multiple researchers have reported that for small solutes, the diffusion coefficient is inversely proportional to the viscosity to a fractional power, and that solutes actually diffuse faster than SE predicts. For other solvent systems, attractive solute-solvent interactions, such as hydrogen bonding, are known to retard the diffusion of a solute. Some researchers have interpreted the slower diffusion due to hydrogen bonding as resulting from the effective diffusion of a larger complex of a solute and solvent molecules. We have developed and used a novel micropipette technique, which can form and hold a single microdroplet of water while it dissolves in a diffusion controlled environment into the solvent. This method has been used to examine the diffusion of water in both n-alkanes and n-alcohols. It was found that the polar solute water, diffusing in a solvent with which it cannot hydrogen bond, closely resembles small nonpolar solutes such as xenon and krypton diffusing in n-alkanes, with diffusion coefficients ranging from 12.5x10(-5) cm(2)/s for water in n-pentane to 1.15x10(-5) cm(2)/s for water in hexadecane. Diffusion coefficients were found to be inversely proportional to viscosity to a fractional power, and diffusion coefficients were faster than SE predicts. For water diffusing in a solvent (n-alcohols) with which it can hydrogen bond, diffusion coefficient values ranged from 1.75x10(-5) cm(2)/s in n-methanol to 0.364x10(-5) cm(2)/s in n-octanol, and diffusion was slower than an alkane of corresponding viscosity. We find no evidence for solute-solvent complex diffusion. Rather, it is possible that the small solute water may be retarded by relatively longer residence times (compared to non-H-bonding solvents) as it moves through the liquid.

Effects of phosphodiesterase (III/IV)-inhibitors and cytokines on mechanical properties of neutrophilic granulocytes in neonates and adults

Sequestration of activated PMN and enrichment in tissues play a key role in tissue damage during septicaemia and after ischemia/reperfusion. Since polymorphonuclear neutrophilic granulocytes (PMN) of term neonates show various functional differences compared to PMN in adults (decreased chemotaxis, decreased intracellular killing, decreased adhesion) we studied the influence of interleukin 8 (IL-8), tumor necrosis factor-alpha (TNF-alpha) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) on the reduction of deformability of PMN in neonates and adults. The following phosphodiesterase (PDE)-inhibitors were applied to ameliorate the reduction in deformability when the PMN were stimulated with fMLP or IL-8: Enoximone, Milrinone (PDE-III-inhibitors), Pentoxifylline (PTX) and Piclamilast (PDE-IV-inhibitors). The micropipette technique and the cell transit analyzer (CTA) were used and compared. Aspiration times into micropipettes with an internal diameter of 5 microm, transit times through 8 microm filter pores and neutrophil elastase concentrations were determined. Despite of the functional differences of PMN in neonates compared to adults the significant decrease of deformability of PMN activated with cytokines compared to passive PMN was not different in both groups. The neutrophil elastase concentrations reflect the activation of the PMN: highest concentrations during activation, decreased concentrations due to PDE-inhibitors, and PMN in a passive state. The neutrophil elastase concentrations were not different from PMN of neonates and adults. These PDE-inhibitors significantly increased the deformability of activated PMN but significant differences between the deformability of PMN in neonates and adults were not found. Despite the functional differences of PMN in neonates PDE-III/IV-inhibitors lead to similar improvement of mechanical properties of activated PMN in neonates and adults. These drugs may ameliorate impaired microcirculation also in neonates during inflammation.

Mechanics of POPC Bilayers in Presence of Alkali Salts

Membranes mechanical properties are affected by solvent properties, i.e. the salt content. In this study, we use POPC GUVs (Giant Unilamellar Vesicles) as model membranes and we measure the membrane mechanical moduli by flickering analysis and micropipette technique for a series of alkali salt solutions. Salt concentration effects and ion specificity are investigated in these measurements. Membrane mechanical moduli are shown to display a complex dependence on the salt solution composition.

Role of Bitumen Components in Stabilizing Water-in-Diluted Oil Emulsions

Role of bitumen components in stabilization of water-in-diluted oil emulsions was studied using the micropipette technique. Naturally occurring components of bitumen, asphaltenes and maltenes were separated (by precipitating asphaltenes with n-pentane) to investigate their influence on the properties of water drop surfaces in Heptol (a mixture of heptane and toluene at a 4:1 volume ratio). The Heptol−water interfacial tension decreased with increasing adsorption of surface active components from both asphaltenes and maltenes. Rigidity of emulsified water droplet surfaces was observed in the presence of asphaltenes dissolved in Heptol. The observed surface crumpling is attributed to the irreversible adsorption of asphaltenes on the emulsified water droplet in Heptol. Further, the results of droplet interaction experiments indicated that the stability of the water-in-diluted oil emulsions was mainly due to the presence of asphaltenes dissolved in Heptol. However, it was observed that the presence of maltenes can also contribute to the emulsion stability even without well-defined skin formation on the emulsified water droplet surfaces.

Isolation of Single Choanoflagellate Cells from Field Samples and Establishment of Clonal Cultures

INTRODUCTIONChoanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. This protocol describes how to enrich freshly collected field samples and isolate single choanoflagellate cells. Two methods are commonly used: isolation by micropipetting single cells and isolation by dilution. The two methods are complementary and each has its advantages and disadvantages. The micropipette technique requires considerable experience. However, provided a single cell has been isolated, any culture arising from it will be clonal. The dilution technique does not require the same level of skill, but single-cell isolation is not ensured. For the latter technique, repeating the isolation procedure using higher dilutions is advised to ensure that only one cell is the basis of an established culture.

BIODIVERSITY OF HYDROCARBON-PRODUCING MICROALGAE FROM OIL CONTAMINATED IN COASTAL ZONE OF BATAM ISLAND

Addressing to the environmental and energy crises in Indonesia, the new alternative of green-economic-energy sources is required. In this study marine microalgae were isolated from oil-polluted coastal zone in Batam Island for alternative energy sources. The research was carried out by collecting the sea water samples from several peninsulas of Batam Island. The abundance of microalgal diversity was observed. The isolation was done by capillary micropipette technique. The method of specific screening for hydrocarbon-producing microalgae was done using selected media, CO2 rate and light intensity period treatments. The results showed that the Cyanobacteria and diatom occupied the Batam's coastal waters. From the isolated samples, 11 strains positively deposit the hydrocarbon in their cells. Further study for determining the isolates and their properties is still in progress.