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ABSTRACT: We compare the PNA-DNA duplex hybridization characteristics of vertically tethered and new horizontally tethered PNA probes on solid surfaces. The horizontal 15-mer PNA probe has been synthesized with linker molecules attached at three locations (γ-points) positioned along the PNA backbone that provides covalent attachment of the probe with the backbone aligned parallel to the surface, which is important for DNA hybridization assays that use electric field effect sensors for detection. A radioactive labeled assay, and real-time surface plasmon resonance (SPR) biosensor are used to assess the probe surface density, non-specific binding, and DNA hybridization affinity, respectively, of the new PNA probe configuration. The estimated equilibrium dissociation constants of the horizontally tethered duplex and the vertically tethered duplex are of the same order of magnitude (KD~5 nM), which indicates a sufficient hybridization affinity for many electronic biosensors that benefit from the horizontal alignment, which minimizes the effects of counter-ion screening.
ACS Applied Materials & Interfaces 05/2013; · 4.53 Impact Factor
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ABSTRACT: We report a new top-down nanofabrication technology to realize large area metal nanowire (m-NW) arrays with tunable sub-20 nm separation nanogaps without the use of chemical etching or milling of the metal layer. The m-NW array nanofabrication technology is based on a self- regulating metal deposition process that is facilitated by closely spaced and isolated heterogeneous template surfaces that confines the metal deposition into two dimensions, and therefore, electrically isolated parallel arrays of m-NW can be realized with uniform and controllable nanogaps. Au-NW and Ag-NW arrays are presented with high-density ~10^5 NWs cm^-1, variable NW diameters down to ~50 nm, variable nanogaps down to ~5 nm, and very large nanogap length density ~1 km cm^-2. The m-NW arrays are designed and implemented as inter-digitated nanoelectrodes for electrochemical applications and as plasmonic substrates where the coupled-mode localized surface plasmon resonance (LSPR) wavelength in the nanogaps between adjacent m-NW dimers can be precisely tuned to match any excitation source in the range from 500 nm to 1000 nm, thus providing optimal local electromagnetic field enhancement. A spatially averaged (n=2500) surface enhanced Raman scattering (SERS) analytical enhancement factor of (1.2±0.1)×10^7 is demonstrated from a benzenethiol monolayer chemisorbed on a Au-NW array substrate with LSPR wavelength matched to a He-Ne laser source.
ACS Nano 05/2013; · 10.77 Impact Factor
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ABSTRACT: We present a label-free (bio)chemical analysis platform that uses all-electrical silicon nanowire sensor arrays integrated with a small volume microfluidic flow-cell for real-time (bio)chemical analysis and detection. The integrated sensing platform contains an automated multi-sample injection system that eliminates erroneous sensor responses from sample switching due to flow rate fluctuations and provides precise sample volumes down to 10 nl. Biochemical sensing is demonstrated with real-time 15-mer DNA-PNA (peptide nucleic acid) duplex hybridization measurements from different sample concentrations in a low ionic strength, and the equilibrium dissociation constant KD ≈ 140 nM has been extracted from the experimental data using the first order Langmuir binding model. Chemical sensing is demonstrated with pH measurements from different injected samples in flow that have sensitivities consistent with the gate-oxide materials. A differential sensor measurement configuration results in a 30× reduction in sensor drift. The integrated label-free analysis platform is suitable for a wide range of small volume chemical and biochemical analyses.
The Analyst 04/2013; · 4.23 Impact Factor
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ABSTRACT: The kinetic activity of individual enzyme molecules was determined in aqueous droplets generated in a nano- and microfluidic device. To avoid high background noise, the enzyme and substrate solution was confined into femtoliter carriers, achieving high product concentrations from single-molecule encapsulation. The tiny droplets (ϕ ∼ 2.5-3 μm) generated from this fluidic system were highly monodisperse, beneficial for an analysis of single enzyme activity. The method presented here allows to follow large numbers of individual droplets over time. The instrumental requirements are furthermore modest, since the small droplet size allows to use of standard microscope and standard Pyrex glass chips as well as the use of relatively high enzyme concentrations (nM range) for single molecule encapsulation.
Lab on a Chip 04/2013; · 5.67 Impact Factor
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ABSTRACT: The kinetic activity of individual enzyme molecules was determined in aqueous droplets generated in a nano- and microfluidic device. To avoid high background noise, the enzyme and substrate solution was confined into femtolitre carriers, achieving high product concentrations from single-molecule encapsulation. The tiny droplets (diameter ~2.5-3 micron) generated from this fluidic system were highly monodisperse, beneficial for an analysis of single enzyme activity. The method presented here allows to follow large numbers of individual droplets over time. The instrumental requirements are furthermore modest, since the small droplet size allows to use of standard microscope and standard Pyrex glass chips as well as the use of relatively high enzyme concentrations (nM range) for single molecule encapsulation.
Lab on a Chip 01/2013; · 5.67 Impact Factor
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ABSTRACT: We present here a novel microchamber sealing valve that is self-actuated by a pressure change during the temperature change in the thermal activation of reactions. Actuation of our valve requires only the use of the same heating device as employed for the reactions. A thermoplastic UV-curable polymer is used as a device material; the polymer allows realization of the temperature-driven valve actuation as well as the fabrication of multi-layered devices. The self-actuated valve achieves effective sealing of the microchamber for the polymerase chain reaction (PCR) even at 90 °C, which is essential for developing highly parallel PCR array devices without the need for complicated peripherals to control the valve operation.
Lab on a Chip 12/2012; · 5.67 Impact Factor
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ABSTRACT: A microfluidic platform is reported for various experimentation schemes on cell membrane models and membrane proteins using a combination of electrical and optical measurements, including confocal microscopy. Bilayer lipid membranes (BLMs) are prepared in the device upon spontaneous and instantaneous thinning of the lipid solution in a 100-μm dry-etched aperture in a 12.5-μm thick Teflon foil. Using this quasi-automated approach, a remarkable 100% membrane formation yield is reached (including reflushing in 4% of the cases), and BLMs are stable for up to 36 h. Furthermore, the potential of this platform is demonstrated for (i) the in-depth characterization of BLMs comprising both synthetic and natural lipids (1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and L-α-phosphatidylcholine (L-α-PC)/cholesterol, respectively) in terms of seal resistance, capacitance, surface area, specific capacitance, and membrane hydrophobic thickness; (ii) confocal microscopy imaging of phase separation in sphingomyelin/L-α-PC/cholesterol ternary membranes; (iii) electrical measurements of individual nanopores (α-hemolysin, gramicidin); and (iv) indirect assessment of the alteration of membrane properties upon exposure to chemical stimuli using the natural nanopore gramicidin as a sensor.
Small 11/2012; · 8.35 Impact Factor
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ABSTRACT: This paper reports a novel design of a miniaturized three-electrode electrochemical cell, the purpose of which is aimed at generating drug metabolites with a high conversion efficiency. The working electrode and the counter electrode are placed in two separate channels to isolate the reaction products generated at both electrodes. The novel design includes connecting channels between these two electrode channels to provide a uniform distribution of the current density over the entire working electrode. In addition, the effect of ohmic drop is decreased. Moreover, two flow resistors are included to ensure an equal flow of analyte through both electrode channels. Total conversion of fast reacting ions is achieved at flow rates up to at least 8 μL/min, while the internal chip volume is only 175 nL. Using this electrochemical chip, the metabolism of mitoxantrone is studied by microchip electrospray ionization-mass spectrometry. At an oxidation potential of 700 mV, all known metabolites from direct oxidation are observed. The electrochemical chip performs equally well, compared to a commercially available cell, but at a 30-fold lower flow of reagents.
Analytical Chemistry 09/2012; · 5.86 Impact Factor
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ABSTRACT: Large-area (∼8000 mm(2)) Au nanogap plasmon resonator array substrates manufactured using maskless laser interference lithography (LIL) with high uniformity are presented. The periodically spaced subwavelength nanogap arrays are formed between adjacent nanopyramid (NPy) structures with precisely defined pitch and high length density (∼1 km cm(-2)), and are ideally suited as scattering sites for surface enhanced Raman scattering (SERS), as well as refractive index sensing. The two-dimensional grid arrangement of NPy structures renders the excitation of the plasmon resonators minimally dependent on the incident polarization. The SERS average enhancement factor (AEF) has been characterized using over 30 000 individual measurements of benzenethiol (BT) chemisorbed on the Au NPy surfaces. From the 1(a(1)), β(CCC) + ν(CS) ring mode (1074 cm(-1)) of BT on surfaces with pitch λ(g) = 200 nm, AEF = 0.8 × 10(6) and for surfaces with λ(g) = 500 nm, AEF = 0.3 × 10(7) from over 99% of the imaged spots. Maximum AEFs > 10(8) have been measured in both cases.
Nanoscale 06/2012; 4(15):4712-8. · 5.91 Impact Factor
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ABSTRACT: In this work, we present a chip-integrated amperometric sensor targeted at the detection of hydrogen peroxide (H(2) O(2) ) in the gaseous phase. Electrode chips are manufactured in a series of microfabrication steps and characterized electrochemically. Using such devices detection of H(2) O(2) in an aqueous phase is shown by means of cyclic voltammetry and amperometry. Furthermore, it is discussed that variation of conditions such as the composition of the supporting electrolyte largely influences the obtained electrochemical signal. Additionally, electrochemical pretreatment of platinum working electrodes aiming at surface oxidation improves the limit of detection of the sensor and the linearity of the calibration curve at low H(2) O(2) concentrations (<10 μM). Agarose-coated electrode chips are used for the measurement of H(2) O(2) in the gaseous phase. Detection of H(2) O(2) is shown in a static and in a flow-through setup. We find a limit of detection of approximately 42 ppb. Current work focuses on expanding the presented device to detection of H(2) O(2) in exhaled breath condensate.
Electrophoresis 06/2012; · 3.30 Impact Factor
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ABSTRACT: In this article high-yield (77%) and high-speed (2700 cells s(-1)) single cell droplet encapsulation is described using a Dean-coupled inertial ordering of cells in a simple curved continuous microchannel. By introducing the Dean force, the particles will order to one equilibrium position after travelling less than 1 cm. We use a planar curved microchannel structure in PDMS to spatially order two types of myeloid leukemic cells (HL60 and K562 cells), enabling deterministic single cell encapsulation in picolitre drops. An efficiency of up to 77% was reached, overcoming the limitations imposed by Poisson statistics for random cell loading, which yields only 37% of drops containing a single cell. Furthermore, we confirm that > 90% of the cells remain viable. The simple planar structure and high throughput provided by this passive microfluidic approach makes it attractive for implementation in lab on a chip (LOC) devices for single cell applications using droplet-based platforms.
Lab on a Chip 06/2012; 12(16):2881-7. · 5.67 Impact Factor
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ABSTRACT: ObjectiveGastrointestinal ischemia is always accompanied by an increased luminal CO2. Currently, air tonometry is used to measure luminal CO2. To improve the response time a new sensor was developed, enabling continuous CO2 measurement. It consists of a pH-sensitive hydrogel which swells and shrinks in response to luminal CO2, which is measured by the pressure sensor. We evaluated the potential clinical value of the sensor during an in vitro and
in vivo study.
MethodsThe response time to immediate, and stepwise change in pCO2 was determined between 5 and 15kPa, as well as temperature sensitivity between 25 and 40°C at two pCO2 levels. Three sensors were compared to air tonometry (Tonocap®) in healthy volunteers using a stepwise incremental exercise test, followed by a period of hyperventilation and an artificial
CO2-peak.
ResultsThe in vitro response time to CO2 increase and decrease was mean 5.9 and 6.6min. The bias, precision and reproducibility were +5%, 3% and 2%, resp. Increase
of 1°C at constant pCO2 decreased sensor signal by 8%.
In vivo tests: The relation with the Tonocap was poor during the exercise test. The response time of the sensor was 3min
during hyperventilation and the CO2 peak.
ConclusionThe hydrogel carbon dioxide sensor enabled fast and accurate pCO2 measurement in a controlled environment but is very temperature dependent. The current prototype hydrogel sensor is still
too unstable for clinical use, and should therefore be improved.
KeywordsGastrointestinal tract-mucosal perfusion-gastrointestinal ischemia-carbon dioxide-measurement techniques-air tonometry-hydrogel-based CO2 sensor-mesenteric-hydrogel.
Journal of Clinical Monitoring and Computing 04/2012; 21(2):83-90. · 0.89 Impact Factor
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ABSTRACT: We demonstrate a robust method to produce monodisperse femtoliter to attoliter droplets by using a nano-microfluidic device.
Two immiscible liquids are forced through a nanochannel where a steady nanoscopic liquid filament forms, thinning close to
the nanochannel exit to a microchannel due to the capillary focusing. When the nanoscopic filament enters the microchannel,
monodisperse droplets are formed by capillary instability. In a certain range of physical parameters and geometrical configurations,
the droplet size is only determined by the nanochannel height and independent of liquid flow rates and ratios, surfactants,
and continuous phase viscosity. By using nanochannels with a height of 100–900nm, 0.4–3.5μm diameter droplets (volume down
to 30aL) have been produced. The generated droplets are stable for at least weeks.
KeywordsNano-microfluidics–Droplet–Attoliter–Monodisperse–Capillary focusing
Microfluidics and Nanofluidics 04/2012; 11(1):87-92. · 3.37 Impact Factor
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ABSTRACT: In this paper we describe a compact fluorescence detection system for on-chip analysis of beads, comprising a low-cost optical HD-DVD pickup. The complete system consists of a fluorescence detection unit, a control unit and a microfluidic chip containing microchannels and optical markers. With these markers the laser beam of the optical pickup can be automatically focused at the centre of the microchannel. With the complete system a two-dimensional fluorescent profile across the channel width can be obtained such that there is no need for hydrodynamic or electrokinetic focusing of the particles in a specific part of the channel. Fluorescent μm sized beads suspended in medium have been detected with the system. Since on both sides of the main beam two additional laser beams at a known distance are generated, also the velocity of individual beads has been determined.
Lab on a Chip 03/2012; 12(10):1780-3. · 5.67 Impact Factor
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ABSTRACT: Electroporation is a powerful technique to increase the permeability of cell membranes and subsequently introduce foreign materials into cells. Pores are created in the cell membrane upon application of an electric field (kV/cm). Most applications employ bulk electroporation, at the scale of 1 mL of cells (ca. one million cells). However, recent progresses have shown the interest to miniaturize the technique to a single cell. Single cell electroporation is achieved either using microelectrodes which are placed in close vicinity to one cell, or in a microfluidic format. We focus here on this second approach, where individual cells are trapped in micrometer-size structures within a microchip, exposed in situ to a high electric field and loaded with either a dye (proof-of-principle experiments) or a plasmid. Specifically, we present one device that includes an array of independent electroporation sites for customized and successive poration of nine cells. The different steps of the single cell electroporation protocol are detailed including cell sample preparation, cell trapping, actual cell poration and on-chip detection of pore formation. Electroporation is illustrated here with the transport of dyes through the plasma membrane, the transfection of cells with GFP-encoding plasmids, and the study of the ERK1 signaling pathway using a GFP-ERK1 protein construct expressed by the cells after their transfection with the corresponding plasmid. This last example highlights the power of microfluidics with the implementation of various steps of a process (cell poration, culture, imaging) performed at the single cell level, on a single device.
Methods in molecular biology (Clifton, N.J.) 01/2012; 853:65-82.
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ABSTRACT: We show that the performance of a streaming-potential based microfluidic energy conversion system can be strongly enhanced by the use of two phase flow. Injection of gas bubbles into a liquid-filled channel increases both the maximum output power and the energy conversion efficiency. In single-phase systems the internal conduction current induced by the streaming potential limits the output power, whereas in a two-phase system the bubbles reduce this current and increase the power. In our system the addition of bubbles enhanced the maximum output power of the system by a factor of 74 and the efficiency of the system by a factor of 163 compared with single phase flow.
Lab on a Chip 12/2011; 11(23):4006-11. · 5.67 Impact Factor
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ABSTRACT: We report a high-throughput clog-free microfluidic deoxyribonucleic acid (DNA) fragmentation chip that is based on hydrodynamic shearing. Salmon sperm DNA has been reproducibly fragmented down to ∼ 5k bp fragment lengths by applying low hydraulic pressures (≤1 bar) across micromachined constrictions positioned in larger microfluidic channels that create point-sink flow with large velocity gradients near the constriction entrance. Long constrictions (100 µm) produce shorter fragment lengths compared to shorter constrictions (10 µm), while increasing the hydrodynamic pressure requirement. Sample recirculation (10 ×) in short constrictions reduces the mean fragment length and fragment length variation, and improves yield compared to single-pass experiments without increasing the hydrodynamic pressure.
Nanotechnology 11/2011; 22(49):494013. · 3.98 Impact Factor
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ABSTRACT: This work reports a new method to hydrophobize glass-based micro- and nanofluidic networks. Conventional methods of hydrophobizing glass surfaces often create particulate debris causing clogging especially in shallow nanochannels or require skilful handling. Our novel method employs oxygen plasma, silicone oil and ultraviolet (UV) light. The contact angle of the modified bare glass surface can reach 100° whilst the inner channels after treatment facilitate stable and durable water-in-oil droplet generation. This modified surface was found to be stable for more than three weeks. The use of UV in principle enables in-channel hydrophobic patterning.
Lab on a Chip 11/2011; 11(24):4260-6. · 5.67 Impact Factor
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ABSTRACT: This article describes the development and full characterization of a microfluidic chip for electrofusion of human peripheral blood B-cells and mouse myeloma (NS-1) cells to generate hybridomas. The chip consists of an array of 783 traps, with dimensions that were optimized to obtain a final cell pairing efficiency of 33±6%. B cells were stained with a cytoplasmic stain CFDA to assess the different stages of cell fusion, i.e. dye transfer to NS-1 cells (initiating fusion) and membrane reorganization (advanced fusion). Six DC pulses of 100 μs (2.5 kV/cm) combined with an AC field (30 s, 2 MHz, 500 V/cm) and pronase treatment resulted in the highest electrofusion efficiency of paired cells (51±11%). Hybridoma formation, with a yield of 0.33 and 1.2%, was observed after culturing the fused cells for 14 days in conditioned medium. This work provides valuable leads to improve the current electrofusion protocols for the production of human antibodies for diagnostic and therapeutic applications.
Electrophoresis 11/2011; 32(22):3138-46. · 3.30 Impact Factor
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ABSTRACT: We report a PDMS microfluidic platform for parallel single-cell analysis (PaSCAl) as a powerful tool to decipher the heterogeneity found in cell populations. Cells are trapped individually in dedicated pockets, and thereafter, a number of invasive or non-invasive analysis schemes are performed. First, we report single-cell trapping in a fast (2-5 min) and reproducible manner with a single-cell capture yield of 85% using two cell lines (P3x63Ag8 and MCF-7), employing a protocol which is scalable and easily amenable to automation. Following this, a mixed population of P3x63Ag8 and MCF-7 cells is stained in situ using the nucleic acid probe (Hoechst) and a phycoerythrin-labeled monoclonal antibody directed at EpCAM present on the surface of the breast cancer cells MCF-7 and absent on the myeloma cells P3x63Ag8 to illustrate the potential of the device to analyze cell population heterogeneity. Next, cells are porated in situ using chemicals in a reversible (digitonin) or irreversible way (lithium dodecyl sulfate). This is visualized by the transportation of fluorescent dyes through the membrane (propidium iodide and calcein). Finally, an electrical protocol is developed for combined cell permeabilization and electroosmotic flow (EOF)-based extraction of the cell content. It is validated here using calcein-loaded cells and visualized through the progressive recovery of calcein in the side channels, indicating successful retrieval of individual cell content.
Electrophoresis 11/2011; 32(22):3094-100. · 3.30 Impact Factor
