Turbo-mixing in microplates

How to effectively mix small volumes of liquids within microplate wells is a still underestimated and often neglected challenge. The method the authors introduce here relies on violent turbulent motion within a liquid caused by spotting an organic solvent drop onto its surface. The amount needed, less than 1 to 3 microL, is generally small enough not to alter bioactive molecules. Moreover, a solvent may be selected for its compatibility with assay components. The method was tested with layers of aqueous liquids that differ in pH and concentration of a pH-dependent dye, allowing mixing to be monitored optically. Rapid mixing was caused by spotting drops of alcohols, acetone, acetonitrile, and aqueous solutions of these, as long as the difference of surface tension between the drop and the uppermost layer of the bulk liquid surpassed 30 dynes/cm. Along with this difference, position and velocity of spotting, as well as viscosity and geometry of the bulk liquid volume, may influence the turbulence evoked. No significant difference was found for the activity of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase when measured after mixing by shaking and after mixing by spotting 1 microL of methanol onto assays within 96-well microplates.     

Impedance analysis of MDCK cells measured by electric cell-substrate impedance sensing.

Transepithelial impedance of Madin-Darby canine kidney cell layers is measured by a new instrumental method, referred to as electric cell-substrate impedance sensing. In this method, cells are cultured on small evaporated gold electrodes, and the impedance is measured in the frequency range 20-50,000 Hz by a small probing current. A model for impedance analysis of epithelial cells measured by this method is developed. The model considers three different pathways for the current flowing from the electrode through the cell layer: (1) in through the basal and out through the apical membrane, (2) in through the lateral and out through the apical membrane, and (3) between the cells through the paracellular space. By comparing model calculation with experimental impedance data, several morphological and cellular parameters can be determined: (1) the resistivity of the cell layer, (2) the average distance between the basal cell surface and substratum, and (3) the capacitance of apical, basal, and lateral cell membranes. This model is used to analyze impedance changes on removal of Ca2+ from confluent Mardin-Darby canine kidney cell layers. The method shows that reduction of Ca2+ concentration causes junction resistance between cells to drop and the distance between the basal cell surface and substratum to increase.     

Assessment of intraluminal impedance for the detection of pharyngeal bolus flow during swallowing in healthy adults

Intraluminal impedance, a nonradiological method for assessing bolus flow within the gut, may be suitable for investigating pharyngeal disorders. This study evaluated an impedance technique for the detection of pharyngeal bolus flow during swallowing. Patterns of pharyngoesophageal pressure and impedance were simultaneously recorded with videofluoroscopy in 10 healthy volunteers during swallowing of liquid, semisolid, and solid boluses. The timing of bolus head and tail passage recorded by fluoroscopy was correlated with the timing of impedance drop and recovery at each recording site. Bolus swallowing produced a drop in impedance from baseline followed by a recovery to at least 50% of baseline. The timing of the pharyngeal and esophageal impedance drop correlated with the timing of the arrival of the bolus head. In the pharynx, the timing of impedance recovery was delayed relative to the timing of clearance of the bolus tail. In contrast, in the upper esophageal sphincter (UES) and proximal esophagus, the timing of impedance recovery correlated well with the timing of clearance of the bolus tail. Impedance-based estimates of pharyngoesophageal bolus clearance time correlated with true pharyngoesophageal bolus clearance time. Patterns of intraluminal impedance recorded in the pharynx during bolus swallowing are therefore more complex than those in the esophagus. During swallowing, mucosal contact between the tongue base and posterior pharyngeal wall prolongs the duration of pharyngeal impedance drop, leading to overestimation of bolus tail timing. Therefore, we conclude that intraluminal impedance measurement does not accurately reflect the bolus transit in the pharynx but does accurately reflect bolus transit across the UES and below.     

Improved frequency response of pneumotachometers by digital compensation

To measure impedance one measures or estimates flow, which is commonly done by measuring the pressure drop across a pneumotachometer. The frequency response characteristics of standard pneumotachometer/pressure transducers (PPT) limit their use to relatively low frequencies. Also, the frequency response of PPTs has been reported to be "load" dependent. Thus, the frequency response characteristics measured under "no-load" conditions, which theoretically could be used to compensate subsequent measurements, may not be appropriate for measurements made under loaded conditions. Another method of measuring impedance exists which depends on a reference impedance element other than a pneumotachometer. In this method, an oscillatory flow signal with known amplitude is generated and used to force the system being tested. Unlike PPTs, this oscillatory flow generator (OFG) is a closed system that allows measurements to be made only during breath holding. Our objective was to determine whether the frequency response of a PPT could be compensated using measurements made under no-load conditions, such that it accurately measured an impedance load. The frequency response of the PPT under no-load conditions was measured by the OFG and used to compensate the output of the PPT in subsequent impedance measurements. The compensated PPT was used to measure the impedance of a mechanical structure and the impedances of four human subjects. The impedances of the mechanical structure and the subjects were also measured using the OFG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adsorption of peptide nucleic acid and DNA decamers at electrically charged surfaces.

Adsorption behavior of peptide nucleic acid (PNA) and DNA decamers (GTAGATCACT and the complementary sequence) on a mercury surface was studied by means of AC impedance measurements at a hanging mercury drop electrode. The nucleic acid was first attached to the electrode by adsorption from a 5-microliter drop of PNA (or DNA) solution, and the electrode with the adsorbed nucleic acid layer was then washed and immersed in the blank background electrolyte where the differential capacity C of the electrode double layer was measured as a function of the applied potential E. It was found that the adsorption behavior of the PNA with an electrically neutral backbone differs greatly from that of the DNA (with a negatively charged backbone), whereas the DNA-PNA hybrid shows intermediate behavior. At higher surface coverage PNA molecules associate at the surface, and the minimum value of C is shifted to negative potentials because of intermolecular interactions of PNA at the surface. Prolonged exposure of PNA to highly negative potentials does not result in PNA desorption, whereas almost all of the DNA is removed from the surface at these potentials. Adsorption of PNA decreases with increasing NaCl concentration in the range from 0 to 50 mM NaCl, in contrast to DNA, the adsorption of which increases under the same conditions.     

Influence of intermediate aminodextran layers on the signal response of surface acoustic wave biosensors

Surface acoustic wave (SAW) devices based on horizontally polarized surface shear waves enable direct and label-free detection of proteins in real time. Binding reactions on the sensor surface are detected by determining changes in surface wave velocity caused mainly by mass adsorption or change of viscoelasticity in the sensing layer. Intermediate hydrogel layers have been proven to be useful to immobilize capture molecules or ligands corresponding to the analyte. However, the SAW signal response strongly depends on the morphology of the hydrogel due to different relative changes of its acoustomechanical parameters such as viscoelasticity and density. In this work five aminodextrans (AMD) and one diamino polyethylene glycol (DA-PEG) were used as intermediate hydrogel layers. Sensors with immobilized streptavidin and samples containing biotinylated bovine serum albumin were used to exemplify affinity assays based on immobilized capture molecules for protein detection. The effects of the three-dimensional AMDs and the two-dimensional (2D) DA-PEG on the SAW signal response were investigated. The signal height decreased with increasing molar mass and increasing amount of immobilized AMD. Consequently, thin hydrogel layers are ideal to obtain optimum signal responses in this type of assay, whereas it is not necessarily a 2D hydrogel that gives the best results.     

A modified method for determination of diffusivities of low molecular substances in non-Newtonian liquids

A modified method for determination of diffusivities of low molecular substances in non-Newtonian liquids described by the power-law model has been proposed. It is based on the dissolution of Geiss body, with a parameter m=1/3 rotating in an infinite fluid. In this case, the solution of the differential equations of motion and mass transfer is available as an analytical formula for calculating the diffusivity coefficient.The method allows the extension of the variety of media and diffusing species. It has been illustrated with dissolving of gypsum in water and five non-Newtonian liquids. The results obtained have been interpreted taking into account the interaction between calcium ions and polymer molecules of the non-Newtonian system, as well as the heterogeneity of the system near to the dissolving surface.     

Real-time cellular impedance measurements detect Ca(2+) channel-dependent oscillations of morphology in human H295R adrenoma cells

Endocrine cells, such as H295R have been widely used to study secretion of steroid and other hormones. Exocytosis-dependent hormone release is accompanied by an increase in plasma membrane surface area and a decrease in vesicle content. Recovery of vesicles and decrease in plasma membrane area is achieved by endocytotic processes. These changes in the extent of the surface area lead to morphological changes which can be determined by label-free real-time impedance measurements. Exo- and endocytosis have been described to be triggered by activation of L-type Ca(2+) channels. The present study demonstrates that activation of L-type calcium channels induces prolonged oscillating changes in cellular impedance. The data support the hypothesis that a tight regulation of the intracellular Ca(2+) concentration is a prerequisite for the observed cellular impedance oscillations. Furthermore evidence is presented for a mechanism in which the oscillations depend on a Ca(2+)-triggered calmodulin-dependent cascade involving myosin light chain kinase, nonmuscle myosin II and ultimately actin polymerization, a known determinant for cell shape changes and exocytosis in secretory cells. The described assay provides a method to determine continuously prolonged changes in cellular morphology such as exo/endocytosis cycles. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Impedance analysis of the organ of corti with magnetically actuated probes

An innovative method is presented to measure the mechanical driving point impedance of biological structures up to at least 40 kHz. The technique employs an atomic force cantilever with a ferromagnetic coating and an external magnetic field to apply a calibrated force to the cantilever. Measurement of the resulting cantilever velocity using a laser Doppler vibrometer yields the impedance. A key feature of the method is that it permits measurements for biological tissue in physiological solutions. The method was applied to measure the point impedance of the organ of Corti in situ, to elucidate the biophysical basis of cochlear amplification. The basilar membrane was mechanically clamped at its tympanic surface and the measurements conducted at different radial positions on the reticular lamina. The tectorial membrane was removed. The impedance was described by a generalized Voigt-Kelvin viscoelastic model, in which the stiffness was real-valued and independent of frequency, but the viscosity was complex-valued with positive real part, which was dependent on frequency and negative imaginary part, which was independent of frequency. There was no evidence for an inertial component. The magnitude of the impedance was greatest at the tunnel of Corti, and decreased monotonically in each of the radial directions. In the absence of inertia, the mechanical load on the outer hair cells causes their electromotile displacement responses to be reduced by only 10-fold over the entire range of auditory frequencies.     

Double-mode impedance analysis of epithelial cell monolayers cultured on shear wave resonators

The viscoelastic behavior of epithelial cells (MDCK-I and MDCK-II) grown on AT-cut quartz crystals with a fundamental resonance at 5 MHz was investigated by impedance spectroscopy. Using the electromechanical model recently derived by Martin et al. [(1991) Anal Chem 63: 2272 – 2281] for Newtonian liquids in contact with shear wave resonators we quantified the viscous damping arising from the adherent cells by fitting the impedance data with a modified Butterworth-Van Dyke circuit in the region of the resonance frequency. Impedance spectroscopy was additionally performed in the frequency range from 1 Hz to 1 MHz to scrutinize the passive electrical properties of the epithelial cell layers using an additional platinum electrode. These data allow one to document the cell layers' integrity as well as the electrode coverage. We were able to confirm that the presence of a cell-layer mainly increases damping of the shear wave and does not exhibit a pure mass-load behavior. These findings were supported by the discovery that the inductance L in the electromechanical model was less influenced by the cell-layer than the resistance R. The apparent cell-viscosities determined by our method are 0.097 poise for MDCK-I and 0.142 poise for MDCK-II cell-layers. These low apparent viscosities may be explained in terms of a considerable spacing between the cells immobilized via their focal contacts and the quartz surface. Received: 5 June 1996 / Accepted: 6 August 1996     

The method of contact angle measurements and estimation of work of adhesion in bioleaching of metals

In this paper, we present our method for the measurement of contact angles on the surface of minerals during the bioleaching process because the standard deviation obtained in our measurements achieved unexpectedly low error. Construction of a goniometer connected with a specially prepared computer program allowed us to repeat measurements several times over a short time course, yielding excellent results. After defining points on the outline of the image of a drop and its baseline as well of the first approximation of the outline of the drop, an iterative process is initiated that is aimed at fitting the model of the drop and baseline. In turn, after defining the medium for which measurements were made, the work of adhesion is determined according to Young-Dupré equation. Calculations were made with the use of two methods named the L-M and L-Q methods.     

Real-time cellular impedance measurements detect Ca channel-dependent oscillations of morphology in human H295R adrenoma cells

Endocrine cells, such as H295R have been widely used to study secretion of steroid and other hormones. Exocytosis-dependent hormone release is accompanied by an increase in plasma membrane surface area and a decrease in vesicle content. Recovery of vesicles and decrease in plasma membrane area is achieved by endocytotic processes. These changes in the extent of the surface area lead to morphological changes which can be determined by label-free real-time impedance measurements. Exo- and endocytosis have been described to be triggered by activation of L-type Ca²⁺ channels. The present study demonstrates that activation of L-type calcium channels induces prolonged oscillating changes in cellular impedance. The data support the hypothesis that a tight regulation of the intracellular Ca²⁺ concentration is a prerequisite for the observed cellular impedance oscillations. Furthermore evidence is presented for a mechanism in which the oscillations depend on a Ca²⁺-triggered calmodulin-dependent cascade involving myosin light chain kinase, nonmuscle myosin II and ultimately actin polymerization, a known determinant for cell shape changes and exocytosis in secretory cells. The described assay provides a method to determine continuously prolonged changes in cellular morphology such as exo/endocytosis cycles.     

Studies of excitable membrane formed on the surface of protoplasmic drops isolated from Nitella III. Impedance of the surface membrane

A protoplasmic drop isolated from an internodal cell of Nitella in an initial solution composed of 70 mM KNO₃, 50 mM NaNO₃ and 5 mM CaCl₂ became electrically excitable when the drop was placed in the final solution containing 0.5 mM KNO₃, 0.5 mM NaCl, 1 mM Ca(NO₃)₂ and 2 mM Mg(NO₃)₂. The electrical impedance of the surface membrane of the drop was measured both in the initial and final solutions at frequencies between 60 Hz and 100 kHz.The impedance and admittance loci of the surface membrane fell on circular arcs. The d.c. resistance R_m°, and the d.c. capacitance C_m° were determined by extrapolating the circular arcs to the low frequency limit. R_m° thus determined was in the range of 50–200 Ω·cm² in the initial solution, and increased to a steady value of 0.4–4.0 kΩ·cm² when the external solution was replaced by the final solution. After the protoplasmic drop was isolated from the internodal cell of Nitella, C_m° decreased monotonically from about 1.5 μF/cm² within 20 min and approached $\text{1.25±0.1 μ}\text{F/cm}{}^2$ both in the initial and final solutions. No appreciable difference was observed for C_m° in these two solutions.The impedance data were discussed in relation to the process of formation of the membrane at the surface of the protoplasmic drop. After the excitable stage was reached, the drop membrane impedance was found to decrease by a factor of 10 during excitation.     

Application of a pull on a disk method to measure surface tension of liquids

The intrinsic property of liquids is a vital indicator of formulation performance and stability. Therefore, investigation of the interfacial phenomenon of surface tension is a routine procedure in the development of products in a wide variety of areas including foods, pharmaceuticals, cosmetics, and painting technologies. We hypothesize that studies related to the maximum pull on a rod can be extrapolated to disk geometry and applied to measure surface tension using a texture analyzer. A glass disk probe was attached to the arm of a texture analyzer and pulled from the liquid surface. The maximum force of detachment was used to calculate surface tension extrapolating from the theory of maximum pull on a rod. The surface tension of water, ethanol, and a hydroalcoholic solution was measured and compared with literature values to validate this hypothesis. The calculated values of surface tension for the liquids studied were within 5% of the reported values. Probe diameter appears to have an important role on surface tension accuracy compared with literature values. Slight discrepancies can be attributed to temperature control and leveling of liquid surface, although still in accordance with the reported values of surface tension measured using different methods. This study presents a simple, precise, and quick method to determine the surface tension of liquids from the maximum pull on a disk. Further studies are warranted to determine the optimum glass disk probe diameter for better accuracy.     

Significance of exine shedding in Cupressaceae-type pollen

In conifers, which have non-saccate Cupressaceae-type pollen, the pollen must land on a pollination drop or be picked up by the pollination drop from the surface of the cone near the ovule before it can be taken into the ovule. After contact with the drop, the pollen intine absorbs moisture from the drop, expands and the exine is shed. In this study the significance of the shedding of the exine is interpreted from experiments in which simulated pollination drops and micropyles were used to determine the movement of pollen and other particles in suspension. The non-expanded pollen, which can be observed upon contact with the pollination drop, sheds the exine, which then functions as a non-elastic particle, while the pollen from which the exine was shed swells and functions as an elastic particle because it is enclosed by the flexible intine. Non-elastic particles are not easily transferred through narrow passages (the micropyle and micropylar canal) and tend to plug these passages. However, elastic particles, such as the swollen pollen, are easily transferred along narrow passages even when non-elastic particles are present. The simulated experiments demonstrate that exine shedding is an important feature in getting pollen through the narrow micropyle and micropylar canal to the nucellus of the ovule.     

Measuring electrical impedance of organs--instrumental equipment for research and clinical use

An apparatus for measuring the impedance of intact biological organs or parts of organs in the frequency range of 10 Hz to 10 MHz is described. In this range impedance exhibits a large dispersion, which is dependent on tissue structures. The time course of alterations of electrical impedance such as occur during ischemia can be recorded with this equipment. Five specimens in five measuring chambers can be examined simultaneously at different temperatures. In the second part of the article, a portable impedance meter for measuring the modulus of impedance near 200 Hz, the phase of impedance at 5 kHz and the local temperature at the measuring point, is described. These parameters permit an intra-operative evaluation of the changing state of ischemic organs. Sterilizable probes with four surface electrodes and an integrated temperature sensor permit atraumatic measurements at the organ surface. The measurement itself is harmless to the tissue.     

A one-step electrochemical method for DNA detection that utilizes a peroxidase-mimicking DNAzyme amplified through PCR of target DNA

A novel one-step electrochemical method for DNA detection is described. The procedure utilizes a reaction catalyzed by a peroxidase-mimicking DNAzyme to produce a product, which forms an insoluble precipitation layer on the surface of an electrode. A rationally designed forward primer, conjugated with a peroxidase DNAzyme complementary sequence at its 5′-end, is used for PCR amplification of target DNA. As a result, the DNAzyme sequence is produced by amplification only when the target DNA is present in the sample. The PCR product is then subjected to the precipitation reaction on the electrode surface using an electrolyte assay buffer containing 4-chloronaphthol, hydrogen peroxide, ferrocenemethanol, hemin, and 5′-lambdaexonuclease. Finally, analysis is carried out using Faradaic impedance spectroscopy. The impedance value was found to greatly increase when target DNA is present owing to the formation of a precipitation layer on the electrode surface caused by the catalytic action of the DNAzyme. In contrast, no impedance increase is observed when a control sample not containing target DNA is utilized. By employing this strategy, target DNA from Chlamydia trachomatis was reliably detected within a 10 min period following precipitation without the need for complicated secondary procedures. This effort has led to the development of a highly convenient electrochemical one-step method for DNA detection that utilizes a peroxidase-mimicking DNAzyme, which is specifically designed to undergo amplification during PCR of target DNA.     

Axisymmetric Drop Shape Analysis for Estimating the Surface Tension of Cell Aggregates by Centrifugation

Biological tissues behave in certain respects like liquids. Consequently, the surface tension concept can be used to explain aspects of the in vitro and in vivo behavior of multicellular aggregates. Unfortunately, conventional methods of surface tension measurement cannot be readily applied to small cell aggregates. This difficulty can be overcome by an experimentally straightforward method consisting of centrifugation followed by axisymmetric drop shape analysis (ADSA). Since the aggregates typically show roughness, standard ADSA cannot be applied and we introduce a novel numerical method called ADSA-IP (ADSA for imperfect profile) for this purpose. To examine the new methodology, embryonic tissues from the gastrula of the frog, Xenopus laevis, deformed in the centrifuge are used. It is confirmed that surface tension measurements are independent of centrifugal force and aggregate size. Surface tension is measured for ectodermal cells in four sample batches, and varies between 1.1 and 7.7 mJ/m². Surface tension is also measured for aggregates of cells expressing cytoplasmically truncated EP/C-cadherin, and is approximately half as large. In parallel, such aggregates show a reduction in convergent extension-driven elongation after activin treatment, reflecting diminished intercellular cohesion.     

Growth models of cultures with two liquid phases. IV. Cell adsorption, drop size distribution, and batch growth

In this work, a mathematical model which can be used to describe butch growth in fermentations with two liquid phases present is developed for systems in which the growth limiting substrate is dissolved in the dispersed phase. The model takes into account the drop size distribution, the rate of adsorption of cells on the drop surface, the rate of desorption of cells from the drop surface, substrate transport between phases, phase equilibrium, and growth kinetics. The model also considers the effect, of coalescence and redispersion of oil drops in the system. It is assumed that the composition of the dispersed phase is such that substrate utilization from it causes little or no change in the interfacial area. A discrete uniform distribution and a discrete normal distribution which is obtained from an experimental distribution curve are used as drop size distributions. Simulation results are obtained for a wide range of parameter values using the IBM S/360 Continuous System Modeling Program.