Showing papers by "Gerald Urban published in 2020"

Zero-Consumption Clark-Type Microsensor for Oxygen Monitoring in Cell Culture and Organ-on-Chip Systems

Abstract: Clark-type oxygen microsensors promise zero analyte consumption if the feedback mode (Ross principle) is successfully implemented. Our approach was a microsensor with platinum as working and counter electrode material, a pHEMA hydrogel layer containing buffer solution as electrolyte and PDMS as gas-permeable membrane. We were able to demonstrate successful implementation of the Ross principle by measurement of the counter electrode potential. The microsensors could be stored dry and activated by immersion into aqueous analyte. Chronoamperometric protocols were applied to enable long-term stability for more than one week without recalibration. The microsensors were integrated in conventional tissue culture flasks for cell measurements. Respiration monitoring was done in T-47D breast cancer monolayer culture. The unique feature combination of zero analyte consumption, 1-point calibration and sufficient long-term stability make these sensors an ideal candidate to monitor oxygenation and respiration in different cell culture and organ-on-chip systems.

Enhanced Protein Immobilization on Polymers—A Plasma Surface Activation Study

Abstract: Over the last years, polymers have gained great attention as substrate material, because of the possibility to produce low-cost sensors in a high-throughput manner or for rapid prototyping and the wide variety of polymeric materials available with different features (like transparency, flexibility, stretchability, etc.). For almost all biosensing applications, the interaction between biomolecules (for example, antibodies, proteins or enzymes) and the employed substrate surface is highly important. In order to realize an effective biomolecule immobilization on polymers, different surface activation techniques, including chemical and physical methods, exist. Among them, plasma treatment offers an easy, fast and effective activation of the surfaces by micro/nanotexturing and generating functional groups (including carboxylic acids, amines, esters, aldehydes or hydroxyl groups). Hence, here we present a systematic and comprehensive plasma activation study of various polymeric surfaces by optimizing different parameters, including power, time, substrate temperature and gas composition. Thereby, the highest immobilization efficiency along with a homogenous biomolecule distribution is achieved with a 5-min plasma treatment under a gas composition of 50% oxygen and nitrogen, at a power of 1000 W and a substrate temperature of 80 °C. These results are also confirmed by different surface characterization methods, including SEM, XPS and contact angle measurements.

Digital DNA microarray generation on glass substrates.

Abstract: In this work we show how DNA microarrays can be produced batch wise on standard microscope slides in a fast, easy, reliable and cost-efficient way. Contrary to classical microarray generation, the microarrays are generated via digital solid phase PCR. We have developed a cavity-chip system made of a PDMS/aluminum composite which allows such a solid phase PCR in a scalable and easy to handle manner. For the proof of concept, a DNA pool composed of two different DNA species was used to show that digital PCR is possible in our chips. In addition, we demonstrate that DNA microarray generation can be realized with different laboratory equipment (slide cycler, manually in water baths and with an automated cartridge system). We generated multiple microarrays and analyzed over 13,000 different monoclonal DNA spots to show that there is no significant difference between the used equipment. To show the scalability of our system we also varied the size and number of the cavities located in the array region up to more than 30,000 cavities with a volume of less than 60 pL per cavity. With this method, we present a revolutionary tool for novel DNA microarrays. Together with new established label-free measurement systems, our technology has the potential to give DNA microarray applications a new boost.

New life for old wires: electrochemical sensor method for neural implants

Abstract: OBJECTIVE Electrochemical microsensors based on noble metals can give essential information on their microenvironment with high spatio-temporal resolution. However, most advanced chemo- and biosensors lack the long-term stability for physiological monitoring of brain tissue beyond an acute application. Noble metal electrodes are widely used as neural interfaces, particularly for stimulating in the central nervous system. Our goal was to recruit already deployed, unmodified noble metal electrodes (Pt, Pt/Ir) as in situ chemical sensors. APPROACH With advanced electrochemical sensor methods, we investigated electrode surface processes, oxidizable species and oxygen as an indicator for tissue mass transport. We developed a unique, multi-step, amperometric/potentiometric sensing procedure derived from the investigation of Pt surface processes by chronocoulometry providing fundamental characterization of the electrode itself. MAIN RESULTS The resulting electrochemical protocol preconditions the electrode, measures oxidizable and reducible species, and the open circuit potential (OCP). A linear, stable sensor performance was demonstrated, also in the presence of proteins, validating signal stability of our cyclic protocol in complex environments. We investigated our sensor protocol with microelectrodes on custom Pt/Ir-wire tetrodes by in vivo measurements in the rat brain for up to four weeks. Results showed that catalytic activity of the electrode is lost over time, but our protocol is repeatedly able to both quantify and restore electrode sensitivity in vivo. SIGNIFICANCE Our approach is highly relevant because it can be applied to any existing Pt electrode. Current methods to assess the brain/electrode microenvironment mainly rely on imaging techniques, histology and analysis of explanted devices, which are often end-point methods. Our procedure delivers online and time-transient information on the chemical microenvironment directly at the electrode/tissue interface of neural implants, gives new insight into the charge transfer processes, and delivers information on the state of the electrode itself addressing long-term electrode degradation.

A lab-on-a-chip for free-flow electrophoretic preconcentration of viruses and gel electrophoretic DNA extraction

Abstract: Nucleic acid amplification techniques such as real-time PCR are essential instruments for the identification and quantification of viruses. They are fast, very sensitive and highly specific, but often require elaborate and labor intensive sample preparation to achieve successful amplification of the target sequence. In this work we demonstrate the complete microfluidic preparation of amplifiable virus DNA from dilute specimens. Our approach combines free-flow electrophoretic preconcentration of viral particles with thermal lysis and gel-electrophoretic nucleic acid extraction on a single device. The on-chip preconcentration achieves a capture efficiency of >99% for dilute suspensions of bacteriophage PhiX174. Following preconcentration, phages are thermally lysed and released DNA is recovered after 40 s of on-chip gel-electrophoresis with a recovery rate of ∼73%. Furthermore we demonstrate a detection limit of ∼1 PFU ml-1 (∼0.02 DNA copies per μl) for the detection of bacteriophage PhiX174 by PCR. To simplify operation of the device, we describe the development of a custom-made chip holder as well as a compact peristaltic pump and power supply, which enable user-friendly operation with low risk of cross-contamination and high potential for automation in the field of point-of-care diagnostics.

A linearized expiration flow homogenizes the compartmental pressure distribution in a physical model of the inhomogeneous respiratory system.

Abstract: OBJECTIVE Flow-controlled expiration (FLEX) and flow-controlled ventilation (FCV) imply a linearized expiration, and were suggested as new approaches for lung-protective ventilation, especially in the case of an inhomogeneous lung. We hypothesized that a linearized expiration homogenizes the pressure distribution between compartments during expiration, compared to volume-controlled (VCV) and pressure-controlled (PCV) ventilation. APPROACH We investigated the expiratory pressure decays in a physical model of an inhomogeneous respiratory system. The model contained four compartments of which two had a high (25 ml cmH2O-1) and two a low compliance (10 ml cmH2O-1). These were combined with either a high (6.5 cmH2O s l-1) or low resistance (2.8 cmH2O s l-1), respectively. The model was ventilated in all modes at various tidal volumes and peak pressures, and we determined in each compartment the expiratory time at which the pressure declined to 50% (t50) of the end-inspiratory pressure, and the maximal differences of t50 (Δt50) and pressure (Δpmax) between all compartments. MAIN RESULTS During FLEX and FCV, t50 was 6- to 7-fold higher compared to VCV and PCV (all P < 0.001). During VCV and PCV, Δt50 was higher (128 ± 18 ms) compared to FLEX and FCV (49 ± 19 ms; all P < 0.001). Δpmax reached up to 3.8 ± 0.2 cmH2O during VCV and PCV, but only 0.6 ± 0.1 cmH2O during FLEX and FCV (P < 0.001). SIGNIFICANCE FLEX and FCV provide a more homogeneous expiratory pressure distribution between compartments with different mechanical properties compared with VCV and PCV. This may reduce shear stress within inhomogeneous lung tissue.

Microsensor electrodes for 3d inline process monitoring in multiphase microreactors

Abstract: We present an electrochemical microsensor for the monitoring of hydrogen peroxide direct synthesis in a membrane microreactor environment by measuring the hydrogen peroxide and oxygen concentrations. In prior work, for the first time, we performed in situ measurements with electrochemical microsensors in a microreactor setup. However, the sensors used were only able to measure at the bottom of the microchannel. Therefore, only a limited assessment of the gas distribution and concentration change over the reaction channel dimensions was possible because the dissolved gases entered the reactor through a membrane at the top of the channel. In this work, we developed a new fabrication process to allow the sensor wires, with electrodes at the tip, to protrude from the sensor housing into the reactor channel. This enables measurements not only at the channel bottom, but also along the vertical axis within the channel, between the channel wall and membrane. The new sensor design was integrated into a multiphase microreactor and calibrated for oxygen and hydrogen peroxide measurements. The importance of measurements in three dimensions was demonstrated by the detection of strongly increased gas concentrations towards the membrane, in contrast to measurements at the channel bottom. These findings allow a better understanding of the analyte distribution and diffusion processes in the microreactor channel as the basis for process control of the synthesis reaction.

Direct EPR detection of atomic nitrogen in an atmospheric nitrogen plasma jet.

Abstract: In this study, an atmospheric nitrogen plasma jet generated by a custom-built micro-plasma device was analyzed at room temperature by continuous wave and pulse EPR spectroscopy in real time. Transiently formed nitrogen atoms were detected without the necessity to use spin-traps or other reagents for their stabilization. In contrast to results from optical emission spectroscopy, only signals from the 4S ground state of 14N and 15N could be detected. EPR data analysis revealed an isotropic g value of 1.9971 and isotropic hyperfine coupling constants of a(14N) = (10.47 ± 0.02) MHz and a(15N) = (14.69 ± 0.02) MHz. Moreover, lifetime and relaxation data could be determined; both are discussed in terms of spectral widths and actual concentrations of the transiently formed nitrogen species within the plasma jet. The data show that the lifetimes of atomic nitrogen and charged particles such as N+ must be different, and for the latter below the observation time window of EPR spectroscopy. We demonstrate that the real-time (pulsed) EPR technique is a fast and reliable alternative to detect atomic nitrogen in atmospheric pressure plasma jets. The method may be used for a continuous monitoring of the quality of plasma jets.

Multichannel cell detection in microcompartments by means of true parallel measurements using the Solartron S-1260

Abstract: Designing proper frontend electronics is critical in the development of highly sophisticated electrode systems. Multielectrode arrays for measuring electrical signals or impedance require multichannel readout systems. Even more challenging is the differential or ratiometric configuration with simultaneous assessment of measurement and reference channels. In this work, an eight-channel frontend was developed for contacting a 2×8 electrode array (8 measurement and 8 reference electrodes) with a large common electrode to the impedance gain-phase analyzer Solartron 1260 (S-1260). Using the three independent and truly parallel monitor channels of the S-1260, impedance of trapped cells and reference material was measured at the same time, thereby considerably increasing the performance of the device. The frontend electronics buffers the generator output and applies a potentiostatic signal to the common electrode of the chip. The applied voltage is monitored using the current monitor of the S-1260 via voltage/current conversion. The frontend monitors the current through the electrodes and converts it to a voltage fed into the voltage monitors of the S-1260. For assessment of the 8 electrode pairs featured by the chip, a relay-based multiplexer was implemented. Extensive characterization and calibration of the frontend were carried out in a frequency range between 100 Hz and 1 MHz. Investigating the influence of the multiplexer and the frontend electronics, direct measurement with and without frontend was compared. Although differences were evident, they have been negligible below one per cent. The significance of measurement using the complex S-1260-frontend-electrode was tested using Kohlrausch's law. The impedance of an electrolytic dilution series was measured and compared to the theoretical values. The coincidence of measured values and theoretical prediction serves as an indicator for electrode sensitivity to cell behavior. Monitoring of cell behavior on the microelectrode surface will be shown as an example.

Long-term in vivo Monitoring of Gliotic Sheathing of Ultrathin Entropic Coated Brain Microprobes with Fiber-based Optical Coherence Tomography

Abstract: Microfabricated neuroprosthetic devices have made possible important observations on neuron activity; however, long-term high-fidelity recording performance of these devices has yet to be realized. Tissue-device interactions appear to be a primary source of lost recording performance. The current state of the art for visualizing the tissue response surrounding brain implants in animals is Immunohistochemistry + Confocal Microscopy, which is mainly performed after sacrificing the animal. Monitoring the tissue response as it develops could reveal important features of the response which may inform improvements in electrode design. Optical Coherence Tomography (OCT), an imaging technique commonly used in ophthalmology, has already been adapted for imaging of brain tissue. Here, we use OCT to achieve real-time, in vivo monitoring of the tissue response surrounding chronically implanted neural devices. The employed tissue- response-provoking implants are coated with a plasma-deposited nanofilms, which have been demonstrated as a biocompatible and anti-inflammatory interface for indwelling devices. We evaluate the method by comparing the OCT results to traditional histology qualitatively and quantitatively. The differences in OCT signal across the implantation period between the plasma group and the control reveal that the Parylene-type coating of otherwise rigid brain probes (glass and silicon) does not improve the glial encapsulation in the brain parenchyma.

Capacitive Properties of Ceramic Humidity Sensors Made from Porous Perovskite Films

Abstract: In this research, ceramic-based capacitive humidity sensors based on the barium strontium titanate perovskite nanocomposite and doped with the various concentrations of magnesia nanoparticles were fabricated and investigated. The particle size of the sensing elements is varied from 56 nm to 35 nm per dopant surcharges. The interaction between bulk perovskites (pellet) and water vapor was studied by impedance spectroscopy. Presence of the ionic transport even at low RH values is observed from the bulk frequency-capacitance spectra. The EIS results of the bulk sample confirm that the proton transfer operates only by charge transfer kinetics and not diffusion process to metals (up to 90% RH). Among all the proposed sensors, the device contains of 3 mol% magnesia exhibits the most capacitance change (21 pF – 25200 pF) with the sensitivity of 335 pF/RH% in the range of 20–95% RH, and a maximum hysteresis of 5.2% RH at 60% RH. The impact of rest of dopant values on the main perovskite is negative.