Development of a novel, multi-analyte biosensor system for assaying cell division: identification of cell proliferation/death precursor events.
A novel, miniaturized biosensor system was created by combining the electrophysiological response of immobilized cells with superoxide-sensing technology, optical and fluorescence microscopy. Vero cells were immobilized in a calcium alginate matrix (at a density of 1.7 x 10(6) cells ml(-1)). A 0.5 cm x 0.5 cm piece of cell-containing gel matrix was aseptically adhered on a glass microscope slide with a microfabricated gold electrode array, sealed with a cover slip and provided with Dulbecco's medium +10% (v/v) fetal calf serum every day by means of a capillary feeding tube. During a culture period of 7 days, the membrane potential of immobilized cells was continuously monitored, while cell division was assayed with an optical microscope. In addition, daily measurements of immobilized cell membrane potential, viability, RNA and calcium concentration, radical oxygen species (ROS) and glutathione accumulation, were conducted by fluorescence microscopy after provision of an appropriate dye. Superoxide accumulation was assayed by covering the electrodes with superoxide dismutase (SOD). Maximum cell membrane potential values and superoxide production were observed upon initiation of cell division. Using the novel biosensor, we were able to correlate seven different cell physiological parameters to each other and formulate a model for ROS-mediated signaling function on cell division and death. In addition, we were able to predict cell proliferation or death by comparing the relative response of the electrophysiological and superoxide sensor during the culture period.
Laboratory of Plant Physiology, Agricultural University of Athens, 75 Iera Odos, 11855 Athens, Greece. skin@aua.gr
S
S
Kintzios
I
I
Marinopoulou
G
G
Moschopoulou
O
O
Mangana
K
K
Nomikou
K
K
Endo
I
I
Papanastasiou
A
A
Simonian
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods; D002455:Cell Division / Q000502:physiology*; D018914:Cells, Immobilized / Q000502:physiology; D002522:Chlorocebus aethiops; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D008564:Membrane Potentials / Q000502:physiology*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D018384:Oxidative Stress / Q000502:physiology; D017382:Reactive Oxygen Species / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000737:chemistry; D018511:Systems Integration; D014709:Vero Cells
D017382:Reactive Oxygen Species; D013482:Superoxide Dismutase