Contraction and extension of Vorticella and its mechanical characterization under flow loading

We have studied the contraction and extension of Vorticella convallaria and its mechanical properties with a microfluidic loading system. Cells of V. convallaria were injected to a microfluidic channel (500 μm in width and 100 μm in height) and loaded by flow up to ∼350 mm s⁻¹. The flow produced a drag force on the order of nanonewton on a typical vorticellid cell body. We gradually increased the loading force on the same V. convallaria specimen and examined its mechanical property and stalk motion of V. convallaria. With greater drag forces, the contraction distance linearly decreased; the contracted length was close to around 90% of the stretched length. We estimated the drag force on Vorticella in the channel by calculating the force on a sphere in a linear shear flow.     

Studying enzymatic bioreactions in a millisecond microfluidic flow mixer

In this study, the pre-steady state development of enzymatic bioreactions using a microfluidic mixer is presented. To follow such reactions fast mixing of reagents (enzyme and substrate) is crucial. By using a highly efficient passive micromixer based on multilaminar flow, mixing times in the low millisecond range are reached. Four lamination layers in a shallow channel reduce the diffusion lengths to a few micrometers only, enabling very fast mixing. This was proven by confocal fluorescence measurements in the channel’s cross sectional area. Adjusting the overall flow rate in the 200 μm wide and 900 μm long mixing and observation channel makes it possible to investigate enzyme reactions over several seconds. Further, the device enables changing the enzyme/substrate ratio from 1:1 up to 3:1, while still providing high mixing efficiency, as shown for the enzymatic hydrolysis using β-galactosidase. This way, the early kinetics of the enzyme reaction at multiple enzyme/substrate concentrations can be collected in a very short time (minutes). The fast and easy handling of the mixing device makes it a very powerful and convenient instrument for millisecond temporal analysis of bioreactions.     

Nanoparticle image velocimetry at topologically structured surfaces

Nanoparticle image velocimetry (nano-PIV), based on total internal reflection fluorescent microscopy, is very useful to investigate fluid flows within ∼100 nm from a surface; but so far it has only been applied to flow over smooth surfaces. Here we show that it can also be applied to flow over a topologically structured surface, provided that the surface structures can be carefully configured not to disrupt the evanescent-wave illumination. We apply nano-PIV to quantify the flow velocity distribution over a polydimethylsiloxane surface, with a periodic gratinglike structure (with 215 nm height and 2 μm period) fabricated using our customized multilevel lithography method. The measured tracer displacement data are in good agreement with the computed theoretical values. These results demonstrate new possibilities to study the interactions between fluid flow and topologically structured surfaces.     

Rapid magnetic heating treatment by highly charged maghemite nanoparticles on Wistar rats exocranial glioma tumors at microliter volume

One of the most significant challenges implementing colloidal magnetic nanoparticles in medicine is the efficient heating of microliter quantities by applying a low frequency alternating magnetic field. The ultimate goal is to accomplish nonsurgically the treatment of millimeter size tumors. Here, we demonstrate the synthesis, characterization, and the in vitro as well as in vivo efficiency of a dextran coated maghemite (γ-Fe₂O₃) ferrofluid with an exceptional response to magnetic heating. The difference to previous synthetic attempts is the high charge of the dextran coating, which according to our study maintains the colloidal stability and good dispersion of the ferrofluid during the magnetic heating stage. Specifically, in vitro 2 μl of the ferrofluid gives an outstanding temperature rise of 33 °C within 10 min, while in vivo treatment, by infusing 150 μl of the ferrofluid in animal model (rat) glioma tumors, causes an impressive cancer tissue dissolution.     

Efficient manipulation of microparticles in bubble streaming flows

Oscillating microbubbles of radius 20–100 μm driven by ultrasound initiate a steady streaming flow around the bubbles. In such flows, microparticles of even smaller sizes (radius 1–5 μm) exhibit size-dependent behaviors: particles of different sizes follow different characteristic trajectories despite density-matching. Adjusting the relative strengths of the streaming flow and a superimposed Poiseuille flow allows for a simple tuning of particle behavior, separating the trajectories of particles with a size resolution on the order of 1 μm. Selective trapping, accumulation, and release of particles can be achieved. We show here how to design bubble microfluidic devices that use these concepts to filter, enrich, and preconcentrate particles of selected sizes, either by concentrating them in discrete clusters (localized both stream- and spanwise) or by forcing them into narrow, continuous trajectory bundles of strong spanwise localization.     

On-chip microfluidic systems for determination of L-glutamate based on enzymatic recycling of substrate

Two microfluidic systems have been developed for specific analysis of L-glutamate in food based on substrate recycling fluorescence detection. L-glutamate dehydrogenase and a novel enzyme, D-phenylglycine aminotransferase, were covalently immobilized on (i) the surface of silicon microchips containing 32 porous flow channels of 235 μm depth and 25 μm width and (ii) polystyrene Poros™ beads with a particle size of 20 μm. The immobilized enzymes recycle L-glutamate by oxidation to 2-oxoglutarate followed by the transfer of an amino group from D-4-hydroxyphenylglycine to 2-oxoglutarate. The reaction was accompanied by reduction of nicotinamide adenine dinucleotide (NAD⁺) to NADH, which was monitored by fluorescence detection (ε_ex=340 nm, ε_em=460 nm). First, the microchip-based system, L-glutamate was detected within a range of 3.1–50.0 mM. Second, to be automatically determined, sequential injection analysis (SIA) with the bead-based system was investigated. The bead-based system was evaluated by both flow injection analysis and SIA modes, where good reproducibility for L-glutamate calibrations was obtained (relative standard deviation of 3.3% and 6.6%, respectively). In the case of SIA, the beads were introduced and removed from the microchip automatically. The immobilized beads could be stored in a 20% glycerol and 0.5 mM ethylenediaminetetraacetic acid solution maintained at a pH of 7.0 using a phosphate buffer for at least 15 days with 72% of the activity remaining. The bead-based system demonstrated high selectivity, where L-glutamate recoveries were between 91% and 108% in the presence of six other L-amino acids tested.     

Field-free particle focusing in microfluidic plugs

Particle concentration is a key unit operation in biochemical assays. Although there are many techniques for particle concentration in continuous-phase microfluidics, relatively few are available in multiphase (plug-based) microfluidics. Existing approaches generally require external electric or magnetic fields together with charged or magnetized particles. This paper reports a passive technique for particle concentration in water-in-oil plugs which relies on the interaction between particle sedimentation and the recirculating vortices inherent to plug flow in a cylindrical capillary. This interaction can be quantified using the Shields parameter (θ), a dimensionless ratio of a particle’s drag force to its gravitational force, which scales with plug velocity. Three regimes of particle behavior are identified. When θ is less than the movement threshold (region I), particles sediment to the bottom of the plug where the internal vortices subsequently concentrate the particles at the rear of the plug. We demonstrate highly efficient concentration (∼100%) of 38 μm glass beads in 500 μm diameter plugs traveling at velocities up to 5 mm/s. As θ is increased beyond the movement threshold (region II), particles are suspended in well-defined circulation zones which begin at the rear of the plug. The length of the zone scales linearly with plug velocity, and at sufficiently large θ, it spans the length of the plug (region III). A second effect, attributed to the co-rotating vortices at the rear cap, causes particle aggregation in the cap, regardless of flow velocity. Region I is useful for concentrating/collecting particles, while the latter two are useful for mixing the beads with the solution. Therefore, the two key steps of a bead-based assay, concentration and resuspension, can be achieved simply by changing the plug velocity. By exploiting an interaction of sedimentation and recirculation unique to multiphase flow, this simple technique achieves particle concentration without on-chip components, and could therefore be applied to a range of heterogeneous screening assays in discrete nl plugs.     

Manipulating particle trajectories with phase-control in surface acoustic wave microfluidics

We present a 91 MHz surface acoustic wave resonator with integrated microfluidics that includes a flow focus, an expansion region, and a binning region in order to manipulate particle trajectories. We demonstrate the ability to change the position of the acoustic nodes by varying the electronic phase of one of the transducers relative to the other in a pseudo-static manner. The measurements were performed at room temperature with 3 μm diameter latex beads dispersed in a water-based solution. We demonstrate the dependence of nodal position on pseudo-static phase and show simultaneous control of 9 bead streams with spatial control of −0.058 μm/deg ± 0.001 μm/deg. As a consequence of changing the position of bead streams perpendicular to their flow direction, we also show that the integrated acoustic-microfluidic device can be used to change the trajectory of a bead stream towards a selected bin with an angular control of 0.008 deg/deg ± 0.000(2) deg/deg.     

Microliter-bioreactor array with buoyancy-driven stirring for human hematopoietic stem cell culture

This work presents the development of an array of bioreactors where finely controlled stirring is provided at the microliter scale (100–300 μl). The microliter-bioreactor array is useful for performing protocol optimization in up to 96 parallel experiments of hematopoietic stem cell (HSC) cultures. Exploring a wide range of experimental conditions at the microliter scale minimizes cost and labor. Once the cell culture protocol is optimized, it can be applied to large-scale bioreactors for stem cell production at the clinical level. The controlled stirring inside the wells of a standard 96-well plate is provided by buoyancy-driven thermoconvection. The temperature and velocity fields within the culture volume are determined with numerical simulations. The numerical results are verified with experimental velocity measurements using microparticle image velocimetry (μPIV) and are used to define feasible experimental conditions for stem cell cultures. To test the bioreactor array’s functionality, human umbilical cord blood-derived CD34⁺ cells were cultured for 7 days at five different stirring conditions (0.24–0.58 μm∕s) in six repeated experiments. Cells were characterized in terms of proliferation, and flow cytometry measurements of viability and CD34 expression. The microliter-bioreactor array demonstrates its ability to support HSC cultures under stirred conditions without adversely affecting the cell behavior. Because of the highly controlled operative conditions, it can be used to explore culture conditions where the mass transport of endogenous and exogenous growth factors is selectively enhanced, and cell suspension provided. While the bioreactor array was developed for culturing HSCs, its application can be extended to other cell types.     

Micromixing visualization and quantification in a microscale multi-inlet vortex nanoprecipitation reactor using confocal-based reactive micro laser-induced fluorescence

A technique for visualizing and quantifying reactive mixing for laminar and turbulent flow in a microscale chemical reactor using confocal-based microscopic laser induced fluorescence (confocal μ-LIF) was demonstrated in a microscale multi-inlet vortex nanoprecipitation reactor. Unlike passive scalar μ-LIF, the reactive μ-LIF technique is able to visualize and quantify micromixing effects. The confocal imaging results indicated that the flow in the reactor was laminar and steady for inlet Reynolds numbers of 10, 53, and 93. Mixing and reaction were incomplete at each of these Reynolds numbers. The results also suggested that although mixing by diffusion was enhanced near the midplane of the reactor at Re_j = 53 and 93 due to very thin bands of acidic and basic fluid forming as the fluid spiraled towards the center of the reactor, near the top, and bottom walls of the reactor, the lower velocities due to fluid friction with the walls hindered the formation of these thin bands, and, thus, resulted in large regions of unmixed and unreacted fluid. At Re_j = 240, the flow was turbulent and unsteady. The mixing and reaction processes were still found to be incomplete even at this highest Reynolds number. At the reactor midplane, the flow images at Re_j = 240 showed unmixed base fluid near the center of the reactor, suggesting that just as in the Re_j = 53 and 93 cases, lower velocities near the top and bottom walls of the reactor hinder the mixing and rection of the acidic and basic streams. Ensemble averages of line-scan profiles for the Re_j = 240 were then calculated to provide statistical quantification of the microscale mixing in the reactor. These results further demonstrate that even at this highest Reynolds number investigated, mixing and reaction are incomplete. Visualization and quantification of micromixing using this reactive μ-LIF technique can prove useful in the validation of computational fluid dynamics models of micromixing within microscale chemical reactors.     

Effect of a dual inlet channel on cell loading in microfluidics

Unwanted sedimentation and attachment of a number of cells onto the bottom channel often occur on relatively large-scale inlets of conventional microfluidic channels as a result of gravity and fluid shear. Phenomena such as sedimentation have become recognized problems that can be overcome by performing microfluidic experiments properly, such as by calculating a meaningful output efficiency with respect to real input. Here, we present a dual-inlet design method for reducing cell loss at the inlet of channels by adding a new “ upstream inlet ” to a single main inlet design. The simple addition of an upstream inlet can create a vertically layered sheath flow prior to the main inlet for cell loading. The bottom layer flow plays a critical role in preventing the cells from attaching to the bottom of the channel entrance, resulting in a low possibility of cell sedimentation at the main channel entrance. To provide proof-of-concept validation, we applied our design to a microfabricated flow cytometer system (μFCS) and compared the cell counting efficiency of the proposed μFCS with that of the previous single-inlet μFCS and conventional FCS. We used human white blood cells and fluorescent microspheres to quantitatively evaluate the rate of cell sedimentation in the main inlet and to measure fluorescence sensitivity at the detection zone of the flow cytometer microchip. Generating a sheath flow as the bottom layer was meaningfully used to reduce the depth of field as well as the relative deviation of targets in the z-direction (compared to the x-y flow plane), leading to an increased counting sensitivity of fluorescent detection signals. Counting results using fluorescent microspheres showed both a 40% reduction in the rate of sedimentation and a 2-fold higher sensitivity in comparison with the single-inlet μFCS. The results of CD4⁺ T-cell counting also showed that the proposed design results in a 25% decrease in the rate of cell sedimentation and a 28% increase in sensitivity when compared to the single-inlet μFCS. This method is simple and easy to use in design, yet requires no additional time or cost in fabrication. Furthermore, we expect that this approach could potentially be helpful for calculating exact cell loading and counting efficiency for a small input number of cells, such as primary cells and rare cells, in microfluidic channel applications.     

Mixing enhancement in T-junction microchannel with acoustic streaming induced by triangular structure

The T-shaped microchannel system is used to mix similar or different fluids, and the laminar flow nature makes the mixing at the entrance junction region a challenging task. Acoustic streaming is a steady vortical flow phenomenon that can be produced in the microchannel by oscillating acoustic transducer around the sharp edge tip structure. In this study, the acoustic streaming is produced using a triangular structure with tip angles of 22.62°, 33.4°, and 61.91°, which is placed at the entrance junction region and mixes the inlets flow from two directions. The acoustic streaming flow patterns were investigated using micro-particle image velocimetry (μPIV) in various tip edge angles, flow rate, oscillation frequency, and amplitude. The velocity and vorticity profiles show that a pair of counter-rotating streaming vortices were created around the sharp triangle structure and raised the Z vorticity up to 10 times more than the case without acoustic streaming. The mixing experiments were performed by using fluorescent green dye solution and de-ionized water and evaluated its performance with the degree of mixing (M) at different amplitudes, flow rates, frequencies, and tip edge angles using the grayscale value of pixel intensity. The degree of mixing characterized was found significantly improved to 0.769 with acoustic streaming from 0.4017 without acoustic streaming, in the case of 0.008 μl/min flow rate and 38 V oscillation amplitude at y = 2.15 mm. The results suggested that the creation of acoustic streaming around the entrance junction region promotes the mixing of two fluids inside the microchannel, which is restricted by the laminar flow conditions.     

Separation by dielectrophoresis of dormant and nondormant bacterial cells of Mycobacterium smegmatis

The dielectrophoretic behavior of active, dead, and dormant Mycobacterium smegmatis bacterial cells was studied. It was found that the 72-h-old dormant cells had a much higher effective particle conductivity (812±10 μS cm⁻¹), almost double that of active cells (560±20 μS cm⁻¹), while that of dead (autoclaved) M. smegmatis cells was the highest (950±15 μS cm⁻¹) overall. It was also found that at 80 kHz, 900 μS cm⁻¹ dead cells were attracted at the edges of interdigitated castellated electrodes by positive dielectrophoresis, but dormant cells were not. Similarly, at 120 kHz, 2 μS cm⁻¹ active cells were attracted and dormant cells were not. Using these findings a dielectrophoresis-based microfluidic separation system was developed in which dead and active cells were collected from a given cell suspension, while dormant cells were eluted.     

Electroosmotic flow in vapor deposited silicon dioxide and nitride microchannels

Electroosmotic flow was studied in thin film microchannels with silicon dioxide and silicon nitride sidewalls formed using plasma-enhanced chemical vapor deposition (PECVD). A sacrificial etching process was employed for channel fabrication allowing for cross-sections with heights of 3 μm, ranging from 2 μm to 50 μm in width. Flow rates were measured for single channels and multichannel electroosmotic pump structures for pH levels ranging from 2.6 to 8.3, and zeta potentials were calculated for both silicon dioxide and silicon nitride surfaces. Flow rates as high as 0.086 μL∕min were measured for nitride multichannel pumps at applied electric fields of 300 V∕mm. The surface characteristics of PECVD nitride were analyzed and compared to more well-known oxide surfaces to determine the density of amine sites compared to silanol sites.     

Construction and operation of a microrobot based on magnetotactic bacteria in a microfluidic chip

Magnetotactic bacteria (MTB) are capable of swimming along magnetic field lines. This unique feature renders them suitable in the development of magnetic-guided, auto-propelled microrobots to serve in target molecule separation and detection, drug delivery, or target cell screening in a microfluidic chip. The biotechnology to couple these bacteria with functional loads to form microrobots is the critical point in its application. Although an immunoreaction approach to attach functional loads to intact MTB was suggested, details on its realization were hardly mentioned. In the current paper, MTB-microrobots were constructed by attaching 2 μm diameter microbeads to marine magnetotactic ovoid MO-1 cells through immunoreactions. These microrobots were controlled using a special control and tracking system. Experimental results prove that the attachment efficiency can be improved to ∼30% via an immunoreaction. The motility of the bacteria attached with different number of loads was also assessed. The results show that MTB can transport one load at a velocity of ∼21 μm/s and still move and survive for over 30 min. The control and tracking system is fully capable of directing and monitoring the movement of the MTB-microrobots. The rotating magnetic fields can stop the microrobots by trapping them as they swim within a circular field with a controllable size. The system has potential use in chemical analyses and medical diagnoses using biochips as well as in nano/microscale transport.     

Investigation on CO(2) laser irradiation inducing glass strip peeling for microchannel formation

The study investigates the use of CO₂ laser to induce glass strip peeling off to form microchannels on soda lime gass substrate. The strip peeling exhibits a strong dependence on the energy deposition rate on the glass surface. In spite of the vast difference in the combination of laser power and scanning speed, when the ratio of the two makes the energy deposition rate in the range 3.0-6.0 J/(cm² s), the temperature rising inside glass will be above the strain point and reach the softening region of the glass. As a result, glass strip peeling is able to occur and form microchannels with dimensions of 20-40 μm in depth and 200-280 μm in width on the glass surface. Beyond this range, higher energy depsotion rate would lead to surface melting associated with solidification cracks and lower energy deposition rate causes the generation of fragment cracks.     

An integrated dielectrophoretic chip for continuous bioparticle filtering, focusing, sorting, trapping, and detecting

Multi-target pathogen detection using heterogeneous medical samples require continuous filtering, sorting, and trapping of debris, bioparticles, and immunocolloids within a diagnostic chip. We present an integrated AC dielectrophoretic (DEP) microfluidic platform based on planar electrodes that form three-dimensional (3D) DEP gates. This platform can continuously perform these tasks with a throughput of 3 μL∕min. Mixtures of latex particles, Escherichia coli Nissle, Lactobacillus, and Candida albicans are sorted and concentrated by these 3D DEP gates. Surface enhanced Raman scattering is used as an on-chip detection method on the concentrated bacteria. A processing rate of 500 bacteria was estimated when 100 μl of a heterogeneous colony of 10⁷ colony forming units ∕ml was processed in a single pass within 30 min.     

Microscale flow propulsion through bioinspired and magnetically actuated artificial cilia

Recent advances in microscale flow propulsion through bioinspired artificial cilia provide a promising alternative for lab-on-a-chip applications. However, the ability of actuating artificial cilia to achieve a time-dependent local flow control with high accuracy together with the elegance of full integration into the biocompatible microfluidic platforms remains remote. Driven by this motive, the current work has constructed a series of artificial cilia inside a microchannel to facilitate the time-dependent flow propulsion through artificial cilia actuation with high-speed (>40 Hz) circular beating behavior. The generated flow was quantified using micro-particle image velocimetry and particle tracking with instantaneous net flow velocity of up to 10¹μm/s. Induced flow patterns caused by the tilted conical motion of artificial cilia constitutes efficient fluid propulsion at microscale. This flow phenomenon was further measured and illustrated by examining the induced flow behavior across the depth of the microchannel to provide a global view of the underlying flow propulsion mechanism. The presented analytic paradigms and substantial flow evidence present novel insights into the area of flow manipulation at microscale.     

Characterization and separation of Cryptosporidium and Giardia cells using on-chip dielectrophoresis

Dielectrophoresis (DEP) has been shown to have significant potential for the characterization of cells and could become an efficient tool for rapid identification and assessment of microorganisms. The present work is focused on the trapping, characterization, and separation of two species of Cryptosporidium (C. parvum and C. muris) and Giardia lambia (G. lambia) using a microfluidic experimental setup. Cryptosporidium oocysts, which are 2-4 μm in size and nearly spherical in shape, are used for the preliminary stage of prototype development and testing. G. lambia cysts are 8–12 μm in size. In order to facilitate effective trapping, simulations were performed to study the effects of buffer conductivity and applied voltage on the flow and cell transport inside the DEP chip. Microscopic experiments were performed using the fabricated device and the real part of Clausius—Mossotti factor of the cells was estimated from critical voltages for particle trapping at the electrodes under steady fluid flow. The dielectric properties of the cell compartments (cytoplasm and membrane) were calculated based on a single shell model of the cells. The separation of C. muris and G. lambia is achieved successfully at a frequency of 10 MHz and a voltage of 3 Vpp (peak to peak voltage).     

Rapid bioparticle concentration and detection by combining a discharge driven vortex with surface enhanced Raman scattering

Rapid concentration and detection of bacteria in integrated chips and microfluidic devices is needed for the advancement of lab-on-a-chip devices because current detection methods require high concentrations of bacteria which render them impractical. We present a new chip-scale rapid bacteria concentration technique combined with surface-enhanced Raman scattering (SERS) to enhance the detection of low bacteria count samples. This concentration technique relies on convection by a long-range converging vortex to concentrate the bacteria into a packed mound of 200 μm in diameter within 15 min. Concentration of bioparticle samples as low as 10⁴ colony forming units (CFU)∕ml are presented using batch volumes as large as 150 μl. Mixtures of silver nanoparticles with Saccharomyces cerevisiae, Escherichia coli F-amp, and Bacillus subtilis produce distinct and noticeably different Raman spectra, illustrating that this technique can be used as a detection and identification tool.