Reversible Stream Drop Transition in a Microfluidic Coflow System via On Demand Exposure to Acoustic Standing Waves.

Transition between stream and droplet regimes in a coflow is typically achieved by adjusting the capillary numbers (Ca) of the phases. Remarkably, we experimentally evidence a reversible transition between the two regimes by controlling exposure of the system to acoustic standing waves, with Ca fixed. By satisfying the ratio of acoustic radiation force to the interfacial tension force, Ca_{ac}>1, experiments reveal a reversible stream drop transition for Ca<1, and stream relocation for Ca≥1. We explain the phenomenon in terms of the pinching, advection, and relocation timescales and a transition between convective and absolute instability from a linear stability analysis [P. Guillot et al., Phys. Rev. Lett. 99, 104502 (2007)PRLTAO0031-900710.1103/PhysRevLett.99.104502].

Acoustic particle trapping driven by axial primary radiation force in shaped traps.

We study particle trapping driven by the axial primary radiation force (A-PRF) in shaped traps exposed to standing bulk acoustic waves (S-BAW) using numerical simulations and experiments. The utilization of the stronger A-PRF as the main retention force is a consequence of standing-wave formation along the flow direction, instead of the orthogonal direction as in the case of traditionally used lateral-PRF S-BAW trapping setups. The study of particle dynamics reveals that the competition between A-PRF and viscous drag force governs particle trajectory. The ratio of the acoustic energy to the viscous work (ß) provides a general criterion for particle trapping at a distinctive off-node site that is spatially controllable. Particles get trapped for ß≥ß_{cr} at some distance away from the nodal plane and the distance varies as ß^{-c} (c=0.6-1.0). The use of A-PRF as the retention force could potentially allow traditional S-BAW trapping systems to envisage high-throughput advancements surpassing the current standards in cell-handling unit operations.

Acoustic resonances in straight micro channels: beyond the 1D-approximation.

Acoustic actuation can be used to perform several tasks in microfluidic systems. In this paper, we investigate an acoustic separator through micro-PIV analysis in stop-flow mode and numerical simulations, and a good agreement between the two is found. Moreover, we demonstrate that it is not sufficient only to characterize devices in flow-through mode, since in these systems much different resonant patterns can result in similarly looking band formations. Furthermore, we conclude that extended 1D approximations of the acoustic radiation force are inadvisable, and instead, a 2D model is preferred. The results presented here provide valuable insight into the nature and functionality of acoustic microdevices, and should be useful in the interpretation and understanding of the same.

Myoelectric control of a computer animated hand: a new concept based on the combined use of a tree-structured artificial neural network and a data glove.

This paper proposes a new learning set-up in the field of control systems for multifunctional hand prostheses. Two male subjects with a traumatic one-hand amputation performed simultaneous symmetric movements with the healthy and the phantom hand. A data glove on the healthy hand was used as a reference to train the system to perform natural movements. Instead of a physical prosthesis with limited degrees of freedom, a virtual (computer-animated) hand was used as the target tool. Both subjects successfully performed seven different motoric actions with the fingers and wrist. To reduce the training time for the system, a tree-structured, self-organizing, artificial neural network was designed. The training time never exceeded 30 seconds for any of the configurations used, which is three to four times faster than most currently used artificial neural network (ANN) architectures.

Classification of motor commands using a modified self-organising feature map.

In this paper, a control system for an advanced prosthesis is proposed and has been investigated in two different biological systems: (1) the spinal withdrawal reflex system of a rat and (2) voluntary movements in two human males: one normal subject and one subject with a traumatic hand amputation. The small-animal system was used as a model system to test different processing methods for the prosthetic control system. The best methods were then validated in the human set-up. The recorded EMGs were classified using different ANN algorithms, and it was found that a modified self-organising feature map (SOFM) composed of a combination of a Kohonen network and the conscience mechanism algorithm (KNC) was superior in performance to the reference networks (e.g. multi-layer perceptrons) as regards training time, low memory consumption, and simplicity in finding optimal training parameters and architecture. The KNC network classified both experimental set-ups with high accuracy, including five movements for the animal set-up and seven for the human set-up.

Protein chips based on recombinant antibody fragments: a highly sensitive approach as detected by mass spectrometry.

With the human genome in a first sequence draft and several other genomes being finished this year, the existing information gap between genomics and proteomics is becoming increasingly evident. The analysis of the proteome is, however, much more complicated because the synthesis and structural requirements of functional proteins are different from the easily handled oligonucleotides, for which a first analytical breakthrough already has come in the use of DNA chips. In comparison with the DNA microarrays, the protein arrays, or protein chips, offer the distinct possibility of developing a rapid global analysis of the entire proteome. Thus, the concept of comparing proteomic maps of healthy and diseased cells may allow us to understand cell signaling and metabolic pathways and will form a novel base for pharmaceutical companies to develop future therapeutics much more rapidly. This report demonstrates the possibilities of designing protein chips based on specially constructed, small recombinant antibody fragments using nano-structure surfaces with biocompatible characteristics, resulting in sensitive detection in the 600-amol range. The assay readout allows the determination of single or multiple antigen-antibody interactions. Mass identity of the antigens, currently with a resolution of 8000, enables the detection of structural modifications of single proteins.

The geometric design of micromachined silicon sieve electrodes influences functional nerve regeneration.

A neural interface could be used to control a limb prosthesis. Such an interface can be created by facilitating axonal regeneration through a sieve electrode and then register nerve signals intended to control the prosthesis. A key question is how to design the electrodes to ensure the best possible regeneration. Our previous studies have indicated that regeneration can be achieved using electrodes with square-shaped, 100 x 100 microm, via holes (holes that axons will regenerate through). Other reports have indicated a suitable range of these holes between 40 and 65 microm. In the present study we used silicon sieve electrodes with via holes of either 30 or 90 microm. The transparency, i.e. the percentage of the total via hole area, of these electrodes was either 20 or 30%. The electrodes were inserted into a silicone chamber which was used to bridge a gap in a rat sciatic nerve. After 12 weeks of nerve regeneration electrodes with a hole size of 30 microm and a 30% transparency had the most favourable result as judged by the regained gastrocnemius muscle force and the formation of reactive tissue inside the chamber. The sieve electrode transparency is crucial for ensuring regeneration.

Rat sciatic nerve regeneration through a micromachined silicon chip.

The capacity of regenerating nerve fibres to grow through a perforated silicon chip was tested using the silicone chamber model for nerve regeneration. The chips were fabricated as circular membranes, 4 mm in diameter, thickness 60 microns, with a perforated area, 2 mm in diameter, in the centre. Three types of chips were fabricated utilizing anisotropic etching. The chips were glued with silicone adhesive between two halves of silicone rubber tubing (total length 8 mm, inner diameter 1.8 mm, outer diameter 3.0 mm) which was used to bridge a 4 mm gap between the proximal and distal nerve stumps of a transected rat sciatic nerve. The capacity of regenerating nerve fibres to grow through the holes of the chip was analysed by light and scanning electron microscopy after 4 or 16 weeks of regeneration. Furthermore, the muscle contractility force of the gastrocnemius muscle was measured after 16 weeks of regeneration and compared as a percentage of the contralateral uninjured side. Nerves generated through chips with hole diameters of 10 or 50 microns were morphological and functional failures. The nerve structures distal to chips with hole diameters of 100 microns contained many myelinated nerve fibres in a minifascicular pattern after both 4 and 16 weeks of regeneration. The muscle contractility force was 56% of that of contralateral control muscles.

Development of silicon microstructures and thin-film MALDI target plates for automated proteomics sample identifications.

Here we report on the development of a proteomic platform utilizing a piezoelectric flow-through dispensing unit made from silicon microstructures. The use of a novel surface coating, where matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI MS) targets were uniformly precoated with a thin film of matrix/nitrocellulose, made the sample preparation straightforward and enabled the enrichment and analysis of proteins at low levels in proteomics samples. We demonstrate this by analyzing excised spots in a biological sample originating from a human fetal fibroblast cell line that was subjected to 2D gel-electrophoresis. Furthermore, a sample deposition rate below 30 Hz results in an increased analyte density on the dispensed sample spot, rendering signal amplification. In general, the sensitivity for proteins and peptides can be enhanced 10-50 times compared to traditional MALDI sample preparation techniques.

A miniaturised electrochemical affinity assay based on a wall-free sample droplet and micro-dispensing of the redox-labelled binding partner.

An affinity-assay was developed that is based on the modulation of the diffusion coefficient of a redox-labelled hapten upon complementary recognition of the analyte leading to an increase of molecular weight and hence to a decrease of the diffusion coefficient. The slower diffusion is monitored by means of cyclic voltammetry. In order to demonstrate the feasibility of this assay format, recognition of biotin by streptavidin has been chosen as a model system. Labelling of biotin was achieved by covalent binding of a ferrocene derivative to the biotin unit. To reduce the consumption of expensive compounds and to allow automatisation of the assay a novel miniaturised set-up was developed based on a wall-free sample droplet which forms the electrochemical cell with typical volumes of up to 10 microl. This droplet is dispensed by means of a step-motor driven syringe pump through a specially designed electrode holder spanning the gap between a micro-working electrode and a macroscopic counter electrode. By means of a piezo-driven micro-dispenser a predefined number of nano-droplets (100 pl volume each) containing the redox-labelled hapten are shot into the sample droplet. By this, any physical contact and hence any cross-contamination between the sample and the reagent solution could be avoided. Signal amplification can be achieved by redox recycling between the micro-electrode and the perpendicular positioned macroscopic counter electrode.

Picodroplet-deposition of enzymes on functionalized self-assembled monolayers as a basis for miniaturized multi-sensor structures.

We are reporting on a novel approach for structured immobilisation of enzymes on gold surfaces modified with monolayers of functionalised alkylthiols. The formation of enzyme spots is achieved by shooting very small volumes of an appropriate enzyme solution (down to 100 pl) onto a thiol-monolayer modified gold surface using a micro-dispenser. Formation of enzyme patterns is obtained by moving the micro-dispenser relative to the modified gold surface using a micro-positioning device. Enzyme spots with typical lateral dimensions of 100 microm are obtained, but also, more complex structures, e.g. lines or meander structures, can be achieved by multiple droplets dispensed during the concomitant movement of the micro-dispenser. The first enzyme layer on top of the functionalised thiol-monolayer is subsequently covalently immobilised using either carbodiimide activation of carboxilic headgroups at the enzyme or via already introduced activated ester functions at the monolayer. Immobilised enzyme activities of glucose oxidase and lactate oxidase patterns have been characterised by means of scanning electrochemical microscopy. The product of the enzyme-catalysed reaction, H(2)O(2), is detected with an micro-electrode in the presence of either or both substrates, glucose and lactate, leading to a visualisation of the corresponding enzyme pattern and the lateral enzymatic activity.

Microfluidic components for protein characterization.

The use of microfluidic components to create an analytical toolbox for the very rapidly growing field of proteomics is described. This toolbox provides novel generic analytical solutions that are highly adaptable for analysis of various biomolecules, ranging from high to low abundant. The components are fabricated using silicon micromachining and consist of a microchip immobilised enzyme reactor (microIMER), a piezoelectric microdispenser and high-density nanovial target plates. This microtechnology based platform interfaces matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI TOF-MS) to a wide range of upstream sample handling and/or analytical techniques. Examples of applications such as rapid on-line digestion (12 s) and sample preparation of proteins, interfacing to capillary liquid chromatography (100 attomol sensitivity), and in-vial chemistry on femtomol amounts of sample are presented.

On-line coupling of microdialysis sampling with liquid chromatography for the determination of peptide and non-peptide leukotrienes.

An automated on-line sampling method was developed using microdialysis as the simultaneous sampling and sample pre-treatment technique. The extraction fraction values of microdialysis probes sampling different eicosanoids were investigated. The impact of cyclodextrins in the perfusion liquid used for sampling hydrophobic eicosanoids in biological systems was also studied. The total time for one analysis was 7.6 min allowing seven measurements per hour for monitoring kinetic changes in biological systems.

Optimal membrane choice for microdialysis sampling of oligosaccharides.

An analytical methodology based on microdialysis sampling, high-performance anion-exchange chromatography and integrated pulsed electrochemical detection for the monitoring of oligosaccharides in bioprocesses is presented. Amylopectin and model maltooligosaccharide standards; glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose were used to demonstrate its versatility in view to sampling in enzymatic bioprocesses. The performance and characteristics of membranes with the same cut-off ranging between 3 and 100 kDa, were evaluated with respect to their extraction fraction (EF), permeability factors, temperature stability and protein (enzyme) interaction. All investigated membranes showed some non-specific interaction with enzymes. The EF and non-specific membrane-enzyme interactions were higher for the polysulfone membranes compared with the polyamide and polyethersulfone membranes. For all saccharides, the EF was independent of the concentration even for a 250-fold change in concentration. The EF and morphology of the membranes in their dehydrated state, as observed using scanning electron microscopy did not show any significant difference between membranes exposed to a 90 degrees C temperature for 3 and 24 h indicating their applicability to the study of high temperature bioprocesses.

Protein identification platform utilizing micro dispensing technology interfaced to matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

An integrated protein microcharacterization/identification platform has been developed. The system has been designed to allow a high flexibility in order to tackle challenging analytical problems. The platform comprises a cooled microautosampler, an integrated system for microcolumn HPLC, and a capillary reversed-phase column that is interfaced to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) system via a low internal volume flow-through microdispenser. The chromatographic separation is continuously transferred onto a MALDI target plate as discrete spots as the dispenser ejects bursts of droplets of the column effluent in a precise array pattern. A refrigerated microfraction collector was coupled to the outlet of the flow-through microdispenser enabling enrichment and re-analysis of interesting fractions. The use of target plates pre-coated with matrix simplified and increased the robustness of the system. By including a separation step prior to the MALDI-TOF-MS analysis and hereby minimizing suppression effects allowed us to obtain higher sequence coverage of proteins compared to conventional MALDI sample preparation methodology. Additionally, synthetic peptides corresponding to autophosphorylated forms of the tryptic fragment 485-496 (ALGADDSYYTAR) of tyrosine kinase ZAP-70 were identified at sensitivities reaching 150 amol.

Perforated silicon nerve chips with doped registration electrodes: in vitro performance and in vivo operation.

An in vitro model was developed for the study of signal transduction between a Cu-wire, miming a neural signal source, and recording electrodes on perforated silicon chips. Phosphorous doped electrodes were used to achieve an all silicon device. The model was used to study signal amplitude as a function of the spatial position, and distance to the signal source. Recordings of the signal crosstalk to neighboring electrodes on the chips were made. It was found that the amplitude decreased by a factor of two at a distance of 50 microns between the electrode surface and the signal source. The chip electrode signal crosstalk was found to be 6 dB using an external reference electrode. Improvements were accomplished with an on chip reference electrode giving a crosstalk suppression of 20 dB. Impedance analysis showed that doped silicon electrodes displayed similar characteristics as Cu-electrodes at frequencies above 3 kHz. Sieve electrodes were implanted in the rat sciatic nerve and following a 10-week nerve regeneration period the dorsal and ventral (L5) roots in the spinal cord were stimulated. Compound action potentials were recorded via the chip. Stimulating the regenerated sciatic nerve via the sieve electrode also induced lower leg muscle contraction activity.

A continuous glucose monitoring system based on microdialysis.

The development of a system for continuous glucose measurement is described. A microdialysis fibre is used to continuously withdraw the glucose sample. After passing through the microdialysis fibre the perfusion liquid enters a micro-enzyme reactor which is filled with immobilized glucose oxidase. The enzyme activity, which varies with the glucose concentration, is monitored by an oxygen electrode. The measuring system can be adapted to a desired dynamic range by varying the perfusion rate. The measurement system has been calibrated for two different perfusion rates and shows a linear response to glucose concentrations between 0-3.5 mmol at 12 microliters/min and 0-8 mmol at 25 microliters/min. A probe, which can be configured with different lengths of the dialysing fibre, has also been designed to enable subcutaneous insertion of the microdialysis fibre in clinical studies. Subcutaneous glucose sampling by means of microdialysis is suggested to be used in the design of a glucose monitoring device for clinical use.

Focusing of sub-micrometer particles and bacteria enabled by two-dimensional acoustophoresis.

Handling of sub-micrometer bioparticles such as bacteria are becoming increasingly important in the biomedical field and in environmental and food analysis. As a result, there is an increased need for less labor-intensive and time-consuming handling methods. Here, an acoustophoresis-based microfluidic chip that uses ultrasound to focus sub-micrometer particles and bacteria, is presented. The ability to focus sub-micrometer bioparticles in a standing one-dimensional acoustic wave is generally limited by the acoustic-streaming-induced drag force, which becomes increasingly significant the smaller the particles are. By using two-dimensional acoustic focusing, i.e. focusing of the sub-micrometer particles both horizontally and vertically in the cross section of a microchannel, the acoustic streaming velocity field can be altered to allow focusing. Here, the focusability of E. coli and polystyrene particles as small as 0.5 µm in diameter in microchannels of square or rectangular cross sections, is demonstrated. Numerical analysis was used to determine generic transverse particle trajectories in the channels, which revealed spiral-shaped trajectories of the sub-micrometer particles towards the center of the microchannel; this was also confirmed by experimental observations. The ability to focus and enrich bacteria and other sub-micrometer bioparticles using acoustophoresis opens the research field to new microbiological applications.

Integrated acoustic immunoaffinity-capture (IAI) platform for detection of PSA from whole blood samples.

On-chip detection of low abundant protein biomarkers is of interest to enable point-of-care diagnostics. Using a simple form of integration, we have realized an integrated microfluidic platform for the detection of prostate specific antigen (PSA), directly in anti-coagulated whole blood. We combine acoustophoresis-based separation of plasma from undiluted whole blood with a miniaturized immunoassay system in a polymer manifold, demonstrating improved assay speed on our Integrated Acoustic Immunoaffinity-capture (IAI) platform. The IAI platform separates plasma from undiluted whole blood by means of acoustophoresis and provides cell free plasma of clinical quality at a rate of 10 uL/min for an online immunoaffinity-capture of PSA on a porous silicon antibody microarray. The whole blood input (hematocrit 38-40%) rate was 50 µl min(-1) giving a plasma volume fraction yield of ≈33%. PSA was immunoaffinity-captured directly from spiked female whole blood samples at clinically significant levels of 1.7-100 ng ml(-1) within 15 min and was subsequently detected via fluorescence readout, showing a linear response over the entire range with a coefficient of variation of 13%.

Ultrasound-induced acoustophoretic motion of microparticles in three dimensions.

We derive analytical expressions for the three-dimensional (3D) acoustophoretic motion of spherical microparticles in rectangular microchannels. The motion is generated by the acoustic radiation force and the acoustic streaming-induced drag force. In contrast to the classical theory of Rayleigh streaming in shallow, infinite, parallel-plate channels, our theory does include the effect of the microchannel side walls. The resulting predictions agree well with numerics and experimental measurements of the acoustophoretic motion of polystyrene spheres with nominal diameters of 0.537 and 5.33 µm. The 3D particle motion was recorded using astigmatism particle tracking velocimetry under controlled thermal and acoustic conditions in a long, straight, rectangular microchannel actuated in one of its transverse standing ultrasound-wave resonance modes with one or two half-wavelengths. The acoustic energy density is calibrated in situ based on measurements of the radiation dominated motion of large 5-µm-diameter particles, allowing for quantitative comparison between theoretical predictions and measurements of the streaming-induced motion of small 0.5-µm-diameter particles.