An immunoassay using an electro-microchip, nanogold probe and silver enhancement

This paper presents a novel immunoassay using an electro-microchip to detect the immuno-reaction signal, gold nanoparticles (ANPs) as a label of antigen or antibody and as a catalyst for silver precipitation, and the silver enhancement reaction to magnify the detection signal. This study is based on the direct immunoassay (two-layer format) and the sandwich immunoassay (three-layer format). The ANPs are introduced to the electro-microchip by the specific binding of the antibodies–ANPs conjugates and then coupled with silver enhancement to produce black spots of silver metal. The silver precipitation constructs a “bridge” between two electrodes of the electro-microchip allowing the electrons to pass, and the variation of the impedance can be easily measured with a commercial LCR meter. Different gap sizes (20, 50, 100, and 200 μm) of the electrodes of electro-microchips were designed for the sensitivity study. The experimental data show that a chip with a 200 μm gap has the highest sensitivity. There is a significant difference in impedance between the experiment and the negative control after 10 min reaction time. The proposed method requires less time and fewer steps than the conventional enzyme-linked immunosorbent assay. In addition, it shows a high detection sensitivity [10 μg/mL of 1st antibody (IgG) immobilized on slides and 10 ng/mL of antigen (protein A)], and there is a clear distinction between the signal intensity and the logarithm of the sample concentration. This new immunoassay has potential applications in proteomics research and clinical diagnosis.     

An integrated flow-cell for full sample stream control

In this study, we present a novel three-dimensional hydrodynamic sheath flow chip that allows full control of a sample stream. The chip offers the possibility to steer each of the four side sheath flows individually. The design of the flow-cell exhibits high flexibility in creating different sample stream profiles (width and height) and allows navigation of the sample stream to every desired position inside the microchannel (vertical and horizontal). This can be used to bring the sample stream to a sensing area for analysis, or to an area of actuation (e.g. for cell sorting). In addition, we studied the creation of very small sample stream diameters. In microchannels (typically 25 × 40 μm2), we created sample stream diameters that were five to ten times smaller than the channel dimensions, and the smallest measured sample stream width was 1.5 μm. Typical flow rates are 0.5 μl/min for the sample flow and around 100 μl/min for the cumulated sheath flows. The planar microfabricated chip, consisting of a silicon–glass sandwich with an intermediate SU-8 layer, is much smaller (6 × 9 mm2) than the previously presented sheath flow devices, which makes it also cost-effective. We present the chip design, fluidic simulation results and experiments, where the size, shape and position of the sample stream have been established by laser scanning confocal microscopy and dye intensity analysis.     

Aliquoting on the centrifugal microfluidic platform based on centrifugo-pneumatic valves

We present a new method for aliquoting liquids on the centrifugal microfluidic platform. Aliquoting is an essential unit operation to perform multiple parallel assays (“geometric multiplexing”) from one individual sample, such as genotyping by real-time polymerase chain reactions (PCR), or homogeneous immunoassay panels. Our method is a two-stage process with an initial metering phase and a subsequent transport phase initiated by switching a centrifugo-pneumatic valve. The method enables aliquoting liquids into completely separated reaction cavities. It includes precise metering that is independent on the volume of pre-stored reagents in the receiving cavities. It further excludes any cross-contamination between the receiving cavities. We characterized the performance for prototypes fabricated by three different technologies: micro-milling, thermoforming of foils, and injection molding. An initial volume of ~90 μl was split into 8 aliquots of 10 μl volume each plus a waste reservoir on a thermoformed foil disk resulting in a coefficient of variation (CV) of the metered volumes of 3.6%. A similar volume of ~105 μl was split into 16 aliquots of 6 μl volume each on micro-milled and injection-molded disks and the corresponding CVs were 2.8 and 2.2%, respectively. Thus, the compatibility of the novel aliquoting structure to the aforementioned prototyping and production technologies is demonstrated. Additionally, the important question of achievable volume precision of the aliquoting structure with respect to the production tolerances inherent to each of these production technologies is addressed experimentally and theoretically. The new method is amenable to low cost mass production, since it does not require any post-replication surface modifications like hydrophobic patches.     

Mixing and hydrodynamic analysis of a droplet in a planar serpentine micromixer

In this work we combined numerical simulation with molecular-diffusion effect, high-tempo micro-particle image velocimetry (μ-PIV), and probability distribution function (PDF) analysis to investigate the chaotic mixing and hydrodynamics inside a droplet moving through a planar serpentine micromixer (PSM). Robust solutions for the distributions of interface and concentration of the droplets were obtained via computational fluid dynamics. The simulated fluid patterns are consistent with those measured with μ-PIV, which serves as a powerful nonintrusive diagnostic approach to observe the droplets. Two mechanisms are proposed to enhance the performance of mixing in a PSM—the deformation of droplets and the asymmetric recirculation within the droplets. On introducing alternating cross sections into a winding channel, this specific design of PSM is found to amplify the fluid disturbance and maximum vorticity difference. Data show that the PDF of the vorticity fields is modified and the fraction with larger vorticity is increased. Accordingly, the PSM is capable of achieving a mixing index 90% within 700 μm (Re = 2), which is eight times better than for a straight microchannel. The results not only demonstrate explicitly the fluid patterns within the droplets but also provide significant insight into the factors dominating the mixing efficiency.     

Replication of precise polymeric microlens arrays combining ultra-precision diamond ball-end milling and micro injection molding

A simple and novel combination of ultra-precision diamond ball-end milling and micro injection molding technique is described to produce precise microlens arrays out of polycarbonate (PC), polymethylmethacrylate (PMMA) as well as polystyrene (PS). The microlens arrays consist of 100 lenses in a 10 × 10 array with a lens radius of 273 μm, a lens diameter of 300 μm and a lens depth of 45 μm. Pitch between the lenses is fixed at 800 μm. The injection molding parameters were optimized to get precise microlens geometries with low surface roughness. The results show a precise diamond milled mold insert and injection molded microlens arrays with minor deviations in radius and surface roughness of the microlenses, particularly for microlens arrays out of PMMA.     

Experiment and simulation of mixed flows in a trapezoidal microchannel

This paper presents experimental and numerical results of mixed electroosmotic and pressure driven flows in a trapezoidal shaped microchannel. A micro particle image velocimetry (μPIV) technique is utilized to acquire velocity profiles across the microchannel for pressure, electroosmotic and mixed electroosmotic-pressure driven flows. In mixed flow studies, both favorable and adverse pressure gradient cases are considered. Flow results obtained from the μPIV technique are compared with 3D numerical predictions, and an excellent agreement is obtained between them. In the numerical technique, the electric double layer is not resolved to avoid expensive computation, rather a slip velocity is assigned at the channel surface based on the electric field and electroosmotic mobility. This study shows that a trapezoidal microchannel provides a tapered-cosine velocity profile if there is any pressure gradient in the flow direction. This result is significantly different from that observed in rectangular microchannels. Our experimental results verify that velocity distribution in mixed flow can be decomposed into pressure and electroosmotic driven components.     

3D visualization of convection patterns in lab-on-chip with open microfluidic outlet

Open-outlet microfluidics is getting more and more attention, thanks to the generation of capillarity-driven flows which simplify the connection with the macro-world. It is known that convection flows are generated at the interface with air, i.e., the meniscus. Several works have investigated evaporation-induced convection, but its effect on particle position control in open-outlet biodevices is still not characterized. In this paper, we present the results of 3D measurement of particle traces near the meniscus in an open-outlet vertical 400 μm micro-channel filled with a water-based saline solution. Using a standard optical microscope and a system of mirrors, we observe the 3D position of individual micro-beads floating in the solution, in a way akin to particle image velocimetry technique. A single vortex is generated at the meniscus and occupies the whole region under observation at a distance of 1.5–2.7 mm from the meniscus. The generation of the convection pattern and the vortex rotational speed are described. The convection patterns disappear when evaporation is inhibited, while both the vortex generation and the rotational speed are faster for highly saline solutions. These results are relevant to the design of biochips which require control of the particle position in a fluid since they emphasize that in open-outlet microfluidic systems not only the gravitational fall but also the convection drag must be counteracted.     

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.     

In-situ fabrication of polymer microsieves for ��TAS by slanted angle holography

We present a simple, versatile method for the in-situ fabrication of membranes inside a microfluidic channel during a chip manufacturing process using only two extra slanted angle holographic exposure steps. This method combines the strengths of both inclined UV exposure and holographic lithography to produce micrometer-sized three-dimensional sieving structures. Using a common chip material, the photoresist material SU-8, together with this method, a leak-free membrane-channel connection is obtained. The resulting membranes are monodisperse, with a very well-defined pore geometry (i.e., microsieves with a pore diameter between 500 nm and 10 μm) that is easily controllable with the holographic set-up. The selectivity of in-situ fabricated microsieves with a pore diameter of 2 μm will be demonstrated using polystyrene beads of 1 and 3 μm.     

Microfluidic inverse phase ELISA via manipulation of magnetic beads

We report a new technique for conducting immuno-diagnostics on a microfluidic platform. Rather than handling fluid reagents against a stationary solid phase, the platform manipulates analyte-coated magnetic beads through stationary plugs of fluid reagents to detect an antigenic analyte. These isolated but accessible plugs are pre-encapsulated in a microchannel by capillary force. We call this platform microfluidic inverse phase enzyme-linked immunosorbent assay (μIPELISA). μIPELISA has distinctive advantages in the family of microfluidic immunoassay. In particular, it avoids pumping and valving fluid reagents during assaying, thus leading to a lab-on-a-chip format that is free of instrumentation for fluid actuation and control. We use μIPELISA to detect digoxigenin-labeled DNA segments amplified from E. coli O157:H7 by polymerase chain reaction (PCR), and compare its detection capability with that of microplate ELISA. For 0.259 ng μl⁻¹ of digoxigenin-labeled amplicon, μIPELISA is as responsive as the microplate ELISA. Also, we simultaneously conduct μIPELISA in two parallel microchannels.     

Decreasing microfluidic evaporation loss using the HMDL method: open systems for nucleic acid amplification and analysis

Evaporation is of great importance when dealing with microfluidic devices with open air/liquid interfaces due to the large surface-to-volume ratio. For devices utilizing a thermal reaction (TR) reservoir to perform a series of biological and chemical reactions, excessive heat-induced microfluidic evaporation can quickly lead to reaction reservoir dry out and failure of the overall device. In this study, we present a simple, novel method to decrease heat-induced fluid evaporation within microfluidic systems, which is termed as heat-mediated diffusion-limited (HMDL) method. This method does not need complicated thermal isolation to reduce the interfacial temperature, or external pure water to be added continuously to the TR chamber to compensate for evaporation loss. The principle of the HMDL method is to make use of the evaporated reaction content to increase the vapor concentration in the diffusion channel. The experimental results have shown that the relative evaporation loss (V _loss/V _ini) based on the HMDL method is not only dependent on the HMDL and TR region’s temperatures (T _HMDL and T _TR), but also on the HMDL and TR’s channel geometries. Using the U-shaped uniform channel with a diameter of 200 μm, the V _loss/V _ini within 60 min is low to 5% (T _HMDL = 105°C, T _TR = 95°C). The HMDL method can be used to design open microfluidic systems for nucleic acid amplification and analysis such as isothermal amplification and PCR thermocycling amplification, and a PCR process has been demonstrated by amplifying a 135-bp fragment from Listeria monocytogenes genomic DNA.     

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.     

Aerosol flow through a long micro-capillary: collimated aerosol beam

A micro-capillary system capable of generating a focused collimated aerosol beam (CAB) is demonstrated both theoretically and experimentally. The approach is based on a manifestation of the Saffman force where high velocity (∼100 m/s) aerosol particles, flowing through a micro-capillary (d ∼ 100 μm and l ∼ 1 cm), migrate perpendicular to the centerline of the capillary. Upon exiting the micro-capillary system, the particles maintain momentum, and when the aerosol is comprised of solid-in-liquid dispersions such as Ag nanoparticle ink, the CAB approach enables printing of advanced materials features with linewidth ≤ 10 μm.     

Chaotic mixing using source�Csink microfluidic flows in a PDMS chip

We present an active fixed-volume mixer based on the creation of multiple source–sink microfluidic flows in a polydimethylsiloxane (PDMS) chip without the need of external or internal pumps. To do so, four different pressure-controlled actuation chambers are arranged on top of the 5 μl volume of the mixing chamber. After the mixing volume is sealed/fixed by microfluidic valves made using ‘microplumbing technology’, a virtual source–sink pair is created by pressurizing one of the membranes and, at the same time, releasing the pressure of a neighboring one. The pressurized air deforms the thin membrane between the mixing and control chambers and creates microfluidic flows from the squeezed region (source) to the released region (sink) where the PDMS membrane is turned into the initial state. Several schemes of operation of virtual source–sink pairs are studied. In the optimized protocol, mixing is realized in just a sub-second time interval, thanks to the implementation of chaotic advection.     

Systematic linearisation of a microfluidic gradient network with unequal solution inlet viscosities demonstrated using glycerol

This paper presents a mathematical and experimental study of the effect of inlet concentration (and therefore viscosity) of glycerol solutions on the performance of a microfluidic network. This was achieved with analytical modelling, implemented in MATLAB, and optical measurement of the entire concentration distribution of the network. A mathematical proposal to improve the linearity of the outlet profile is also implemented and successfully verified experimentally. The concentration gradients of a two inlet–six outlet (2–6) microfluidic network device were obtained with inlet solutions of 10–40 wt% glycerol and flow rates of up to 5 μl/s per inlet. The mathematical model developed gave a good agreement with the experimental results obtained. ‘S’ shaped outlet profiles were obtained for the four glycerol cases studied and the closest results to the model were achieved at an optimised flow rate of 1μl/s for 10 wt% glycerol, 5 μl/s for both 20 and 30 wt% glycerol and 1.5 μl/s for 40 wt% glycerol. The linearity of the outlet profiles for the 20, 30 and 40 wt% inlet glycerol experiments were improved from R ² of 0.977, 0.946 and 0.966, respectively (before linearisation) to their new values of 0.997, 0.995 and 0.974, respectively (after the linearisation). This was performed by application of the mathematical model, at controlled inlet flow rate ratios of 0.77, 0.63 and 0.52 with respect to the viscous inlet, for 20, 30 and 40 wt% glycerol experiments, again with very good agreement of the outlet performance between the experimental and the mathematical results.     

Characterization of an electro-thermal micro gripper and tip sharpening using FIB technique

A micromachined electro-thermal gripper, first introduced by Ivanova et al. (Microelectron Eng 83:1393–1395, 2006), represents a promising candidate for the manipulation and handling of micro or even nano-scaled objects. To further optimize the performance of the device, a detailed electrical and mechanical characterization is needed. Due to the so-called duo-action gripper approach (i.e., a separate actuator for closing and opening action) these investigations focused on the maximum (minimum) opening width being 11.5 μm (3.3 μm), while in rest position a value of 4 μm is feasible. The maximum, electrical input power is limited to 80 mW/actuator element, resulting in a current density of up to 1.27 MA cm⁻² in the corresponding metal layers. When applying, however, larger current densities the probability of device failure increases substantially as in combination with an enhanced temperature of about 200°C electromigration effects occur in the metallization. Furthermore, the cut-off frequency and parasitic effects during actuation such as the z-deflection and the increase in length of each arm both showing values of up to 3 μm have been investigated as a function of operation parameters. Finally, the tips of the gripper were sharpened using Focused Ion Beam technique to a radius of less than 1 μm for gripping operations in space-restricted environments or for the manipulation or handling of sub-μm scaled objects.     

Local abstraction–refinement for the <Emphasis Type="Italic">μ</Emphasis>-calculus

A key technique for the verification of programs is counterexample-guided abstraction–refinement (CEGAR). Grumberg et al. (LNCS, vol 3385, pp. 233–249. Springer, Berlin, 2005; Inf Comput 205(8):1130–1148, 2007) developed a CEGAR-based algorithm for the modal μ-calculus. There, every abstract state is split in a refinement step. In this paper, the work of Grumberg et al. is generalized by presenting a new CEGAR-based algorithm for the μ-calculus. It is based on a more expressive abstract model and applies refinement only locally (at a single abstract state), i.e., the lazy abstraction technique for safety properties is adapted to the μ-calculus. Furthermore, it separates refinement determination from the (3-valued based) model checking. Three different heuristics for refinement determination are presented and illustrated.     

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.     

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.     

Use of a PDMS spring element for ink jet printing applications

Results of the design, microfabrication and testing of a proof-of-concept, diaphragm-type silicone sealing joint are presented. DRIE-etched cavities were filled with a flexible sealing element made of polydimethylsiloxane that supports a silicon piston. A series of sealing joints were produced with variable widths, and the displacement of the piston was measured after applying pressures of up to 1 bar above atmospheric pressure in 0.2 bar increments. Two masks were designed to produce several sets of silicone springs with widths of 2–10.5 μm, each consisting of a 10 μm thick silicon piston that is 2 mm long. Tests performed on the shear spring joints were found to give a displacement of 0.5 μm at 1 bar when the sealing width is 6 μm or more. The sealing joint with a 10 μm width was found to give a displacement of 0.9 μm and an elastic recovery of 88%. The results showed this type of joint in the form of an elastically-deforming seal provides sufficient displacement for propelling liquid droplets as part of a liquid propulsion system.