Publications by authors named "Jochen Kieninger"

21 Publications

  • Page 1 of 1

Electrochemical microelectrode degradation monitoring: in situ investigation of platinum corrosion at neutral pH.

J Neural Eng 2022 Jan 4. Epub 2022 Jan 4.

IMTEK - Department of Microsystems Engineering, University of Freiburg, Georges-Köhler-Allee 103, Freiburg im Breisgau, 79110, GERMANY.

Objective: The stability of platinum and other noble metal electrodes is critical for neural implants, electrochemical sensors, and energy sources. Beyond the acidic or alkaline environment found in most electrochemical studies, the investigation of electrode corrosion in neutral pH and chloride containing electrolytes is essential, particularly regarding the long-term stability of neural interfaces, such as brain stimulation electrodes or cochlear implants. In addition, the increased use of microfabricated devices demands the investigation of thin-film electrode stability.

Approach: We developed a procedure of electrochemical methods for continuous tracking of electrode degradation in situ over the complete life cycle of platinum thin-film microelectrodes in a unique combination with simultaneous chemical sensing. We used chronoamperometry and cyclic voltammetry to measure electrode surface and analyte redox processes, together with accelerated electrochemical degradation.

Main Results: We compared degradation between thin-film microelectrodes and bulk electrodes, neutral to acidic pH, different pulsing schemes, and the presence of the redox active species oxygen and hydrogen peroxide. Results were confirmed by mechanical profilometry and microscopy to determine material changes on a nanometer scale. We found that electrode degradation is mainly driven by repeated formation and removal of the platinum surface oxide, also within the electrochemical stability window of water. There was no considerable difference between thin-film micro- and macroscopic bulk electrodes or in the presence of reactive species, whereas acidic pH or extending the potential window led to increased degradation.

Significance: Our results provide valuable fundamental information on platinum microelectrode degradation under conditions found in biomedical applications. For the first time, we deployed a unified method to report quantitative data on electrode degradation up to a defined endpoint. Our method is a widely applicable framework for comparative long-term studies of sensor and neural interface stability.
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http://dx.doi.org/10.1088/1741-2552/ac47daDOI Listing
January 2022

Standard cochlear implants as electrochemical sensors: Intracochlear oxygen measurements in vivo.

Biosens Bioelectron 2022 Mar 9;199:113859. Epub 2021 Dec 9.

Department of Oto-Rhino-Laryngology, Section of Experimental and Clinical Otology, Neurobiological Research Laboratory, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Germany. Electronic address:

Cochlear implants are the most successful neural prostheses worldwide and routinely restore sensorineural hearing loss by direct electrical stimulation of the auditory nerve. Enhancing this standard implant by chemical sensor functionality opens up new possibilities, ranging from access to the biochemical microenvironment of the implanted electrode array to the long-term study of the electrode status. We developed an electrochemical method to turn the platinum stimulation microelectrodes of cochlear implants into electrochemical sensors. The electrodes showed excellent and stable chemical sensor properties, as demonstrated by in vitro characterizations with combined amperometric and active potentiometric dissolved oxygen and hydrogen peroxide measurements. Linear, stable and highly reproducible sensor responses within the relevant concentration ranges with negligible offset were shown. This approach was successfully applied in vivo in an animal model. Intracochlear oxygen dynamics in rats upon breathing pure oxygen were reproducibly and precisely measured in real-time from the perilymph. At the same time, correct implant placement and its functionality was verified by measurements of electrically evoked auditory brainstem responses with clearly distinguishable peaks. Acute measurements indicated no adverse influence of electrical stimulation on electrochemical measurements and vice versa. Our work is ground-breaking towards advanced implant functionality, future implant lifetime monitoring, and implant-life-long in situ investigation of electrode degradation in cochlear implant patients.
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http://dx.doi.org/10.1016/j.bios.2021.113859DOI Listing
March 2022

Microfluidic organ-on-chip system for multi-analyte monitoring of metabolites in 3D cell cultures.

Lab Chip 2021 Dec 1. Epub 2021 Dec 1.

Laboratory for Sensors, IMTEK - Department of Microsystems Engineering, University of Freiburg, Georges-Köhler-Allee 103, 79110 Freiburg, Germany.

Three-dimensional cell cultures using patient-derived stem cells are essential models for a more efficient and individualized cancer therapy. Currently, culture conditions and metabolite concentrations, especially hypoxia, are often not accessible continuously and within microphysiological systems. However, understanding and standardizing the cellular microenvironment are the key to successful models. We developed a microfluidic organ-on-chip platform for matrix-based, heterogeneous 3D cultures with fully integrated electrochemical chemo- and biosensor arrays for the energy metabolites oxygen, lactate, and glucose. Advanced microstructures allow straightforward cell matrix integration with standard laboratory equipment, compartmentalization, and microfluidic access. Single, patient-derived, triple-negative breast cancer stem cells develop into tumour organoids in a heterogeneous spheroid culture on-chip. Our system allows unprecedented control of culture conditions, including hypoxia, and simultaneous verification by integrated sensors. Beyond previous works, our results demonstrate precise and reproducible on-chip multi-analyte metabolite monitoring under dynamic conditions from a matrix-based culture over more than one week. Responses to alterations in culture conditions and cancer drug exposure, such as metabolite consumption and production rates, could be accessed quantitatively and in real-time, in contrast to endpoint analyses. Our approach highlights the importance of continuous, metabolite monitoring in 3D cell cultures regarding the standardization and control of culture conditions, and drug screening in cancer research. Overall, the results underline the potential of microsensors in organ-on-chip systems for successful application, in personalized medicine.
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http://dx.doi.org/10.1039/d1lc00689dDOI Listing
December 2021

Electrochemical methods for neural interface electrodes.

J Neural Eng 2021 10 5;18(5). Epub 2021 Oct 5.

Laboratory for Sensors, IMTEK-Department of Microsystems Engineering, University of Freiburg, Freiburg, Germany.

. Neural interfaces often rely on charge transfer processes between electrodes and the tissue or electrolyte. Electrochemical processes are at the core of electrode function and, therefore, the key to neural interface stability, electrode performance characterization, and utilization of electrodes as chemical sensors. Electrochemical techniques offer a variety of options to investigate the charge transfer and electrocatalytic properties of electrodes.. In this tutorial, we present various experiments to illustrate the power of electrochemical methods, serve as a reference and guideline, and stimulate deeper understanding of the subject.As a basis for the following experiments, we discuss the platinum cyclic voltammogram and focus on understanding surface processes and roughness determination. We highlight the importance of appropriate instrumentation using potentiostats and how strongly it can influence results. We then discuss a number of potential-controlled and current-controlled methods for electrode characterization, including chronocoulometry, chronoamperometry, (active) potentiometry, and chronopotentiometry. They illustrate charge transfer caused by both electrode surface processes and the presence of redox-active species, such as dissolved oxygen and hydrogen, or hydrogen peroxide. We also discuss the electrode potential with respect to a reference electrode under various conditions and how it affects its electrochemical properties like surface state, catalytic properties and capability to transfer charge.Electrochemical methods are still underutilized in neural engineering, and valuable information is therefore often not accessed. Many studies on electrode characterization would benefit from a more consistent and target-oriented electrochemical methodology and instrumentation. That ranges from the investigation of new materials and processes, over electrode performance assessment to the development of more long-term stable and biocompatible neural interfaces. Ultimately, standardization, consistency and comparability will play a key role in the translation of microtechnology into biomedical and clinical applications.
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http://dx.doi.org/10.1088/1741-2552/ac28d5DOI Listing
October 2021

Electrochemical Microsensor for Microfluidic Glyphosate Monitoring in Water Using MIP-Based Concentrators.

ACS Sens 2021 07 13;6(7):2738-2746. Epub 2021 Jul 13.

Laboratory for Sensors, IMTEK-Department of Microsystems Engineering, University of Freiburg, 79110 Freiburg, Germany.

Glyphosate (GLY) is a broad-spectrum herbicide and is the most used pesticide worldwide. This vast usage has raised strong interest in the ecotoxicological impacts and human risks, with contamination of water being a major concern. Decentralized analytical techniques for water monitoring are of high importance. In this work, we present a small, low-cost, and time-effective electrochemical, chip-based microfluidic device for direct electrochemical detection of GLY downstream of a molecularly imprinted polymer (MIP) concentrator. We studied the electrochemical behavior of GLY and its metabolite aminomethylphosphonic acid (AMPA) using cyclic voltammetry with noble metal electrodes in acidic, neutral, and basic media. A chronoamperometric sensor protocol was developed for sensitive and selective GLY measurements on gold electrodes. The optimized protocol was transferred to a chip-based microsensor platform for online and real-time detection of GLY in a microfluidic setup. The results in the range from 0 to 50 μM GLY in 0.5 M HSO show high linearity and a sensitivity of 10.3 ± 0.6 μA mm mM for the chip-based microfluidic platform. Successful recovery of GLY concentrated from untreated tap water and its precise detection from low volumes demonstrates the advantages of our system.
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http://dx.doi.org/10.1021/acssensors.1c00884DOI Listing
July 2021

In Situ Mapping of H, O, and HO in Microreactors: A Parallel, Selective Multianalyte Detection Method.

ACS Sens 2021 04 22;6(4):1583-1594. Epub 2021 Jan 22.

Laboratory for Sensors, IMTEK-Department of Microsystems Engineering, University of Freiburg, 79110 Freiburg, Germany.

Determining local concentrations of the analytes in state-of-the-art microreactors is essential for the development of optimized and safe processes. However, the selective, parallel monitoring of all relevant reactants and products in a multianalyte environment is challenging. Electrochemical microsensors can provide unique information on the reaction kinetics and overall performance of the hydrogen peroxide synthesis process in microreactors, thanks to their high spatial and temporal resolution and their ability to measure in situ, in contrast to other techniques. We present a chronoamperometric approach which allows the selective detection of the dissolved gases hydrogen and oxygen and their reaction product hydrogen peroxide on the same platinum microelectrode in an aqueous electrolyte. The method enables us to obtain the concentration of each analyte using three specific potentials and to subtract interfering currents from the mixed signal. While hydrogen can be detected independently, no potentials can be found for a direct, selective measurement of oxygen and hydrogen peroxide. Instead, it was found that for combined signals, the individual contribution of all analytes superimposes linearly additive. We showed that the concentrations determined from the subtracted signals correlate very well with results obtained without interfering analytes present. For the first time, this approach allowed the mapping of the distribution of the analytes hydrogen, oxygen, and hydrogen peroxide inside a multiphase membrane microreactor, paving the way for online process control.
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http://dx.doi.org/10.1021/acssensors.0c02509DOI Listing
April 2021

Microsensor Electrodes for 3D Inline Process Monitoring in Multiphase Microreactors.

Sensors (Basel) 2020 08 28;20(17). Epub 2020 Aug 28.

Laboratory for Sensors, IMTEK – Department of Microsystems Engineering, University of Freiburg, 79110 Freiburg, Germany.

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.
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http://dx.doi.org/10.3390/s20174876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7506731PMC
August 2020

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

J Neural Eng 2019 12 11;17(1):016007. Epub 2019 Dec 11.

Laboratory for Sensors, IMTEK-Department of Microsystems Engineering, University of Freiburg, Freiburg, Germany. BrainLinks-BrainTools Center, University of Freiburg, Freiburg, Germany. Author to whom any correspondence should be addressed.

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.
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http://dx.doi.org/10.1088/1741-2552/ab4c69DOI Listing
December 2019

On-chip photodynamic therapy - monitoring cell metabolism using electrochemical microsensors.

Lab Chip 2018 11;18(22):3353-3360

Laboratory for Sensors, IMTEK - Department of Microsystem Engineering, University of Freiburg, Germany.

We introduce a new system which combines metabolic monitoring using electrochemical microsensors with photodynamic therapy on-chip for the first time. Oxygen consumption of T-47D breast cancer cells was measured during therapy with protoporphyrin IX. We determined the efficacy of the therapy and revealed its recovery effects, which underlines the high relevance of continuous monitoring.
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http://dx.doi.org/10.1039/c8lc00799cDOI Listing
November 2018

Active Potentiometry for Dissolved Oxygen Monitoring with Platinum Electrodes.

Sensors (Basel) 2018 Jul 24;18(8). Epub 2018 Jul 24.

Laboratory for Sensors, IMTEK-Department of Microsystems Engineering, University of Freiburg, Georges-Köhler Allee 103, D-79110 Freiburg, Germany.

Potentiometric oxygen monitoring using platinum as the electrode material was enabled by the combination of conventional potentiometry with active prepolarization protocols, what we call . The obtained logarithmic transfer function is well-suited for the measurement of dissolved oxygen in biomedical applications, as the physiological oxygen concentration typically varies over several decades. We describe the application of active potentiometry in phosphate buffered salt solution at different pH and ion strength. Sensitivity was in the range of 60 mV/dec oxygen concentration; the transfer function deviated from logarithmic behavior for smaller oxygen concentration and higher ion strength of the electrolyte. Long-term stability was demonstrated for 60 h. Based on these measurement results and additional cyclic voltammetry investigations a model is discussed to explain the potential forming mechanism. The described method of active potentiometry is applicable to many different potentiometric sensors possibly enhancing sensitivity or selectivity for a specific parameter.
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http://dx.doi.org/10.3390/s18082404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6111276PMC
July 2018

Sensor Access to the Cellular Microenvironment Using the Sensing Cell Culture Flask.

Biosensors (Basel) 2018 Apr 26;8(2). Epub 2018 Apr 26.

Laboratory for Sensors, IMTEK-Department of Microsystems Engineering, University of Freiburg, Georges-Köhler Allee 103, D-79110 Freiburg, Germany.

The Sensing Cell Culture Flask (SCCF) is a cell culture monitoring system accessing the cellular microenvironment in 2D cell culture using electrochemical microsensors. The system is based on microfabricated sensor chips embedded in standard cell culture flasks. Ideally, the sensor chips could be equipped with any electrochemical sensor. Its transparency allows optical inspection of the cells during measurement. The surface of the sensor chip is in-plane with the flask surface allowing undisturbed cell growth on the sensor chip. A custom developed rack system allows easy usage of multiple flasks in parallel within an incubator. The presented data demonstrates the application of the SCCF with brain tumor (T98G) and breast cancer (T-47D) cells. Amperometric oxygen sensors were used to monitor cellular respiration with different incubation conditions. Cellular acidification was accessed with potentiometric pH sensors using electrodeposited iridium oxide films. The system itself provides the foundation for electrochemical monitoring systems in 3D cell culture.
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http://dx.doi.org/10.3390/bios8020044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6022884PMC
April 2018

Microsensor systems for cell metabolism - from 2D culture to organ-on-chip.

Lab Chip 2018 05;18(9):1274-1291

Laboratory for Sensors, IMTEK - Department of Microsystems Engineering, University of Freiburg, Germany.

Microsensor systems for cell metabolism are essential tools for investigation and standardization in cell culture. Electrochemical and optical read-out schemes dominate, which enable the marker-free, continuous, online recording of transient effects and deliver information beyond microscopy and end-point tests. There has been much progress in microfluidics and microsensors, but the translation of both into standard cell culture procedures is still limited. Within this critical review, we discuss different cell culture formats ranging from standard culture vessels to dedicated microfluidic platforms. Key aspects are the appropriate supply of cells, mass transport of metabolites to the sensors and generation of stimuli. Microfluidics enable the transition from static to dynamic conditions in culture and measurement. We illustrate the parameters oxygen (respiration), pH (acidification), glucose and lactate (energy metabolism) as well as short-lived reactive species (ROS/RNS) from the perspective of microsensor integration in 2D and 3D cell culture. We discuss different sensor principles and types, along with their limitations, microfabrication technologies and materials. The state-of-the-art of microsensor platforms for cell culture is discussed with respect to sensor performance, the number of parameters and timescale of application. That includes the advances from 2D culture to the increasingly important 3D approaches, with specific requirements for organotypic microtissues, spheroids and solid matrix cultures. We conclude on the current progress, potential, benefits and limitations of cell culture monitoring systems from monolayer culture to organ-on-chip systems.
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http://dx.doi.org/10.1039/c7lc00942aDOI Listing
May 2018

In-vivo monitoring of infection via implantable microsensors: a pilot study.

Biomed Tech (Berl) 2018 Jul;63(4):421-426

Department of Plastic, Reconstructive and Aesthetic Surgery, Handsurgery, Alfried Krupp Krankenhaus, Essen, Germany.

The most common complication after implantation of foreign material is infection, leading to implant failure and severe patient discomfort. Smoldering-infections proceed inapparently and might not get verified by radiological diagnostics. Early identification of this type of infection might significantly reduce the rate of complications. Therefore, we manufactured a microsensor strip in a hybrid of thin-film and laminate technology in a wafer-level process. It comprises electrochemical, amperometric microsensors for glucose, oxygen and lactate as well as an integrated reference electrode. Microsensors have been implanted in the mouse dorsal skin fold chamber, which got inoculated with a human-pathogen bacterial strain. A selective signal could be measured for all parameters and time points. The infection led to measurable changes of the wound environment as given by a decrease of the oxygen- as well as the glucose-concentration while the lactate concentration increased markedly over time. The given results in this study are the first hints on a promising new tool and should therefore be interpreted as a proof of the principle to show the functionality of the microsensors in an in vivo setting. These microsensors could be used to monitor smoldering infections of implantable foreign materials reducing foreign implant associated complications.
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http://dx.doi.org/10.1515/bmt-2016-0250DOI Listing
July 2018

Multianalyte Antibiotic Detection on an Electrochemical Microfluidic Platform.

Anal Chem 2016 10 1;88(20):10036-10043. Epub 2016 Aug 1.

University of Freiburg , Department of Microsystems Engineering, Georges-Koehler-Allee 103, DE-79110 Freiburg, Germany.

The excessive use of antibiotics in human and veterinary medicine causes the emergence of multidrug resistant bacteria. In this context, the surveillance of many different antibiotics provokes a worldwide challenge. Hence, fast and versatile multianalyte single-use biosensors are of increasing interest for many fields such as medical analysis or environmental and food control. Here we present a microfluidic platform enabling the electrochemical readout of up to eight enzyme-linked assays (ELAs), simultaneously. To demonstrate the applicability of this platform for the surveillance and monitoring of antibiotics, we used highly sensitive biomolecular sensor systems for the simultaneous detection of two commonly employed antibiotic classes tetracycline and streptogramin. Thus, microfluidic channel networks are designed, comprising distinct numbers of immobilization sections with a very low volume of 680 nL each. These passively metered sections can be actuated separately for an individual assay procedure. The limits of detection (LOD) are determined, with high precision, to 6.33 and 9.22 ng mL for tetracycline and pristinamycin, respectively. The employed channel material, dry film photoresist (DFR), allows an easy storage of preimmobilized assays with a shelf life of at least 3 months. Multianalyte measurements in a complex medium are demonstrated by the simultaneous detection of both antibiotics in spiked human plasma within a sample-to-result time of less than 15 min.
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http://dx.doi.org/10.1021/acs.analchem.6b02294DOI Listing
October 2016

Accessing 3D microtissue metabolism: Lactate and oxygen monitoring in hepatocyte spheroids.

Biosens Bioelectron 2017 Jan 28;87:941-948. Epub 2016 Jul 28.

Laboratory for Sensors, IMTEK - Department of Microsystems Engineering, University of Freiburg, Freiburg, Germany.

3D hepatic microtissues, unlike 2D cell cultures, retain many of the in-vivo-like functionalities even after long-term cultivation. Such 3D cultures are increasingly applied to investigate liver damage due to drug exposure in toxicology. However, there is a need for thorough metabolic characterization of these microtissues for mechanistic understanding of effects on culture behaviour. We measured metabolic parameters from single human HepaRG hepatocyte spheroids online and continuously with electrochemical microsensors. A microsensor platform for lactate and oxygen was integrated in a standard 96-well plate. Electrochemical microsensors for lactate and oxygen allow fast, precise and continuous long-term measurement of metabolic parameters directly in the microwell. The demonstrated capability to precisely detect small concentration changes by single spheroids is the key to access their metabolism. Lactate levels in the culture medium starting from 50µM with production rates of 5µMh were monitored and precisely quantified over three days. Parallel long-term oxygen measurements showed no oxygen depletion or hypoxic conditions in the microwell. Increased lactate production by spheroids upon suppression of the aerobic metabolism was observed. The dose-dependent decrease in lactate production caused by the addition of the hepatotoxic drug Bosentan was determined. We showed that in a toxicological application, metabolic monitoring yields quantitative, online information on cell viability, which complements and supports other methods such as microscopy. The demonstrated continuous access to 3D cell culture metabolism within a standard setup improves in vitro toxicology models in replacement strategies of animal experiments. Controlling the microenvironment of such organotypic cultures has impact in tissue engineering, cancer therapy and personalized medicine.
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http://dx.doi.org/10.1016/j.bios.2016.07.094DOI Listing
January 2017

Microfabricated, amperometric, enzyme-based biosensors for in vivo applications.

Anal Bioanal Chem 2016 Jul 2;408(17):4503-21. Epub 2016 Mar 2.

Laboratory for Sensors, Department of Microsystems Engineering - IMTEK, University of Freiburg, Georges-Köhler-Allee 103, 79110, Freiburg, Germany.

Miniaturized electrochemical in vivo biosensors allow the measurement of fast extracellular dynamics of neurotransmitter and energy metabolism directly in the tissue. Enzyme-based amperometric biosensing is characterized by high specificity and precision as well as high spatial and temporal resolution. Aside from glucose monitoring, many systems have been introduced mainly for application in the central nervous system in animal models. We compare the microsensor principle with other methods applied in biomedical research to show advantages and drawbacks. Electrochemical sensor systems are easily miniaturized and fabricated by microtechnology processes. We review different microfabrication approaches for in vivo sensor platforms, ranging from simple modified wires and fibres to fully microfabricated systems on silicon, ceramic or polymer substrates. The various immobilization methods for the enzyme such as chemical cross-linking and entrapment in polymer membranes are discussed. The resulting sensor performance is compared in detail. We also examine different concepts to reject interfering substances by additional membranes, aspects of instrumentation and biocompatibility. Practical considerations are elaborated, and conclusions for future developments are presented. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s00216-016-9420-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4909808PMC
July 2016

Lift-Off Free Fabrication Approach for Periodic Structures with Tunable Nano Gaps for Interdigitated Electrode Arrays.

ACS Nano 2016 Jan 4;10(1):1086-92. Epub 2015 Dec 4.

Department of Microsystem Engineering (IMTEK), University of Freiburg , 79110 Freiburg, Germany.

We report a simple, low-cost and lift-off free fabrication approach for periodic structures with adjustable nanometer gaps for interdigitated electrode arrays (IDAs). It combines an initial structure and two deposition process steps; first a dielectric layer is deposited, followed by a metal evaporation. The initial structure can be realized by lithography or any other structuring technique (e.g., nano imprint, hot embossing or injection molding). This method allows the fabrication of nanometer sized gaps and completely eliminates the need for a lift-off process. Different substrate materials like silicon, Pyrex or polymers can be used. The electrode gap is controlled primarily by sputter deposition of the initial structure, and thus, adjustable gaps in the nanometer range can be realized independently of the mask or stamp pattern. Electrochemical characterizations using redox cycling in ferrocenemethanol (FcMeOH) demonstrate signal amplification factors of more than 110 together with collection factors higher than 99%. Furthermore, the correlation between the gap width and the amplification factor was studied to obtain an electrochemical performance assessment of the nano gap electrodes. The results demonstrate an exponential relationship between amplification factor and gap width.
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http://dx.doi.org/10.1021/acsnano.5b06405DOI Listing
January 2016

Targeting tumour hypoxia to prevent cancer metastasis. From biology, biosensing and technology to drug development: the METOXIA consortium.

J Enzyme Inhib Med Chem 2015 27;30(5):689-721. Epub 2014 Oct 27.

a Department of Physics , University of Oslo , Oslo , Norway .

The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation-deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009-2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [(18)F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (<1% O(2)), and the unfolded protein response (UPR) activated by endoplasmatic reticulum (ER) stress and operating at more severe hypoxia (<0.2%). The prioritised targets were the HIF-regulated proteins carbonic anhydrase IX (CAIX), the lactate transporter MCT4 and the PERK/eIF2α/ATF4-arm of the UPR. The METOXIA project has developed patented compounds targeting CAIX with a preclinical documented effect. Since hypoxia-specific treatments alone are not curative they will have to be combined with traditional anti-cancer therapy to eradicate the aerobic cancer cell population as well.
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http://dx.doi.org/10.3109/14756366.2014.966704DOI Listing
May 2016

Polymer-based, flexible glutamate and lactate microsensors for in vivo applications.

Biosens Bioelectron 2014 Nov 14;61:192-9. Epub 2014 May 14.

Laboratory for Sensors, Department of Microsystems Engineering - IMTEK, University of Freiburg, Germany.

We present a flexible microsensor, based on a polymer substrate, for multiparametric, electrochemical in vivo monitoring. The sensor strip with a microelectrode array at the tip was designed for insertion into tissue, for fast and localized online monitoring of physiological parameters. The microsystem fabrication on a wafer-level is based on a polyimide substrate and includes the patterning of platinum microelectrodes as well as epoxy and dry-film-resist insulation in a cost-effective thin-film and laminate process. A stable, electrodeposited silver/silver chloride reference electrode on-chip and a perm-selective membrane as an efficient interference rejection scheme are integrated on a wafer-level. Amperometric, electrochemical, enzyme-based biosensors for the neurotransmitter L-glutamate and the energy metabolite L-lactate have been developed. Hydrogel membranes or direct cross-linking as stable concepts for the enzyme immobilization are shown. Sensor performance including high selectivity, tailoring of sensitivity and long-term stability is discussed. For glutamate, a high sensitivity of 2.16 nAmm(-2) µM(-1) was found. For lactate, a variation in sensitivity between 2.6 and 32 nAmm(-2)mM(-1) was achieved by different membrane compositions. The in vivo application in an animal model is demonstrated by glutamate measurements in the brain of rats. Local glutamate alterations in the micromolar range and in nanoliter-range volumes can be detected and quantified with high reproducibility and temporal resolution. A novel, versatile platform for the integration of various electrochemical sensors on a small, flexible sensor strip for a variety of in vivo applications is presented.
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http://dx.doi.org/10.1016/j.bios.2014.05.014DOI Listing
November 2014

Cell culture monitoring for drug screening and cancer research: a transparent, microfluidic, multi-sensor microsystem.

Lab Chip 2014 Jan 11;14(1):138-46. Epub 2013 Nov 11.

Laboratory for Sensors, Department of Microsystems Engineering (IMTEK), University of Freiburg, Georges-Köhler-Allee 103, 79110 Freiburg, Germany.

We present a novel, multiparametric microphysiometry system for the dynamic online monitoring of human cancer cell metabolism. The optically transparent, modular, hybrid microsystem is based on a glass chip and combines a cell cultivation chamber, microfluidics and metabolic monitoring with fully integrated chemo- and biosensors. pH and oxygen are measured in the cell culture area, and biosensors for lactate and glucose are connected downstream by microfluidics. The wafer-level fabrication features thin-film platinum and iridium oxide microelectrodes on a glass chip, microfluidics in an epoxy resist, a hybrid assembly and an on-chip reference electrode. The reliable analytical performance of the sensors in cell culture medium was demonstrated. The pH sensors exhibit a long-term stable, linear response. The oxygen sensors show a linear behaviour, which is also observed for low oxygen concentrations. Glucose and lactate measurements show a linear, long-term stable, selective and reversible behaviour in the desired range. T98G human brain cancer cells were cultivated and cell culture metabolism was measured on-chip. Stop/flow cycles were applied and extracellular acidification, respiration, glucose consumption and lactate production were quantified. Long-term metabolic rates were determined and all parameters could be measured in the outlet channel. A placement downstream of the cell cultivation area for biosensors was realised. A highly effective medium exchange and undiluted sampling from the cell culture chamber with low flow rates (2 μl min(-1)) and low volumes (15 μl per cycle) were achieved. The drug screening application was demonstrated by detecting alteration and recovery effects of cellular metabolism induced by the addition of substances to the medium.
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http://dx.doi.org/10.1039/c3lc50759aDOI Listing
January 2014

Taking advantage of tumor cell adaptations to hypoxia for developing new tumor markers and treatment strategies.

J Enzyme Inhib Med Chem 2009 Apr;24 Suppl 1:1-39

Laboratory for Stem Cell Research, Aalborg University, Aarhus, Denmark.

Cancer cells in hypoxic areas of solid tumors are to a large extent protected against the action of radiation as well as many chemotherapeutic drugs. There are, however, two different aspects of the problem caused by tumor hypoxia when cancer therapy is concerned: One is due to the chemical reactions that molecular oxygen enters into therapeutically targeted cells. This results in a direct chemical protection against therapy by the hypoxic microenvironment, which has little to do with cellular biological regulatory processes. This part of the protective effect of hypoxia has been known for more than half a century and has been studied extensively. However, in recent years there has been more focus on the other aspect of hypoxia, namely the effect of this microenvironmental condition on selecting cells with certain genetic prerequisites that are negative with respect to patient prognosis. There are adaptive mechanisms, where hypoxia induces regulatory cascades in cells resulting in a changed metabolism or changes in extracellular signaling. These processes may lead to changes in cellular intrinsic sensitivity to treatment irrespective of oxygenation and, furthermore, may also have consequences for tissue organization. Thus, the adaptive mechanisms induced by hypoxia itself may have a selective effect on cells, with a fine-tuned protection against damage and stress of many kinds. It therefore could be that the adaptive mechanisms may take advantage of for new tumor labeling/imaging and treatment strategies. One of the Achilles' heels of hypoxia research has always been the exact measurements of tissue oxygenation as well as the control of oxygenation in biological tumor models. Thus, development of technology that can ease this control is vital in order to study mechanisms and perform drug development under relevant conditions. An integrated EU Framework project 2004-2009, termed EUROXY, demonstrates several pathways involved in transcription and translation control of the hypoxic cell phenotype and evidence of cross-talk with responses to pH and redox changes. The carbonic anhydrase isoenzyme CA IX was selected for further studies due to its expression on the surface of many types of hypoxic tumors. The effort has led to marketable culture flasks with sensors and incubation equipment, and the synthesis of new drug candidates against new molecular targets. New labeling/imaging methods for cancer diagnosing and imaging of hypoxic cancer tissue are now being tested in xenograft models and are also in early clinical testing, while new potential anti-cancer drugs are undergoing tests using xenografted tumor cancers. The present article describes the above results in individual consortium partner presentations.
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http://dx.doi.org/10.1080/14756360902784425DOI Listing
April 2009
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