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Free-flow electrophoresis (FFE) has the ability to continuously separate charged solutes from complex biological mixtures. Recently, a free-flow counterflow gradient focusing mechanism has been introduced to FFE, and it offers the potential for improved resolution and versatility. However, further investigation is needed to understand the solute dispersion at the focal position. Therefore, the goal of this work is to model the impact of electroosmotic flow, which is found to produce a pressure-driven backflow to maintain the fixed counterflow inputs. Like the counterflow, this backflow has a parabolic velocity profile that must be considered when predicting the concentration distribution of a given solute. After the model is established, preliminary experimental results are presented for a qualitative comparison. Results demonstrate a reasonable agreement at low applied voltages and provide a strong framework for future experimental validation., (© 2022 Wiley-VCH GmbH.)

Free-flow electrophoresis (FFE) enables the continuous separation and collection of charged solutes, and as a result, it has drawn interest as both a preparative and an analytical tool for biological applications. Recently, a free-flow counterflow gradient focusing (FF-CGF) mechanism has been proposed with the goal of improving the resolution and versatility of FFE. To realize this potential, the factors that influence solute dispersion deserve further attention, including the gradient strength and the parabolic profile of the counterflow. Therefore, the goal of this work is to develop a theoretical model to study the interplay between these factors and molecular diffusion. Overall, an asymmetric solute distribution emerges for a wide range of parameters, and this behavior can be characterized with an exponentially modified Gaussian function. Results show that FF-CGF can achieve high-resolution separations, with the potential for high-throughput protein purification. Moreover, this work provides a practical guide for optimizing experimental conditions, as well as a strong framework for understanding and developing FF-CGF further., (© 2021 Wiley-VCH GmbH.)

With its ability to continuously separate and collect charged analytes, free-flow electrophoresis (FFE) has become a useful tool for the purification and real-time analysis of biological mixtures. This work presents a new free-flow counterflow gradient focusing (FF-CGF) mechanism that uses a novel velocity gradient to counterbalance electrophoretic migration. This counterflow gradient is created by simply introducing fluid flow through the sidewalls of the FFE chamber. The theoretical foundation and device design for FF-CGF are provided in this work, followed by implementation and validation, with a detailed discussion on future opportunities and challenges. Initial results show promise, with the potential to improve FFE resolution and offer versatility. Compared to existing focusing techniques, such as free-flow isotachophoresis and isoelectric focusing, no complex buffer compositions are required.

Deposition of colloidal- and nano-scale particles on surfaces is critical to numerous natural and engineered environmental, health, and industrial applications ranging from drinking water treatment to semi-conductor manufacturing. Nano-scale surface roughness-induced hydrodynamic impacts on particle deposition were evaluated in the absence of an energy barrier to deposition in a parallel plate system. A non-linear, non-monotonic relationship between deposition surface roughness and particle deposition flux was observed and a critical roughness size associated with minimum deposition flux or "sag effect" was identified. This effect was more significant for nanoparticles (<1 μm) than for colloids and was numerically simulated using a Convective-Diffusion model and experimentally validated. Inclusion of flow field and hydrodynamic retardation effects explained particle deposition profiles better than when only the Derjaguin-Landau-Verwey-Overbeek (DLVO) force was considered. This work provides 1) a first comprehensive framework for describing the hydrodynamic impacts of nano-scale surface roughness on particle deposition by unifying hydrodynamic forces (using the most current approaches for describing flow field profiles and hydrodynamic retardation effects) with appropriately modified expressions for DLVO interaction energies, and gravity forces in one model and 2) a foundation for further describing the impacts of more complicated scales of deposition surface roughness on particle deposition.

This study reports a new method for establishing an open tubular IPG in a microchip coupled with a whole column image detection (WCID) system for protein separation applications. This method allows a wider range of immobilized pH (2.6-9.5) to be established in a PDMS/quartz channel by controlling the diffusion of acidic and basic polymer solutions into the channel through well-designed channel dimensions. The developed pH gradient was experimentally validated by performing the separation of a mixture of standard pI markers. It was further validated by the separation of the hemoglobin control AFSC sample. This method is advantageous over existing IPG methods because it has a wider range of pH and maintains the open tubular feature that matches the UV WCID to improve the sensitivity., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Counter-flow gradient electrofocusing allows the simultaneous concentration and separation of analytes by generating a gradient in the total velocity of each analyte that is the sum of its electrophoretic velocity and the bulk counter-flow velocity. In the scanning format, the bulk counter-flow velocity is varying with time so that a number of analytes with large differences in electrophoretic mobility can be sequentially focused and passed by a single detection point. Studies have shown that nonlinear (such as a bilinear) velocity gradients along the separation channel can improve both peak capacity and separation resolution simultaneously, which cannot be realized by using a single linear gradient. Developing an effective separation system based on the scanning counter-flow nonlinear gradient electrofocusing technique usually requires extensive experimental and numerical efforts, which can be reduced significantly with the help of analytical models for design optimization and guiding experimental studies. Therefore, this study focuses on developing an analytical model to evaluate the separation performance of scanning counter-flow bilinear gradient electrofocusing methods. In particular, this model allows a bilinear gradient and a scanning rate to be optimized for the desired separation performance. The results based on this model indicate that any bilinear gradient provides a higher separation resolution (up to 100%) compared to the linear case. This model is validated by numerical studies., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Droplet-based microfluidics is an emerging high-throughput screening technology finding applications in a variety of areas such as life science research, drug discovery and material synthesis. In this paper we present a cost-effective, scalable microwave system that can be integrated with microfluidic devices enabling remote, simultaneous sensing and heating of individual nanoliter-sized droplets generated in microchannels. The key component of this microwave system is an electrically small resonator that is able to distinguish between materials with different electrical properties (i.e. permittivity, conductivity). The change in these properties causes a shift in the operating frequency of the resonator, which can be used for sensing purposes. Alternatively, if microwave power is delivered to the sensing region at the frequency associated with a particular material (i.e. droplet), then only this material receives the power while passing the resonator leaving the surrounding materials (i.e. carrier fluid and chip material) unaffected. Therefore this method allows sensing and heating of individual droplets to be inherently synchronized, eliminating the need for external triggers. We confirmed the performance of the sensor by applying it to differentiate between various dairy fluids, identify salt solutions and detect water droplets with different glycerol concentrations. We experimentally verified that this system can increase the droplet temperature from room temperature by 42 °C within 5.62 ms with an input power of 27 dBm. Finally we employed this system to thermally initiate the formation of hydrogel particles out of the droplets that are being heated by this system.

Micellar affinity gradient focusing (MAGF) is a microfluidic counterflow gradient focusing technique that combines the favorable features of MEKC and temperature gradient focusing. MAGF separates analytes on the basis of a combination of electrophoretic mobility and partitioning with the micellar phase. A temperature gradient is produced along the separation channel containing an analyte/micellar system to create a gradient in interaction strength (retention factor) between the analytes and micelles. Combined with a bulk counterflow, species concentrate at a unique point where their total velocity sums to zero. MAGF can be used in scanning mode by varying the bulk flow so that a large number of analytes can be sequentially focused and passed by a single detection point. In this work, we develop a bilinear temperature gradient along the separation channel that improves separation performance over the conventional linear designs. The temperature profile along the channel consists of a very sharp gradient used to preconcentrate the sample followed by a shallow gradient that increases resolution. We fabricated a hybrid PDMS/glass microfluidic chip with integrated micro heaters that generate the bilinear profile. Performance is characterized by separating several different samples including fluorescent dyes using SDS surfactant and pI markers using both SDS and poly-SUS surfactants as the micellar phase. The new design shows a nearly two times improvement in peak capacity and resolution in comparison to the standard linear temperature gradient., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This study extends our previous work on droplet generation in microfluidic T-junction generators to include dynamic interfacial tension effects created by the presence of surfactants. In Paper I [T. Glawdel, C. Elbuken, and C. L. Ren, Phys. Rev. E 85, 016322 (2012)], we presented experimental findings regarding the formation process in the squeezing-to-transition regime, and in Paper II [T. Glawdel, C. Elbuken, and C. L. Ren, Phys. Rev. E 85, 016323 (2012)] we developed a theoretical model that describes the performance of T-junction generators without surfactants. Here we study dynamic interfacial tension effects for two surfactants, one with a small molecular weight that adsorbs quickly, and the other with a large molecular weight that adsorbs slowly. Using the force balance developed in Paper II we extract the dynamic interfacial tension from high speed videos obtained during experiments. We then develop a theoretical model to predict the dynamic interfacial tension in microfluidic T-junction generators as a function of the surfactant properties, flow conditions, and generator design. This model is then incorporated into the overall model for generator performance to effectively predict the size of droplets produced when surfactants are present.

Temperature gradient focusing (TGF) is a counterflow gradient focusing technique, which utilizes a temperature gradient across a microchannel or capillary to separate analytes. With an appropriate buffer, the temperature gradient creates a gradient in both the electric field and electrophoretic velocity. Combined with a bulk counter flow, ionic species concentrate at a unique point where the total velocity sums to zero and separate from each other. Scanning TGF uses varying bulk flow so that a large number of analytes that have large differences in electrophoretic mobility can be sequentially focused and passed by a single detection point. Up to now, scanning TGF examples have been performed using a linear temperature gradient which has limitations in improving peak capacity and resolution at the same time. In this work, we develop a bilinear temperature gradient along the separation channel that improves both peak capacity and separation resolution simultaneously. The temperature profile along the channel consists of a very sharp gradient used to preconcentrate the sample followed by a shallow gradient that increases separation resolution. A specialized design is developed for the heaters to achieve the bilinear profile using both analytical and numerical modeling. The heaters are integrated onto a hybrid PDMS/glass chip fabricated using conventional sputtering and soft-lithography techniques. Separation performance is characterized by separating several different dyes and amino acids that have close electrophoretic mobilities. Experiments show a dramatic improvement in peak capacity and resolution in comparison to the standard linear temperature gradient.

This is the second part of a two-part study on the generation of droplets at a microfluidic T-junction operating in the transition regime. In the preceding paper [Phys. Rev. E 85, 016322 (2012)], we presented our experimental observations of droplet formation and decomposed the process into three sequential stages defined as the lag, filling, and necking stages. Here we develop a model that describes the performance of microfluidic T-junction generators working in the squeezing to transition regimes where confinement of the droplet dominates the formation process. The model incorporates a detailed geometric description of the drop shape during the formation process combined with a force balance and necking criteria to define the droplet size, production rate, and spacing. The model inherently captures the influence of the intersection geometry, including the channel width ratio and height-to-width ratio, capillary number, and flow ratio, on the performance of the generator. The model is validated by comparing it to speed videos of the formation process for several T-junction geometries across a range of capillary numbers and viscosity ratios.

This is the first part of a two-part study on the generation of droplets at a microfluidic T-junction operating in the transition regime where confinement of the droplet creates a large squeezing pressure that influences droplet formation. In this regime, the operation of the T-junction depends on the geometry of the intersection (height-to-width ratio, inlet width ratio), capillary number, flow ratio, and viscosity ratio of the two phases. Here in paper I we presented our experimental observations through the analysis of high-speed videos of the droplet formation process. Various parameters are tracked during the formation cycle such as the shape of the droplet (penetration depth and neck), interdroplet spacing, production rate, and flow of both phases across several T-junction designs and flow conditions. Generally, the formation process is defined by a two-stage model consisting of an initial filling stage followed by a necking stage. However, video evidence suggests the inclusion of a third stage, which we term the lag stage, at the beginning of the formation process that accounts for the retraction of the interface back into the injection channel after detachment. Based on the observations made in this paper, a model is developed to describe the formation process in paper II, which can be used to understand the design and operation of T-junction generators in the transition regime.

When droplets enter a junction they sort to the channel with the highest flow rate at that instant. Transport is regulated by a discrete time-delayed feedback that results in a highly periodic behavior where specific patterns can continue to cycle indefinitely. Between these highly ordered regimes are chaotic structures where no pattern is evident. Here we develop a model that describes droplet sorting under various asymmetries: branch geometry (length, cross-section), droplet resistance and pressures. First, a model is developed based on the continuum assumption and then, with the assistance of numerical simulations, a discrete model is derived to predict the length and composition of the sorting pattern. Furthermore we derive all unique sequences that are possible for a given distribution and develop a preliminary estimation of why chaotic regimes form. The model is validated by comparing it to numerical simulations and results from microfluidic experiments in PDMS chips with good agreement.

This study presents a microfluidic system that incorporates electroosmotic pumps, a concentration gradient generator and a fish cell line (rainbow trout gill) to perform toxicity testing on fish cells seeded in the system. The system consists of three mechanical components: (1) a toxicity testing chip containing a microfluidic gradient generator which creates a linear concentration distribution of toxicant in a cell test chamber, (2) an electroosmotic (EO) pump chip that controls the flow rate and operation of the toxicity chip, and (3) indirect reservoirs that connect the two chips allowing for the toxicant solution to be pumped separately from the electroosmotic pump solution. The flow rate and stability of the EO pumps was measured and tested by monitoring the gradient generator using fluorescence microscopy. Furthermore, a lethality test was performed with this system setup using a rainbow trout gill cell line (RTgill-W1) as the test cells and sodium dodecyl sulfate as a model toxicant. A gradient of sodium dodecyl sulfate, from 0 to 50 microg mL(-1), was applied for 1 hr to the attached cells, and the results were quantified using a Live/Dead cell assay. This work is a preliminary study on the application of EO pumps in a living cell assay, with the potential to use the pumps in portable water quality testing devices with RTgill-W1 cells as the biosensors.

A poly(dimethylsiloxane)(PDMS)/SU-8/quartz hybrid chip was developed and applied in the isoelectric focusing (IEF) of proteins with ultraviolet (UV) absorbance-based whole-channel imaging detection (UV-WCID). Each hybrid chip was made of three layers: a PDMS flat top substrate, a bottom quartz substrate and a middle layer of SU-8 photoresist. The SU-8 serves two purposes: it contains the microchannel used for IEF separation, and acts as an optical slit that absorbs UV light below 300 nm improving detection sensitivity in WCID. The novel hybrid design demonstrates a two to three times improvement in sensitivity over a comparable PDMS/PDMS design. In addition, the hybrid chip exhibits increased heat dissipation due to the superior thermal conductivity of the bottom quartz substrate allowing for larger electric fields to be used in separations. The hybrid design with IEF-UV-WCID was successful in resolving a complicated sample, hemoglobin control, with high fidelity.

Rhodamine B based fluorescence thermometry is commonly used in microfluidics to measure fluid temperatures in microchannels. Notable absorption of Rhodamine B into PDMS channel walls, however, causes difficulties in obtaining accurate temperature measurements due to a steady increase in the overall fluorescent signal. A simple and effective technique is reported that removes the fluorescent signal from absorbed Rhodamine B dye by means of photobleaching with a high intensity light source before taking images for thermometry analysis. The temperature field at the convergence of hot and cold streams in a Y-channel fabricated in PDMS/glass microfluidic chip is studied to demonstrate the execution of the photobleaching technique.

Simple-structured, well-functioned disposable poly(dimethylsiloxane) (PDMS) microchips were developed for capillary isoelectric focusing with whole column imaging detection (CIEF-WCID). Side-by-side comparison of the developed microchips with well-established commercial capillary cartridges demonstrated that the disposable microchips have comparable performance as well as advantages such as absence of lens effect and possibility of high-aspect-ratio accompanied with a dramatic reduction in cost.

A poly(dimethylsiloxane) microfluidic chip-based cartridge is developed and reported here for protein analysis using isoelectic focusing (IEF)-whole-channel imaging detection (WCID) technology. In this design, commercial dialysis membranes are integrated to separate electrolytes and samples and to reduce undesired pressure-driven flow. Fused-silica capillaries are also incorporated in this design for sample injection and channel surface preconditioning. This structure is equivalent to that of a commercial fused-silica capillary-based cartridge for adapting to an IEF analyzer (iCE280 analyzer) to perform IEF-WCID. The successful integration of dialysis membranes into a microfluidic chip significantly improves IEF repeatability by eliminating undesired pressure-driven hydrodynamics and also makes sample injection much easier than that using the first-generation chip as reported recently. In this study, two microfluidic chips with a 100-microm-high, 100-microm-wide and a 200-microm-high, 50-microm-wide microchannel, respectively, were applied for qualitative and quantitative analysis of proteins. The mixture containing six pI markers with a pH range of 3-10 was successfully separated using IEF-WCID. The pH gradient exhibited a good linearity by plotting the pI value versus peak position, and the correlation coefficient reached 0.9994 and 0.9995 separately for the two chips. The separation of more complicated human hemoglobin control sample containing HbA, HbF, HbS, and HbC was also achieved. Additionally, for the quantitative analysis, a good linearity of IEF peak value versus myoglobin concentration in the range of 20-100 microg/mL was obtained.

A novel method is presented for on-chip temperature measurements using a poly(dimethylsiloxane) (PDMS) thin film dissolved with Rhodamine B dye. This thin film is sandwiched between two glass substrates (one of which is 150 microm thick) and bonded to a microchannel molded in a PDMS substrate. Whole-chip (liquid and substrate) temperature measurements can be obtained via fluorescent intensity visualization. For verification purposes, the thin film was tested with a tapered microchannel subjected to Joule heating, with resulting axial temperature gradients comparing well with numerical simulations. Errors induced by the definite film thickness are discussed and accounted for during experimental and analytical analysis. Alternative validation using the traditional in-channel Rhodamine B injection method was also attempted. The thin film has several advantages over traditional methods. First, false intensity readings due to adsorption and absorption of Rhodamine B into PDMS channels are eliminated. Second, whole-chip temperature measurements are possible. Third, separation of working liquid from Rhodamine B dye prevents possible electrophoresis effects.