Improved electrochemical analysis of neuropathy target esterase activity by a tyrosinase carbon paste electrode modified by 1-methoxyphenazine methosulfate.

A graphite-paste tyrosinase biosensor was improved by adding 1-methoxyphenazine methosulfate as a mediator. Mediator modification enhanced sensitivity to phenol 4-fold and long-term stability 3-fold. Phenol could be detected at 25 nM (S/N = 2) using an Ag/AgCl reference electrode. The biosensor was used to measure the activity of a toxicologically significant enzyme, neuropathy target esterase (NTE), which yields phenol by hydrolysis of the substrate, phenyl valerate. Using the new biosensor, blood and brain NTE inhibition by organophosphorus (OP) compounds with different neuropathic potencies were well correlated (r = 0.990, n = 7), supporting the use of blood NTE as a biochemical marker of exposure to neuropathic OP compounds.

Bioelectrochemical analysis of neuropathy target esterase activity in blood.

Bioelectrochemical analysis of neuropathy target esterase (NTE) and its inhibitors is based on the combination of the NTE-catalyzed hydrolysis of phenyl valerate and phenol detection by a tyrosinase carbon-paste electrode. The use of the tyrosinase electrode improves 10-fold the sensitivity of NTE detection in comparison with a spectrophotometric method. The tyrosinase electrode was found to be suitable for measurements in whole human blood where spectrophotometric detection is considerably restricted. The specificity of NTE in blood for mipafox and di-2-propyl phosphorofluoridate was close to that for neuronal NTE. The NTE-like activity in blood was determined to be 0.19 +/- 0.02 nmol/min/mg of protein.

A new approach for determination of neuropathy target esterase activity.

Neuropathy target esterase (NTE) was shown to be an excellent biochemical marker for screening of organophosphates (OPs) with respect to their ability to result in organophosphate induced delayed neurotoxicity (OPIDN). This paper describes a new biosensor approach to the analysis of NTE and its inhibitors. The method is based on the combination of NTE enzymatic hydrolysis of phenyl valerate (PV) with phenol detection by the Clark-type oxygen electrode modified by immobilized tyrosinase. The validity of this biosensor method is confirmed by the facts that the calibration curves for NTE obtained by colorimetric and flow-through electrochemical methods were nearly identical and the titration of NTE by test inhibitor mipafox was shown to yield the same pI50 values. The developed electrochemical methods can be considered as a promising approach both for serial express NTE analysis and for kinetic characteristics of NTE.

Automated amplified flow immunoassay for cocaine.

An amplified flow immunoassay (AFIA) was developed for cocaine, which combines a noncompetitive immunoenzymometric assay (IEMA) with an on-line detection of the enzyme label alkaline phosphatase (ALP) by a substrate-recycling biosensor. In the IEMA, the analyte cocaine first binds to a labeled polyclonal anti-cocaine antibody. Then, the excess labeled antibody is separated on an affinity column that contains a perfusion chromatography carrier modified by immobilized cocaine. The unbound complexes of the analyte cocaine with the ALP-labeled antibody are detected postcolumn. The detector senses phenol produced by ALP from phenyl phosphate. As detector, an amperometric substrate-recycling biosensor was used, which consists of a Clark-type oxygen electrode covered by tyrosinase and pyrroloquinoline quinone-dependent glucose dehydrogenase. The lower limit of detection is 380 pM (38 fmol) for cocaine. The sampling rate is 26/h. Cocaine could be detected from "real samples" with an imprecision of +/- 10% (n = 3) and with a recovery of 49 +/- 3% for various concentrations. AFIA is generally important as a new approach for the fast detection of picomolar concentrations of haptens.

Zeptomole-detecting biosensor for alkaline phosphatase in an electrochemical immunoassay for 2,4-dichlorophenoxyacetic acid.

A bienzyme substrate-recycling biosensor in a flow injection analysis system is described for the sensitive measurement of alkaline phosphatase (ALP) and applied to the fast readout of a competitive immunoassay for the widely used pesticide 2,4-dichlorophenoxyacetic acid (2,4-D). The phenol-indicating biosensor consists of a Clark-type electrode covered by a membrane with coentrapped tyrosinase and quinoprotein glucose dehydrogenase. ALP dephosphorylates phenyl phosphate to phenol (K(m) = 36 microM) outside the flow system. Phenol is oxidized in the sensor membrane by the oxygen-consuming tyrosinase via catechol to o-quinone. The quinone is reconverted to catechol by glucose dehydrogenase. This substrate cycling results in a 350-fold amplified sensor response to phenol. The oxygen consumption of the enzyme couple in the presence of phenol is monitored as a decrease in current. A total of 3.2 fM ALP (320 zmol/ 100 microL) has been detected after a 57.5 min incubation with phenyl phosphate. All involved reagents are stable over the time of measurement. The sensor does not produce any measurable blank signals. The immunoassay detects 0.1 microgram/L 2,4-D, the maximum concentration for pesticides allowed in drinking water by European Community regulations. The applicability of this biosensor for fast immunoassay readout is demonstrated by a 2 min incubation. By comparison, a standard photometric method (p-nitrophenyl phosphate) requires overnight incubation.

New catalytic properties of monoamine oxidase immobilized in Langmuir-blodgett films with amphiphilic polyelectrolytes.

Langmuir-Blodgett (LB) films of monoamine oxidase (MAO) have been formed on the surface f a polypropylene membrane using amphiphilic polyelectrolytes. The enzyme activity of such protein-polyelectrolyte films was measured by a Clark electrodes. It was shown that in LB films thus formed the use of amphiphilc polyelectrolytes, MAO activity was higher than in polyelectrolyte-free LB films. Immobilization of MAO with branched polyethylenimine modified on 12% by laurylchain led to pronounced changes in its catalytic properties. The dependence of the enzyme's kinetic parameters on amphiphilic polyelectrolyte structures was discussed. (c) 1994 John Wiley & Sons, Inc.

Affinity biosensors based on preconcentration/voltammetric analysis. Detection of phenothiazine drugs at Langmuir-Blodgett films of tyrosine hydroxylase.

A new route for operating affinity biosensors based on the voltammetric monitoring of the accumulated guest (analyte) is described. High sensitivity and selectivity accrue from the coupling of the specific receptor binding process and the inherent sensitivity of the preconcentration/pulse-voltammetric scheme. The redox (measurement) process results in dissociation of the receptor-guest complex, thus allowing multiple analytical determinations. The receptor layer also serves as an effective barrier that excludes interfering species. The new concept of preconcentration/voltammetric affinity biosensors is illustrated in connection with the detection of phenothiazine drugs using Langmuir-Blodgett films of their receptor, the enzyme tyrosine hydroxylase. The effect of various experimental variables upon the sensor performance is described.

[Tyrosine hydroxylase activity in two-dimensional monomolecular films]. / Aktivnost' tirozingidroksilazy v dvukhmernykh monomolekuliarnykh plenkakh.

The activity of a neurospecific enzyme of tyrosine hydroxylase (TH) in monomolecular films formed onto solid surface was studied. The data obtained show that the formation of two-dimensional films onto negative-charge surfaces by the Langmuir-Schafer technology does not lead to the inactivation of the enzyme. Neuroleptic trifluoperazine increased the activity of TH. Monomolecular films of TH may be used as a sensitive element of biosensors for primary monitoring of neuroleptic-like compounds.

[The effect of delta-9-tetrahydrocannabinol on the receptor and physicochemical properties of the membranes in the rat brain]. / Vliianie del'ta-9-tetragidrokannabinola na retseptornye i fiziko-khimicheskie svoistva membran golovnogo krys.

It has been demonstrated that delta-9-THC does not affect the specific binding of 3H-IQNB, 3H-DAGO and 3H-dihydroalprenolol, decreases the level of specific binding 3H-LSD and 3H-spiperone, a 2-3-fold increase in the total and nonspecific binding being observed in this case, and also increases the microviscosity of the rat obtain membranes and disrupts lipid-protein interactions. Increasing the microviscosity of membranes by other method (lipid peroxidation) differently affects the binding of radioactively labeled ligands with the membranes from rat brain.

[Dynamics of synaptosomal membranes in experimental neurosis in animals using donor-acceptor pairs of luminophores]. / Izuchenie dinamiki sinaptosomal'nykh membran pri éksperimental'noi nevrotizatsii zhivotnykh s ispol'zovaniem donorno--aktseptornykh par liuminoforov.

Dynamic of synaptosomal membrane's structural parameters (fluidity, protein clusterization, thickness of lipid bilayer) during chronic (15 days) psychogenic stress was compared with kinetic of membrane bound enzyme--Na,K-ATPase. For evaluation of structural changes in membranes a special multiprobe method, based on the use of fluorescent probes ANSA and pyrene, fluorescence of endogenous tryptophane and inductive-resonance energy transfer was designed. The data obtained indicates correlation between structural and functional changes in synaptosomal membranes. It was also concluded that the multiprobe procedure used is a sensitive and adequate tool for investigation of structural changes in biomembranes.

Changes in synaptosomal membranes from cerebral cortex due to psychogenic stress in rats.

The changes in synaptosomal membranes in comparison with those of macrophages and mitochondria during repeated psychogenic stress were evaluated using a specially designed multiprobe procedure. Activity of Na,K-ATPase activity was measured as a functional marker for synaptosomal membranes. The interaction of peritoneal macrophages with nitrobluetetrazole (NBT-test) was also evaluated. Some bioenergetic parameters were measured in mitochondria using spectrophotometric techniques. The data revealed profound structural and functional changes in synaptosomal membranes on 15th day of stress. Those in macrophage membranes, present on 3rd and 6th day were not accompanied by functional ones. In conclusion the present data suggest that stress repeated for 15 days leads to a modelled psychopathology in rats and induce selective structural and functional changes in synaptosomal membranes from the cerebral cortex. This model may be used for investigations of the effects of psychotropic drugs.

[Effect of aminazine and triftazin on the synaptosome membranes of the rat cerebral cortex]. / Vliianie aminazina i triftazina na sinaptosomnye membrany kory golovnogo mozga krys.

Fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and pyrene were employed in studying the effect of aminazine and triftazin versus that of imipramine on microviscosity of rat brain cortex synaptosomal membranes. Unlike imipramine, the neuroleptics decrease microviscosity of membrane's lipid bilayer. All drugs decrease fluorescence of endogenous tryptophan, but fail to change fluorescence of L-tryptophan in the solution. It is concluded that neuroleptics induce conformational perturbations in membrane-bound proteins modifying microviscosity of lipid bilayer whereas imipramine changes the surface electric charge of lipid bilayer of synaptosomal membranes.

[Interaction of psychotropic preparations with the synaptic membranes of the cerebral cortex in rats]. / Vzaimodeistvie psikhotropnykh preparatov s sinapticheskimi membranamikory golovnogo mozga krys.

Interaction of psychotropic drugs with synaptosomal membranes from rat brain cortex was investigated by fluorescent probes. The data obtained indicate that all studied drugs change the state of membrane's surface. Tranquilizers, unlike antidepressants and neuroleptics, decreased fluorescence of the probe. Neuroleptics, unlike other drugs, penetrated deeper into the membranes hydrophobic core.

[Effect of subchronic administration of phenazepam and synthetic antioxidants on the functional status of synaptic membranes in the cerebral cortex of rats subjected to prolonged stress]. / Vliianie subkhronicheskogo vvedeniia fenazepama i sinteticheskikh antioksidantov na funktsional'noe sostoianie sinapticheskikh membran kory golovnogo mozga krys, podvergnutykh dlitel'nomu stress-vozdeistviiu.

Monoamine oxidase activity, lipid peroxidation and membrane structure were tested after chronic stress and 7-day treatment with drugs in the brain cortex synaptosomal membranes of rats. The most potent corrector of stress-induced changes, as compared to hydrophobic antioxidant dibunol and tranquilizer phenazepam, was compound 3-OP, a hydrophilic antioxidant.

[Effects of pharmacological substances structurally close to gamma-aminobutyric acid on the synthesis, metabolism and membrane transport systems in the cerebral cortex of rats in vitro]. / Vliianie farmakologicheskikh veshchestv, strukturno blizkikh gamma-aminomaslianoi kislote, na sintez, metabolizm i membrannye transportnye sistemy v kore mozga krys in vitro.

Effects of pantogam (calcium homopantotenate), piracetam, phenibut (beta-phenyl-GABA), sodium hydroxybutyrate and sodium valproate (depakine) at concentrations of 100 and 500 microM on the activities of glutamic acid decarboxylase, GABA-transaminase, Na, K- and Mg-ATPases, synaptosomal uptake and K+-stimulated release of 3H-GABA were studied in vitro. None of the compounds influenced the 3H-GABA uptake and activity of Mg-ATPase. Piracetam and phenibut stimulated slightly Na, K-ATPase. Phenibut and hydroxybutyrate exerted the inhibitory effect on GABA-transaminase activity. Hydroxybutyrate produced a moderate decrease of 3H-GABA release and suppressed the activity of glutamic acid decarboxylase.

[Interaction of psychotropic preparations with model lipid membranes]. / Vzaimodeistvie psikhotropnykh preparatov s model'nymi lipidnymi membranami.

Interaction of psychotropic drugs with model phosphatidylcholine membranes was investigated by fluorescent probes. The data obtained indicate different affinity of the drugs for phosphatidylcholine. The tranquilizers were not bound to the model membranes. The antidepressants were localized in the lipid polar groups area whereas the neuroleptics in the lipid polar groups area and deeper regions of the lipid bilayer.

[Changes in the synaptosomal membranes in the chronic neurotization of rats]. / Izmeneniia sinaptosomal'nykh membran pri khronicheskoi nevrotizatsii krys.

A study was made of the changes in synaptosomal membranes and in some synaptic processes under the development of experimental neurosis in rats. Neurotic rats demonstrated changes in the protein/lipid correlation and in the interaction of the fluorescent ANS probe and synaptosomal membranes. This can be accounted for by an increase in the membrane water repellency. The activity of Na, K-ATPase remains unchanged. The rate of noradrenaline, serotonin, dopamine and GABA synaptosomal reverse uptake in neurotic rats was found to be increased.