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.

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].

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.

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.

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%.

Acoustofluidics 5: Building microfluidic acoustic resonators.

Acoustophoresis is getting more attention as an effective and gentle non-contact method of manipulating cells and particles in microfluidic systems. A key to a successful assembly of an acoustophoresis system is a proper design of the acoustic resonator where aspects of fabrication techniques, material choice, thickness matching of involved components, as well as strategies of actuation, all have to be considered. This tutorial covers some of the basics in designing and building microfluidic acoustic resonators and will hopefully be a comprehensive and advisory document to assist the interested reader in creating a successful acoustophoretic device.

Emerging clinical applications of microchip-based acoustophoresis.

Acoustophoresis is currently in a state of transition from the academic laboratories, moving into the biomedical laboratories and industries. Clear areas of interest are seen in clinical diagnostics and therapeutics, where new approaches to cell handling and purification are emphasized as highly potent areas. This article outlines some of the basic unit operations of acoustophoresis, where applications as cell washing, binary separation, free-flow acoustophoresis, and affinity acoustophoresis are highlighted. The most recent steps to move acoustophoresis into clinical and preclinical applications are also presented.

Review of cell and particle trapping in microfluidic systems.

The ability to obtain ideal conditions for well-defined chemical microenvironments and controlled temporal chemical and/or thermal variations holds promise of high-resolution cell response studies, cell-cell interactions or e.g. proliferation conditions for stem cells. It is a major motivation for the rapid increase of lab-on-a-chip based cell biology research. In view of this, new chip-integrated technologies are at an increasing rate being presented to the research community as potential tools to offer spatial control and manipulation of cells in microfluidic systems. This is becoming a key area of interest in the emerging lab-on-a-chip based cell biology research field. This review focuses on the different technical approaches presented to enable trapping of particles and cells in microfluidic system.

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

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

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.

Reverse-phase versus sandwich antibody microarray, technical comparison from a clinical perspective.

Protein microarrays are powerful tools to quantify and characterize proteins in multiplex assays. They have great potential within clinical diagnostics and prognostics, as they minimize consumption of both analyte and biological sample. Assays that do not require labeling of the biological specimen, henceforth called label-free, are vital for ease of clinical sample processing. Here, we evaluate two label-free techniques, reverse-phase and sandwich antibody assays, using microarrays on high-performance porous silicon surfaces and fluorescence detection. In view of increasing interest in reverse microarrays, this paper focuses on analytical sensitivity of the reverse assays compared to the more complex but highly sensitive sandwich assay. Sensitivity, linear range, and reproducibility of the two assays were compared using prostate-specific antigen (PSA) in buffer. The sandwich assay displayed 5 orders of magnitude lower detection limit (0.7 ng/mL) compared to the reverse assay (70 microg/mL). PSA at 50 nM (1.5 microg/mL) in cell lysates was detected by the sandwich assay but not by the reverse assay, demonstrating again a far lower detection limit for sandwich microarrays. In independent assay runs of PSA spiked in female serum, the sandwich assay had good linearity (R2 > 0.99) and reproducibility (coefficient of variation < or =15%), and the detection limit could be improved to 0.14 ng/mL. Without further signal amplification, the sandwich assay would be our choice for PSA analysis of clinical samples using a microarray technology platform.

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.

Integrated protein microchip assay with dual fluorescent- and MALDI read-out.

A pore chip protein array (PCPA) concept based on a dual readout configuration, fluorescence imaging, and MALDI-TOF MS has been developed. Highly packed, (>4000 spots/cm2), antibody arrays were dispensed on the porous chip by using a piezo-electric microdispenser. Sandwich assay was made after blocking by addition of a secondary antibody either IgG-FITC-labeled or anti-Ang II. The antigen in the first system was a large protein (IgG), and in the other system, a FITC marked peptide Angiotensin II (Ang II) was used. Ang II antibodies showed specificity for Ang II, while the Ang I antibodies showed binding properties for Ang I, II, and Renin. Fluorescence and MALDI TOF MS read-out was made for IgG and Ang II. A major advantage of the dual read-out PCPA approach is that both affinity binding and mass identity are derived. Detection limits for Ang II on the chip is as low as 500 zmol (Ang II).

Disposable polymeric high-density nanovial arrays for matrix assisted laser desorption/ionization-time of flight-mass spectrometry: II. Biological applications.

A novel disposable high-density matrix assisted laser desorption/ionization (MALDI) target plate made either from polymethylmethacrylate (PMMA) or polycarbonate (PC) is presented where thousands (1,200-1,600) of samples can be deposited and subsequently analyzed by MALDI-time of flight (TOF) mass spectrometry. Good reproducibility was obtained across the plate regardless of position on the target plate with a relative standard deviation (RSD) on the peak intensity of typically 30% calculated from data generated by analysis of a 10 nm peptide mixture of angiotensin I, II, III and bradykinin. The nanovial array format combined with microdispensing technology makes it possible to carry out in-vial chemistry on deposited samples. This is demonstrated by the analysis of peptides from beta-casein and subsequent in-vial dephosphorylation of its phosphopeptides at 10 fmol levels by microdispensing of alkaline phosphatase, into the nanovial. The mass spectra obtained from these polymeric targets provides can also be used in high sensitivity applications as shown by peptide mass fingerprinting of human fibroblast proteins separated by two-dimensional gel electrophoresis.

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.

A method for the design and study of enzyme microstructures formed by means of a flow-through microdispenser.

Micrometer-sized enzyme grids were fabricated on gold surfaces using a novel method based on a flow-through microdispenser. The method involves dispensing very small droplets of enzyme solution (approximately 100 pL) during the concomitant relative movement of a gold substrate with respect to the nozzle of a microdispenser, resulting in enzyme patterns with a line width of approximately 100 microm. Different immobilization methods have been evaluated, yielding either enzyme monolayers using functionalized self-assembled thiol monolayers for covalent binding of the enzyme or enzyme multilayers by cross-linking or entrapping the enzymes in a polymer film. The latter immobilization techniques allow the formation of coupled multienzyme structures. On the basis of this feature, coupled bienzyme (glucose oxidase and catalase) or three-enzyme (alpha-glucosidase, mutarotase, and glucose oxidase) microstructures consisting of line patterns of one enzyme intersecting with the patterned lines of the other enzyme(s) were fabricated. By means of scanning electrochemical microscopy (SECM) operated in the generator-collector mode, the enzyme microstructures and their integrity were visualized using the localized detection of enzymatically produced/consumed H2O2. A calibration curve for glucose could be obtained by subsequent SECM line scans over a glucose oxidase microstructure for increasing glucose concentrations, demonstrating the possibility of obtaining localized quantitative data from the prepared microstructures. Possible applications of these enzyme microstructures for multianalyte detection and interference elimination and for screening of different biosensor configurations are highlighted.

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.

Determination of diffusion coefficients of electroactive species in time-of-flight experiments using a microdispenser and microelectrodes.

Two novel methods for the determination of diffusion coefficients of redox species combining the special properties of microdispensing devices and microelectrodes are presented. Both are based on the local application of tiny volumes of the redox-active species by means of a dispenser nozzle at a defined distance from the surface of a microelectrode. The microelectrode, which is inserted through the bottom into an electrochemical cell, is held at a constant potential sufficient to oxidize or reduce the electro-active species under diffusional control. The dispenser, which is filled with the electro-active species, can be positioned by means of micrometer screws over the microelectrode. After dispensing a defined number of droplets near the microelectrode surface, the current through the microelectrode is recorded, usually yielding a peak-shaped curve having a defined time delay between the shooting of the droplets and the maximum current. The time that is necessary to attain maximum current, together with the known distance between two dispensing points, can be used to determine the diffusion coefficient of the electroactive species without knowledge of any system parameters, such as concentration of the redox species, diameter of the electroactive surface or number of transferred electrons. A similar method for the determination of diffusion coefficient of redox species involves a second redox species for calibration purposes. A mixture of both species is shot close to the microelectrode surface. Due to the different formal potentials of the redox species that are used, they can be distinguished in sequential experiments by variation of the potentials that are applied to the microelectrode, and it is thus possible to determine the individual transit times of the redox species independently. The difference in the transit times, together with the known diffusion coefficient of one of the redox species, can be used to calculate the unknown diffusion coefficient of the second one.