Nanoliter droplet array for microRNA detection based on enzymatic stem-loop probes ligation and SYBR Green real-time PCR

In this paper, a nanoliter droplet array based on enzymatic stem-loop probes ligation and SYBR Green real-time PCR for quantification of microRNA was developed. By employing T4 RNA ligase 2 instead of T4 DNA ligase, we designed simplified stem-loop probes to perform microRNA-templated DNA ligation and reduced the non-specific ligation of T4 DNA ligase. SYBR green I dye was employed instead of TaqMan probes in present miniaturized real-time PCR systems. Specifically, we optimized the dosage of SYBR Green I dye in nanoliter droplet and verified the performance of this system by detecting synthetic mir-122 with a 6 logs dynamic range (from 1.5 × 10⁵ to 1.5 × 10¹⁰ copies). Linear relationship of the standard curve (R² = 0.9997) and high PCR amplification efficiency (96.83%) were obtained under the optimized conditions. We detected the expression of mir-122 across five mouse tissues and the result was consistent with that TaqMan microRNA assay. We think this miniaturized real-time PCR platform reduced the detection cost considerably, thus showing the great potential to quantitative biology.     

Highly efficient capillary polymerase chain reaction using an oscillation droplet microreactor

The current work presents the development of a capillary-based oscillation droplet approach to maximize the potential of a continuous-flow polymerase chain reaction (PCR). Through the full utilization of interfacial chemistry, a water-in-oil (w/o) droplet was generated by allowing an oil–water plug to flow along a polytetrafluoroethylene (PTFE) capillary. The w/o droplet functioned as the reactor for oscillating-flow PCR to provide a stable reaction environment, accelerate reagent mixing, and eliminate surface adsorption. The capillary PCR approach proposed in the current research offers high amplification efficiency, fast reaction speed, and easy system control attributable to the oscillation droplet reactor. Experimental results show that the droplet-based micro-PCR assay requires lower reaction volume (2 μL) and shorter reaction time (12 min) compared with conventional PCR methods. Taking the amplification of the New Delhi metallo-beta-lactamase (NDM-1) gene as an example, the present work demonstrates that the oscillation droplet PCR assay is capable of achieving high efficiency up to 89.5% and a detection limit of 10 DNA copies. The miniature PCR protocol developed in the current work is fast, robust, and low-cost, thus exhibiting the potential for expansion into various practical applications.     

Experimental study of evaporation of sessile water droplet on PDMS surfaces

Evaporation of sessile water droplet on polydimethylsiloxane （PDMS） surfaces with three different curing ratios （5：1, 10：1, and 20：1） was experimentally investigated in this paper. We show that the constant contact radius （CCR） evaporation on surface with high curing ratio lasts longer than that with low curing ratio. We also measured Young＇s moduli of PDMS films by using atomic force microscopy （AFM） and simulated surface deformation of PDMS films induced by sessile water droplet. With increasing curing ratio of PDMS film, Young＇s modulus of PDMS film is getting lower, and then there will be larger surface deformation and more elastic stored energy. Since such energy acts as a barrier to keep the three-phase contact line pinned, thus it will result in longer CCR evaporation on PDMS surface with higher curing ratio.     

A collision algorithm for anisotropic disperse flows based on ellipsoidal parcel representations

In this study, a new approach to the prediction of collision probabilities is proposed, which is specifically derived for Lagrangian Monte Carlo simulations of highly anisotropic disperse flows. The collision probability formulation eliminates the influence of the collision mesh by introduction of a parcel diameter. Two non-parametric parcel diameter estimators are given, an isotropic parcel diameter estimator for homogeneous droplet clouds, and an anisotropic parcel diameter estimator for inhomogeneous droplet clouds of reduced dimensionality, which obtains an ellipsoidal parcel representation from a weighted principal component analysis of the parcel population. The new formulation effectively reduces the parameters for spatial resolution from two (mesh size, number of parcels) to one (number of parcels only). For validation, simple synthetic test cases are run that allow a comparison of the collision probability formulation to purely stochastic and purely deterministic schemes. Dependence on numerical parameters is tested exemplary with a highly anisotropic hollow-cone spray for gasoline direct injection.     

Electrochemical Detection of Droplets in Microfluidic Devices: Simultaneous Determination of Velocity,Size and Content

Microfluidic devices were designed to electrochemically detect in a two‐phase flow the velocity, size and content of aqueous droplets containing redox species. The principle of these determinations is based on the analysis of a unique chronoamperometric response recorded during the passage of a droplet over channel microelectrodes. Two configurations of electrochemical cell with different geometries were investigated both theoretically and experimentally. Velocity and size of droplets, as well as internal recirculating convection within droplets, were evaluated from chronoamperometric curves by specific transition times depending on the cell configuration. In addition, the droplet content was probed from the Faradaic current controlled by mass transport and by internal hydrodynamic regime. For droplet velocity and size, experimental data were systematically compared to optical measurements. All the results demonstrated the high performance of the electrochemical detection reached under these conditions. They successfully validate the concept of self‐consistent electrochemical detections of aqueous droplets within microchannels for the simultaneous determination of their velocity, size and content.     

Modelling and simulation of micro-well formation

Physico-chemical processes on the micro-scale require new modelling concepts because some effects become dominating that are negligible for macroscopic systems. This is illustrated by a new method for the production of micro-wells based on the placement of a small drop of toluene on a plate of polystyrene. After droplet evaporation, a micro-well is left. A mathematical model has been developed to understand the elementary processes of the micro-well formation. The model accounts for: (1) growth of the drop on the substrate, (2) evaporation process of the solvent, (3) dissolution of the substrate, (4) flow rate in the evaporating drop caused by the pinning effect, including the vertical velocity profile, and (5) increase in the concentration of dissolved material followed by precipitation. In the modelling and simulation process, it could be shown that the method of drop production also has a significant influence on the shape of the micro-wells.     

Spark-generated bubble collapse near or inside a circular aperture and the ensuing vortex ring and droplet formation

This paper aims to study the oscillation of a sparkgenerated submerged bubble located near or inside a circular aperture made in a flat plate using high-speed visualization technique. In the case of a bubble oscillating near an aperture the initial free surface of the water was set at the bottom surface of the plate. The effects of aperture size and bubblefree surface distance on the bubble behavior as well as on the ensuing droplet dynamics are investigated. It was found that the direction of the bubble reentrant jet was towards the aperture or away from it respectively when the normalized aperture size was smaller or greater than a certain critical value. In addition, a toroidal vortex ring was observed to form, which rotated inwards as it moved away from the aperture. It was also found that if the bubble was incepted at a distance sufficiently away from a supercritical size aperture a single droplet could be produced. In the case of a bub- ble initiated in the middle of a circular aperture submerged just beneath the water free surface, the bubble was found to take the shape of an ellipsoid during its expansion. Then a reentrant jet was initiated and pierced the bubble from its top side.     

Droplet coalescence in the shear flow of model emulsions

During the flow of an emulsion, droplets of the dispersed phase can deform, break up, coalesce or migrate to other regions within the flow field. Understanding these different processes is relevant to morphology development in immiscible polymer blends. Here, emulsions of castor oil in silicone oil were employed to study shear-induced coalescence alone; the conditions chosen were such that drop breakup and drop migration did not occur. A cone-and-plate device and tubes of varying length were used to examine the influence of the average shear rate, the time of shearing, concentration of the dispersed phase, and temperature on the average droplet size. It was found that the extent of “demixing” was not influenced by the spatially non-homogeneous nature of flow in a tube; results correlated very well with the average shear rate. On the other hand, coalescence was significant even when the concentration of the dispersed phase was as low as 0.5%, and it became more important as the concentration was increased. Other results were that the extent of coalescence could be promoted by lowering the shear rate. In quantitative terms, it was found that available coalescence theory gave the correct order of magnitude for the average steady-state droplet size as a function of the imposed shear rate, but the actual variation of drop size with shear rate was gentler than that predicted by theory. An unusual observation was that, under some circumstances, the droplets did not coalesce but simply stuck to each other and maintained their separate identity. Received: 25 March 1999/Accepted: 22 July 1999     

Absorption of thermal radiation in a semi-transparent spherical droplet: a simplified model

The boundary-value problem for calculation of differential absorption of thermal radiation is formulated based on the modified DP₀ approximation. The solution of this problem is supplemented by simple analytical approximations for the normalised absorbed radiation power. The latter is used together with the analytical approximation for the efficiency factor of absorption, suggested earlier. The resulting simplified model is applied to the specific problem of absorption of thermal radiation by a diesel fuel droplet. Two types of diesel fuel have been considered. It is pointed out that the radial distribution of absorbed thermal radiation power is non-monotonic. The power absorbed in the droplet core is shown to be rather large and almost homogeneous. Also, the absorbed power is large in the vicinity of the droplet surface, but is minimal in the intermediate region. It is pointed out that the variations of the refractive index of diesel fuel with wavelengths can smooth the predicted radial dependence of the thermal radiation power, absorbed in diesel fuel droplets.