Exploring operational boundaries for acoustic concentration of cell suspensions.

The development of a standardized, generic method for concentrating suspensions in continuous flow is challenging. In this study, we developed and tested a device capable of concentrating suspensions with an already high cell concentration to meet diverse industrial requirements. To address typical multitasking needs, we concentrated suspensions with high solid content under a variety of conditions. Cells from Saccharomyces cerevisiae, Escherichia coli, and Chinese hamster ovary cells were effectively focused in the center of the main channel of a microfluidic device using acoustophoresis. The main channel bifurcates into three outlets, allowing cells to exit through the central outlet, while the liquid evenly exits through all outlets. Consequently, the treatment separates cells from two-thirds of the surrounding liquid. We investigated the complex interactions between parameters. Increasing the channel depth results in a decrease in process efficiency, attributed to a decline in acoustic energy density. The study also revealed that different cell strains exhibit distinct acoustic contrast factors, originating from differences in dimensions, compressibility, and density values. Finally, a combination of high solid content and flow rate leads to an increase in diffusion through a phenomenon known as shear-induced diffusion. KEY POINTS: • Acoustic focusing in a microchannel was used to concentrate cell suspensions • The parameters influencing focusing at high concentrations were studied • Three different cell strains were successfully concentrated.

Scanning defocusing particle tracking for the experimental characterization of flows in demanding microfluidic systems.

A novel scanning particle image velocimetry technique, to the best of our knowledge, is proposed to characterize flows in microfluidic applications. Three-dimensional information is acquired by oscillating the target sample over a fixed focal plane, allowing the reconstruction of particle trajectories with micrometer accuracy over an extended depth. This technology is suited for investigating acoustic flows with unprecedented precision in microfluidic applications. In this contribution, we describe the experimental setup and the data processing pipeline in detail; we study the technique's performance by reconstructing pressure-driven flow; and we report the three-dimensional trajectory of a 2 µm particle in an acoustic flow in a 525µm×375µm microchannel with micrometric accuracy.

C-Term Reduction in 3 µm Open-Tubular High-Aspect-Ratio Channels in AC-EOF Vortex Chromatography Operation.

The performance of liquid chromatography operation in open-tubular channels, the ideal chromatographic column format, is limited by slow mass transport between the mobile and stationary phase. We recently introduced a lateral mixing methodology ("vortex chromatography") to reduce Taylor-Aris dispersion by employing (small) AC-EOF (alternating current electroosmotic flow) fields oriented perpendicular to the conventionally applied, axially oriented pressure gradient, resulting in the reduction of the C-term by a factor of 3, studied in 40 × 20 µm2 (aspect ratio (AR) = 2) channels under unretained conditions. In the present contribution, a further increased performance gain for channel dimensions relevant for chromatographic applications is demonstrated. The impact of the applied voltage and salt concentration is studied for 3 × 20 and 5 × 20 µm2 channels in ARs of up to 6.7, revealing a C-term reduction potential of a factor of up to 5 for large molecules (dextran) under unretained conditions. The decrease in κaris in a 5 µm channel (reduction of 80%) was larger than the decrease in a 3 µm channel (reduction of 44%).

Concepts and recent advances in microchip electrophoresis coupled to mass spectrometry: Technologies and applications.

The online coupling of microchip electrophoresis (ME) as a fast, highly efficient, and low-cost miniaturized separation technique to mass spectrometry (MS) as an information-rich and sensitive characterization technique results in ME-MS an attractive tool for various applications. In this paper, we review the basic concepts and latest advances in technology for ME coupled to MS during the period of 2016-2021, covering microchip materials, structures, fabrication techniques, and interfacing to electrospray ionization (ESI)-MS and matrix-assisted laser desorption/ionization-MS. Two critical issues in coupling ME and ESI-MS include the electrical connection used to define the electrophoretic field strength along the separation channel and the generation of the electrospray for MS detection, as well as, a miniaturized ESI-tip. The recent commercialization of ME-MS in zone electrophoresis and isoelectric focusing modes has led to the widespread application of these techniques in academia and industry. Here we summarize recent applications of ME-MS for the separation and detection of antibodies, proteins, peptides, carbohydrates, metabolites, and so on. Throughout the paper these applications are discussed in the context of benefits and limitations of ME-MS in comparison to alternative techniques.

Rail configuration for lateral particle transport across intact co-flows: effect of wall and rail geometry on liquid and particle transport.

Layer-by-Layer coating technology is of great importance for many applications of microparticles whereby exposure of the particles to various reagents is needed. Mutual contamination of the reagents during this process is a key challenge, and this undesired effect should be avoided. Here we introduce a device that provides subsequent exposure of particles to various liquids and minimizes mixing of the liquids at the same time. The key element of the device is a rail (groove) at the bottom of a microfluidic channel. The rail forms an angle (between 0 and 90 degrees) and thus enables passive transport of particles through the intact co-flows of the different fluids. To avoid the undesirable effect of reagent stream mixing, internal walls are introduced to separate the different flows. Various designs of the proposed device are considered, and their performance is experimentally analyzed.

Selecting active matter according to motility in an acoustofluidic setup: self-propelled particles and sperm cells.

Active systems - including sperm cells, living organisms like bacteria, fish, birds, or active soft matter systems like synthetic "microswimmers" - are characterized by motility, i.e., the ability to propel using their own "engine". Motility is the key feature that distinguishes active systems from passive or externally driven systems. In a large ensemble, motility of individual species can vary in a wide range. Selecting active species according to their motility represents an exciting and challenging problem. We propose a new method for selecting active species based on their motility using an acoustofluidic setup where highly motile species escape from the acoustic trap. This is demonstrated in simulations and in experiments with self-propelled Janus particles and human sperm. The immediate application of this method is selecting highly motile sperm for medically assisted reproduction (MAR). Due to the tunable acoustic trap, the proposed method is more flexible than the existing passive microfluidic methods. The proposed selection method based on motility can also be applied to other active systems that require selecting highly motile species or removing immotile species.

Performance of laterally elongated pillar array columns in capillary electrochromatography mode.

In the present study, cylindrical and laterally elongated pillar array columns were investigated for use in capillary electrochromatography. Minimal theoretical plate heights of H = 1.90 and 1.46 µm (in absence of sidewall effect) were obtained for coumarin C440 under unretained conditions for cylindrical and rectangular (laterally elongated, aspect ratio 4) pillar array columns, respectively. By comparing dispersion at the entire channel width to that at the central zone only, it appears that sidewall related dispersion significantly contributes to overall dispersion. A 40% reduction of the plate height was observed by taking into account only the central channel zone. A kinetic plot analysis was performed to evaluate the potential of the studied geometries by considering a maximum operating voltage of 20 kV as limiting parameter. It was demonstrated that rectangular radially elongated pillars produce a higher efficiency than cylindrical pillars and other microfabricated column structures for microchip capillary electrochromatography previously studied.

Anisotropic Exclusion Effect between Photocatalytic Ag/AgCl Janus Particles and Passive Beads in a Dense Colloidal Matrix.

Synthetic nano- and micromotors interact with each other and their surroundings in a complex manner. Here, we report on the anisotropy of active-passive particle interaction in a soft matter system containing an immobile yet photochemical Ag/AgCl-based Janus particle embedded in a dense matrix of passive beads in pure water. The asymmetry in the chemical gradient around the Janus particle, triggered upon visible light illumination, distorts the isotropy of the surrounding electric potential and results in the repulsion of adjacent passive beads to a certain distance away from the Janus particle. This exclusion effect is found to be anisotropic with larger distances to passive beads in front of the Ag/AgCl cap of the Janus particle. We provide insight into this phenomenon by performing the angular analysis of the radii of exclusion and tracking their time evolution at the level of a single bead. Our study provides a novel fundamental insight into the collective behavior of a complex mixture of active and passive particles and is relevant for various application scenarios, e.g., particle transport at micro- and nanoscale and local chemical sensing.

Achieving a Peak Capacity of 1800 Using an 8 m Long Pillar Array Column.

In the present study, the peak capacity potential of ultralong porous cylindrical pillar array columns is investigated. Coupling 4 columns of 2 m long allows for working near the minimal separation impedance of small molecules under retained conditions at a maximal pressure load of 250 bar. Minimal plate heights of H = 5.0 µm, H = 6.3 µm, and H = 7.7 µm were obtained for uracil (unretained), butyrophenone (k = 0.85), and valerophenone (k = 1.94), respectively, corresponding to a number of theoretical plates of N = 1.6 × 106, N = 1.2 × 106, and N = 1.0 × 106. The optimal linear velocities were 0.60 mm/s for a retained compound and 0.74 mm/s for an unretained compound. Based on a mixture of 9 compounds, the peak capacity nc was determined as a function of gradient time (tG). Peak capacities (tG-based) of 1103 and 1815 were obtained when applying 650 min and 2050 min gradients (tG/t0 = 4.5 and 14, respectively, with tG as the gradient time and t0 as the void time). These values are much higher than earlier reported peak capacity values for small molecules.

Study of peak capacities generated by a porous layered radially elongated pillar array column coupled to a nano-LC system.

The performance of a porous-layered radially elongated pillar (PLREP) array column in a commercial nano-LC system was examined by performing separation of alkylphenones and peptides. The mesoporous silica layer was prepared by sol-gel processing of a mixture of tetramethoxysilane and methyltrimethoxysilane on REPs filling a 16.5 cm long, 1 mm wide channel (three lanes of 5.5 cm long channels connected by turns). The minimum plate height of 1.4 µm for octanophenone (k = 2.21) observed in isocratic mode is 5 times smaller than the smallest off-column plate height previously reported for porous pillar array columns for a retained component. This advantage is related to the earlier introduced shape of the radially elongated pillar bed that outperforms the cylindrically shaped pillar bed in terms of the plate height. In gradient mode, maximum conditional peak capacities of 220 (for a mixture of thiourea and 7 alkylphenones, tG = 180 min) and 160 (for a cytochrome c digest, tG = 150 min) were obtained. These results indicate excellent potential for implementation of this sol-gel layer in pillar array column formats.

Chip-Based Multicapillary Column with Maximal Interconnectivity to Combine Maximum Efficiency and Maximum Loadability.

On the basis of our previous work on the design of pillar array columns for liquid chromatography, we report on a new pillar array design for high-efficiency, high volumetric loadability gas chromatography columns. The proposed pillar array configuration leads to a column design which can either be considered as a packed bed with perfectly ordered and uniform flow paths or as multicapillary columns (8 parallel tracks) with a maximal interconnectivity between the flow paths to avoid the so-called polydispersity effect (dispersion arising from the inevitable differences in migration velocity between parallel flow paths). Despite our relative inexperience with column coating, and most probably (not supported by data) suffering from the same problem of stationary phase pooling in the right-angled corners of the flow-through channels as other chip-based GC devices, the efficiencies obtained in a L = 70 cm long and 75 µm deep and 6.195 mm wide chip for, respectively, quasi-unretained and retained components (k = 7) went up to N = 60 000 and 12 500 under isothermal conditions using H2 as carrier gas and a downstream restriction. Under programmed temperature conditions (Ti = 80 °C, Tf = 175 °C at 30 °C/min, and a H2 flow of 0.4 mL/min), a peak capacity of 170 was obtained in 3.6 min. For retained compounds, the optimal flow rate is found to be on the order of 0.4 mL/min, achieved at an operating pressure of 2.3 bar. Intrinsically, the column combines the efficiency of a 75 µm capillary with the volumetric loadability of a 240 µm capillary.

Chromatographic Properties of Minimal Aspect Ratio Monolithic Silica Columns.

We report on a study wherein we synthesized TMOS-based silica monolithic skeletons in capillaries with an i.d. of 5 and 10 µm to produce skeleton structures with very low capillary-to-domain size aspect-ratios. These structures include the absolute minimal aspect-ratio case of a monolithic structure whose cross-section only contains a single node point. With domain-sized based reduced plate heights running as low as hmin = 1.3-1.5 for retained coumarin dyes providing a retention factor of k = 0.6-1.0, the study confirms the classic observation that ultralow aspect ratio columns generate a markedly lower dispersion than columns with a larger aspect ratio made in the past by Knox, Jorgenson, and Kennedy for the packed bed of spheres, but now for silica monoliths. The course of the reduced van Deemter curves, and more specifically the ratio of A-term versus C-term band broadening, could be interpreted in terms of the width and persistence length of the velocity bias zones in the columns. Considering the overall kinetic performance, it is found that the two best performing structures are also the structures with the lowest number of domains or node points, that is, with the lowest capillary-to-domain size aspect-ratio and, hence, resembling closest to the open-tubular format, which remains confirmed as the column format with the best kinetic performance. This is quantified by the fact that the minimal impedance values (order of Emin = 100) of the best performing ultralow aspect ratio monolithic columns are still significantly larger than the Emin values for the reference open-tubular columns (order of Emin = 15-20).

Very High Efficiency Porous Silica Layer Open-Tubular Capillary Columns Produced via in-Column Sol-Gel Processing.

It is demonstrated that 5 µm i.d. capillaries can be coated with mesoporous silica layers up to 550 nm thickness. All the columns produced using in-column sol-gel synthesis with tetramethoxysilane provide plate height curves that closely follow the Golay-Aris theory. In 60 cm long columns, efficiencies as high as N = 150 000 and N = 120 000 were obtained, respectively, for a 300 and 550 nm thick porous layer. An excellent retention and plate height reproducibility was obtained when the recipes were subsequently applied to produce very long (1.9 and 2.5 m) capillaries. These columns produced efficiencies up to N = 600 000 plates for a retained and around N = 1 000 000 plates for an unretained component. Given the good reproducibility on the long capillaries, and considering that mesoporous silica is still the preferred support for LC, it is believed the present study could spur a renewed interest in open-tubular LC.

Merging Open-Tubular and Packed Bed Liquid Chromatography.

Experimental and theoretical proof is provided for the fact that a microfabricated packed bed column, which is uniformly filled with radially elongated pillars (REPs), can produce the same separation performance as nonpacked, open-tubular columns. These are generally recognized as the best possible chromatographic column format, offering the highest conceivable separation speed and efficiency. It is also demonstrated both experimentally and theoretically that, as long as pressure is not a limiting factor, the REP column format can even outperform the open-tubular column format, with significant gains in either speed or efficiency proportional to the tortuosity, τ, of the bed. Conducting chromatographic experiments on 4 cm long micromachined packed bed columns filled with radially elongated pillars, separation efficiencies corresponding to N = 160,000 theoretical plates (unretained analytes) and N = 70,000 theoretical plates were achieved, despite the relatively large interpillar distance (2.5 µm).

Mass-manufacturable polymer microfluidic device for dual fiber optical trapping.

We present a microfluidic chip in Polymethyl methacrylate (PMMA) for optical trapping of particles in an 80µm wide microchannel using two counterpropagating single-mode beams. The trapping fibers are separated from the sample fluid by 70µm thick polymer walls. We calculate the optical forces that act on particles flowing in the microchannel using wave optics in combination with non-sequential ray-tracing and further mathematical processing. Our results are compared with a theoretical model and the Mie theory. We use a novel fabrication process that consists of a premilling step and ultraprecision diamond tooling for the manufacturing of the molds and double-sided hot embossing for replication, resulting in a robust microfluidic chip for optical trapping. In a proof-of-concept demonstration, we show the trapping capabilities of the hot embossed chip by trapping spherical beads with a diameter of 6µm, 8µm and 10µm and use the power spectrum analysis of the trapped particle displacements to characterize the trap strength.

In situ measurement of the transversal dispersion in ordered and disordered two-dimensional pillar beds for liquid chromatography.

Using a fully transparent micropillar array chip and an optical "injection" setup capable of writing pulsed and continuous patterns into the flow by uncaging a fluorescent dye, highly detailed measurements of the transversal dispersion process in two-dimensional (2D) chromatographic beds could be made. With the use of water-glycerol mobile phase spanning a wide range of viscosities, the obtained data cover a broad range of reduced velocities (0 < ν < 3000) and show a clear leveling-off of the transversal dispersion coefficient at large reduced velocities. With dependence on the packing density, this leveling-off occurs at a value of about Dtrans/Dmol = 10 (ε = 0.4), Dtrans/Dmol = 4 (ε = 0.6) and Dtrans/Dmol = 2.5 (ε = 0.8). Another interesting observation that could be made is that the effect of the bed order on the observed transversal dispersion process is relatively small. The observed leveling-off in the relation between the measured Dtrans values and the reduced liquid velocity furthermore clearly invalidates the classical Galton-board model, predicting a linear increase of Dtrans with the reduced velocity. On the other hand, it corroborates a recently proposed series-connection transport model for Dtrans in 2D porous media.

Integration of uniform porous shell layers in very long pillar array columns using electrochemical anodization for liquid chromatography.

Electrochemical anodization has been applied to grow porous shell layers of 300 nm (30 nm pores) in 5 µm diameter pillar array columns (PACs) with a spacing of 2.5 µm. Using turn structures preceded and followed by the flow distributor structures recently introduced by our group and filled with radially elongated pillars, columns with quasi unlimited channel lengths could be conceived. The uniformity of the porous PAC was assessed by determining local plate heights along the channel, which appeared to be constant. Minimal (absolute) plate heights (H) between 4 and 6 µm were obtained at optimal flow rates when imposing increasing retention factors. Upon measuring the surface area involved in chromatographic retention as an indicator of the available surface area, an increase in the surface area by a factor of about 30 compared to that of non-anodized pillars was found. On reconfiguring a commercial HPLC instrument to enable on-chip injections, 90% of the performance (expressed in theoretical plates) could be maintained for a 1 m column, while for a 25 cm column severe losses were still observed. As the corresponding pressure drop for optimal operation of retained components is on the order of 10 bar per m only, portable and cheaper HPLC devices with high efficiencies become realistically conceivable.

A numerical study of the effects of lateral flow and retention in open tubular vortex chromatography.

Axial dispersion in chromatographic columns is responsible for a reduced separation efficiency. In the present research macrotransport theory is used to predict the phenomenological constants related to axial dispersion. We evaluate the efficacy of lateral flow induced by alternating current (AC) in the presence of retaining walls on the separation resolution. Results show that lateral flows induced by laterally applied potentials as low as 0.3 V reduce C-term dispersion by a factor of 5.0 for unretained conditions (k = 0) and 2.7 for retained (k = 5) conditions, with a diffusion coefficient (Dm) of 10-11m2/s. The present paper further contributes to the understanding of the use of secondary lateral flows for dispersion reduction and offers practical guidance for designing future vortex chromatographic columns. It appears that a maximal performance gain is attained at low aspect ratios (AR=1), with the gain reduced from a factor of 5 to 1.6 for AR=4 for unretained conditions, and from 2.7 to 1.4 for retained conditions (k = 5).

Enhanced Performance of an Acoustofluidic Device by Integrating Temperature Control.

Acoustofluidics is an emerging research field wherein either mixing or (bio)-particle separation is conducted. High-power acoustic streaming can produce more intense and rapid flow patterns, leading to faster and more efficient liquid mixing. However, without cooling, the temperature of the piezoelectric element that is used to supply acoustic power to the fluid could rise above 50% of the Curie point of the piezomaterial, thereby accelerating its aging degradation. In addition, the supply of excessive heat to a liquid may lead to irreproducible streaming effects and gas bubble formation. To control these phenomena, in this paper, we present a feedback temperature control system integrated into an acoustofluidic setup using bulk acoustic waves (BAWs) to elevate mass transfer and manipulation of particles. The system performance was tested by measuring mixing efficiency and determining the average velocity magnitude of acoustic streaming. The results show that the integrated temperature control system keeps the temperature at the set point even at high acoustic powers and improves the reproducibility of the acoustofluidic setup performance when the applied voltage is as high as 200 V.

Characterizing Acoustic Behavior of Silicon Microchannels Separated by a Porous Wall.

Lateral flow membrane microdevices are widely used for chromatographic separation processes and diagnostics. The separation performance of microfluidic lateral membrane devices is determined by mass transfer limitations in the membrane, and in the liquid phase, mass transfer resistance is dependent on the channel dimensions and transport properties of the species separated by the membrane. We present a novel approach based on an active bulk acoustic wave (BAW) mixing method to enhance lateral transport in micromachined silicon devices. BAWs have been previously applied in channels for mixing and trapping cells and particles in single channels, but this is, to the best of our knowledge, the first instance of their application in membrane devices. Our findings demonstrate that optimal resonance is achieved with minimal influence of the pore configuration on the average lateral flow. This has practical implications for the design of microfluidic devices, as the channels connected through porous walls under the acoustic streaming act as 760 µm-wide channels rather than two 375 µm-wide channels in the context of matching the standing pressure wave criteria of the piezoelectric transducer. However, the roughness of the microchannel walls does seem to play a significant role in mixing. A roughened (black silicon) wall results in a threefold increase in average streaming flow in BAW mode, suggesting potential avenues for further optimization.