Sensorchip based impedance spectroscopy can detect inhibitory effects of human neuropeptide Y (hNPY) on living cells in a non-invasive labelling free way in real time without the need of supporting reagents. Since the discovery that neoplasmatic transformations in breast cancer are correlated with a change of the receptor subtype expression of hNPY in the affected tissue, the hNPY receptor-ligand system has come to the fore of cancer research. Today there are different methods detecting hNPY receptor interactions like fluorescent and radioactive labelling or detecting hNPY-pathway activation like cyclic adenosine monophosphate (cAMP) and G protein-coupled receptor (GPCR)-assays. For all these assays it is necessary to either label related proteins with additional substances, which can affect the nature state of the cell, or the need of producing cell lysate which allows only a snapshot of the investigated cells. To overcome these problems we established a new method to detect hNPY-receptor interactions. Therefore, we monitor the complex electric resistance (impedance) of cells attached to a microelectrode over a wide frequency range. Cell alterations are detected as changes in the impedance spectra. After application of the adenylyl cyclase-stimulating reagent forskolin, impedance is decreased at 5 kHz frequency within minutes. This effect can be inhibited by preincubating the cells with hNPY for a time range of 20 min. The inhibitory effect of hNPY can be washed out and the same cells can be stimulated by forskolin again.

For a feasible and cost-effective impedance measurement of cellular alterations in real-time, we combined commercially available microelectrode arrays (MEAs), consisting of 60 microelectrodes, with a conventional impedance analyzer. For proof of principle, a breast carcinoma cell line (MCF-7) was cultured on MEAs, and cellular alterations were measured by impedance spectroscopy at a frequency ranging from 10 Hz to 1 MHz. Cells were stimulated with phorbol 12-myristate 13-acetate (PMA) at different concentrations to activate protein kinase C (PKC)-mediated extra- and intracellular changes. By addition of 0.03 microM PMA, an increase of the relative impedance (Z(rel)) was observed after 10 min with a maximum at 1 kHz. Moreover a gradual elevation of the impedance was measured 60 min after stimulation with PMA. If 0.3 microM PMA was applied, the maximal amplitude of the relative impedance after 60 min shifted from 1 kHz (0.03 microM PMA) to 150 Hz. Subsequently, the impedance was further increased up to 90 min after PMA application, after which the impedance reduced after 240 min. Since we could use MEAs for at least 10 times without affecting the sensitivity, our study revealed that commercially available MEAs comprising nanocolumnar titanium nitrite electrodes are suitable microstructures for a highly reproducible and cost-effective multisite measurement of intracellular processes by impedance spectroscopy.