Mirko Lehmann

Microelectronic sensor system for microphysiological application on living cells

Abstract Living cells can be considered as complex biochemical plants. Biochemical and biophysical processes enable a cell to maintain itself, to grow, to reproduce and to communicate with the environment. Getting more information about the multifunctional cellular processing of input- and output-signals in different cellular plants is essential for basic research as well as for various fields of biomedical applications. For in-vitro investigations on living cells, the cellular environment differs from the native environment found in vivo. As a first approach for on-line monitoring of cellular reactions under well controlled experimental conditions we have developed the so called Cell Monitoring System (CMS®). It allows parallel and non-invasive measurement of different parameters from cellular systems by the use of microsensors. Microelectronic sensors are the adequate choice for the non-invasive measurement of environmental—as well as in- and output—parameters of cells. In this paper we present a measurement system with pH-sensitive ISFETs (ionsensitive fieldeffect transistors) for the measurement of extracellular pH-related signals on cells and tissues.

Non-invasive measurement of cell membrane associated proton gradients by ion-sensitive field effect transistor arrays for microphysiological and bioelectronical applications.

The pH in the cellular microenvironment (pH(M)) is an important regulator of cell-to-cell and cell-to-host interactions. Additionally the extracellular acidification rate of a cell culture is an important indicator of global cellular metabolism. In a new approach a biocompatible ion-sensitive field effect transistor (ISFET)-array was developed to measure the pH(M) close to a surface and the global extracellular acidification rate at the same time. This ISFET-array is part of a new multiparametric microsensor chip. The paper highlights some basic applications of this method for in-vitro measurements. Using a fluid perfusion system for cell culture media, it is possible to measure the pH(M) of few (five to ten) adherent tumor cells in a distance of 10-100 nm from the cell plasma membrane. Experiments showed a pH(M)-value of 6.68 +/- 0.06 pH. Further experiments suggest that both the low pH, and the extracellular acidification rate of the examined tumor cell line are mainly built up by glycolysis.

Approach to a multiparametric sensor-chip-based tumor chemosensitivity assay.

Although not widely practiced by oncologists, in vitro tumor chemosensitivity assays (TCA) have proved to increase the lifetime of tumor patients in prospective clinical trials. By individualizing cancer therapy, they can support the clinicians decision which is usually based on empirically retrieved data and thereby prevent inadequate chemotherapy. We present the first results of a new sensor-chip-based technology which might be useful for a multiparametric TCA. In particular, the aspect of dynamic on-line data generation on intact cellular specimens is a major difference to alternative assays. A series of experiments has been performed on cell lines and human tumor explants. Cell cultures and tumor tissue explants were placed on miniaturized silicon and glass sensor chips. The sensor data currently analyze metabolic profiles (rates of extracellular acidification and cellular oxygen consumption) and changes in cell morphology (monitoring of electric impedance). With the cell lines, drug-associated cellular signals have been detected with all three parameters, while primary explants so far caused metabolic responses only. In particular, cellular respiration or mitochondrial activity seems to be a most sensitive indicator of acute cytotoxic effects. The experimental results were achieved using different test versions. Besides giving a status report, the theoretical potential and current problems of sensor chip technology in TCA is discussed.

Biofunctional hybrid structures—cell–silicon hybrids for applications in biomedicine and bioinformatics

Abstract Cell–silicon hybrids represent miniaturized analytical systems which are qualified by means of the cellular signal processing network to identify biomolecules and biophysical signals in a highly specific and quantitative manner. Such biofunctional hybrid structures are also appropriate tools for a number of experimental approaches where specific reaction patterns and signal transducing processes of living cells are tested in response to defined environmental stimuli. The paper describes how cellular systems can function as multi-potent signal discriminators and interfaces to a variety of physical detection arrays. The analysis is based on recent results regarding the understanding of the intracellular signal transduction network. Our systems analytical approach gives an idea how the biological component of a cellular biosensor works and facilitates the directed design of new families of biofunctional hybrid structures. After a brief review of the construction lines of cell–silicon hybrids, novel improvements of their design and their applicability in tumor biology will be discussed.

An ISFET-algal (Chlamydomonas) hybrid provides a system for eco-toxicological tests.

A cellular sensoring system was designed in which metabolism-dedicated pH-ISFETs and the unicellular green alga Chlamydomonas reinhardtii as a biological component, were combined. The system permits on-line detection of pH changes caused by the metabolic and photosynthetic activities of the cells. Photosynthetic activity results in a basification of the medium caused by uptake of CO2. In darkness, an acidification of the medium, resulting from the production of CO2 by degradation of starch was observed. Both, acidification and basification, are sensitive indicators for the physiological activity of the alga. Experiments using inhibitors of energy metabolism or photosynthesis illustrate the utility of this system for an on-line monitoring of substances of eco-toxicological importance.

Microsensor-Aided Measurements of Cellular Signalling and Metabolism on Tumor Cells

Microsensors provide instruments particularly suited for the rapid, noninvasive and on-line analysis of cell and tissue cultures. The microsensor system presented in this paper is a modular arrangement of various planar and nonplanar sensor elements for the measurement of physiological parameters of cell cultures. An optic access to the cultures (e.g. for light microscopy and spectrophotometric techniques) is also provided for a parallel and comparative data acquisition. The system was originally designed for biomedical research in chemotherapy (predicative chemotherapy assays) and pharmacology but it turned out to be also an effective tool for toxicological and environmental research.

Polysaccharide microarrays with a CMOS based signal detection unit

Microarray based test assays have become increasingly important tools in diagnostics for fast multi-parameter detection especially where sample volumes are limited. We present here a simple procedure to create polysaccharide microarrays, which can be used to analyze antibodies using an integrated, complementary metal-oxide-semiconductor (CMOS) based electric signal readout process. To accomplish this chips are used which consist of an array of silicon photodiodes and where different types of polysaccharides from the bacteria Streptococcus pneumoniae are printed on the (silicon dioxide) chip surface. Typical amounts of polysaccharide deposited in the printing process are around 12 attomol/spot. In a subsequent reaction step the polysaccharide microarrays were used for the measurement of IgG antibody concentrations in human blood sera using either chemiluminescence or fluorescence based detection. To understand the device performance the influence of surface density of the immobilized polysaccharide molecules and other parameters on the assay performance are investigated. The dynamic measurement range of the sensor is shown to reach over more than 3 decades of concentration and covers the whole physiologically relevant range for the analysis of antibodies against a large panel of pneumococcal polysaccharides.

Investigation of Cell-Sensor Hybrid Structures by Focused Ion Beam (FIB) Technology

For the investigation of the adhesion of mammalian cells on a semiconductor biosensor structure, nerve cells on silicon neurochips were prepared for scanning electron microscopy investigations (SEM) and cross-sectional preparation by focused ion beam technology (FIB). The cross-sectional pattern demonstrates the focal adhesion points of the nerve cells on the chip. Finally, SEM micrographs were taken parallel to the FIB ablation to investigate the cross section of the cells slice by slice in order to demonstrate the spatial distribution of focal contact positions for a possible three-dimensional reconstruction of the cell-silicon interface.

The Physiocontrol-Microsystem (PCM): Analysis of Cellular Behaviour for Biomedical Research

Microsensors provide instruments particularly suited for the noninvasive analysis of cell and tissue cultures. Their outstanding benefit is the passive behaviour of continuously working transducers, which allows the dynamic recording of function-specific cellular processes. The microsensor system presented is a modular arrangement of various planar and non-planar sensor elements arranged in small cell culture chambers. An optic access to the cultures (e.g. for high resolution light microscopy and spectro-photometric techniques) enables a parallel and comparative data acquisition. The system was originally designed for biomedical research in chemotherapy and pharmacology but it turned out to be an effective device for toxicological and environmental research as well.

Cell meets silicon - Biomedizinische Sensorik für Diagnostik und Therapie

Die Analyse von Alterationen der zellulären Signalverarbeitung in neoplastischen Epithelzellen unter experimentell therapeutischen Bedingungen durch unsere Arbeitsgruppe hat gezeigt, daß mittels mikrosensorischer Techniken, bereits lange vor mikroskop-optisch nachweisbaren Veränderungen, fiinktionsassoziierte Signalmuster detektiert werden können [1,2,3]. Zu diesem Zweck haben wir Meßsysteme entwickelt und gebaut, die es ermöglichen, Tumorzellen auf elektrisch aktiven Silizi-