Cytosensor

Fig. 20.8. P -gp-ATPase activation profiles obtained by measurements of extracellular acidification rates in living cells by means of a cytosensor physiometer. (A) Cyclosporin A ( ), progesterone (0)> trifluoperazine (A) and verapamil ( ) in LLC-MDR1 cells (B) amitriptyline ( ), calcein-AM (A), diltiazem ( ) and vinblastine (T) in LLC-MDR1 cells.

Cytosensor Microphysiometer technology has been used to detect perturbation in mammalian cells (Hafner, 2000). The system measures small changes in extracellular acidification using a light addressable potentiometric sensor. If the metabolism is interfered with, acid excretion will be affected which could be sensitively measured by LAPS. In principle, this system should be suitable for monitoring pathogen interaction with mammalian cells. 

Hafner, E. (2000). Cytosensor(R) microphysiometer Technology and recent applications. Biosens. Bioelectron. 15,149-158. 

McConnell et al. Tlte Cytosensor Microphysiometer Biological Applications of Silicon Technology, Science, 1906 (September 25, 1992). 

Fig. 5.9. Picture of the commercial Cytosensor microphysiometer from Molecular Devices. Four fluid pathways with (1) analyte container, (2) displacement pump, (3) debubbler, (4) valves, (5) LAPS sensor chamber and (6) reference electrode.

Besides this remarkably fast commercial development, other research groups designed their own laboratory-compatible LAPS systems for similar purposes. The use of, e.g., a nicotinic acetylcholine receptor was reported to create a LAPS-receptor biosensor capable of detecting receptor ligands (acetylcholine, carbamylcholine, succinylcholine, sub-eryldicholine, nicotine as well as d-tubocurarine, a-bungarotoxin and a-Naja toxin) [85]. Another system quite similar to the Cytosensor setup was introduced, where mouse fibroblast fine 3T6 cells were chosen to demonstrate the determination of metabolic processes of these cells [86]. 

H.M. McConnell, J. Owicki, J. Parce, D. Miller, G. Baxter, H. Wada and S. Pitchford, The Cytosensor microphysiometer biological applications of silicon technology, Science, 257(5078) (1992) 1906-1912. 

McConnell HM, Owicki JC, Parce JW, Miller DL, Baxter GT, Wada HG, et al. The cytosensor microphysiometer Biological applications of silicon technology. Science 1992 257 1906-12. 

Figure 18.4 Binding from water to the transporter (1) is divided into two steps membrane partitioning (2) and transporter binding (3). These processes are fast and can be considered (to a first approximation) as equilibrium processes in inside-out vesicles as well as in cells under steady-state conditions in a Cytosensor this is indicated by the double arrows. The free energy of binding of the drug from water to the transporter (AGj jj) was...

Cytosensor microphysiometer (CM) Validated in 2009, Draft OECD TG Applicable to water-soluble substances and mixtures. 

The Cytosensor Microphysiometer (CM) test method is currently under discussion (draft test guideline on the Cytosensor Microphysiometer) at OECD level [34], It is a cytotoxicity and cell-function based in vitro assay that is performed on a sub-confluent monolayer of adherent mouse L929 fibroblasts cultured in a sensor chamber using a pH-meter to detect changes in acidity [35]. The CM test method serves as an in vitro model system for the cytotoxic action of a test chemical on the cell membranes of the corneal and conjunctival epithelium where the irritant chemical would be... 

Cytosensor Microphysiometer Test Method an in vitro method for identifying ocular 3 corrosive and severe irritant chemicals as well as chemicals not classified as ocular irritants. Available at http //www.oecd.org/env/... 

The effects of selected compounds on the cellular metabolic activity (rate of excretion of acidic metabolites) of CHO cells transfected with human muscarinic receptor subtypes were determined utilizing a Cytosensor micro-physiometer (McConnell, 1992). 

McConnell. H.M., 1992. The cytosensor microphysiometer biological applications of silicon technology. Science 257, 1906-1912. 

Chaplen, F.W., Upson, R., McFadden, P.N., and Kolodziej, W.J., Fish chromatophores as cytosensors in a microscale device detection of environmental toxins and microhial pathogens. Pigment Cell Res., 15 (1), 19-26, 2002. 

Pacut, A., Kolodziej, W., and Chaplen, F.W., Cytosensors for early detection of biological and chemical threats-statistical approach, in Neuronal Networks and Expert Systems in Medicine and Healthcare, Proceedings of the fourth International Conference — NNESMED, Milos Island, Greece, June 20-22, Technological Education Institute of Crete, 2001, pp. 437-442. 

Eklund SE, Cliffel DE, Kozlov E, Prokop A, Wikswo J, Baudenbacher F (2003) Modification of the cytosensor(TM) microphysiometer to simultaneously measure extracellular acidification and oxygen consumption rates. Analytica Chimica Acta 496 93-101... 

One of the simplest possible applications of the LAPS/microflow chamber combination is the measurement of enzyme activity. One way to demonstrate this is to immobilize an enzyme in a chamber, and provide it with its substrate in the flow medium. As an example, let s consider acetyl cholinesterase, which catalyzes the hydrolysis of acetylcholine to acetate and choline, liberating protons in the process. Acetyl cholinesterase-coated agarose beads (Sigma) were immobilized between two thin polycarbonate membranes in a Cytosensor [4] chamber, and the pH response measured when an acetylcholine-containing medium is flowed through. Figure 5 shows the data from this experiment. Rates of about 120 pV/s are obtained. Note that the presence of the membranes slows down the time constant of the return to baseline of the pH during the flow-on periods. This demonstrates the measurement of enzyme activity, with possible applications to immunoassays. 

Figure 5 Sensor response to flow on/flow off cycles with a Cytosensor chamber loaded with acetyl cholinesterase coated beads. The flow medium contains 8 mM acetylcholine.

Genosensors [deoxyribonucleic acid (DNA)-based biosensors], immunosensors, cytosensors, and aptamer-based biosensors (aptasensors) are types of affinity-based biosensors. 

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