Botrytis cinerea causes gray mold on a great number of host plants. Infection is initiated by airborne conidia that invade the host tissue, often by penetration of intact epidermal cells. To mimic the surface properties of natural plant surfaces, conidia were incubated on apple wax-coated surfaces, resulting in rapid germination and appressorium formation. Global changes in gene expression were analyzed by microarray hybridization between conidia incubated for 0 h (dormant), 1 h (pregermination), 2.5 h (postgermination), 4 h (appressoria), and 15 h (early mycelium). Considerable changes were observed, in particular between 0 h and 1 h. Genes induced during germination were enriched in those genes encoding secreted proteins, including lytic enzymes. Comparison of wild-type and a nonpathogenic MAP kinase mutant (bmp1) revealed marked differences in germination-related gene expression, in particular related to secretory proteins. Using promoter-GFP reporter strains, we detected a strictly germination-specific expression pattern of a putative chitin deacetylase gene (cda1). In contrast, a cutinase gene (cutB) was found to be expressed only in the presence of plant lipids, in a developmentally less stringent pattern. We also identified a coregulated gene cluster possibly involved in secondary metabolite synthesis which was found to be controlled by a transcription factor also encoded in this cluster. Our data demonstrate that early conidial development in B. cinerea is accompanied by rapid shifts in gene expression that prepare the fungus for germ tube outgrowth and host cell invasion.

We introduce a novel technique of impedimetric sensing of cellular adhesion, which might have the potential to supplement the well-known technique of Electrical Cell-substrate Impedance Sensing (ECIS) in cell culture assays. In contrast to the already commercialized ECIS method, we are using ion-sensitive field-effect transistor (ISFET) devices. The standard gold microelectrode size in ECIS is in the range of 100-250 μm in diameter. Reason for this limitation is that when downscaling the sensing electrodes, their effective impedance governed by the metal-liquid interface impedance is becoming very large and hence the currents to be measured are becoming very small reaching the limit of standard instrumentation. This is the main reason why typical assays with ECIS are focusing on applications like cell-cell junctions in confluent cultures. Single cell resolution is barely reachable with these systems. Here we use impedance spectroscopy with ISFET devices having gate dimensions of only 16 × 2 μm(2), which is enabling a real single cell resolution. We introduce an electrically equivalent circuit model, explain the measured effects upon single cell detachment, and present different cellular detachment scenarios. Our approach might supplement the field of ECIS with an alternative tool opening up a route for novel cell-substrate impedance sensing assays with so far unreachable lateral resolution.

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Graphene, with its unique electrical properties and biocompatibility, has become a material of choice for the development of biosensor platforms. In this study, a microelectrode array based on reduced graphene oxide (rGO) was constructed and used as a platform for electrical monitoring of cell-substrate adhesion. The rGO-based sensor arrays were designed in order to facilitate sensor pads comparable to the size of individual cells. The sensor chips were fabricated in a scalable manner via site-specific immobilization of graphene oxide flakes onto microelectrode pairs followed by reduction to rGO. The sensor chips were mounted on a measurement platform equipped with a fluidic cell. Electrical characteristics were recorded and fieldeffect behavior was confirmed. Sensors reacted to changes of pH value in the solution. Finally, as a proof-of-concept, the graphene oxide-based sensing platform was used for electrical cell-substrate impedance sensing of individual HEK293 cells in culture.Schematic view of the rGO-based sensor chip for electrical cell-substrate adhesion assays.

In this study we describe how we were able to use an equivalent‐electrical circuit for the cell–transistor contact to optimize a future chip design for better performance in cell–substrate adhesion experiments. From our simulations we found that higher capacitances of the source and drain contact lines are able to shift the cell adhesion effects to more moderate frequencies in the range of up to 200 kHz. For larger cell–substrate adhesion effects, the transconductance value of the FET devices needs to be increased as well. We used these simulation results to design a new generation of FET devices. These devices were successfully fabricated in our clean room facilities. In electronic cell–substrate adhesion experiments this new generation of devices showed superior performance compared to an earlier version. Most importantly, our new devices have now an almost flat topology enabling future single‐cell migration experiments. HEK293 cells adhered with a flat morphology to a newly fabricated FET sensor device (scale bar 50 µm)

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