An optimal three-dimensional focusing technique for micro-flow cytometers

This study presents a novel three-dimensional (3-D) hydrodynamic focusing technique for micro-flow cytometers. In the proposed approach, the sample stream is initially compressed in the horizontal direction by two sheath flows such that it is constrained in the central region of the microchannel. The sample stream is then focused in the vertical direction by a second pair of sheath flows and subsequently passes over a micro-weir structure positioned directly beneath an optical detection system. The microchannel configuration and operational parameters are optimized by performing a series of numerical simulations to examine the effects on the sample stream distribution of the vertical and horizontal focusing ratios, the entrance angle of the second set of sheath flow channels, and the width and depth of the second set of sheath flow channels. The results indicate that the horizontal and vertical sheath flows successfully constrain the sample stream within a narrow, well-defined region of the microchannel. Furthermore, the micro-weir structure results in the separation of the cells/particles in the vertical direction and ensures that they flow in a sequential fashion through the detection region of the microchannel and can therefore be reliably counted. It is shown that the 3-D focusing technique can achieve a focused sample stream width of between 6 and 15 μm given an appropriate value of the horizontal focusing ratio. Thus, the viability of the microflow cytometer for the counting and detection of individual biological cells is confirmed.     

Novel continuous particle sorting in microfluidic chip utilizing cascaded squeeze effect

This article presents a novel technique for the continuous sorting and collection of microparticles in a microfluidic chip using a cascaded squeeze effect. In the proposed approach, microparticles of different sizes are separated from the sample stream using sheath flows and are then directed to specific side channels for collection. The sheath flows required to separate the particles are generated using a single high voltage supply integrated with a series of variable resistors designed to create electric fields of different intensities at different points of the microchip. Numerical simulations are performed to analyze the electrical potential contours and flow streamlines within the microchannel. Experimental trials show that the microchip is capable of continuously separating microparticles with diameters of 5, 10 and 20 μm, respectively. To further evaluate the performance of the microchip, a sample composed of yeast cells and polystyrene beads is sorted and collected. The results indicate that the microchip achieves a recovery ratio of 87.7% and a yield ratio of 94.1% for the yeast cells and therefore attains a comparable performance to that of a large-scale commercial flow cytometer. Importantly, the high performance of the microchip is achieved without the need for a complex control system or for sophisticated actuation mechanisms such as embedded microelectrodes, ultrasonic generators, or micropumps, and so forth.     

Microflow cytometer incorporating sequential micro-weir structure for three-dimensional focusing

A novel microflow cytometer is proposed in which the sample stream is focused initially in the horizontal (X–Y) plane by two sheath flows and is then focused in the vertical (Y–Z) plane by a sequential micro-weir structure before entering the detection region of the device. The proposed device is fabricated using a modified glass fabrication process, and is then characterized both numerically and experimentally using fluorescent polystyrene beads with diameters of 7 and 15 μm, respectively. The experimental and numerical results confirm the effectiveness of the hydrodynamic sheath flows in centralizing the particles in the horizontal plane prior to entering the sequential micro-weir structure. Furthermore, it is shown numerically that the micro-weir structure confines the particle stream to the center of the vertical plane such that the particles pass through the detection region in a one-by-one fashion can be counted with a high degree of reliability. The experimental results confirm that the proposed 3-D focusing scheme enables the fluorescent beads with different diameters to be reliably sorted and counted.     

In-channel focusing of flowing microparticles utilizing hydrodynamic filtration

We present herein microfluidic systems to continuously focus the positions of flowing particles onto the center of a microchannel, which is indispensable to various applications for manipulating particles or cells such as flow cytometry and particle-based bioassay. A scheme called ‘hydrodynamic filtration’ is employed to repeatedly split fluid flows from a main stream, while remaining particles in the main stream. By re-injecting the split flows into the main channel, these flows work as sheath flows, focusing the positions of the particles onto the center of the microchannel without the help of sheath flows or complicated devices generating physical forces. In this study, we proposed two schemes, and compared the focusing efficiencies between the two schemes using particles 5.0 μm in diameter. Also, we confirmed that the flow speed did not affect the focusing efficiency, demonstrating the versatility and applicability of the presented systems. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Blood flow driven by surface tension in a microchannel

This study aims to identify distinct blood flow characteristics in a microchannel at different sloping angles. The channel is determined by a bottom hydrophilic stripe on a glass substrate and a fully covered hydrophobic glass substrate. The channel has a height of 3 μm, and a width of 100 μm. It is observed that increasing the sloping angle from −90° (downward flow) to 90° (upward flow) increases the blood flow rate monotonically. These peculiar behaviors on the micro scale are explained by a dynamic model that establishes the balance among the inertial, surface tension, gravitational, and frictional forces. The frictional force is further related to the effective hematocrit. The model is used to calculate the frictional force, and thus the corresponding hematocrit, which is smaller when the blood flows upward, reducing the frictional force.     

A power-free deposited microbead plug-based microfluidic chip for whole-blood immunoassay

We present a deposited microbead plug (DMBP)-based microfluidic chip capable of performing plasma extraction and on-chip immunoassay. The DMBP used as a porous blood filter provides pure blood plasma without the contamination of blood cells or beads. Capillary-driven flow eliminates the requirement of external pumps. The human IgG and goat anti-human IgG sample-to-answer assay was performed in this chip within 600 s using only a 10 μl whole-blood sample. This easy-to-use, rapid, inexpensive, and disposable DMBP-based chip holds a great promise for point-of-care application.     

High-throughput electrokinetic bioparticle focusing based on a travelling-wave dielectrophoretic field

This study presents a sheathless and portable microfluidic chip that is capable of high-throughput focusing bioparticles based on 3D travelling-wave dielectrophoresis (twDEP). High-throughput focusing is achieved by sustaining a centralized twDEP field normal to the continuous through-flow direction. Two twDEP electrode arrays are formed from upper and lower walls of the microchannel and extend to its center, which induce twDEP forces to provide the lateral displacements in two directions for focusing the bioparticles. Bioparticles can be focused to the center of the microchannel effectively by twDEP conveyance when the characteristic time due to twDEP conveying in the y direction is shorter than the residence time of the particles within twDEP electrode array. Red blood cells can be effectively focused into a narrow particle stream (~10 μm) below a critical flow rate of 10 μl/min (linear flow velocity ~5 mm/s), when under a voltage of 14 V_p–p at a frequency of 500 kHz is applied. Approximately 90% focusing efficiency for red blood cells can be achieved within two 6-mm-long electrode arrays when the flow rate is below 12 μl/min. Blood cells and Candida cells were also focused and sorted successfully based on their different twDEP mobilities. Compared to conventional 3D-paired DEP focusing, velocity is enhanced nearly four folds of magnitude. 3D twDEP provides the lateral displacements of particles and long residence time for migrating particles in a high-speed continuous flow, which breaks through the limitation of many electrokinetic cell manipulation techniques.     

Scaling the formation of slug bubbles in microfluidic flow-focusing devices

The present study aims at scaling the formation of slug bubbles in flow-focusing microfluidic devices using a high-speed digital camera and a micro particle image velocimetry (μ-PIV) system. Experiments were conducted in two different polymethyl methacrylate square microchannels of respectively 600 × 600 and 400 × 400 μm. N₂ bubbles were generated in glycerol–water mixtures with several concentrations of surfactant sodium dodecyl sulfate. The influence of gas and liquid flow rates, the viscosity of the liquid phase and the width of the microchannel on the bubble size were explored. The bubble size was correlated as a function of the width of the microchannel W _c, the ratio of the gas/liquid flow rates Q _g/Q _l and the liquid Reynolds number. During the pinch-off stage, the variation of the minimum width of the gaseous thread W _m with the remaining time could be scaled as _boxclose_boxclose ()^ - 0.15 (T - t)^1/3 . W_{\text{m}} \propto ({\frac{{Q_{\text{g}} }}{{Q_{\text{l}} }}})^{ - 0.15} (T - t)^{1/3} . The velocity fields in the liquid phase around the thread, determined by μ-PIV measurements, were obtained around a forming bubble to reveal the role of the liquid phase.     

Microchannel emulsification for mass production of uniform fine droplets: integration of microchannel arrays on a chip

We present a novel microchannel emulsification (MCE) system for mass-producing uniform fine droplets. A 60 × 60-mm MCE chip made of single-crystal silicon has 14 microchannel (MC) arrays and 1.2 × 10⁴ MCs, and each MC array consists of many parallel MCs and a terrace. A holder with two inlet through-holes and one outlet through-hole was also developed for simply infusing each liquid and collecting emulsion products. The MCE chip was sealed well by physically attaching it to a flat glass plate in the holder during emulsification. Uniform fine droplets of soybean oil with an average diameter of 10 μm were reliably generated from all the MC arrays. The size of the resultant fine droplets was almost independent of the dispersed-phase flow rate below a critical value. The continuous-phase flow rate was unimportant for both the droplet generation and the droplet size. The MCE chip enabled mass-producing uniform fine droplets at 1.5 ml h⁻¹ and 1.9 × 10⁹ h⁻¹, which could be further increased using a dispersed phase of low viscosity.     

Design and simulation of a normally closed glucose sensitive hydrogel based microvalve

In drug delivery systems microvalves are the key components that have been developed for active control of drugs. In this research a normally closed microvalve with a glucose sensitive hydrogel actuating system is designed and simulated. Swelling of the hydrogel forces a silicone rubber membrane to deflect and causes the valve to be opened. The component of the valve that can be opened because of the hydrogel pressure is a silicon nitride cantilever beam which is sealed with a parylene layer. Simulations have been done by FEM analysis and the results show that membrane deflection is large enough to enable the valve to be opened and the fluid to flow through the microchannel. For both rectangular and trapezoidal microchannels with various hydraulic diameters, output flow rates less than 50 μl/min to several hundreds of μl/min can be achieved. Final design has been optimized for the opening point of microvalve at glucose concentration of 15 mM. Overall investigation has been done for a microvalve with specific dimensions and with 4 kPa input pressure the output flow rate of 100 μl/min has been generated which is in the desired range.     

Towards a continuous microfluidic rheometer

In a previous paper we presented a way to measure the rheological properties of complex fluids on a microfluidic chip (Guillot et al., Langmuir 22:6438, 2006). The principle of our method is to use parallel flows between two immiscible fluids as a pressure sensor. In fact, in a such flow, both fluids flow side by side and the size occupied by each fluid stream depends only on both flow rates and on both viscosities. We use this property to measure the viscosity of one fluid knowing the viscosity of the other one, both flow rates and the relative size of both streams in a cross-section. We showed that using a less viscous fluid as a reference fluid allows to define a mean shear rate with a low standard deviation in the other fluid. This method allows us to measure the flow curve of a fluid with less than 250 μL of fluid. In this paper we implement this principle in a fully automated set up which controls the flow rate, analyzes the picture and calculates the mean shear rate and the viscosity of the studied fluid. We present results obtained for Newtonian fluids and complex fluids using this set up and we compare our data with cone and plate rheometer measurements. By adding a mixing stage in the fluidic network we show how this set up can be used to characterize in a continuous way the evolution of the rheological properties as a function of the formulation composition. We illustrate this by measuring the rheological curve of four formulations of polyethylene oxide solution with only 1.3 mL of concentrated polyethylene oxide solution. This method could be very useful in screening processes where the viscosity range and the behavior of the fluid to an applied stress must be evaluated.     

Single-cell-based measurement of supraphysiological thermal injury in human carcinoma cells utilizing a micropatterned hydrogel chip

Hyperthermia affects certain regulatory proteins, kinases or cyclins, resulting in alternations to the cell cycle and even to apoptosis. Damage to the cell plasma membrane is a key factor in the killing of a cell by hyperthermia. Analysis at the single-cell level is necessary for understanding the fundamental mechanisms of hyperthermia-induced cell death and the generation of thermotolerance in surviving cells. Engineering approaches achieving precise control of cellular micropatterning provide the potential for investigating the mechanisms of thermal injury to cells at the single-cell level. The main purpose of this study is to fabricate a hydrogel chip with microwells for cellular patterning and to demonstrate the feasibility of measurement of supraphysiological thermal injury in human carcinoma cells (HeLa cells) at the single-cell level. To accomplish this, measurement of membrane injury by dye leakage post-thermal insult was performed and reported in this work. A hydrogel chip with microwells with different diameters was fabricated. For cell concentrations at 0.5 × 10⁶ cells/mL, the occupancy of cells on the microchip with 40 μm microwells was up to 86.6%, a value far higher than that found on the 30 μm microwells (approximately 78.5%). Most microwells of 30 μm in diameter (about 70%) were occupied by a single cell; hence, the hydrogel chip with 30 μm microwells was suitable for the applications of single-cell-based analysis. The fluorescent images showed that calcein leakage occurred when cell membranes were damaged under supraphysiological temperatures between 43 and 50°C. The normalized intensity of calcein decreased to 32% under a supraphysiological temperature of 43°C for 20 min. The intensity of calcein in cells was less than 20% under a supraphysiological temperature of 50°C. The feasibility of the single-cell-based experiment of thermal injury in the microchip with hydrogel microwells was therefore successfully demonstrated.     

Fabrication of a guide block for measuring a device with fine pitch area-arrayed solder bumps

This paper describes the design and fabrication of a guide block and micro probes, which were used for a vertical probe card to test a chip with area-arrayed solder bumps. The size of the fabricated guide block was 10 mm × 6 mm. The guide block consisted of 172 holes to insert micro probes, 2 guide holes for exact alignment, and 4 holes for bolting between the guide block and the housing of a PCB. Pitch and size of the inserting holes were 80 μm, and 90 μm × 30 μm, respectively. A silicon on insulator wafer was used as the substrate of the guide block to reduce micro probes insertion error. The micro probes were made of nickel–cobalt (Ni–Co) alloy using an electroplating method. The length and thickness of the micro probes were 910 and 20 μm, respectively. A vertical probe card assembled with the fabricated guide block and micro probes showed good x–y alignment and planarity errors within ±4 and ±3 μm, respectively. In addition, average leakage current and contact resistance were approximately 0.35 nA and 0.378 ohm, respectively. The proposed guide block and micro probes can be applied to a vertical probe card to test a chip with area-arrayed solder bumps.     

High-current density DC magenetohydrodynamics micropump with bubble isolation and release system

One of the major challenges for integrated Lab-on-a-Chip (LOC) systems is the precise control of fluid flow in a micro-flow cell. Magnetohydrodynamics (MHD) micropumps which contain no moving parts and capable of generating a continuous flow in any ionic fluid offer an ideal solution for biological applications. MHD micropumping has been demonstrated by using both AC and direct current (DC) currents by a number of researchers with varying degrees of success. However, current MHD designs based on DC do not meet the flow rate requirements for fully automated LOC applications (>100 μl/min). In this research, we introduce a novel DC-based MHD micropump which effectively increases flow rate by limiting the effects of electrolysis generated bubbles at the electrode–electrolyte interface through isolation and a mechanism for their release. Gas bubbles, particularly, hydrogen generated by high current density at the electrodes are the main culprit in low experimental flow rate compared with theoretical values. These tiny bubbles coalesce in the flow channel thus obstructing the flow of fluid. Since hydrolysis is inevitable with DC excitation, compartmentalized electrode channels with bubble isolating and coalescence retarding mechanisms and bubble release systems are implemented to prevent the coalescence of these bubbles and minimize their effects on flow rate. In this novel design called bubble isolation and release system, flow rate of up to 325 μl/min is achieved using 1 M NaCl solution in DC mode with potentials of 5 V and current density of about 5,000 A/m² for a main channel of 800 μm × 800 μm cross-section and 6.4 mm length.     

Viscosity-difference-induced asymmetric selective focusing for large stroke particle separation

We developed a new approach for particle separation by introducing viscosity difference of the sheath flows to form an asymmetric focusing of sample particle flow. This approach relies on the high-velocity gradient in the asymmetric focusing of the particle flow to generate a lift force, which plays a dominated role in the particle separation. The larger particles migrate away from the original streamline to the side of the higher relative velocity, while the smaller particles remain close to the streamline. Under high-viscosity (glycerol–water solution) and low-viscosity (PBS) sheath flows, a significant large stroke separation between the smaller (1.0 μm) and larger (9.9 μm) particles was achieved in a sample microfluidic device. We demonstrate that the flow rate and the viscosity difference of the sheath flows have an impact on the interval distance of the particle separation that affects the collected purity and on the focusing distribution of the smaller particles that affects the collected concentration. The interval distance of 293 μm (relative to the channel width: 0.281) and the focusing distribution of 112 μm (relative to the channel width: 0.107) were obtained in the 1042-μm-width separation area of the device. This separation method proposed in our work can potentially be applied to biological and medical applications due to the wide interval distance and the narrow focusing distribution of the particle separation, by easy manufacturing in a simple device.     

Rapid quantification of bio-particles based on image visualisation in a dielectrophoretic microfluidic chip

We studied an imaging-based technique for the rapid quantification of bio-particles in a dielectrophoretic (DEP) microfluidic chip. Label-free particles could be successively sorted and trapped in a continuous flow manner under the applied alternating current (AC) conditions. Both 2 and 3 μm polystyrene beads at a concentration of 1.0 × 10⁷ particles ml⁻¹ could be rapidly quantified within 5 min in our DEP system. Capturing efficiencies higher than 95% could be 2 μm polystyrene beads with a linear flow speed, applied voltage and frequency of 0.89 mm s⁻¹, 20 V_p-p and 5 MHz. Yeast cells (Candida glabrata and Candida albicans) could also be captured even at a lower concentration of 2.5 × 10⁵ cells ml⁻¹. Images of aggregative particles taken from the designed trapping area were further processed based on the intensity of relative greyscale followed by correction of the particle numbers. The imaging-based quantification method showed higher agreement than that of the conventional counting chamber method and proved the stability and feasibility of our AC DEP system.     

Pre-programmable polymer transformers as on-chip microfluidic vacuum generators

This paper develops novel polymer transformers using thermally actuated shape memory polymer (SMP) materials. This paper applies SMPs with thermally induced shape memory effect to the proposed novel polymer transformers as on-chip microfluidic vacuum generators. In this type of SMPs, the morphology of the materials changes when the temperature of materials reaches its glass transition temperature (T _g). The structure of the polymer transformer can be pre-programmed to define its functions, which the structure is reset to the temporary shape, using shape memory effects. When subjected to heat, the polymer transformer returns to its pre-memory morphology. The morphological change can produce a vacuum generation function in microfluidic channels. Vacuum pressure is generated to suck liquids into the microfluidic chip from fluidic inlets and drive liquids in the microchannel due to the morphological change of the polymer transformer. This study adopts a new smart polymer with high shape memory effects to achieve fluid movement using an on-chip vacuum generation source. Experimental measurements show that the polymer transformer, which uses SMP with a T _g of 40°C, can deform 310 μm (recover to the permanent shape from the temporary shape) within 40 s at 65°C. The polymer transformer with an effective cavity volume of 155 μl achieved negative pressures of −0.98 psi. The maximum negative up to −1.8 psi can be achieved with an effective cavity volume of 268 μl. A maximum flow rate of 24 μl/min was produced in the microfluidic chip with a 180 mm long channel using this technique. The response times of the polymer transformers presented here are within 36 s for driving liquids to the end of the detection chamber. The proposed design has the advantages of compact size, ease of fabrication and integration, ease of actuation, and on-demand negative pressure generation. Thus, this design is suitable for disposable biochips that need two liquid samples control. The polymer transformer presented in this study is applicable to numerous disposable microfluidic biochips.     

A microfluidic device for array patterning by perpendicular electrokinetic focusing

This paper describes a microfluidic chip in which two perpendicular laminar-flow streams can be operated to sequentially address the surface of a flow-chamber with semi-parallel sample streams. The sample streams can be controlled in position and width by the method of electrokinetic focusing. For this purpose, each of the two streams is sandwiched by two parallel sheath flow streams containing just a buffer solution. The streams are being electroosmotically pumped, allowing a simple chip design and a setup with no moving parts. Positioning of the streams was adjusted in real-time by controlling the applied voltages according to an analytical model. The perpendicular focusing gives rise to overlapping regions, which, by combinatorial (bio) chemistry, might be used for fabrication of spot arrays of immobilized proteins and other biomolecules. Since the patterning procedure is done in a closed, liquid filled flow-structure, array spots will never be exposed to air and are prevented from drying. With this device configuration, it was possible to visualize an array of 49 spots on a surface area of 1 mm². This article describes the principle, fabrication, experimental results, analytical modeling and numerical simulations of the microfluidic chip.     

Interfacing microfluidics to LDI-MS by automatic robotic spotting

We developed a method of interfacing microfluidics with mass spectrometry (MS) using a robotic spotting system to automate the contact spotting process. We demonstrate that direct and automated spotting of analyte from multichannel microfluidic chips to a custom microstructured MALDI target plate was a simple, robust, and high-throughput method for interfacing parallel microchannels using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Using thermoplastic cyclic olefin copolymer (COC) polymer microfluidic chips containing eight parallel 100 μm × 46 μm microchannels connected to a single input port, spotting volume repeatability and MALDI-MS signal uniformity are evaluated for a panel of sample peptides. The COC microfluidic chips were fabricated by hot embossing and solvent bonding techniques followed by chip dicing to create open ends for MS interfacing. Using the automatic robotic spotting approach, microfluidic chip-based reversed-phase liquid chromatography (RPLC) separations were interfaced with electrochemically etched nanofilament silicon (nSi) target substrate, demonstrating the potential of this approach toward chip-based microfluidic separation coupled with matrix-free laser desorption/ionization mass spectrometry.     

Particle streaming and separation using dielectrophoresis through discrete periodic microelectrode array

This study presents a particle manipulation and separation technique based on dielectrophoresis principle by employing an array of isosceles triangular microelectrodes on the bottom plate and a continuous electrode on the top plate. These electrodes generate non-uniform electric fields transversely across the microchannel. The particles within the flowing fluid experience a dielectrophoretic force perpendicular to the fluid flow direction due to the non-uniform electric fields. The isosceles triangular microelectrodes were designed to continuously exert a small dielectrophoretic force on the particles. Particles experiencing a larger dielectrophoretic force would move further in the perpendicular direction to the fluid flow as they traveled past each microelectrode. Polystyrene microspheres were used as the model particles, with particles of ∅20 μm employed for studying the basic characteristics of this technique. Particle separation was subsequently demonstrated on ∅10 and ∅15 μm microspheres. Using an applied sinusoidal voltage of 20 V_pp and frequency of 1 MHz, a mean separation distance of 0.765 mm between them was achieved at a flow rate of 3 μl/min (~1 mm/s), an important consideration for high throughput separation capability in a micro-scale technology device. This unique isosceles triangular microelectrodes design allows heterogeneous particle populations to be separated into multiple streams in a single continuous operation.