Rudolf Heer

Electric impedance sensing in cell-substrates for rapid and selective multipotential differentiation capacity monitoring of human mesenchymal stem cells.

Biosensor systems which enable impedance measurements on adherent cell layers under label-free conditions are considered powerful tools for monitoring specific biological characteristics. A radio frequency identification-based sensor platform was adopted to characterize cultivation and differentiation of human bone marrow-derived multipotent stem cells (bmMSC) over periods of up to several days and weeks. Electric cell-substrate impedance sensing was achieved through fabrication of sensitive elements onto glass substrates which comprised two comb-shaped interdigitated gold electrodes covering an area of 1.8 mm×2 mm. The sensing systems were placed into the wells of a 6-well tissue culture plate, stacked onto a reader unit and could thus be handled and operated under sterile conditions. Continuous measurements were carried out with a sinusoidal voltage of 35 mV at a frequency of 10 kHz. After seeding of human bmMSC, this sensor was able to trace significant impedance changes contingent upon cell spreading and adhesion. The re-usable system was further proven suitable for live examination of cell-substrate attachment or continuous cell monitoring up to several weeks. Induction of either osteogenic or adipogenic differentiation could be validated in bmMSC cultures within a few days, in contrast to state-of-the-art protocols, which require several weeks of cultivation time. In the context of medical cell production in a GMP-compliant process, the here presented interdigitated electric microsensor technology allows the documentation of MSC quality in a fast, efficient and reliable fashion.

Interdigitated impedance sensors for analysis of biological cells in microfluidic biochips

ZusammenfassungEin neuartiges miniaturisiertes Analysesystem zur quantitativen Zellanalyse, auf Basis dielektrischer Mikrosensoren und Mikrofluidik, wird in dieser Arbeit vorgestellt. Das realisierte Lab-on-a-Chip beinhaltet in Kammern eingebettete, passivierte interdigitale Elektrodensysteme. Die Einführung einer Multilagen-Passivierung ermöglicht, im Gegensatz zu herkömmlichen Bioimpedanz-Systemen, die Isolation und somit die räumliche Trennung der dielektrischen Mikrosensoren von der Flüssigkeitsumgebung in den Analysekammern. Anhand von unterschiedlichen, in vitro cultivated cells. The overall performance of the system is demonstrated on various bacterial and yeast strains. Due to the high sensitivity of the contact-less dielectric microsensors it is possible to directly identify microbial strains, based on morphological differences and biological composition in the absence of any indicators or labels. Additionally, dielectric changes occurring in sub-cellular structures such as membranes can be directly monitored over a wide frequency range. As a result, microfluidic biochips are developed to continuously monitor cell morphology changes in a non-invasive manner over long periods of time.SummaryIn the presented work we describe the novel combination of contact-less dielectric microsensors and microfluidics for quantitative cell analysis. The lab-on-a-chip system consists of microfluidic channels and chambers together with integrated and passivated interdigitated electrode structures. In contrast to existing bioimpedance methods implemented for cell analysis, the dielectric microsensors are completely insulated and physically removed from the liquid sensing environment using defined multi-passivation layer of distinct size and composition. Consequently, these structures act as contact-less microsensors for the characterization of in vitro kultivierten, Bakterien und Hefezellen wird das Lab-on-a-Chip charakterisiert. Es zeigt sich, dass mikrobiologische Substanzen aufgrund morphologischer Unterschiede bzw. ihrer biologischen Zusammensetzung ohne Verwendung von Markern oder Indikatoren identifiziert werden können. Dielektrische Variationen in subzellularen Strukturen, wie beispielsweise der Membranen, sind über einen weiten Messfrequenzbereich beobachtbar. Der präsentierte mikrofluidische Biochip wurde speziell für die kontinuierliche und nicht-invasive Beobachtung der Zellmorphologie über lange Zeiträume entwickelt.

Magnetoresistive-based real-time cell phagocytosis monitoring.

The uptake of large particles by cells (phagocytosis) is an important factor in cell biology and also plays a major role in biomedical applications. So far, most methods for determining the phagocytic properties rely on cell-culture incubation and end-point detection schemes. Here, we present a lab-on-a-chip system for real-time monitoring of magnetic particle uptake by human fibroblast (NHDF) cells. It is based on recording the time evolution of the average position and distribution of magnetic particles during phagocytosis by giant-magnetoresistive (GMR) type sensors. We employ particles with a mean diameter of 1.2 μm and characterize their phagocytosis-relevant properties. Our experiments at physiological conditions reveal a cellular uptake rate of 45 particles per hour and show that phagocytosis reaches saturation after an average uptake time of 27.7h. Moreover, reference phagocytosis experiments at 4°C are carried out to mimic environmental or disease related inhibition of the phagocytic behavior, and our measurements clearly show that we are able to distinguish between cell-membrane adherent and phagocytosed magnetic particles. Besides the demonstrated real-time monitoring of phagocytosis mechanisms, additional nano-biointerface studies can be realized, including on-chip cell adhesion/spreading as well as cell migration, attachment and detachment dynamics. This versatility shows the potential of our approach for providing a multifunctional platform for on-chip cell analysis.

Thermal switching field distribution of a single domain particle for field-dependent attempt frequency

We present an analytical derivation of the switching field distribution (SFD) at finite temperature for a single domain particle from the Neel-Brown model in the presence of a linearly swept magnetic field. By considering the field dependence of the attempt frequency f0 in the rate equation, we find enhancement of coercivity compared to models using constant f0. The contribution of thermal fluctuations to the standard deviation of the switching field HC derived here reaches values of 10% HC. Considering this contribution, which has been neglected in previous work, is important for the correct interpretation of measurements of switching field distributions.

Long target droplet polymerase chain reaction with a microfluidic device for high-throughput detection of pathogenic bacteria at clinical sensitivity.

In this article we present a long target droplet polymerase chain reaction (PCR) microsystem for the amplification of the 16S ribosomal RNA gene. It is used for detecting Gram-positive and Gram-negative pathogens at high-throughput and is optimised for downstream species identification. The miniaturised device consists of three heating plates for denaturation, annealing and extension arranged to form a triangular prism. Around this prism a fluoropolymeric tubing is coiled, which represents the reactor. The source DNA was thermally isolated from bacterial cells without any purification, which proved the robustness of the system. Long target sequences up to 1.3 kbp from Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa have successfully been amplified, which is crucial for the successive species classification with DNA microarrays at high accuracy. In addition to the kilobase amplicon, detection limits down to DNA concentrations equivalent to 102 bacterial cells per reaction were achieved, which qualifies the microfluidic device for clinical applications. PCR efficiency could be increased up to 2-fold and the total processing time was accelerated 3-fold in comparison to a conventional thermocycler. Besides this speed-up, the device operates in continuous mode with consecutive droplets, offering a maximal throughput of 80 samples per hour in a single reactor. Therefore we have overcome the trade-off between target length, sensitivity and throughput, existing in present literature. This qualifies the device for the application in species identification by PCR and microarray technology with high sample numbers. Moreover early diagnosis of infectious diseases can be implemented, allowing immediate species specific antibiotic treatment. Finally this can improve patient convalescence significantly.

Signal Generator for Wireless Impedance Monitoring of Microbiological Systems

This paper presents a small microcontroller-based sine-wave generator for probe signals of a wide range of amplitudes and frequencies, which requires very little energy so that it can be used for wireless applications in biosensing. The presented realization generates a 10-kHz sine wave with a 100-mV peak-to-peak voltage. Its total harmonic distortion is less than 0.15%, and the spurious free dynamic range excluding harmonics is about 85 dB. The generators total power consumption at a 3.3-V supply voltage and without output buffers is less than 3.5 mW. This value includes the power requirement of the microcontrollers internal 8-MHz clock generator and is below the power needs of current direct-digital-synthesis-based signal generators. The presented concept bases on commercially available electronic components and is implemented for probe signal generation in wireless batteryless tags for long-term impedance measurements on standard microbiological cell cultures. The included microcontroller enables the use of a phase-controlled trigger signal and other simultaneous control functions, e.g., for peripheral components and data communication.

Wireless powered electronic sensors for biological applications

Radio frequency identification technology is used to power a novel platform of sensor devices. The employed energy harvesting system of the individual sensors enables a blanking of the radio frequency field for a defined period, while supplying the sensor electronics with a highly stable voltage. This guarantees interference free operation of the electronic circuitry during measurements. The implementation of this principle is demonstrated for a sensor system which is based on insets for state-of-the-art micro-titer-plates. Each inset is carrying electronic circuitry and an interdigitated electrode system which is acting as sensor for recording alterations of the cell metabolism. The presented sensor devices work without batteries and are designed for impedance measurements on microbiological cell cultures under physiological relevant conditions.

Contemporaneous cell spreading and phagocytosis: magneto-resistive real-time monitoring of membrane competing processes.

Adhesion and spreading of cells strongly depend on the properties of the underlying surface, which has significant consequences in long-term cell behavior adaption. This relationship is important for the understanding of both biological functions and their bioactivity in disease-related applications. Employing our magnetic lab-on-a-chip system, we present magnetoresistive-based real-time and label-free detection of cellular phagocytosis behavior during their spreading process on particle-immobilized sensor surfaces. Cell spreading experiments carried out on particle-free and particle-modified surfaces reveal a delay in spreading rate after an elapsed time of about 2.2h for particle-modified surfaces due to contemporaneous cell membrane loss by particle phagocytosis. Our associated magnetoresistive measurements show a high uptake rate at early stages of cell spreading, which decreases steadily until it reaches saturation after an average elapsed time of about 100 min. The corresponding cellular average uptake rate during the entire cell spreading process accounts for three particles per minute. This result represents a four times higher phagocytosis efficiency compared to uptake experiments carried out for confluently grown cells, in which case cell spreading is already finished and, thus, excluded. Furthermore, other dynamic cell-surface interactions at nano-scale level such as cell migration or the dynamics of cell attachment and detachment are also addressable by our magnetic lab-on-a-chip approach.

Ballistic electron emission microscopy on spin valve structures

Spin valve structures, as employed in base layers of spin valve transistor devices, are characterized by ballistic electron emission microscopy (BEEM). In detail, Co–Cu–Permalloy–Au layers sputtered onto n-type GaAs bulk substrates were studied. BEEM spectra taken on these multilayers show that magnetocurrents on the order of 600% can be achieved even at room temperature. Small area images (400nm×400nm) show that the spin filtering effect of the spin valves is quite homogeneous on the submicron scale. On larger scales, magnetic domains were imaged close to the switching field of the spin valve structure.

Multifrequency impedance measurement technique for wireless characterization of microbiological cell cultures

An impedance measurement system with probe signal frequencies up to 50 kHz with AC-probe voltages below 30 mV rms was integrated for wireless and battery-free monitoring of microbiological cell cultures. The here presented modular design and the use of state-of-the-art components greatly eases adoptions to a wide range of biotechnological applications without the need of bulky LCR-meters or potentiostats. The device had a power consumption of less than 2.5 mA at a 3.3 V single power supply and worked trouble-free within the humid environment of a cell culture incubator. Measurements on lumped RC-elements showed an error of less than 1% for absolute values and less than 1° regarding the phase of the complex impedance. The performance of sensor devices with interdigitated electrode structures for the measurement of adherent cell cultures was tested in the presence of phosphate-buffered saline solution in the humid atmosphere of an incubator for biological cell cultures.