Numerical investigation of cell encapsulation for multiplexing diagnostic assays using novel centrifugal microfluidic emulsification and separation platform

In the present paper, we report a novel centrifugal microfluidic platform for emulsification and separation. Our design enables encapsulation and incubation of multiple types of cells by droplets, which can be generated at controlled high rotation speed modifying the transition between dripping-to-jetting regimes. The droplets can be separated from continuous phase using facile bifurcated junction design. A three dimensional (3D) model was established to investigate the formation and sedimentation of droplets using the centrifugal microfluidic platform by computational fluid dynamics (CFD). The simulation results were compared to the reported experiments in terms of droplet shape and size to validate the accuracy of the model. The influence of the grid resolution was investigated and quantified. The physics associated with droplet formation and sedimentation is governed by the Bond number and Rossby number, respectively. Our investigation provides insight into the design criteria that can be used to establish centrifugal microfluidic platforms tailored to potential applications, such as multiplexing diagnostic assays, due to the unique capabilities of the device in handling multiple types of cells and biosamples with high throughput. This work can inspire new development of cell encapsulation and separation applications by centrifugal microfluidic technolog

DEVELOPMENT OF LONG-TERM STABLE MIXED SODIUM CASEINATE AND PEA PROTEIN ISOLATE-STABILIZED NANOEMULSIONS FOR THE DELIVERY OF CURCUMIN

Nanoemulsions (NEs) with extremely small droplet size (radius <100 nm) were found to possess characteristics that have many advantages over conventional emulsion systems. These nano-sized droplets were found to contribute to higher stability of the NEs and also found to improve the bioavailability of poorly water-soluble bioactive components. The overall aim of this thesis is to develop oil-in-water (O/W) NEs stabilized by a mixture of sodium caseinate (a dairy protein) and pea protein isolates (a pulse protein). The mixed protein stabilized NEs were utilized to encapsulate a bioactive compound, curcumin, where the goal is to investigate its stability, delivery and bioavailability through in vitro digestion studies. Various concentrations (2.5 – 10 wt%) of sodium caseinate (SC) were used as the sole emulsifier in the development of 5 wt% O/W NEs and their long-term storage stability for 6 months was investigated. The sodium caseinate stabilized NEs (SCEs) developed in this work displayed an average droplet diameter less than 200 nm, which remained unchanged for an experimental time fame of 6 months. However, all of them displayed rapid creaming, which increased with an increase in protein concentration, in accordance with previous studies. It was postulated that excess unabsorbed protein caused depletion flocculation leading to creaming of oil droplets, which was confirmed using confocal laser scanning microscopy. Calculation of depletion interaction energy showed an increase in attraction with protein concentration and decrease with a reduction in droplet size, making NEs more resistant to flocculation than conventional emulsions. Next, pea protein isolate (PPI), was utilized to partially replace SC and thereby PPIs efficacy in the formation and long-term stabilization of mixed protein NEs (MPEs) was investigated. Total aqueous phase-protein concentration of 5, 7.5 and 10 wt%, with SC and PPI in a 1:1 ratio, was used. As a control individual PPI-stabilized NEs (PPIE) were also prepared. PPI failed to produce stable flowable NEs displaying excessive droplet and protein aggregation. At higher concentrations of PPI (7.5 and 10 wt%), the emulsion transformed into viscoelastic gels. Interestingly, the mixed SC and PPI-stabilized NE did not display any creaming or aggregation and remained stable throughout the experimental timeframe of 6 months with average droplet diameter <200 nm. Results from interfacial protein composition (surface load) and SDS-PAGE indicated the presence of PPI at the interface along with SC confirming PPI’s ability to take part in droplet formation and stabilization. It was hypothesized that the mutual presence of SC and PPI during high-pressure homogenization led to interactions between the proteins, which was confirmed by FTIR spectroscopy, intrinsic fluorescence and surface hydrophobicity measurements. Interactions between the proteins not only prevented depletion flocculation effect of SC, but also interfered with PPI aggregation thereby preventing both the destabilizing mechanisms seen in individual protein-stabilized NEs. The mixed-protein stabilization could be a novel way to utilize plant proteins in the development of NEs. In the final part, the efficacy of the MPE for stability, delivery and bioavailability of an encapsulated bioactive compound, curcumin was investigated and compared to the SCE . It was seen that over 54% of encapsulated curcumin was degraded in the MPE, while only ~42% was degraded in the SCE over a period of 8 weeks. In vitro digestion studies indicated that the amount of bioavailable curcumin from SCE was slightly higher (although not statistically significant) compared to that from MPE, which was attributed to a thicker droplet interface in the mixed protein NE (due to presence of globular protein PPI), thereby making the droplet less susceptible towards protein hydrolysis by pepsin in the stomach. Overall, it was concluded that it is possible to develop mixed protein NEs utilizing PPI and SC, which displayed better stability when compared to the individual protein-stabilized NEs. The presented approach not only utilized pulse protein, PPI, in the development of NEs, but also showed good applicability in terms of encapsulating bioactive ingredients for prospective applications in food and pharmaceutical industries

Strategies for developing lentil protein stabilized canola oil in water nanoemulsions

The overall goal of this research is to utilize the lentil protein isolate (LPI), prepared with isoelectric precipitation by POS Bio-Sciences (Saskatoon, SK, Canada), in the development of canola oil-in-water nanoemulsions. The effect of LPI concentration and the effect of high-pressure treatment of LPI on the formation, stability and rheological behaviour of canola oil-in-water nanoemulsions was investigated. According to a previous study of coarse emulsions, LPI showed the best emulsifying properties at pH 3; therefore, all nanoemulsions were prepared at this pH. In the first study, nanoemulsions were prepared by adding 5 wt% canola oil to the LPI solutions at various concentrations (0.5 – 5 wt% LPI) in a citric acid buffer at pH 3 and homogenized at 20,000 psi pressure. The storage stability of the nanoemulsions was recorded for 28 days. All nanoemulsions showed bimodal droplet size distribution, where the smaller peak was attributed to the oil droplets while the larger peak was attributed to unadsorbed protein aggregates in the continuous phase which grew in the size with increasing LPI concentration. The protein aggregation was also confirmed with confocal microscopy. Concentration of 0.5 wt% LPI was not sufficient for long term stabilization of oil droplets therefore the nanoemulsion separated out over the 28 days. The best stability of the nanoemulsions was observed with 1, 1.5 and 2 wt% LPI, confirmed by a photocentrifuge, which evaluates oil droplet movement and hence emulsion stability under accelerated gravitational force. Nanoemulsions stabilized with 3 and 5 wt% LPI transformed to a thick gel, most likely due to a network formation between the oil droplets and free proteins in the continuous phase. The viscosity and the gel strength of nanoemulsions increased with increasing protein concentration because of increased aggregation of free proteins in the continuous phase of the nanoemulsions. In the second study, the effect of high-pressure homogenization of LPI on the formation and stability of the nanoemulsions were investigated. The most stable and flowable nanoemulsions at 1, 1.5 and 2 wt% LPI concentrations were chosen based on the previous results. Prior to nanoemulsion formation, LPI solutions (1 -2 wt% LPI) were homogenized at 5,000 and 15,000 psi pressure for six cycles. Nanoemulsions were then prepared by adding 5 wt% canola oil to 95 wt% pre-treated LPI solutions at pH 3 and homogenized at 20,000 psi pressure. High-pressure homogenization of LPI significantly improved long term stability of the nanoemulsions by decreasing the large protein particles into small ones, which was confirmed by particle size distribution, light microscopy and photocentrifuge dispersion analysis. Small particles improved migration of proteins to the oil-water interface and facilitated formation of oil droplets and resulted in a decrease in the average oil droplet size from ~ 250 nm to less than 200 nm. No significant difference was observed between 5,000 and 15,000 psi pressure indicating that 5,000 psi homogenization of LPI solution was sufficient to brake large protein particles into small ones. High-pressure homgenization of LPI solutions also decreased protein aggregation in the continuous phase of the nanoemulsions which was confirmed with the confocal microscopic imaging and this might be due to the lower surface hydrophobicity created by high-pressure homogenization of LPI. Results from the interfacial rheology indicated that weaker interfacial film was formed by the high-pressure homogenized LPI solutions compared to un-homogenized proteins. Storage stability of the nanoemulsions prepared with high-pressure homogenized LPI solutions was significantly improved compare to the nanoemulsions prepared without high-pressure treated LPI due to a smaller droplet size and less protein aggregation in the continuous phase. Lipid digestibility showed an increase for nanoemulsions prepared with high-pressure homogenized LPI solutions (1 wt%) due to a smaller droplet size and weaker interfacial film, however no significant difference was observed for 1.5 and 2 wt% LPI homogenized solutions. This might be due to a higher LPI concentration covering the oil droplet surface and preventing digestive enzymes to access the oil. Overall, high-pressure homogenization improved emulsification properties of LPI and shelf life and lipid digestibility of the prepared nanoemulsions thereby increasing the nutritional value of the product. Lentil protein-stabilized nanoemulsions containing low oil volume fractions have many applications in the development of beverage type products due to their increased stability, flowability and longer shelf life

A technology platform for in vitro transcription and translation of enzymes in micro compartments

Enzymes are crucial elements of all living cells. As biological catalysts they accelerate chemical reactions without being consumed in the process. The modern bioeconomy strives to identify new enzymes for improved usage in biotechnological applications such as the production of fine chemicals or pharmaceuticals. A promising source for the discovery of new enzymes are metagenomes. As there is no intermediary cultivation step for the extraction of the genetic material necessary, the genetic pool of a metagenome comprises genes of cultivable and non-cultivable microorganisms, which is beneficial for the detection of new enzymes. Here we introduce a new technology platform towards high efficient screening of whole metagenome libraries by combining in vitro compartmentation with a cell-free protein synthesis approach (Figure 1). A key element of this platform is a centrifugal microfluidic cartridge which encapsulates the metagenome library in up to 100’000 monodisperse droplets with a volume of 520 pl. The micro droplets are generated by centrifugal step emulsification (1) and are further transported into a standard reaction tube, decoupling the emulsification from the downstream processing. Based on substrate specificity, droplets with active enzymes are selected and a subsequent sequencing analysis allows the identification of the DNA sequence of these enzymes. The high number of generated micro droplets enables a high-throughput of large libraries and the high coverage increases the chance of finding new or rare enzymes. Compared to traditional approaches, the introduced all-in-one metagenome screening platform decreases screening time to a large extend by replacing heterologous expression with in vitro protein synthesis and massive screening. Further, the selection process minimizes the sequencing and annotation effort. Please click Additional Files below to see the full abstract

Development of nanogels from nanoemulsions and investigation of their rheology and stability

Nanoemulsions with extremely small droplet sizes (<100 nm) have shown several advantages over conventional emulsions. However, almost all nanoemulsions in usage are liquids that restrict their use in many soft materials. The aim of this thesis is to understand the formation and long-term stability of viscoelastic nanogels developed from liquid nanoemulsions. At first, gelation in 40 wt% canola oil-in-water nanoemulsions were investigated as a function of emulsifier type (anionic sodium dodecyl sulfate (SDS) or nonionic Tween 20) and concentration. Three different regimes of colloidal interactions were observed as a function of SDS concentration. 1) At low SDS concentration (0.5 – 2 times CMC) the counterion shell layer increased the effective volume fraction of the dispersed phase (eff) close to the random jamming, resulting in repulsive gelation. 2) At SDS concentration between 5 – 15 times CMC, micelle induced depletion attractions led to extensive droplet aggregation and gelation. 3) At very high SDS concentration, however, oscillatory structural forces (OSF) due to layered-structuring of excess micelles in the interdroplet regions led to loss of gelation. In repulsive gelation, reduction in droplet size coupled with the electrical double layer resulted in a linear increase of Gʹ. On the contrary, attractive nanoemulsions showed rapid increase in gel strength below a critical droplet radius, and was explained by transformation of OSF into depletion attraction. No gelation was seen in Tween 20 nanoemulsions, due to lack of repulsive interactions and weak depletion attraction. Next the influence of the dispersed phase volume fraction () on repulsive nanoemulsion gelation was investigated and the Gʹ values were modeled using empirical scaling law developed by Mason et al. (1995). It was found that an initial liquid regime transformed into glassy phase at a eff = g ~ 0.58, where droplets are entrapped in a cage of neighbouring droplets due to crowding. It was followed by jamming transition at a critical volume fraction (j), where droplet deformation led to large increase in elasticity. The model predicted j = 0.7, which is close to the predictions for repulsive polydispersed emulsions found in the literature. In the final phase long-term stability of the nanogels was evaluated until 90 days, during which the nanogels remained stable to creaming and coalescence. However, repulsive nanogels showed a significant decrease in Gʹ and the gels converted into flowable liquids over time. For attractive nanogels decrease in Gʹ was much less, although given enough time they would also transformed into weak gels. It was hypothesized that surface active compounds generated due to lipid oxidation altered interfacial charge cloud leading to loss of gel strength for repulsive nanogels. For attractive nanogels slippery bonds in the aggregates permitted rotational and translational diffusion of nanodroplets on the surface of each other leading to network compactness and a decrease in gel strength with time. Overall, it was concluded that it is possible to form nanogels from canola oil nanoemulsions using ionic emulsifiers. The gel strength and stability of the nanogels depends on emulsifier concentration, droplet size, and the chemical stability of the oil used. More investigation is needed in order to improve the long-term stability of the nanogels. The nanogels possess high potential for use in low-fat foods, pharmaceuticals, and cosmetic products

Evaluation of emulsification kinetics of oil in water

Tese de mestrado, Farmacotecnia Avançada, 2009, Universidade de Lisboa, Faculdade de FarmáciaEmulsions are the complex fluids and their stability depends, or is reflected by the size of the emulsion droplets which is considered to be critical. In the design and study of an emulsification process, knowledge of factors governing the particle size and size distribution of the droplets is an important consideration. The study of kinetics of emulsification involves various parameters and the droplet sizing is a significant one. In this work the effects of various experimental factors has been carried out to study the kinetics of emulsification of oil in water (e.g. agitation duration, agitation rate, surfactant concentration, oil concentration, ageing). Sizing of droplets was carried out by Phase Doppler Anemometry (PDA), Photo Correlation Spectroscopy (PCS), Laser Diffraction (LD) and Microscopy. The effect of these parameters during the emulsification process was studied and the reason for imparting significance on these parameters is for the fact that controlling these critical parameters can assist to obtain a desired final product as this paves the path for achieving the quality aspect of being Right at the first time which is a central issue in Quality by Design (QbD). The comparison of size distribution obtained by these sizing methods was studied. Results have shown that by increasing agitation duration and rate a decrease in droplet diameter was observed, surfactant concentration cause a decrease in droplet diameter and an increase in oil concentration increased the droplet diameter. Ageing has also shown an increase in droplet diameter. Regarding the techniques considered, PDA was limited by the imposed dilution on emulsions to allow measurements, PCS was used at the marginal upper limit range thus care had been taken to present accurate measurements, LD was sensitive to dilution of samples prior to measurements and Microscopy, a tedious technique difficult to use and when limited samples are used one may have non representative results. The possibility of using Phase Doppler Anemometry (PDA) as online monitoring equipment (as a Process Analytical Technology) was discussed where the emulsion droplets could be assessed online there by contributing to the real time emulsification control was discussed. The study of viscosity of these emulsions was done as this is one of the significant criteria for the quality control aspects. From the techniques used, PDA seems to be most adequate as online/inline equipment for monitoring the process of emulsification

The effect of interfacial compositions on the dispersed phase-induced gelation and controlled digestion of mono and bilayer nanoemulsions

This thesis examines the role of the interfacial thickness (δ) in controlling the gelation and digestion behaviour of oil-in-water emulsions (oil volume fraction, φ = 0.2 to 0.4)-stabilized by food-grade emulsifiers and polysaccharides. Importance was given to addressing the increase in effective volume fraction (φeff) of oil droplets beyond maximum random jamming (φMRJ) by reducing droplet size, removing excess emulsifier and changing the interfacial composition. In the first study, the gelation in 40 wt% canola oil-in-water nanoemulsions was investigated as a function of excess emulsifier Citrem (citric acid esters of mono and di-glycerides) removal from the aqueous phase. The removal of excess Citrem increased the viscosity, yield stress and storage moduli of nanoemulsions, more significantly at smaller droplet sizes. It was attributed to a change in inter-droplet interaction from non-DLVO oscillatory structural forces to DLVO dominated repulsive forces after removing excess Citrem. This also increased the δ and φeff beyond φMRJ, leading to a self-standing repulsively jammed nanoemulsion gel. Next, the droplet velocity and packing behaviour of Citrem-stabilized nanoemulsions were tracked using an analytical photo-centrifuge to predict their stability and shelf-life. The reduction of droplet size and removal of excess micelles improved the accelerated stability and shelf-life of the nanoemulsions. The droplets’ packing density (φp) was decreased under the applied RCF after removing excess micelles, which we related to strong repulsive forces between nanodroplets. To further increase the δ, a second layer of polysaccharide (chitosan and pectin) with different magnitude of charge was deposited on Citrem and whey protein isolate (WPI)-stabilized nanodroplets, respectively. Two different layer-by-layer (LbL) electrostatic deposition techniques, namely one-step versus two-step, were utilized for Citrem-chitosan and WPI-pectin systems, respectively. It was found that the rheology of bilayer emulsions was affected by the droplet size, presence and absence of excess emulsifier, polysaccharide concentration and charge, and the type of LbL method used. In the one-step LbL method, a liquid-like behaviour of Citrem-stabilized monolayer emulsions transformed into repulsive bilayer weak emulsions gel above a critical chitosan concentration, where electrostatic and steric repulsive forces had a significant contribution in elevating δ and φeff. However, the two-step LbL method and removal of excess emulsifier were more effective in creating well-distributed bilayer nanodroplets with increased interfacial thickness leading to an increase in gel strength compared to the monolayer emulsions at a lower φ. The deposition of the second layer also controlled the lipase action during in vitro digestion leading to lowering of lipid digestibility. Overall, the study showed that the random jamming amongst the nanodroplets could be induced by increasing δand φeff beyond φMRJ where emulsions behave like a viscoelastic gel. The fundamental knowledge developed from this research can be used to develop food-grade low-fat emulsion gels with controlled digestion

Development of stable liquid water-in-oil emulsions by modifying emulsifier-aqueous phase interactions

This research investigated the stabilization mechanism of liquid water-in-oil (W/O) emulsions by modifying emulsifiers, aqueous and continuous phase compositions, and interactions. At first, liquid W/O emulsions were developed to resist multiple thermal cycles for their application in droplet digital polymerase chain reaction (ddPCR), which significantly improved the detection limit of conventional PCR. Thermally stable coarse W/O emulsions as DNA microreactors were developed with polyglycerol polyricinoleate (PGPR) and sodium bis(2-ethylhexyl) sulfosuccinate (AOT) as emulsifiers dissolved in a mixture of light and heavy mineral oil, with a range of viscosities. Coarse emulsions, formed by vortex mixing, were subjected to PCR thermal-cycling, after which AOT-stabilized water droplets remained stable; however, PGPR-stabilized water droplets size significantly increased. Higher AOT molecular packing at the interface was proposed as the mechanism of thermal stability. Next, the (de)-stabilization mechanism of glycerol monooleate (GMO)-stabilized liquid W/O emulsions in mineral (MO) and canola oil (CO) was investigated. It was hypothesized that hydroxyl group donating agents in the aqueous phase would prevent GMO's desorption from the oil-water interface by forming stronger hydrogen bonding. W/O emulsions with 20% aqueous phase were formed by high-pressure homogenizer. Of the three agents, emulsions with low methoxyl pectin (LMP) showed the highest stability in both oils after 7-day storage compared to citric or ascorbic acid with or without sodium chloride. Water and GMO melting behaviour, determined by differential scanning calorimetry, and intermolecular interaction by Fourier transform infrared spectroscopy revealed stronger H-bonding between GMO and LMP, thereby improving emulsion stability. Finally, the viscoelastic behaviour of GMO-stabilized W/O emulsion was improved such that an elastic gel could be formed by increasing the water content (20 to 50 wt%) and incorporating specific ingredients in the continuous and dispersed phases for application in food-grade low-fat tablespreads. Fully hydrogenated soybean oil was incorporated in CO to create a fat crystal network in the continuous phase. Emulsions with LMP in the aqueous phase exhibited self-supported structure without phase-separation while control emulsions, without LMP, showed flow or water separation. All emulsions exhibited strong gel-like properties; however, control emulsions showed structure breakup after 30-day storage. Overall, the studies in liquid W/O emulsions were the basis to improve our understanding of the molecular assembly and interactions at the W-O interface, which subsequently supported the research to enhance emulsion elasticity