Feasibility study of an online toxicological sensor based on the optical waveguide technique.

Morphological properties of the cells often change as an early response to the presence of a pharmacologically acting toxic substance [Etcheverry, S.B., Crans, D.C., Keramidas, A.D., Cortizo, A.M., Arch. Biochem. Biophys. 338 (1997) 7-14]. Recently it has been shown that living animal cell adhesion and spreading can be monitored online and quantitatively via the interaction of the cells with the evanescent electromagnetic field present at the surface of an optical waveguide [Ramsden, J.J., Li, S.Y., Heinzle, E., Prinosil, J.E. Cytometry 19 (1995) 97-102]. In the present study, optical waveguide lightmode spectroscopy (OWLS) and confocal laser scanning microscopy (CLSM), which provides information about the shape of the cells at the surface, were compared under identical experimental conditions. This allowed for the correlation between the cell-shape information from CLSM and the cell-surface interaction measurements from OWLS. The proposed design of the microsystem sensor involves the establishment of a cell layer on the surface of the waveguide and the subsequent online measurement of the morphological response of the cells to various toxic substances. In the present study, the setup was evaluated using cells from an osteoblastic MC 3T3-E1 cell line, and sodium hypochlorite was used as model toxic substance. Comparing the OWLS signal to the morphological response measured by CLSM reveals that OWLS is effective in monitoring not only cell attachment and spreading but also the cellular response to toxic compounds (i.e. by means of change in cell morphology). For the model toxin, the OWLS measurements indicate that, at concentrations above 0.01%, the cells exhibit a clearly discernable morphological effect (i.e. a decrease in average cell contact area). Thus, the potential of an on-line sensor based on OWLS to applications in toxicology, pharmacy and biocompatibility was demonstrated.

Electrochemical glucose and lactate sensors based on "wired" thermostable soybean peroxidase operating continuously and stably at 37 degrees C.

Following a recent report from our laboratory on a thermostable amperometric H2O2 sensor based on "wiring" soybean peroxidase, glucose and lactate sensors maintaining stable output under continuous operation at 37 degrees C for 12 and 8 days, respectively, were built. The vitreous carbon base of the sensor was coated with four polymer layers. The first was made by cross-linking thermostable soybean peroxidase and the redox polymer formed through complexing part of the rings of poly-(vinylpyridine) with [Os(bpy)2Cl]+/2+ (bpy = bipyridine) and quaternizing part of the rings with bromoethylamine. The second was an insulating and H2O2 transport controlling cellulose acetate layer. The third was an immobilized glucose oxidase or lactate oxidase layer. The fourth was a substrate transport controlling cellulose acetate layer In the case of the glucose sensor, the current output was independent of potential between -0.2 and +0.3 V (vs SCE), and the response time (t10/90) was < 2 min when the concentration was raised from 0 to 5 mM glucose. The current was independent of the O2 partial pressure above 15 Torr. The sensor was relatively insensitive to motion and to interferants. Changing the rotation speed of the electrode from 50 to 2500 rpm increased the current by < 10%. At a glucose concentration of 4 mM, the addition of 0.1 mM ascorbate decreased the current by < 1%. The operational stability was glucose oxidase loading dependent. Though the current decreased by 85% after 100 h of operation at 37 degrees C when the 3-mm-diameter electrode was loaded with only 1.3 micrograms of glucose oxidase, it decreased by < 1% after such operation when loaded with 52 micrograms of the enzyme. Similar results were obtained for the lactate sensor, with the exception of a more noticeable oxygen concentration dependence of the lactate response at low oxygen concentrations.

Oligosaccharide dehydrogenase-modified graphite electrodes for the amperometric determination of sugars in a flow injection system.

Enzyme electrodes for the determination of sugars based on solid graphite electrodes modified with oligosaccharide dehydrogenase "wired" with an osmium-based one-electron (no proton) acceptor redox hydrogel were studied as sensors in a flow injection system. The enzyme and a poly(1-vinylimidazole) (PVI) where every tenth mer is complexed with osmium (4,4'-dimethylbpy)(2)Cl, (denoted PVI(10)dmeOs) were cross-linked with poly(ethylene glycol) (diglycidyl) ether. The electrodes were active for l-arabinose, d-xylose, d-galactose, d-fructose, d-glucose, d-mannose, cellobiose, lactose, maltose, and maltooligosaccharides up to a degree of polymerization of 7. The highest relative response found was for glucose (100%) followed by maltose (40.6%) and lactose (40.6%). Fructose and isomaltotriose gave the lowest responses (<1%). Calibration curves for glucose were strictly linear in the range between 30 and 500 µM with sensitivity and apparent Michaelis-Menten constant (K(m)(app)) of 23.0 ± 1.4 µA mM(-)(1)cm(-)(2) and 4.26 ± 0.95 mM, respectively.

Preliminary investigations of an amperometric oligosaccharide dehydrogenase-based electrode for the detection of glucose and some other low molecular weight saccharides.

Biosensors for the determination of sugars were constructed using oligosaccharide dehydrogenase (ODH) and soluble phenazine methosulfate (PMS) or an osmium-based three-dimensional redox hydrogel. In the latter case the enzyme and poly(1-vinylimidazole) complexed with osmium (4,4'-dimethylbpy)2Cl were cross-linked with poly(ethylene glycol) diglycidyl ether. Both electrode configurations showed similar sensitivities for glucose in the range between 8 and 21 muAmM-1 cm-2. The responses for 10 mono and oligosaccharides were studied. There was no response for fructose. In the concentration range 0.1-20 mM the relative sensitivities were determined for arabinose (96%), xylose (3%), mannose (50%), galactose (11%), glucose (100%), maltose (24%), lactose (12%), cellobiose (34%) and maltotriose (10%).

'Wiring' of lactate oxidase within a low-redox potential electron-conducting hydrogel.

Lactate electrodes based on electron-conducting hydrogels made by cross-linking lactate oxidase and the redox polymer formed upon complexing polyvinyl imidazole with [Os(dmo-bpy)2Cl]+/2+ (dmo-bpy = 4,4'-dimethoxy-2,2'-bipyridine) on vitreous carbon electrode surfaces were investigated. The redox potential of the hydrogels was -69 mV, versus the standard calomel electrode (SCE), and their lactate electro-oxidation current reached a plateau at +50 mV (SCE). Urate and acetaminophen were not electro-oxidized at this potential at rates that would interfere with the lactate assays, but ascorbate was catalytically oxidized by the gel. At 6 mM lactate concentration, switching of the atmosphere from argon to O2, reduced the current by 40 percent, showing that the rate of electron transfer from the reduced enzyme to the gel was slow.