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This review shows the recent advances and state of the art in paper-based analytical devices (PADs) through the analysis of their integration with microfluidics and LOC micro- and nanotechnologies, electrochemical/optical detection and electronic devices as the convergence of various knowledge areas. The important role of the paper design/architecture in the improvement of the performance of sensor devices is discussed. The discussion is fundamentally based on μPADs as the new generation of paper-based (bio)sensors. Data about the scientific publication ranking of PADs, illustrating their increase as an experimental research topic in the past years, are supplied. In addition, an analysis of the simultaneous evolution of PADs in academic lab research and industrial commercialization highlighting the parallelism of the technological transfer from academia to industry is displayed. A general overview of the market behaviour, the leading industries in the sector and their commercialized devices is given. Finally, personal opinions of the authors about future perspectives and tendencies in the design and fabrication technology of PADs are disclosed.

As an emerging alternative to the conventional counterpart, surface microfluidics incorporates both intrinsic resistive solid-liquid and elastic frictionless gas-liquid interfaces, leading to unique flow-pressure characteristics. Furthermore, the open-surface microfluidic platforms can be fabricated on a monolithic substrate with high wettability contrast by the previously reported one-step lithographic process of a photosensitive superhydrophobic nanocomposite material, which permits flexible fluidic operations and direct surface modifications. In the paper, we first present three-dimensional microfluidic manipulations utilizing the unconventional gas-liquid interfaces of surface microfluidics, outlined by the micropatterned wetting boundaries (also known as the triple lines). In contrast to the primary linear (resistive) nature of the conventional closed-channel microfluidics, the distinct elastic interface of surface microfluidics enables remarkable three-dimensional (deformable) and time-dependent (capacitive) operations of the flow. Specifically, spatiotemporal dependence of microflow patterns on the planar fluidic surfaces has been theoretically analyzed and experimentally characterized. Utilizing the unconventional interface-enabled flow-pressure relationship, novel surface fluidic operations, including microflow regulation and flow-controlled switching, have been demonstrated and fully investigated. Furthermore, three-dimensional surface microfluidic networks together with analog-to-digital stereo-flow activations have been established, in which miniature capillary bridges form fluidic connections between two independent surface microfluidic circuits.

We present a method called "Lock Release Lithography (LRL)" that utilizes a combination of channel topography, mask design, and pressure-induced channel deformation to form and release particles in a cycled fashion. This technique provides a means for the high-throughput production of particles with complex 3D morphologies and composite particles with spatially configurable chemistries. In this work, we demonstrate a diverse set of functional particles including those displaying heterogeneous swelling characteristics and containing functional entities such as nucleic acids, proteins and beads.

Dielectrophoretic (DEP) droplet dispensing using dielectric-coated coplanar electrode structures provides an ideal platform for testing smart control systems for high-speed microfluidic devices. Open-loop control of DEP droplet dispensing is not sufficiently robust for precision droplet dispensing because unavoidable surface property variations of the substrates and other parameters such as liquid viscosity introduce uncertainty in the motion. Closed-loop systems employing distributed optical sensors and feedback provide flexibility, sensitivity, and reliability. In this new scheme, an array of distributed optical sensors detects fluid motion and, through a programmable control module, triggers application of AC voltage bursts of appropriate magnitude, duration, and frequency to control liquid motion and droplet formation. Reconfiguring the module connections and reprogramming the control module permits testing of a variety of control strategies.

A new method for catalyst deposition on the inner walls of capillary microreactors is proposed which allows exact control of the coating thickness, pore size of the support, metal particle size, and metal loading. The wall-coated microreactors have been tested in a selective hydrogenation reaction. Activity and selectivity reach values close to those obtained with a homogeneous Pd catalyst. The catalyst activity was stable for a period of 1000 h time-on-stream.

Microreactors fabricated with optically transparent inorganic polymers from two types of precursors using a UV-microimprinting process demonstrated reliable solvent resistance and capability for performing three model organic synthetic reactions, which were compared with batch systems and glass based microreactors.

Encapsulation of cells within picolitre-size monodisperse drops provides new means to perform quantitative biological studies on a single-cell basis for large cell populations. Variability in the number of cells per drop due to stochastic cell loading is a major barrier to these techniques. We overcome this limitation by evenly spacing cells as they travel within a high aspect-ratio microchannel; cells enter the drop generator with the frequency of drop formation.

We present a fully planar integrated optofluidic platform that permits single particle detection, manipulation and analysis on a chip. Liquid-core optical waveguides guide both light and fluids in the same volume. They are integrated with fluidic reservoirs and solid-core optical waveguides to define sub-picoliter excitation volumes and collect the optical signal, resulting in fully planar beam geometries. Single fluorescently labeled liposomes are used to demonstrate the capabilities of the optofluidic chip. Liposome motion is controlled electrically, and fluorescence correlation spectroscopy (FCS) is used to determine concentration and dynamic properties such as diffusion coefficient and velocity. This demonstration of fully planar particle analysis on a semiconductor chip may lead to a new class of planar optofluidics-based instruments.

This paper describes a novel technology-an electric field isolator (EFI)-that can be used for achieving isolated and electrophoretic manipulation of charged biomolecules inside a selected microscopic location. The EFI is a ground ring-shaped electrode (RE) surrounding a centre electrode (CE), which is comprised of a functional unit. When the CE is powered, the ground RE can inhibit the electric field from spreading to the neighbouring functional units. Therefore, the electrophoretic movement of the charged molecules in an electric field, which is based on the principle similar to that of electrophoresis, can be isolated inside a selected location. The ground RE causing this phenomenon is referred to as the EFI. In this paper, we clearly show the functionality of the EFI with mathematical and experimental studies.

A simple, fast, and inexpensive masking technology without any photolithographic step to produce glass microchannels is proposed in this work. This innovative process is based on the use of toner layers as mask for wet chemical etching. The layouts were projected in graphic software and printed on wax paper using a laser printer. The toner layer was thermally transferred from the paper to cleaned glass surfaces (microscope slides) at 130 degrees C for 2 min. After thermal transference, the glass channel was etched using 25% (v/v) hydrofluoric acid (HF) solution. The toner mask was then removed by cotton soaked in acetonitrile. The etching rate was approximately 7.1 +/- 0.6 microm min(-1). This process is economically more attractive than conventional methods because it does not require any sophisticated instrumentation and it can be implemented in any chemical/biochemical laboratory. The glass channel was thermally bonded against a flat glass cover and its analytical feasibility was investigated using capacitively coupled contactless conductivity detection (C(4)D) and laser-induced fluorescence (LIF) detection.

We report a microfluidic separation and sizing method of microparticles with hydrophoresis--the movement of suspended particles under the influence of a microstructure-induced pressure field. By exploiting slanted obstacles in a microchannel, we can generate a lateral pressure gradient so that microparticles can be deflected and arranged along the lateral flows induced by the gradient. Using such movements of particles, we completely separated polystyrene microbeads with 9 and 12 microm diameters. Also, we discriminated polystyrene microbeads with diameter differences of approximately 7.3%. Additionally, we measured the diameter of 10.4 microm beads with high coefficient of variation and compared the result with a conventional laser diffraction method. The slanted obstacle as a microfluidic control element in a microchannel is analogous to the electric, magnetic, optical, or acoustic counterparts in that their function is to generate a field gradient. Since our method is based on intrinsic pressure fields, we could eliminate the need for external potential fields to induce the movement of particles. Therefore, our hydrophoretic method will offer a new opportunity for power-free and biocompatible particle control within integrated microfluidic devices.

The utilization of microfluidic "lab-on-a-chip" devices in fundamental medical research, drug discovery and clinical diagnostics has rapidly increased in the past decade. Lab-on-a-chip devices process small volumes of analytes and reagents through on-chip microfluidic signal processing circuits. This paper discusses the implementation of a basic microfluidic circuit block, the concentration digital-to-analog converter (or C-DAC) which produces discretized chemical concentrations in a constant stream of solvent. The chemical concentration is controlled by a time-varying digital word; hence C-DACs are suitable for on-chip generation of arbitrary chemical signals. A 4-bit continuous-flow C-DAC was fabricated in two-level PDMS technology and tested. Several chemical waveforms (sawtooth, cosine, and ramp) were generated at flow rates of 2 microL min(-1) and frequencies of 0.6-4 mHz. The frequency cut off of this C-DAC was approximately 500 mHz.

A multi-component microfluidic electrochemical cell is shown to be a useful analytical tool for probing complex coupled processes in electrolytic systems. We recently reported an enzymatic signal amplification phenomenon that may provide increased sensitivity when detecting bio-analytes (M. S. Hasenbank, E. Fu and P. Yager, Langmuir, 2006, 22, 7451-7453), but to fully harness this method requires an improved understanding of the underlying electrochemical and chemical processes. We use spatial control of electrolyte streams on patterned conductive substrates in a microfluidic platform to elucidate the coupling of homogeneous chemical steps to heterogeneous electrochemical charge transfer processes. Because the gold surface was observable using SPR imaging, electrochemical phenomena could be monitored optically in real time. Based on these and additional results, we propose a mechanism for the novel amplification phenomenon that involves direct electron transfer between surface-immobilized enzyme molecules and the gold surface. This improved understanding of the underlying mechanism should enable the future implementation of this phenomenon in signal amplification schemes for highly sensitive lab-on-a-chip biosensors.

In situ liquid-phase photopolymerization (LP(3)) has been applied to the field of microfluidics to create components within integrated systems. As an extension of LP(3) technology, we present reconfigurable components that utilize the swelling of hydrogels. These components can be conveniently used to enhance microfluidic functions and applications. In order to utilize the swelling characteristic of hydrogels to the fullest, we demonstrate strategies to increase the swelling performance temporally and spatially. To this end, two successful applications using the reconfigurable components were tested: (1) active walls to block or divert flow at different steps in the fabrication or assay process, and (2) delivery pistons to move objects to specific locations within the microchannels after device fabrication.

In this work we present the formation of micrometre-sized lipid vesicles and tubes with perfectly homogeneous diameter and extraordinary length. The method is a novel approach for unconventional fabrication of soft-matter microstructured devices based on the combination of top-down and bottom-up fabrication processes. Photolithography techniques are applied to fabricate microsized apertures that provide the requirements to form lipid structures with predictable size and to align and guide the vesicles and tubes in microstructured channels. The formation is facilitated by self-assembly of polar lipids to a lipid membrane that is afterwards forced to undergo a shape transformation by extrusion through a microsized aperture. Both the geometrical restriction by the small aperture and the pressure difference between the top and bottom sides of the aperture determine the form and length of the vesicles and tubes. A strong pressure difference favors the formation of lipid tubes, while a low pressure difference results in the formation of vesicle bunches with spherical and cylindrical shapes. Potential applications for the formed lipid structures could be as microreactors and transport channels as well as in the construction of flexible microfluidic networks.

The ability to controllably and continuously stretch large DNA molecules in a microfluidic format is important for gene mapping technologies such as Direct Linear Analysis (DLA). We have recently shown that electric field gradients can be readily generated in a microfluidic device and the resulting field is purely elongational. We present a single molecule fluorescence microscopy analysis of T4 DNA (169 kbp) stretching in the electric field gradients in a hyperbolic contraction microchannel. In addition, we are able to selectively pattern a crosslinked gel anywhere inside the microchannel. With an applied electric field, DNA molecules are forced to reptate through the gel and they moderately stretch as they exit the gel. By placing a gel immediately in front of the hyperbolic contraction, we bypass "molecular individualism" and achieve highly uniform and complete stretching of T4 DNA.

Mixing in microscale flows with rotating chains of paramagnetic particles can be enhanced by adjusting the ratio of viscous to magnetic forces so that chains dynamically break and reform. Lattice Boltzmann (LB) simulations were used to calculate the interaction between the fluid and suspended paramagnetic particles under the influence of a rotating magnetic field. Fluid velocities obtained from the LB simulations are used to solve the advection diffusion equation for massless tracer particles. At relatively high Mason numbers, small chains result in low edge velocities, and hence mixing is slower than at other Mason numbers. At low Mason numbers, long, stable chains form and produce little mixing toward the center of the chains. A peak in mixing rate is observed when chains break and reform. The uniformity of mixing is greater at higher Mason numbers because more small chains result in a larger number of small mixing areas.

A honeycomb structure microchannel scrubber was developed to achieve efficient and stable gas collection. A thin porous membrane was pasted on a microchannel by the adhesive force of a fresh polydimethylsiloxane surface. The microchannel scrubber achieved much more efficient gas collection than conventional impingers and diffusion scrubbers. Two sets of the microchannel scrubbers and detectors were integrated in a 10 cm x 9 cm plastic board to create a micro gas analysis system (microGAS) for simultaneous measurements of H2S and SO2. The whole system including a battery was incorporated in a carrying case 34 cm W x 16 cm D x 17 cm H for use in the field. Liquid flows at 30 microl min(-1) were obtained by bimetal micropumps. The estimated detection limits were 0.1 ppbv for H2S and 1 ppbv for SO2. The system was demonstrated for real atmospheric gas analysis, and the results agreed well with data concurrently obtained by ion chromatography coupled with a cylindrical diffusion scrubber. The system we developed allowed automated continuous analyses in the field and achieved a much higher time resolution compared to those by ion chromatographic analysis.

A new lab-on-a-chip compatible binding assay platform is introduced. The platform combines dry-chemistry bioaffinity reagents and the recently introduced ArcDia TPX binding assay technique. The technique employs polymer microspheres as a solid phase reaction carrier, fluorescently labeled antibody conjugates, and detection of fluorescence emission from the surface of individual microspheres by two-photon excitation fluorescence. Signal response of the technique is independent of the reaction volume, thus the technique is particularly well suited for detection of bioaffinity reactions from miniature volumes. Performance of the new assay platform is studied by means of an immunometric assay of human alpha-fetoprotein (hAFP) in 384-plate format, and the results are compared to those of a corresponding wet-chemistry assay method. The results show that the ArcDia TPX detection technique can be combined with dry-chemistry reagents without compromises in assay performance. The microchip field has so far been characterized with a lack of microchip-compatible detection platforms which would allow cost-effective microchip design and sensitive bioaffinity detection. The presented detection technique is expected to provide a solution for this shortage.

Most recent developments in fluorescent molecular sensor devices are based on "ON-OFF" switching, which is an operation with a single measurable parameter, commonly the quenching of total intensity. In the meantime, with this approach self-calibration on the molecular level is not achievable. This calibration is strongly needed in all kinds of microscale applications, including microarrays, microfluidic systems and living cell imaging. Different possibilities are discussed for overcoming this difficulty and an "OR-OR" switching concept is suggested that involves the two-channel detection as a promising solution. For achieving the desired efficiency specific conditions are needed: it should be a single reporter dye exhibiting rapid reversible excited-state reaction and providing two-band wavelength ratiometric response.

As a first step towards a fully disposable stand-alone diagnostic microchip for determination of urinary human serum albumin (HSA), we report the use of a thin-film organic light emitting diode (OLED) as an excitation source for microscale fluorescence detection. The OLED has a peak emission wavelength of 540 nm, is simple to fabricate on flexible or rigid substrates, and operates at drive voltages below 10 V. In a fluorescence assay, HSA is reacted with Albumin Blue 580, generating a strong emission at 620 nm when excited with the OLED. Filter-less discrimination between excitation light and generated fluorescence is achieved through an orthogonal detection geometry. When the assay is performed in 800 microm deep and 800 microm wide microchannels on a poly(dimethylsiloxane)(PDMS) microchip at flow rates of 20 microL min(-1), HSA concentrations down to 10 mg L(-1) can be detected with a linear range from 10 to 100 mg L(-1). This sensitivity is sufficient for the determination of microalbuminuria (MAU), an increased urinary albumin excretion indicative of renal disease (clinical cut-off levels: 15-40 mg L(-1)).

A thin chip polymer-based microsprayer has been coupled to a hybrid quadrupole time-of-flight mass spectrometer (QTOF MS) and introduced in carbohydrate research. The feasibility of the approach is demonstrated for mapping, sequencing and structural elucidation of glycoconjugates originating from human body fluids and tissues such as a glycopeptide mixture from normal human urine and an isolated and purified GT1 ganglioside fraction from normal adult human brain. The optimization procedure required by each glycoconjugate category is described and the advantages of the system in terms of flexibility and adaptability to QTOF MS, stability of the ESI MS signal, carbohydrate ionization and sequencing, sensitivity, speed of analysis and sample consumption are discussed.

We report a Micro Throttle Pump (MTP) which has been shown to pump 5 microm diameter polystyrene beads at a concentration of 4.5 x 10(7) beads ml(-1). This new MTP design is constructed in a straightforward manner and actuated by a single piezoelectric (PZT) element. Maximum flow rates at 800 Hz drive frequency of 132 microl min(-1) with water and 108 microl min(-1) with a bead suspension were obtained. Maximum back-pressures of 6 kPa were observed in both cases. The reported MTP employs specific location of distinct internal microfluid structures cast in a single compliant elastomeric substrate to exploit the opposing directions of flexure of regions of a piezoelectric-glass composite bonded to the elastomer. By this novel means, distinct flexural regions, exhibiting compressive and tensile stresses respectively, allow both the pump's integrated input and output throttles and its pump chamber to be actuated concurrently by a single PZT. To support MTP design we also report the characterisation of an individual throttle's resistance as a function of actuator deflection and discuss the underlying mechanism of the throttling effect.

A microfluidic and optical system was created for the detection and analysis of single molecules in solution. Fluidic channels with submicrometer dimensions were used to isolate, detect and identify individual quantum dots conjugated with organic fluorophores. The channels were fabricated in fused silica with a 500 nm square cross section. The resulting focal volume of approximately 500 aL reduced fluorescent background and increased the signal to noise ratio of single molecule detection. The channels also enabled the rapid detection of 99% of quantum dots and organic fluorophores traversing the focal volume. Conjugates were driven through the channels electrokinetically at 2.3 kV cm(-1), excited with a single 476 nm wavelength laser and detected with a confocal microscope. Fluorescence emission was collected simultaneously from green (500-590 nm) and red (610-680 nm) regions of the spectrum. Signal rejection was minimized by the narrow and symmetric emission spectra of the quantum dots. To demonstrate efficient multicolor detection and characterization of single molecule binding, Qdot 655 Streptavidin Conjugates were bound to Alexa Fluor 488 molecules and individually detected. Photon counting histogram analysis was used to quantify coincident detection and degree of binding. Fluorescence correlation spectroscopy was used to measure the mobility of bound and unbound species. The union of fluidic channels with submicrometer dimensions and quantum dots as fluorescent labels resulted in efficient and rapid multiplexed single molecule detection and analysis.

We show that it is possible to use single layer soft lithography to create deformable polymer membranes within microfluidic chips for performing a variety of microfluidic operations. Single layer microfluidic chips were designed, fabricated, and characterized to demonstrate pumping, sorting, and mixing. Flow rates as high as 0.39 microl min(-1) were obtained by peristaltic pumping using pneumatically-actuated membrane devices. Sorting was attained via pneumatic actuation of membrane units placed alongside the branch channels. An active mixer was also demonstrated using single-layer deformable membrane units.

This paper describes a novel liquid separation technique for chembio extraction by an ultrasonic nanoliter-liquid-droplet ejector built on a PZT sheet. This technique extracts material from an aqueous two-phase system (ATPS) in a precise amount through digital control of the number of nanoliter droplets, without any mixing between the two liquids in the ATPS. The ultrasonic droplet ejector uses an acoustic streaming effect produced by an acoustic beam focused on the liquid surface, and ejects liquid droplets only from the liquid surface without disturbing most of the liquid below the surface. This unique characteristic of the focused acoustic beam is perfect (1) for separating a top-layer liquid (from the bulk of liquid) that contains particles of interest or (2) for recovering a top-layer liquid that has different phase from a bottom-layer liquid. Three kinds of liquid extraction are demonstrated with the ultrasonic droplet ejector: (1) 16 microl of top layer in Dextran-polyethylene glycol-water ATPS (aqueous two-phase system) is recovered within 20 s; (2) micron sized particles that float on water surface are ejected out with water droplets; and (3) oil layer on top of water is separated out.

In this paper the fabrication and electrical characterization of a silicon microreactor for high-temperature catalytic gas phase reactions, like Rh-catalyzed catalytic partial oxidation of methane into synthesis gas, is presented. The microreactor, realized with micromachining technologies, contains silicon nitride tubes that are suspended in a flow channel. These tubes contain metal thin films that heat the gas mixture in the channel and sense its temperature. The metal patterns are defined by using the channel geometry as a shadow mask. Furthermore, a new method to obtain Pt thin films with good adhesive properties, also at elevated temperatures, without adhesion metal is implemented in the fabrication process. Based on different experiments, it is concluded that the electrical behaviour at high temperatures of Pt thin films without adhesion layer is better than that of Pt/Ta films. Furthermore, it is found that the temperature coefficient of resistance (TCR) and the resistivity of the thin films are stable for up to tens of hours when the temperature-range during operation of the microreactor is below the so-called "burn-in" temperature. Experiments showed that the presented suspended-tube microreactors with heaters and temperature sensors of Pt thin films can be operated safely and in a stable way at temperatures up to 700 degrees C for over 20 h. This type of microreactor solves the electrical breakdown problem that was previously reported by us in flat-membrane microreactors that were operated at temperatures above 600 degrees C.

We use microfluidic chips to detect the biologically important cytokine tumor necrosis factor alpha (TNF- alpha) with picomolar sensitivity using sub-microliter volumes of samples and reagents. The chips comprise a number of independent capillary systems (CSs), each of which is composed of a filling port, an appended microchannel, and a capillary pump. Each CS fills spontaneously by capillary forces and includes a self-regulating mechanism that prevents adventitious drainage of the microchannels. Thus, interactive control of the flow in each CS is easily achieved via collective control of the evaporation in all CSs by means of two Peltier elements that can independently heat and cool. Long incubation times are crucial for high sensitivity assays and can be conveniently obtained by adjusting the evaporation rate to have low flow rates of approximately 30 nL min(-1). The assay is a sandwich fluorescence immunoassay and takes place on the surface of a poly(dimethylsiloxane)(PDMS) slab placed across the microchannels. We precoat PDMS with capture antibodies (Abs), localize the capture of analyte molecules using a chip, then bind the captured analyte molecules with fluorescently-tagged detection Abs using a second chip. The assay results in a mosaic of fluorescence signals on the PDMS surface which are measured using a fluorescence scanner. We show that PDMS is a compatible material for high sensitivity fluorescence assays, provided that detection antibodies with long excitation wavelength fluorophores ( > or =580 nm) are employed. The chip design, long incubation times, proper choice of fluorophores, and optimization of the detection Ab concentration all combine to achieve high-sensitivity assays. This is exemplified by an experiment with 170 assay sites, occupying an area of approximately 0.6 mm(2) on PDMS to detect TNF-alpha in 600 nL of a dendritic cell (DC) culture medium with a sensitivity of approximately 20 pg mL(-1)(1.14 pM).

Carboxylic esters were successfully labeled with one of two short-lived positron-emitters, carbon-11 or fluorine-18, within a hydrodynamically-driven micro-reactor. The non-radioactive methyl ester was obtained at room temperature; its yield increased with higher substrate concentration and with reduced infusion rate. Radioactive methyl ester was obtained from the reaction of (10 mM) with in 56% decay-corrected radiochemical yield (RCY) at an infusion rate of 10 microL min(-1), and when the infusion rate was reduced to 1 microL min(-1), the RCY increased to 88%. The synthesis of the non-radioactive fluoroethyl ester from and required heating of the micro-reactor on a heating block at 80 degrees C (14-17% RCY), whilst the corresponding radioactive from and was obtained in 10% RCY. The radioactive 'peripheral' benzodiazepine receptor ligand was obtained from the reaction of acid with labeling agent in 45% RCY at an infusion rate of 10 microL min(-1). When the infusion rate was reduced to 1 microL min(-1), the RCY increased to 65%. The results exemplify a new methodology for producing radiotracers for imaging with positron emission tomography that has many potential advantages, including a requirement for small quantities of substrates, enhanced reaction, rapid reaction optimisation and easy product purification.

A microfluidic apparatus capable of creating continuous microscale cylindrical polymeric structures has been developed. This system is able to produce microstructures (e.g. fibers, tubes) by employing 3D multiple stream laminar flow and "on the fly"in-situ photopolymerization. The details of the fabrication process and the characterization of the produced microfibers are described. The apparatus is constructed by merging pulled glass pipettes with PDMS molding technology and used to manufacture the fibers and tubes. By controlling the sample and sheath volume flow rates, the dimensions of the microstructures produced can be altered without re-tooling. The fiber properties including elasticity, stimuli responsiveness, and biosensing are characterized. Responsive woven fabric and biosensing fibers are demonstrated. The fabrication process is simple, cost effective and flexible in materials, geometries, and scales.

We report on a novel technique of resonant frequency shift measurement based on a nanomechanical cantilever with a PZT actuating layer for label-free detection of a prostate-specific antigen (PSA) in a liquid environment. The nanomechanical PZT thin film cantilever is composed of SiO(2)/Ta/Pt/PZT/Pt/SiO(2) on a SiN(x) supporting layer for simultaneous self-exciting and sensing; it was fabricated using a standard MEMS (micro electromechanical system) process. The specific binding characteristics of the PSA antigen to its antibody, which is immobilized with Calixcrown self-assembled monolayers (SAMs) on a gold surface deposited on a cantilever, are determined to a high sensitivity. For the bioassay in a liquid environment, a liquid test cell with a 20 microl volume reaction chamber has been fabricated, using a bonding technique between poly(dimethyl siloxane) (PDMS) bilayers. An observed trend of resonant frequency change with respect to time could be explained by the binding kinetics due to the Langmuir isotherm and diffusion and by the effects of a small volume reaction chamber. In the saturated regimes, the resonant frequency of the cantilever increased with increase of the PSA concentration in the reaction chamber, showing that the trend of the resonance frequency change was similar to that of the fluorescence results. The saturated resonance frequency shift of the cantilever was proportional to the PSA antigen concentration of analyte solution.

This paper presents a system-oriented model for analyzing the dispersion of electrophoretic transport of charged analyte molecules in a general-shaped microchannel, which is represented as a system of serially connected elemental channels of simple geometry. Parameterized analytical models that hold for analyte bands of virtually arbitrary initial shape are derived to describe analyte dispersion, including both the skew and broadening of the band, in elemental channels. These models are then integrated to describe dispersion in the general-shaped channel using appropriate parameters to represent interfaces of adjacent elements. This lumped-parameter system model offers orders-of-magnitude improvement in computational efficiency over full numerical simulations, and is verified by results from experiments and numerical simulations. The model is used to perform a systematic parametric study of serpentine channels consisting of a pair of complementary turn microchannels, and the results indicate that dispersion in a particular turn can contribute to either an increase or decrease of the overall band broadening. The efficiency and accuracy of the system model is further demonstrated by its application to general-shaped channels that occur in practice, including a serpentine channel with multiple complementary turns and a multi-turn spiral-shaped channel. The results indicate that our model is an accurate and efficient simulation tool useful for designing optimal electrophoretic separation microchips.

The progressive development of a micro-fluidic manifold for the chemiluminescent detection of copper in water samples, based on the measurement of light emitted from the Cu(ii) catalysed oxidation of 1,10-phenanthroline by hydrogen peroxide, is reported. Micro-fluidic manifolds were designed and manufactured from polymethylmethacrylate (PMMA) using three micro-fabrication techniques, namely hot embossing, laser ablation and direct micro-milling. The final laser ablated design incorporated a reagent mixing channel of dimensions 7.3 cm in length and 250 x 250 microm in width and depth (triangular cross section), and a detection channel of 2.1 cm in length and 250 x 250 microm in width and depth (total approx. volume of between 16 to 22 microL). Optimised reagents conditions were found to be 0.07 mM 1,10-phenanthroline, containing 0.10 M cetyltrimethylammonium bromide and 0.075 M sodium hydroxide (reagent 1 delivered at 0.025 mL min(-1)) and 5% hydrogen peroxide (reagent 2 delivered at 0.025 mL min(-1)). The sample stream was mixed with reagent 1 in the mixing channel and subsequently mixed with reagent 2 at the start of the detection channel. The laser ablated manifold was found to give a linear response (R(2) = 0.998) over the concentration ranges 0-150 microg L(-1) and be reproducible (% RSD = 3.4 for five repeat injections of a 75 microg L(-1) std). Detection limits for Cu(ii) were found to be 20 microg L(-1). Selectivity was investigated using a copper selective mini-chelating column, which showed common cations found in drinking waters did not cause interference with the detection of Cu(ii). Finally the optimised system was successfully used for trace Cu(ii) determinations in a standard reference freshwater sample (SRM 1640).

Electrically controlled droplet-based labs-on-a-chip operate under the principles of electro-capillarity and dielectrophoresis. The microfluidic mechanics of manipulating electrified droplets are complex and not entirely understood. In this article, we analyse these operating principles, especially electrowetting on dielectric (a form of electro-capillarity) and dielectrophoresis, under a unified framework of droplet electrohydrodynamics. We differentiate them by their electric origins and their energy transduction mechanisms. Our study shows that both electrowetting on dielectric and dielectrophoresis are effective for droplet generation and manipulation. In addition, our study demonstrates: (1) the presence of a wetting contribution to dielectrophoresis; and (2) contact angle reduction is merely an observable consequence of, not a condition for, the occurrence of electrowetting on dielectric. Simulations are used extensively in this article to illustrate device operation, to expose underlying physics, and to validate our conclusions. Simulations of electrically driven droplet generation, droplet translocation, droplet fusion, and droplet fission are presented.

Clinical diagnostics is one of the most promising applications for microfluidic lab-on-a-chip systems, especially in a point-of-care setting. Conventional microfluidic devices are usually based on continuous-flow in microchannels, and offer little flexibility in terms of reconfigurability and scalability. Handling of real physiological samples has also been a major challenge in these devices. We present an alternative paradigm--a fully integrated and reconfigurable droplet-based "digital" microfluidic lab-on-a-chip for clinical diagnostics on human physiological fluids. The microdroplets, which act as solution-phase reaction chambers, are manipulated using the electrowetting effect. Reliable and repeatable high-speed transport of microdroplets of human whole blood, serum, plasma, urine, saliva, sweat and tear, is demonstrated to establish the basic compatibility of these physiological fluids with the electrowetting platform. We further performed a colorimetric enzymatic glucose assay on serum, plasma, urine, and saliva, to show the feasibility of performing bioassays on real samples in our system. The concentrations obtained compare well with those obtained using a reference method, except for urine, where there is a significant difference due to interference by uric acid. A lab-on-a-chip architecture, integrating previously developed digital microfluidic components, is proposed for integrated and automated analysis of multiple analytes on a monolithic device. The lab-on-a-chip integrates sample injection, on-chip reservoirs, droplet formation structures, fluidic pathways, mixing areas and optical detection sites, on the same substrate. The pipelined operation of two glucose assays is shown on a prototype digital microfluidic lab-on-chip, as a proof-of-concept.

Laser techniques were applied to an acoustically levitated droplet for remote investigation of the diameter, species concentration and temperature of the suspended droplet. To this end, the third and the fourth harmonic of a Nd:YAG laser were used for investigation of elastic, fluorescence and phosphorescence signals from the droplet. The droplet was seeded with thermographic phosphors and acetone for the phosphorescence and fluorescence measurements, respectively. The techniques were applied simultaneously using an imaging stereoscope. The imaging device allowed for an identical visualization of incoming signal through separate optical filters. Temperature measurements in droplets is important in the study of e.g. exothermic chemical reactions, spray processes, combustion, and in bioanalytical applications where the biological material is temperature sensitive or dependent on optimal temperature for function. Results from these investigations showed that temperature measurements in acoustically levitated droplets using laser-induced phosphorescence are feasible. The results also show the potential of simultaneous laser based measurements on levitated droplets. Diameter variation (surface area), mixture concentration and temperature were measured simultaneously.

A three-dimensional microchannel network with two-level crossings of channels was constructed in a glass microchip by sandwiching an insulating glass plate between two glass plates with microchannels followed by thermal bonding. Pressure-driven stable multi-phase laminar flows inside the three-dimensional channel network were realized by balancing flow rates of syringe pumps. Micro unit operations for mixing, reaction, solvent extraction, and detection were properly arranged in the multi-phase laminar flows, so that four parallel analyses, comprising twenty unit operations in total, could be integrated onto a single chip. Two chelating reagents and two sample solutions containing heavy metal ions (Fe(ii) or Co(ii)) were mixed and reacted in four different combinations using the three-dimensional channel network. After chelating reactions were completed, post processing (solvent extraction or addition of acid) was applied to each solution stream to remove the interferences of coexisting metal ions. Finally, target metal complexes were detected using a thermal lens microscope (TLM). Integrity of the micro system was confirmed by qualitative analysis of Fe(ii) and Co(ii). This is the first example of continuous flow chemical processing utilizing multi-phase laminar flow realized in a three-dimensional channel network.

This study presents a novel method for rapid prototyping of polymer microsystems. The method is based on excimer laser ablation of a thermally and mechanically stable polymer, such as PEEK (poly-ether-ether-ketone). A negative of the desired microsystem is laser machined in PEEK, which can then be used directly for hot embossing or injection moulding of a series of prototypes. This approach is very rapid and considerably cheaper than more traditional approaches to toolmaking, while still performing well in terms of reproduction of tool dimensions. The reduction in time and cost for a master tool using this method opens up new possibilities for testing small series in the R&D phase of a microsystem. Finally, two particular applications of the technique are presented.

The high surface to volume ratio of microchannel components offers many advantages in micro chemical engineering. It is obvious, however, that the reactor material and corrosion phenomena play an important role when applying these components. For chemical reactions at very high temperatures or/and with corrosive reactants involved, microchannel components made of metals or polymers are not suited. Hence, a modular microreactor system made of alumina was developed and fabricated using a rapid prototyping process chain. With exchangeable inserts the system can be adapted to the requirements of various reactions. Two heterogeneously catalysed gas-phase reactions (oxidative coupling of methane, isoprene selective oxidation to citraconic anhydride) were investigated to check the suitability of the system at temperatures of up to 1000 degrees C. Apart from the high thermal and chemical resistance, the lack of any blind activity was found to be another advantage of ceramic components.

Flow rates of up to 50 microm s(-1) have been successfully achieved in a closed-loop channel using an AC electroosmotic pump. The AC electroosmotic pump is made of an interdigitated array of unequal width electrodes located at the bottom of a channel, with an AC voltage applied between the small and the large electrodes. The flow rate was found to increase linearly with the applied voltage and to decrease linearly with the applied frequency. The pump is expected to be suitable for circular chromatography for the following reasons: the driving forces are distributed over the channel length and the pumping direction is set by the direction of the interdigitated electrodes. Pumping in a closed-loop channel can be achieved by arranging the electrode pattern in a circle. In addition the inherent working principle of AC electroosmotic pumping enables the independent optimisation of the channel height or the flow velocity.

In spite of considerable efforts, flow control in micro-channels remains a challenge owing to the very small ratio of channel/supply-system volumes, as well as the induction of spurious flows by extremely small pressure or geometry changes. We present here an inexpensive and robust system for flow control in a microchannel system, based on a dynamic control of reservoir pressures at the end of each channel. This system allows flow equilibration with a time constant smaller than one second, and is also able to maintain stable flux from stopped flow to many microl min(-1) range over several hours. It is robust to changes in ambient pressure and temperature. This system further includes a feature for sub-microliter sample injection during the experiment. We quantify flow control in elastomer and thermoplastic channels, and demonstrate the impact on one application of the system, namely the reproducible, automated separation of large DNA by electrophoresis in a self-organized magnetic bead matrix in a microchannel.

We use micro particle image velocimetry (microPIV) and fluorescence microscopy techniques to characterize microscale segmented gas-liquid flow at low superficial velocities relevant for chemical reactions with residence times of up to several minutes. Different gas-liquid microfluidic channel networks of rectangular cross section are fabricated in poly(dimethylsiloxane) (PDMS) using soft lithography techniques. The recirculation motion in the liquid segments associated with gas-liquid flows as well as the symmetry characteristics of the recirculations are quantified for straight and meandering channel networks. Even minor surface roughness effects and the compressibility of the gas phase induce loss of symmetry and enhance mixing across the centerline in straight channels. Mixing is further accelerated in meandering channels by the periodic switching of recirculation patterns across the channel center. We demonstrate a new, piezoelectrically activated flow injection technique for determining residence time distributions (RTDs) of fluid elements in multiphase microfluidic systems. The results confirm a narrowed liquid phase RTD in segmented flows in comparison to their single-phase counterparts. The enhanced mixing and narrow RTD characteristics of segmented gas-liquid flows are applied to liquid mixing and in sol-gel synthesis of colloidal nanoparticles.

We propose a novel technique for patterning active proteins on a glass substrate using a perforated polydimethylsiloxane (PDMS) sheet-sieve. The sieve, which has tapering holes, is fabricated by spin-coating PDMS on a pyramidal-shaped mold. By means of this sieve, FITC (fluorescent isothiocyanate, bovine)-albumin was successfully spotted in a 5 x 5 microm(2) area in an array. The patterned spots were perfectly isolated, which eliminates the problem of non-specific binding of proteins to undesired areas. To show that proteins maintained their activity after the patterning, we used F(1)-ATPase biomolecular motors; their activity can easily be verified by observing their rotary motion after patterning. Selective patterning with three kinds of fluorescent micro beads indicated the possibility of patterning of different proteins on the same substrate by using the sieve.

We have devised a microfluidic mixer design that produces all the mixture combinations of a given number of dilutions of the input compounds. As proof of the concept, we present a device that generates four titrations of two dye solutions, blue and yellow, and combinatorially mixes the blue titrations with the yellow titrations to deliver the sixteen mixture combinations in separate outlet microchannels. Our device features four different flow levels made by stacking nine laser-cut Mylar laminates. The fluidic network has a symmetric design that guarantees that the flow rates are the same at all the outlets, with deviations attributable to imperfections in the fabrication, assembly, or perfusion processes. Design rules for scaling up the number of compounds and/or dilutions are presented. The mixing scheme has broad applicability in high-throughput combinatorial testing applications such as drug screening, cell-based biochemical assays, lab-on-a-chip devices, and biosensors.

A hybrid multiplex microarray microsystem has been developed that consists of 32 individually addressable array reaction chambers, supporting the use of multichannel pipettes for addition of up to 8 samples simultaneously. Discrimination between Campylobacter jejuni and Campylobacter coli bacteria was observed in DNA samples containing Campylobacter spp., with the same specificity and sensitivity as when compared to a full-size microarray. The spinloaded multiplex microarray microsystem described provides a novel and convenient test format for simultaneous low-density microarray analysis and is universally adaptable to other DNA, protein or small molecule microarray based applications.

Simulation and experimental demonstration of the in vitro gene delivery enhancement using electrostatic forces and electroporation (EP) microchips were conducted. Electroporation is a technique with which DNA molecules can be delivered into cells using electric field pulses. This study demonstrates that plasmid DNA can be attracted to the cell surfaces at the specific regions using an electrostatic force. Therefore, the DNA concentration on the cell surface is dramatically increased, which highly enhances the gene transfection efficiency compared to that without an attracting-electric field. The electrostatic force can be designed into specific regions, where the DNA plasmids are attracted to, to provide the region-targeting function. In this micro-device, the top electrode and the interdigitated electrodes provided the DNA attracting-electric field, and the interdigitated electrodes provided adequate electric fields for the electroporation process on the chip surface. Using the EP microchip, cells could be manipulated in situ without detachment if adherent cells were used for electroporation. Five different cells of two different types, primary cell and cell line, were successfully transfected under multi-pulse or single pulse electric field stimulation without applying an attracting-electric field. This study simulated and analyzed the electric field distributions at the DNA attracting and electroporation processes, and successfully demonstrated that the electrostatic force attracted DNA plasmids to specific regions and highly enhanced the gene delivery. In summary, this EP microchip should provide many potential applications for gene therapy.

We report the use of a thin-film polymer light emitting diode as an integrated excitation source for microfabricated capillary electrophoresis. The polyfluorene-based diode has a peak emission wavelength of 488 nm, an active area of 40 microm x 1000 microm and a thickness of similar 2 mm. The simple layer-by-layer deposition procedures used to fabricate the polymer component allow facile integration with planar chip-based systems. To demonstrate the efficacy of the approach, the polyfluorene diode is used as an excitation source for the detection of fluorescent dyes separated on-chip by electrophoresis. Using a conventional confocal detection system the integrated pLED is successfully used to detect fluorescein and 5-carboxyfluorescein at concentrations as low as 10(-6) M with a mass detection limit of 50 femtomoles. The drive voltages required to generate sufficient emission from the polymer diode device are as low as 3.7 V.

Diagnostic PCR has been used to analyse a wide range of biological materials. Conventional PCR consists of several steps such as sample preparation, template purification, and PCR amplification. PCR is often inhibited by contamination of DNA templates. To increase the sensitivity of the PCR, the removal of PCR inhibitors in sample preparation steps is essential and several methods have been published. The methods are either chemical or based on filtering. Conventional ways of filtering include mechanical filters or washing e.g. by centrifugation. Another way of filtering is the use of electric fields. It has been shown that a cell will experience a force when an inhomogeneous electric field is applied. The effect is called dielectrophoresis (DEP). The resulting force depends on the difference between the internal properties of the cell and the surrounding fluid. DEP has been applied to manipulate cells in many microstructures. In this study, we used DEP as a selective filter for holding cells in a microsystem while the PCR inhibitors were flushed out of the system. Haemoglobin and heparin - natural components of blood - were selected as PCR inhibitors, since the inhibitory effects of these components to PCR have been well documented. The usefulness of DEP in a microsystem to withhold baker's yeast (Saccharomyces cerevisiae) cells while the PCR inhibitors haemoglobin and heparin are removed will be presented and factors that influence the effect of DEP in the microsystem will be discussed. This is the first time dielectrophoresis has been used as a selective filter for removing PCR inhibitors in a microsystem.

Microfluidic devices are a promising new tool for studying and optimizing (bio)chemical reactions and analyses. Many (bio)chemical reactions require accurate temperature control, such as for example thermocycling for PCR. Here, a new integrated temperature control system for microfluidic devices is presented, using chemical and physical processes to locally regulate temperature. In demonstration experiments, the evaporation of acetone was used as an endothermic process to cool a microchannel. Additionally, heating of a microchannel was achieved by dissolution of concentrated sulfuric acid in water as an exothermic process. Localization of the contact area of two flows in a microfluidic channel allows control of the position and the magnitude of the thermal effect.