Intracellular protein determination using droplet-based immunoassays

This paper describes the implementation of a sensitive, on-chip immunoassay for the analysis of intracellular proteins, developed using microdroplet technology. The system offers a number of analytical functionalities, enabling the lysis of low cell numbers, as well as protein detection and quantification, integrated within a single process flow. Cells were introduced into the device in suspension and were electrically lysed in situ. The cell lysate was subsequently encapsulated together with antibody-functionalized beads into stable, water-in-oil droplets, which were stored on-chip. The binding of intracellular proteins to the beads was monitored fluorescently. By analyzing many individual droplets and quantifying the data obtained against standard additions, we measured the level of two intracellular proteins, namely, HRas-mCitrine, expressed within HEK-293 cells, and actin-EGFP, expressed within MCF-7 cells. We determined the concentrations of these proteins over 5 orders of magnitude, from ~50 pM to 1 µM. The results from this semiautomated method were compared to those for determinations made using Western blots, and were found not only to be faster, but required a smaller number of cells. © 2011 American Chemical Society.

Time scale analysis for fluidized bed melt granulation I: granule-granule and granule-droplet collision rates

Fluidized bed spray granulators (FBMG) are widely used in the process industry for particle size growth; a desirable feature in many products, such as granulated food and medical tablets. In this paper, the first in a series of four discussing the rate of various microscopic events occurring in FBMG, theoretical analysis coupled with CFD simulations have been used to predict granule–granule and droplet–granule collision time scales. The granule–granule collision time scale was derived from principles of kinetic theory of granular flow (KTGF). For the droplet–granule collisions, two limiting models were derived; one is for the case of fast droplet velocity, where the granule velocity is considerable lower than that of the droplet (ballistic model) and another for the case where the droplet is traveling with a velocity similar to the velocity of the granules. The hydrodynamic parameters used in the solution of the above models were obtained from the CFD predictions for a typical spray fluidized bed system. The granule–granule collision rate within an identified spray zone was found to fall approximately within the range of 10-2–10-3 s, while the droplet–granule collision was found to be much faster, however, slowing rapidly (exponentially) when moving away from the spray nozzle tip. Such information, together with the time scale analysis of droplet solidification and spreading, discussed in part II and III of this study, are useful for probability analysis of the various event occurring during a granulation process, which then lead to be better qualitative and, in part IV, quantitative prediction of the aggregation rate.

Precision emulsification for droplet and capsule production

Emulsions and microcapsules are typical structures in various dispersion formulations for pharmaceutical, food, personal and house care applications. Precise control over size and size distribution of emulsion droplets and microcapsules are important for effective use and delivery of active components and better product quality. Many emulsification technologies have been developed to meet different formulation and processing requirements. Among them, membrane and microfluidic emulsification as emerging technologies have the feature of being able to precisely manufacture droplets in a drop-by-drop manner to give subscribed sizes and size distributions with lower energy consumption. This paper reviews fundamental sciences and engineering aspects of emulsification, membrane and microfluidic emulsification technologies and their use for precision manufacture of emulsions for intensified processing. Generic application examples are given for single and double emulsions and microcapsules with different structure features. © 2013 The Society of Powder Technology Japan. Published by Elsevier B.V.

Design of experiments to study the impact of process parameters on droplet size and development of non-invasive imaging techniques in tablet coating

Atomisation of an aqueous solution for tablet film coating is a complex process with multiple factors determining droplet formation and properties. The importance of droplet size for an efficient process and a high quality final product has been noted in the literature, with smaller droplets reported to produce smoother, more homogenous coatings whilst simultaneously avoiding the risk of damage through over-wetting of the tablet core. In this work the effect of droplet size on tablet film coat characteristics was investigated using X-ray microcomputed tomography (XμCT) and confocal laser scanning microscopy (CLSM). A quality by design approach utilising design of experiments (DOE) was used to optimise the conditions necessary for production of droplets at a small (20 μm) and large (70 μm) droplet size. Droplet size distribution was measured using real-time laser diffraction and the volume median diameter taken as a response. DOE yielded information on the relationship three critical process parameters: pump rate, atomisation pressure and coating-polymer concentration, had upon droplet size. The model generated was robust, scoring highly for model fit (R2 = 0.977), predictability (Q2 = 0.837), validity and reproducibility. Modelling confirmed that all parameters had either a linear or quadratic effect on droplet size and revealed an interaction between pump rate and atomisation pressure. Fluidised bed coating of tablet cores was performed with either small or large droplets followed by CLSM and XμCT imaging. Addition of commonly used contrast materials to the coating solution improved visualisation of the coating by XμCT, showing the coat as a discrete section of the overall tablet. Imaging provided qualitative and quantitative evidence revealing that smaller droplets formed thinner, more uniform and less porous film coats.

Scale-up in emulsion polymerisation

The procedure for successful scale-up of batchwise emulsion polymerisation has been studied. The relevant literature on liquid-liquid dispersion on scale-up and on emulsion polymerisation has been crit1cally reviewed. Batchwise emulsion polymerisation of styrene in a specially built 3 litre, unbaffled, reactor confirmed that impeller speed had a direct effect on the latex particle size and on the reaction rate. This was noted to be more significant at low soap concentrations and the phenomenon was related to the depletion of micelle forming soap by soap adsorption onto the monomer emulsion surface. The scale-up procedure necessary to maintain constant monomer emulsion surface area in an unbaffled batch reactor was therefore investigated. Three geometrically similar 'vessels of 152, 229 and 305mm internal diameter, and a range of impeller speeds (190 to 960 r.p.m.) were employed. The droplet sizes were measured either through photomicroscopy or via a Coulter Counter. The power input to the impeller was also measured. A scale-up procedure was proposed based on the governing relationship between droplet diameter, impeller speed and impeller diameter. The relationships between impeller speed soap concentration, latex particle size and reaction rate were investigated in a series of polymerisations employing an amended commercial recipe for polystyrene. The particle size was determined via a light transmission technique. Two computer models, based on the Smith and Ewart approach but taking into account the adsorption/desorption of soap at the monomer surface, were successful 1n predicting the particle size and the progress of the reaction up to the end of stage II, i.e. to the end of the period of constant reaction rate.

Liquid-liquid extraction in a pilot scale rotating disc contactor

A study of the hydrodynamics and mass transfer characteristics of a liquid-liquid extraction process in a 450 mm diameter, 4.30 m high Rotating Disc Contactor (R.D.C.) has been undertaken. The literature relating to this type of extractor and the relevant phenomena, such as droplet break-up and coalescence, drop mass transfer and axial mixing has been revjewed. Experiments were performed using the system C1airsol-350-acetone-water and the effects of drop size, drop size-distribution and dispersed phase hold-up on the performance of the R.D.C. established. The results obtained for the two-phase system C1airso1-water have been compared with published correlations: since most of these correlations are based on data obtained from laboratory scale R.D.C.'s, a wide divergence was found. The hydrodynamics data from this study have therefore been correlated to predict the drop size and the dispersed phase hold-up and agreement has been obtained with the experimental data to within +8% for the drop size and +9% for the dispersed phase hold-up. The correlations obtained were modified to include terms involving column dimensions and the data have been correlated with the results obtained from this study together with published data; agreement was generally within +17% for drop size and within +14% for the dispersed phase hold-up. The experimental drop size distributions obtained were in excellent agreement with the upper limit log-normal distributions which should therefore be used in preference to other distribution functions. In the calculation of the overall experimental mass transfer coefficient the mean driving force was determined from the concentration profile along the column using Simpson's Rule and a novel method was developed to calculate the overall theoretical mass transfer coefficient Kca1, involving the drop size distribution diagram to determine the volume percentage of stagnant, circulating and oscillating drops in the sample population. Individual mass transfer coefficients were determined for the corresponding droplet state using different single drop mass transfer models. Kca1 was then calculated as the fractional sum of these individual coefficients and their proportions in the drop sample population. Very good agreement was found between the experimental and theoretical overall mass transfer coefficients. Drop sizes under mass transfer conditions were strongly dependant upon the direction of mass transfer. Drop Sizes in the absence of mass transfer were generally larger than those with solute transfer from the continuous to the dispersed phase, but smaller than those with solute transfer in the opposite direction at corresponding phase flowrates and rotor speed. Under similar operating conditions hold-up was also affected by mass transfer; it was higher when solute transfered from the continuous to the dispersed phase and lower when direction was reversed compared with non-mass transfer operation.

The drying of small drops of particulate slurries

The literature on the evaporation of drops of pure liquids, drops containing solids and droplet sprays has been critically reviewed. An experimental study was undertaken on the drying of suspended drops of pure water and aqueous sodium sulphate decahydrate with concentrations varying from 5 to 54. 1 wt. %. Individual drops were suspended from a glass filament balance in a 26 mm I.D. vertical wind tunnel, designed and constructed to supply hot de-humidified air, to simulate conditions encountered in commercial spray driers. A novel thin film thermocouple was developed to facilitate the simultaneous measurement of drop weight and core temperature. The heat conduction through the thermocouple was reduced because of its unique design; using essentially a single 50μ diameter nickel wire. For pure water drops, the Nusselt number was found to be a function of the Reynolds, Prandtl and Transfer numbers for a temperature range between 19 to 79°C.                  Nu = 2 + 0.19 (1/B)0.24 Re0.5 Pr0.33 Two distinct periods were observed during the drying of aqueous sodium sulphate decahydrate. The first period was characterised by the evaporation from a free liquid surface, whilst drying in the second period was controlled by the crust resistance. Fracturing of the crust occurred randomly but was more frequent at higher concentrations and temperatures. A model was proposed for the drying of slurry drops, based on a receding evaporation interface. The model was solved numerically for the variation of core temperature, drop weight and crust thickness as a function of time. Experimental results were in excellent agreement with the model predictions although at higher temperatures modifications to the model had to be made to accommodate the unusual behaviour of sodium sulphate slurries, i.e. the formation of hydrates.

Synthesis and expression of a gene encoding a 48-residue repeat in the Pseudomonas syringae ice nucleation protein

The aim of this work was to construct short analogues of the repetitive water-binding domain of the Pseudomonas syringae ice nucleation protein, InaZ. Structural analysis of these analogues might provide data pertaining to the protein-water contacts that underlie ice nucleation. An artificial gene coding for a 48-mer repeat sequence from InaZ was synthesized from four oligodeoxyribonucleotides and ligated into the expression vector, pGEX2T. The recombinant vector was cloned in Escherichia coli and a glutathione S-transferase fusion protein obtained. This fusion protein displayed a low level of ice-nucleating activity when tested by a droplet freezing assay. The fusion protein could be cleaved with thrombin, providing a means for future recovery of the 48-mer peptide in amounts suitable for structural analysis by nuclear magnetic resonance spectroscopy.

Evolution of non-uniformly seeded warm clouds in idealized turbulent conditions

We present a mean-field model of cloud evolution that describes droplet growth due to condensation and collisions and droplet loss due to fallout. The model accounts for the effects of cloud turbulence both in a large-scale turbulent mixing and in a microphysical enhancement of condensation and collisions. The model allows for an effective numerical simulation by a scheme that is conservative in water mass and keeps accurate count of the number of droplets. We first study the homogeneous situation and determine how the rain-initiation time depends on the concentration of cloud condensation nuclei (CCN) and turbulence level. We then consider clouds with an inhomogeneous concentration of CCN and evaluate how the rain initiation time and the effective optical depth vary in space and time. We argue that over-seeding even a part of a cloud by small hygroscopic nuclei, one can substantially delay the onset and increase the amount of precipitation.

Mechanisms of heat and mass transfer to and from single drops freely-suspended in an air stream

A specially-designed vertical wind tunnel was used to freely suspend individual liquid drops of 5 mm initial diameter to investigate drop dynamics, terminal velocity and heat and mass transfer rates. Droplets of distilled, de-ionised water, n-propanol, iso-butanol, monoethanolamine and heptane were studied over a temperature range of 50oC to 82oC. The effects of substances that may provide drop surface rigidity (e.g. surface active agents, binders and polymers) on mass transfer rates were investigated by doping distilled de-ionised water drops with sodium di-octyl sulfo-succinate surfactant. Mass transfer rates decreased with reduced drop oscillation as a result of surfactant addition, confirming the importance of droplet surface instability. Rigid naphthalene spheres and drops which formed a skin were also studied; the results confirmed the reduced transfer rates in the absence of drop fluidity. Following consideration of fundamental drop dynamics in air and experimental results from this study, a novel dimensionless group, the Oteng-Attakora, (OT), number was included in the mass transfer equation to account for droplet surface behaviour and for prediction of heat and mass transfer rates from single drops which exhibit surface instability at Re>=500. The OT number and the modified mass transfer equation are respectively: OT=(ava2/d).de1.5(d/) Sh = 2 + 0.02OT0.15Re0.88Sc0.33 Under all conditions drop terminal velocity increased linearly with the square root of drop diameter and the drag coefficient was 1. The data were correlated with a modified equation by Finlay as follows: CD=0.237.((Re/P0.13)1.55(1/We.P0.13) The relevance of the new model to practical evaporative spray processes is discussed.