Continuous-flow Mixing Research Articles

BLENDING RESPIRATORY GAS MIXTURES

The binary or ternary gas mixtures used in diving can be blended either by a successive and controlled injection of the component gases into containers of constant volume, following the pressure in the containers, or by a continuous flow mixing, with the simultaneous injection of the component gases to the desired proportion, using for this devices equipped with calibrated nozzles working in the critical (sonic) range, which ensure the delivery of gaseous components at constant mass flow rates.

Determination of micromixing times in commercially available continuous-flow mixers: evaluation of the incorporation and interaction by exchange with the mean model

Determination of micromixing times in commercially available continuous-flow mixers: evaluation of the incorporation and interaction by exchange with the mean model

Numerical study of the mixing of micron particles in a vane mixer

As the research and application of micron particles are intensified, higher requirements for continuous flow mixing of multiple micron particles have been put forward. This study is based on rotating blades in the flow field to generate a vortex through which various micron particles are mixed. The particle mixing device designed in this study has a simple structure and small size advantages. The particle mixing model for coupled flow field study is established by using the finite element analysis method to realize the study of multiple micron particle mixing. The effects of blade shape, blade size, blade rotation speed, and inlet particle entry interval on the mixing impact are analyzed in the study. In this study, the factors affecting the particle mixing effect were analyzed by counting the number of outlet particles and plotting the flow lines in the mixing chamber. Optimization of the mixer scheme based on the influencing factors obtained from the study resulted in a high mixing effect of the designed particle mixer, which can provide a significant reference value for the subsequent micron particle mixing scheme design.

A Salmonella Microfluidic Chip Combining Non-Contact Eddy Heater and 3D Fan-Shaped Mixer with Recombinase Aided Amplification.

Foodborne pathogenic bacteria have become a worldwide threat to human health, and rapid and sensitive bacterial detection methods are urgently needed. In this study, a facile microfluidic chip was developed and combined with recombinase-aided amplification (RAA) for rapid and sensitive detection of Salmonella typhimurium using a non-contact eddy heater for dynamic lysis of bacterial cells and a 3D-printed fan-shaped active mixer for continuous-flow mixing. First, the bacterial sample was injected into the chip to flow through the spiral channel coiling around an iron rod under an alternating electromagnetic field, resulting in the dynamic lysis of bacterial cells by this non-contact eddy heater to release their nucleic acids. After cooling to ~75 °C, these nucleic acids were continuous-flow mixed with magnetic silica beads using the fan-shaped mixer and captured in the separation chamber using a magnet. Finally, the captured nucleic acids were eluted by the eluent from the beads to flow into the detection chamber, followed by RAA detection of nucleic acids to determine the bacterial amount. Under the optimal conditions, this microfluidic chip was able to quantitatively detect Salmonella typhimurium from 1.1 × 102 to 1.1 × 105 CFU/mL in 40 min with a detection limit of 89 CFU/mL and might be prospective to offer a simple, low-cost, fast and specific bacterial detection technique for ensuring food safety.

Advances in Mixer Design and Detection Methods for Kinetics Studies of Macromolecular Folding and Binding on the Microsecond Time Scale.

Many important biological processes such as protein folding and ligand binding are too fast to be fully resolved using conventional stopped-flow techniques. Although advances in mixer design and detection methods have provided access to the microsecond time regime, there is room for improvement in terms of temporal resolution and sensitivity. To address this need, we developed a continuous-flow mixing instrument with a dead time of 12 to 27 µs (depending on solution viscosity) and enhanced sensitivity, sufficient for monitoring tryptophan or tyrosine fluorescence changes at fluorophore concentrations as low as 1 µM. Relying on commercially available laser microfabrication services, we obtained an integrated mixer/flow-cell assembly on a quartz chip, based on a cross-channel configuration with channel dimensions and geometry designed to minimize backpressure. By gradually increasing the width of the observation channel downstream from the mixing region, we are able to monitor a reaction progress time window ranging from ~10 µs out to ~3 ms. By combining a solid-state UV laser with a Galvano-mirror scanning strategy, we achieved highly efficient and uniform fluorescence excitation along the flow channel. Examples of applications, including refolding of acid-denatured cytochrome c triggered by a pH jump and binding of a peptide ligand to a PDZ domain, demonstrate the capability of the technique to resolve fluorescence changes down to the 10 µs time regime on modest amounts of reagents.

Microsecond Dynamics During the Binding-induced Folding of an Intrinsically Disordered Protein

Tau is an intrinsically disordered protein implicated in many neurodegenerative diseases. The repeat domain fragment of tau, tau-K18, is known to undergo a disorder to order transition in the presence of lipid micelles and vesicles, in which helices form in each of the repeat domains. Here, the mechanism of helical structure formation, induced by a phospholipid mimetic, sodium dodecyl sulfate (SDS) at sub-micellar concentrations, has been studied using multiple biophysical probes. A study of the conformational dynamics of the disordered state, using photoinduced electron transfer coupled to fluorescence correlation spectroscopy (PET-FCS) has indicated the presence of an intermediate state, I, in equilibrium with the unfolded state, U. The cooperative binding of the ligand (L), SDS, to I has been shown to induce the formation of a compact, helical intermediate (IL5) within the dead time (∼37 µs) of a continuous flow mixer. Quantitative analysis of the PET-FCS data and the ensemble microsecond kinetic data, suggests that the mechanism of induction of helical structure can be described by a U ↔ I ↔ IL5 ↔ FL5 mechanism, in which the final helical state, FL5, forms from IL5 with a time constant of 50–200 µs. Finally, it has been shown that the helical conformation is an aggregation-competent state that can directly form amyloid fibrils.

Determination of the yield, mass and structure of silver patches on colloidal silica using multiwavelength analytical ultracentrifugation

Anisotropic nanoparticles offer considerable promise for applications but also present significant challenges in terms of their characterization. Recent developments in the electroless deposition of silver patches directly onto colloidal silica particles have opened up a simple and scalable synthesis method for patchy particles with tunable optical properties. Due to the reliance on patch nucleation and growth, however, the resulting coatings are distributed in coverage and thickness and some core particles remain uncoated. To support process optimization, new methods are required to rapidly determine patch yield, thickness and coverage. Here we present a novel approach based on multiwavelength analytical ultracentrifugation (MWL-AUC) which permits simultaneous hydrodynamic and spectroscopic characterization. The patchy particle colloids are produced in a continuous flow mixing process that makes use of a KM-type micromixer. By varying the process flow rate or metal precursor concentration we show how the silver to silica mass ratio distribution derived from the AUC-measured sedimentation coefficient distribution can be influenced. Moreover, through reasoned assumptions we arrive at an estimation of the patch yield that is close to that determined by arduous analysis of scanning electron microscopy (SEM) images. Finally, combining MWL-AUC, electrodynamic simulations and SEM image analysis we establish a procedure to estimate the patch thickness and coverage.

Improving the efficiency of the process of continuous flow mixing of bulk components

Modern industrial and agricultural processing almost always implies mixing loose material on a variety of equipment. At present, there are known mixers of various designs, principles, and techniques to implement the technological process. One of the existing mixing techniques is a continuous flow method that has significant advantages – reducing energy intensity while improving the quality of the process of the mixture continuous preparation and distribution. However, the continuous-flow technique of mixing loose materials has been paid little attention to. This prevents the application of well-known analytical models of the process of moving loose components to substantiate the structural and technological parameters for the working bodies of a continuous flow mixer. The result of the analytical study of the continuous-flow mixing technique is the constructed system of differential equations of the movement of a bulk material’s components in the airflow under the influence of the working bodies’ surfaces of the designed mixer. The reported system of differential equations underlies the physical-mathematical apparatus for the numerical modeling of the specified process employing the software package StarCCM+ (USA). The result of the numerical modeling is the established dependences of the dynamics of change in the concentration of components in the mixture and the homogeneity of the mixture in the zones of a continuous flow mixer depending on the study factors (the frequency of rotations, the angle of attack of the blade mixer, the performance of feeding the first and second components). The optimal structural and technological parameters for a continuous flow mixer involved in the process of mixing a two-component feed mixture (stem and concentrated feed) have been determined, at which the uniformity of the resulting forage mixture is maximal

A New Modeling Approach of Mixing Quality and Axial Hold-up Distribution in Agitated Liquid-Liquid Flow Mixers for Enabling Process Intensification

Liquid-liquid mixing, extraction, reaction, separation, and beneficiation have found a wide range of industrial applications. New theoretical based models have been developed and validated to enable process intensification by properly predicting the mixing quality and the local dispersed phase hold-up distributions in the axial direction of a continuous liquid-liquid flow mixer. The models relate the mixing index and axial hold-up distributions to a Peclet Number, the average dispersed phase hold-up, the input flow rates and the physical properties of the dispersion. The developed models offer a new understanding of the nature of the mixing process in continuous mixers with two liquid-liquid immiscible phases in flow. Experimental work was also performed to investigate the mixing index and axial dispersed phase hold-up distribution using Laser Transmission Technique (LTT) in a mixer setup. The experimental results were used in validating the models. The experimental and the predicted data agreed well for a wide range of impeller speeds/flow ratios.

Networks of electrostatic and hydrophobic interactions modulate the complex folding free energy surface of a designed βα protein

The successful de novo design of proteins can provide insights into the physical chemical basis of stability, the role of evolution in constraining amino acid sequences, and the production of customizable platforms for engineering applications. Previous guanidine hydrochloride (GdnHCl; an ionic denaturant) experiments of a designed, naturally occurring βα fold, Di-III_14, revealed a cooperative, two-state unfolding transition and a modest stability. Continuous-flow mixing experiments in our laboratory revealed a simple two-state reaction in the microsecond to millisecond time range and consistent with the thermodynamic results. In striking contrast, the protein remains folded up to 9.25 M in urea, a neutral denaturant, and hydrogen exchange (HDX) NMR analysis in water revealed the presence of numerous high-energy states that interconvert on a time scale greater than seconds. The complex protection pattern for HDX corresponds closely with a pair of electrostatic networks on the surface and an extensive network of hydrophobic side chains in the interior of the protein. Mutational analysis showed that electrostatic and hydrophobic networks contribute to the resistance to urea denaturation for the WT protein; remarkably, single charge reversals on the protein surface restore the expected urea sensitivity. The roughness of the energy surface reflects the densely packed hydrophobic core; the removal of only two methyl groups eliminates the high-energy states and creates a smooth surface. The design of a very stable βα fold containing electrostatic and hydrophobic networks has created a complex energy surface rarely observed in natural proteins.

Polymeric microfluidic continuous flow mixer combined with hyperspectral FT-IR imaging for studying rapid biomolecular events.

Early reaction intermediates in protein folding, such as those resulting in β-amyloid formation due to transient misfolding, emerge within a few hundred microseconds. Here, we report a method to obtain sub-millisecond temporal resolution and molecular structural information of protein (mis-)folding events by using a microfluidic continuous-flow mixer (MCFM) in combination with Fourier transform infrared (FT-IR) imaging. The MCFMs are made out of cyclic olefin copolymer (COC) films, because this approach allows for rapid prototyping of different mixer designs. Furthermore, COC offers high IR transparency between 1500 and 2500 cm-1, thus maximizing the signal to noise ratio of the IR data obtained from a sample of interest. By combining narrow and wide channel widths in MCFM design, the platform provides fast mixing (460 μs) to induce protein (mis-)folding, and it maximizes the residence time in the observing area, so a wide range of reaction timescales can be captured in a single image. We validated the platform for its ability to induce and observe sub-millisecond processes by studying two systems: (i) the mixing of H2O and D2O and (ii) the mixing induced deprotonation of carboxylic acid. First, we observed excellent agreement between simulated and experimental data of the on-chip mixing of H2O and D2O, which verifies the distance-reaction time relationships based on simulation. Second, deprotonation of carboxylic acid by on-chip mixing with sodium hydroxide solution validates the ability of the platform to induce rapid pH jump that is needed for some biomolecular reactions. Finally, we studied the methanol-induced partial-unfolding of ubiquitin to show that our platform can be used to study biomolecular events 'on-pathway' using FT-IR imaging. We successfully extracted kinetic and structural details of the conformational changes along the channel. Our results are in agreement with prior studies that required more elaborate stopped flow approaches to acquire data for different time points. In summary, the reported method uses an easy-to-fabricate microfluidic mixer platform integrated with hyperspectral FT-IR imaging for rapid acquisition of structural details and kinetic parameters of biomolecular reactions. This approach does not need stopped flow or molecular imaging probes, as required respectively for alternative FT-IR spectroscopy and fluorescence approaches.

Refolding kinetics of cytochrome c studied with microsecond timescale continuous-flow UV–vis spectroscopy and rapid freeze-quench EPR

The study of the structure, function, folding and conformational transitions of cytochrome c is of great interest because this protein plays an important role in biological electron transport and apoptosis. The different native and non-native conformations have been studied extensively under equilibrium conditions at different pH values, however, kinetic studies are rare because they require technically challenging rapid mixing and spectroscopic monitoring techniques. Here we present the refolding kinetics of acid denatured cytochrome c using the pH jump technique from pH 2 to pH 4.7 in combination with a new ultrafast continuous flow mixing device that allows time resolved measurements to the microsecond time scale. Our results show that the initial refolding of denatured oxidized cytochrome c occurs very rapidly with a time constant τ = 10 μs, and is followed by discrete refolding steps with time constants of 56 and 208 μs. Electron paramagnetic resonance analysis of the different intermediates, obtained by microsecond freeze hyper quenching showed that the first two intermediates are predominantly high spin, and the third intermediate is the low spin species with complete His/Met coordination. The initial rapid phase is characterized by the formation of high spin species distinct from the completely unfolded state. We interpret this as the formation of a five coordinate species with His18 as the axial ligand or six coordinate with water and His18 as the axial ligands.

Continuous Flow Type Gas Blending Facility Used for Autonomous and System Diving

The present paper, deals with a new gas blending method, in order to achieve a continuous flow mixing, and describes the associated facility for manufacturing of breathable mixtures used in the professional diving field. The facility is an original equipment conceived by the authors, specially designed to achieve the blending of two or three gases in order to form binary or ternary breathable gas mixtures used for activities which assume hyperbaric processes. The working principle of the equipment is based exclusively on gas dynamics phenomena which ensure the injection of the pure gases at a constant mass flow rate, independent of the pressure value at the consumer. As a particularity, the system facilitates the blending of any type of gases irrespective of the ratios of the compounds that forms the mixture. In order to understand both the proposed blending method and the manner in which the facility operates, as a first subject the paper describes the technological scheme of the blending facility. Starting this point on the gas mixture preparation procedure is described together with the associated computing relationships. The last subject is related to the designing process of the main parts of the facility. At the end of the paper, some general conclusions regarding the facility conceived for gas mixture manufacturing are presented, and how it relates to facilities from the same category.

Application of continuous mixing technology in ethanol precipitation process of Salvia miltiorrhiza by using micromixer

Continuous pharmaceutical manufacturing is one of the development directions in international pharmaceutical technology. In this study, a continuous mixing technology of ethanol and concentrated extract in the ethanol precipitation of Salvia miltiorrhiza was realized by using a membrane dispersion method. The effects of ethanol flowrate, concentrated extract flowrate, and flowrate ratio on ethanol precipitation results were investigated. With the increase of the flowrates of ethanol and concentrated extract, retention rate of active phenolic acids components was increased, and the total solid removal rate was decreased. The purity of active components in supernatants was mainly affected by the ratio of ethanol flowrate and concentrated extract flowrate. The mixing efficiency of adding ethanol under continuous flow mixing mode in this study was comparable to that of industrial ethanol precipitation. Continuous adding ethanol by using a membrane dispersion mixer is a promising technology with many advantages such as easy enlargement, large production per unit volume, and easy control.

Controllable Acoustic Mixing of Fluids in Microchannels for the Fabrication of Therapeutic Nanoparticles.

Fifteen years ago, surface acoustic waves (SAW) were found to be able to drive fluids and numerous applications in microfluidics have been developed since. Here, we review the progress made and report on new approaches in setting-up microfluidic, continuous flow acoustic mixing. In a microchannel, chaotic advection is achieved by generation of a SAW driven fluid jet perpendicular to the mean flow direction. Using a high speed video camera and particle image velocimetry, we measure the flow velocities and show that mixing is achieved in a particularly controllable and fast way. The mixing quality is determined as a function of system parameters: SAW power, volume flux and fluid viscosity. Exploring the parameter space of mixing provides a practical guide for acoustic mixing in microchannels and allows for adopting conditions to different solvents, as e.g., required for the generation of nanoscale particles from alcoholic phases. We exemplarily demonstrate the potential of SAW based continuous flow mixing for the production of therapeutic nucleic acid nanoparticles assembled from polymer and lipid solutions.

Mass spectrometric real-time monitoring of an enzymatic phosphorylation assay using internal standards and data-handling freeware.

Continuous-flow reaction detection systems (monitoring enzymatic reactions with mass spectrometry (MS)) lack quantitative values so far. Therefore, two independent internal standards (IS) are implemented in a way that the online system stability can be observed, quantitative conversion values for substrate and product can be obtained and they can be used as mass calibration standards for high MS accuracy. An application previously developed for the MS detection of peptide phosphorylation by cAMP-dependent protein kinase A (PKA) (De Boer et al., Anal. Bioanal. Chem. 2005, 381, 647-655) was transferred to a continuous-flow reaction detection system. This enzymatic reaction, involving enzyme activation as well as the transfer of a phosphate group from ATP to a peptide substrate, was used to prove the compatibility of a quantitative enzymatic assay in a continuous-flow real-time system (connected to MS). Moreover (using internal standards), the critical parameter reaction temperature (including solution density variations depending on temperature) was studied in the continuous-flow mixing system. Furthermore, two substrates (malantide and kemptide), two enzyme types (catalytic subunit of PKA and complete PKA) and one inhibitor were tested to determine system robustness and long-term availability. Even spraying solutions that contained significant amount of MS contaminants (e.g. the polluted catalytic subunit) resulted in quantifiable MS signal intensities. Subsequent recalculations using the internal standards led to results representing the power of this application. The presented methodology and the data evaluation with available Achroma freeware enable the direct coupling of biochemical assays with quantitative MS detection. Monitoring changes such as temperature, reaction time, inhibition, or compound concentrations can be observed quantitatively and thus enzymatic activity can be calculated.

Sandwich-format 3D printed microfluidic mixers: a flexible platform for multi-probe analysis

We report on a microfluidic mixer fabrication platform that increases the versatility and flexibility of mixers for biomolecular applications. A sandwich-format design allows the application of multiple spectroscopic probes to the same mixer. A polymer spacer is ‘sandwiched’ between two transparent windows, creating a closed microfluidic system. The channels of the mixer are defined by regions in the polymer spacer that lack material and therefore the polymer need not be transparent in the spectral region of interest. Suitable window materials such as CaF2 make the device accessible to a wide range of optical probe wavelengths, from the deep UV to the mid-IR. In this study, we use a commercially available 3D printer to print the polymer spacers to apply three different channel designs into the passive, continuous-flow mixer, and integrated them with three different spectroscopic probes. All three spectroscopic probes are applicable to each mixer without further changes. The sandwich-format mixer coupled with cost-effective 3D printed fabrication techniques could increase the applicability and accessibility of microfluidic mixing to intricate kinetic schemes and monitoring chemical synthesis in cases where only one probe technique proves insufficient.

Enzymatic Assays Coupled with Mass Spectrometry with or without Embedded Liquid Chromatography.

This article reviews monitoring strategies for enzymatic assays coupled with mass spectrometric detection. This coupling has already been shown to be helpful in providing versatile and detailed knowledge about enzyme kinetics. Various available publications address two general approaches. 1) The continuous-flow setup allows real-time determination of substrate degradation. Simultaneously, resulting product or potential intermediates can be detected. 2) The online coupled continuous-flow mixing assay allows the direct coupling of an enzymatic assay to chromatographic separation of complex mixtures. The latest efforts in improving the methodology have been made with regard to miniaturization. This is especially advantageous with regard to reducing costly consumption of chemicals. Finally, these developments are applicable for diverse bioanalytical purposes in the realms of pharmaceutical, biotechnological, food, and environmental research.

Effect of rheological parameters on non-ideal flows in the continuous-flow mixing of biopolymer solutions

A novel study on exploring the effect of the rheological parameters of the yield-pseudoplastic fluids on the non-ideal flows in a continuous-flow mixer was performed. The rheological behavior of the xanthan gum solution, a yield-pseudoplastic fluid, was modeled by the Herschel–Bulkley model. In fact, varying the xanthan gum concentration changes the values of all rheological parameters (i.e. consistency index (K), power law index (n) and fluid yield stress (τy)) simultaneously. Thus, merely studying the effect of the xanthan gum concentration on the flow nonideality represents the combined effect of all Herschel–Bulkley model parameters. The core objective of this research was to investigate the effects of K (3–33Pasn), n (0.11–0.99), τy (1.7–20.6Pa), xanthan gum mass concentration (0.5–1.5w/v%), and feed flow rate (Q) (03.20–14.17Lmin−1) on the percentage of channeling (f) and fully mixed volume (Vfully mixed/Vtotal) in the continuous-flow reactor through computational fluid dynamics (CFD). The validated CFD model predicted that the percentage of the parameter f increased and Vfully mixed/Vtotal decreased as n was increased from 0.11 to 0.99. This result revealed that the mixing was improved when the extent of the shear thinning was increased. Moreover, the mixing effectiveness of the continuous-flow mixer was enhanced by decreasing the consistency index, decreasing the fluid yield stress, enhancing the residence time of the fluid in the tank (TR), and the reducing the solution mass concentration.

An improved rapid mixing device for time-resolved electrospray mass spectrometry measurements.

Time series data can provide valuable insight into the complexity of biological reactions. Such information can be obtained by mass-spectrometry-based approaches that measure pre-steady-state kinetics. These methods are based on a mixing device that rapidly mixes the reactants prior to the on-line mass measurement of the transient intermediate steps. Here, we describe an improved continuous-flow mixing apparatus for real-time electrospray mass spectrometry measurements. Our setup was designed to minimize metal–solution interfaces and provide a sheath flow of nitrogen gas for generating stable and continuous spray that consequently enhances the signal-to-noise ratio. Moreover, the device was planned to enable easy mounting onto a mass spectrometer replacing the commercial electrospray ionization source. We demonstrate the performance of our apparatus by monitoring the unfolding reaction of cytochrome C, yielding improved signal-to-noise ratio and reduced experimental repeat errors.