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.

In vitro system for the prediction of hepatotoxic effects in primary hepatocytes.

Prediction of liver toxicity and compound responses continues to be a major challenge for the pharmaceutical industry. In vitro studies on liver cells have been developed to reduce or replace animal experiments. However, most of the tests in use are based on cell lines which do not necessarily represent normal cell physiology. We compared the response of primary human hepatocytes from two donors with primary rat hepatocytes and the cell line HepG2 to the test compound acetaminophen (AAP) by measuring oxygen consumption, extracellular acidification and cell adhesion as dynamic parameters of cell metabolism. Primary human hepatocytes were cultured on collagen pre-coated sensor chips or in conventional two-dimensional cultures in chemically defined Human Hepatocyte Maintenance Medium. This medium allows cultivation of functionally differentiated hepatocytes for several weeks. Sensor chip based results were compared with conventional assays for hepatocytes like albumin release and urea release. The hepatocytes were exposed to AAP (50-2815 mg/l) for 24 h. Cell respiration was inhibited by AAP concentrations of 500 mg/l and more in all three cell types, whereas only the cellular acidification rates and cell adhesion of the rat hepatocytes and the HepG2 cells were affected by AAP. In conventional cultures of human hepatocytes, AAP had no effect on cellular viability. Whereas high doses of AAP (2815 mg/l) diminished albumin secretion by 70-80%.

Online monitoring of cell metabolism for studying pharmacodynamic effects.

To characterize modes of action of substances and their cytotoxic effects Bionas GmbH has developed a new screening system to allow the continuous recording of how an active substance can act (Bionas 2500 analyzing system). In the pharmaceutical industry it is important to acquire as much information as possible about the metabolic effects of an active substance. Most classical pre-clinical studies are very expensive and time-consuming. Often they are so-called end-point tests which require many individual tests before approximate statements can be made about how an effect takes its course. With the Bionas 2500 analyzing system metabolically relevant data including oxygen consumption, acidification rate and the adhesion (cell impedance) of cells can be measured in parallel, online and label-free. Using e.g. ion-sensitive field effect-transistors (ISFET) and electrode structures it is possible to observe metabolic parameters non-invasively and continuously over longer periods of time. The system has already been established for several cell models, cell lines as well as primary cells. It also offers the advantage that regenerative effects can be observed during the same test run.

Measurement of electrical activity of long-term mammalian neuronal networks on semiconductor neurosensor chips and comparison with conventional microelectrode arrays.

Based on complementary metal-oxide semiconductor (CMOS) technology a neurosensor chip with passive palladium electrodes was developed. The CMOS technology allows a high reproducibility of the sensors as well as miniaturization and the on-chip integration of electronics. Networks of primary neurones were taken from murine foetal spinal cord (day 14) and frontal cortex (day 15) tissues and cultured on the silicon surface in a chamber volume of 200 microl with 7 mm diameter. Measurements were performed between days 15 and 59 in vitro. Signals were recorded from both types of cultures. To test the capability of the system to detect pharmacologically induced activity changes two established neuromodulators were applied. The GABA(A)-receptor blocker bicuculline was applied to both tissue cultures, the glycine-receptor blocker strychnine to spinal cord cultures. Four network frequency parameters were analysed: spike rate (SR), burst rate (BR), frequency in bursts (FiB) and peak frequency in bursts (PFiB). Significant changes of spike rate and burst rate were measured with spinal cord cultures after bicuculline application. Significant changes of frequency in bursts and peak frequency in bursts were observed with frontal cortex cultures after bicuculline application. Significant changes of spike rate and frequency in bursts were recorded with spinal cord cultures after strychnine application. These results were compared with results achieved in the same laboratory by using glass-microelectrode arrays (MEAs). This comparison showed for spinal cord similar native spike and burst rate, but higher mean frequency and peak frequency in bursts, whereas frontal cortex activity had higher spike and burst rate and peak frequency in bursts. Application of bicuculline or strychnine to spinal cord networks showed stronger effects on MEAs, whereas with frontal cortex networks the modulation of activity was similar after application of bicuculline.