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Introduction to CAN-Spectroscopy

Venneos' Cell Adhesion Noise-Spectroscopy is a new technique that brings cell-based assays to the next level. We use semiconductor chips to interface human cells directly and thereby enable an unprecedented readout. 

Cell-based assays

Cell-based assays are used to study and quantify the behavior of human cells or tissue under the influence of substances. They are widely used in basic research, drug discovery and regulatory affairs within the cosmetic and chemical industry, for example as a cheap yet meaningful alternative for animal studies. Thus, over the last decades, a multitude of cell-based assays for different applications and questions have been developed.

Technologies, which facilitate the investigation of cell behavior in an in-vivo like environment, are a prerequisite for understanding biological processes. Methods to investigate cell-based assays can are classified into optical, labeling technologies and label-free technologies. As both technologies have advantages and disadvantages, there is still a huge demand for techniques, which enable the analysis and quantification of physiological relevant information from cell-based assays.

Optical technologies require the addition of an exogenous label (usually fluorescent dye) and involve the disclosure of phenotypical changes on single cell level via a microscope. In the case of a live-cell staining these approaches may suffer from interferences with the dye, e.g. several dyes show toxic effects after a relatively short incubation time. Furthermore, the incorporation of labels can be very heterogeneous and/or unspecific, leading to incorrect results. They also have the tendency to be biased by choice of the label for only one defined parameter. Also, the assays are most of the time only a one-endpoint measurement and thereby neglecting important information about the kinetics of the cellular response. 

As alternative, label-free methods have been emerged, whereas impedance spectroscopy is the most common. In comparison to microscopy, it allows the continuous monitoring of cellular parameters over a longer period without the use of a label. Impedance spectroscopy yields a direct quantification for a broad spectrum of biological processes such as adhesion, proliferation, cell death, and GPCR activity. Although, the readout represents only an average response of the whole cell population and thereby neglecting single cell information and cell heterogeneity. Furthermore, changes in the readout curve can be caused by several cellular changes and cannot be directly connected to phenotypical changes, leading to excessively large scope of data interpretation.

Cell Adhesion Noise-Spectroscopy

CAN-Spectroscopy overcomes the limitation of both previously mentioned methods. Here is how it works: If a cell attaches to any kind of surface, a tiny gap in the range of 10 nm to 100 nm remains between the surface and cell membrane. This is due to polymers sticking out of the cell membrane and avoiding direct contact of the bilipid layer to the surface. The gap remains filled with the surrounding culture media, which has properties of an electrical conductor. The ions move around within the gap and are not equally distributed. Accordingly, the local ion concentration between cell membrane and chip surface is permanently fluctuating and thereby causing temporal electrical signals – the cell adhesion noise.

 

What if one would be capable of detecting these small signals? At Venneos, we have developed a semiconductor chip (the CAN-Q Chip) to capture these signals. The CAN-Q Chip consists of nearly 100,000 electrolyte-oxide-metal-oxide-semiconductor field-effect transistors (EO-MOSFETs) or ‘measuring pixels’ with a diameter of 6 µm. Cells are isolated from the semiconductor by a thin layer of oxide and can live up to several weeks or even months on the CAN-Q Chip. The sensitive area on the CAN-Q Chip is 1.6 x 2.5 mm², so up to several hundred cells can be analyzed in parallel.

 

Due to the high density of measurement pixels, CAN-Spectroscopy facilitates the visualization of phenotypical changes on sub-cellular level. Applying smart interpolation algorithms and taking into account, that already only partially covered measuring pixels cause detectable signals, the resolution can be improved further.

 

Such an image can be acquired within seconds. That way different biological processes such as attachment, proliferation, cell death, morphological changes and many others can be investigated in real time up to several days. In contrast to other label-free technologies, these processes can be clearly discriminated from each other. 

 

But CAN-Spectroscopy is more than another imaging method: Analysis of the statistical properties of the cell adhesion noise reveals further information about the cell-chip interface. Data about cell adhesion, cell membrane properties like membrane integrity and even intercellular changes are acquired. All these parameters are equally detected on single cell level and can be correlated with phenotypic cellular behavior to open up a large spectrum of new cell-based assays. This information-rich readout, in combination with the minimal required cell count, makes CAN Spectroscopy the perfect tool for investigating highly valuable cells such as primary cells or stem cells. For sample data regarding cell adhesion please have a look here

Key advantages of CAN-Spectroscopy

  • Using CAN-Spectroscopy, different biological processes such as adhesion, proliferation, cell death, cell signaling, morphological changes and many others can be monitored in real time up to several days. It does not require any dye, so cells remain in their native state during measurements. In contrast to other label-free technologies, biological processes can be clearly discriminated from each other.
  • CAN-Spectroscopy data allows for analysis on single cell or even subcellular level. Consequently, cell heterogeneity is revealed, providing new insights. Furthermore, all obtained parameters can be correlated on single cell level.
  • Compared to other technologies, the required cell count is low. This makes CAN-Spectroscopy a perfect tool for the analysis of highly valuable cells such as stem cells or primary cells.
  • Application specific analysis software apps allow and automated thus user independent evaluation of the experiment. Consequently, analysis takes less time and yields better results.

Sources

  • The Thermal Voltage Fluctuations in the Planar Core-Coat Conductor of a Neuron-Semiconductor Interface, Zeitler, 2013
  • Current-Induced Transistor Sensorics with Electrogenic Cells, Fromherz, 2016