Claude Burstein

Increased paraoxon detection with solvents using acetylcholinesterase inactivation measured with a choline oxidase biosensor

Abstract Inactivation by paraoxon of free and immobilized acetylcholinesterase, in the presence of various amounts of different organic solvents, and with choline oxidase immobilized on an oxygen electrode was studied. With acetylcholinesterase in solution it was found that 5% cyclohexane enhances paraoxon detection when compared to the detection without solvent. In these conditions, as low as 10 −9 m paraoxon can be detected. Free acetylcholinesterase was more sensitive to paraoxon than immobilized acetylcholinesterase, depending on the nature and concentration of solvent (500-fold in the presence of 5% cyclohexane). The stability of the biosensor in continuous use and stored at room temperature was at least 3–4 weeks with triton X100 and bovine serum albumin addition. The sensitivity of paraoxon detection with free acetylcholinesterase in the presence of 5% cyclohexane corresponds to 0·2 ppb paraoxon, 10-fold below the legal limit admitted in the European Economic Community. The same approach could be applied for detection of low amounts of different organophosphorus or carbamate pesticides.

Detection of heavy metal salts with biosensors built with an oxygen electrode coupled to various immobilized oxidases and dehydrogenases

Abstract Immobilized oxidases were bound on the surface of an affinity membrane and mounted on an oxygen electrode. These biosensors were used for heavy metal salt measurements. After immobilization on the enzymes, first order kinetics of inactivation were observed. Surface immobilization increases the sensitivity by a factor of 10, compared to reticulation of the enzyme in a gelatin matrix. After immobilization, 50% inactivation was observed with 20 μ m HgCl 2 for L-glycerophosphate oxidase and 50 n m for pyruvate oxidase. Restoration of activity after HgCl 2 treatment is feasible, but neither complete nor reproducible. To reuse the biosensor, L-lactate dehydrogenase (LDH) from rabbit muscle in solution was coupled to immobilized L-lactate oxidase (insensitive to heavy metal salts). LDH (particularly inexpensive) was replaced after each measurement. The I 50 in phosphate buffer was 1 μ m for HgCl 2 and 0·1 μ m for AgNO 3 ; with other heavy metal salts, no inhibition was observed below 500 μ m . In Tris buffer, the I 50 was 10 μ m for CdCl 2 and ZnCl 2 , 50 μ m for Pb-acetate and 250 μ m for CuSO 4 . The use of different enzymes and buffers may allow measurement of specific heavy metal salts.

Kinetic study of heavy metal salt effects on the activity of L-lactate dehydrogenase in solution or immobilized on an oxygen electrode

A sensitive and convenient biosensor for detection of heavy metal salts has been developed. The method is based on the effects of heavy metal salts on the catalytic activity of L-lactate dehydrogenase (LDH) in solution or coimmobilized with L-lactate oxidase (LOD) on an oxygen electrode. At metal concentrations below 100 microM, the kinetic behavior, with the LDH substrate NADH, showed a competitive inhibition with high affinity during the first 10 s. With increased incubation time, irreversible first order inactivation with respect to enzyme concentration was observed. This irreversible inactivation of LDH in solution was dose dependent. The efficiencies obtained for the different heavy metal salts were: HgGl2 > AgNO3 > Pb(COOCH3)2 > CuSO4 > ZnCl2. HgCl2 and AgNO3 were effective in the nanomolar range while the other metal salts acted at the micromolar level. LDH is protected by saturating amounts of substrate NADH against the effects of the heavy metal salts studied. The pKs for LDH catalytic activity and inactivation by heavy metal salts were similar. The results suggest binding of the heavy metal salts to the enzyme active site. Except for lead acetate, all heavy metal detection was in the range of European norms. For AgNO3, CuSO4 and HgCl2, the sensor limit of detection reached the European norm values whereas with ZnCl2 it was well below. The immobilization of LDH considerably decreased the amount of enzyme consumed by permitting repetitive assays. The efficiency of inactivation by the heavy metal salts was reduced in comparison with LDH in solution. Restoration of activity of the inactivated immobilized enzyme was obtained with DTT, EDTA, KCN and NADH treatment. This opens up possibilities for detection of toxic compounds using simple procedures suitable for assays in a variety of monitoring conditions in environmental and food pollution control.

5-Aryl-1,3,4-oxadiazol-2(3H)-one derivatives and sulfur analogues as new selective and competitive monoamine oxidase type B inhibitors

Eighteen new 5-aryl-1,3,4-oxadiazol-2(3H)-one derivatives and sulfur analogues were prepared and evaluated in vitro for their inhibitory properties on monoamine oxidase (MAO) types A and B. The most active compounds in these series acted preferentially against MAO B with IC50 values in the range of 1.8-0.056 μM. The 5-(4-biphenylyl)-3-(2-cyanoethyl)-1,3,4-oxadiazol-2(3H)-one 23 and its oxadiazolethione analogue 33 were found to act as potent, selective and competitive MAO B inhibitors with a slight slow-binding character. Both compounds inhibited MAO A in a classical competitive manner. According to their Ki (MAO B) values of 2.6 and 4 × 10−8 M, respectively, and their Ki (MAO A)/Ki (MAO B) ratios of 270 and 500, respectively, 23 and 33 can be placed among the most active and selective competitive MAO B inhibitors known up to now. The structure-activity relationships are discussed.

Inhibition of monoamine oxidase types A and B by 2-aryl-4H-1,3,4-oxadiazin-5(6H)-one derivatives

Abstract Monoamine oxidase (MAO) assay specificity based on substrate specificity was investigated by substrate competition experiments. 10 μM serotonin (5-HT) and 5 μM β-phenylethylamine (PEA) were found to ensure total substrate specificity for, respectively, MAO types A and B. Twenty-five 2-aryl-4 H -1,3,4-oxadiazin-5(6 H )-one derivatives were synthesized and tested in vitro for their inhibitory effects on MAO A and B. Most of them inhibited preferentially MAO B. The 2-(4-biphenylyl)-4-(2-cyanoethyl)-4 H -1,3,4-oxadiazin-5(6 H )-one 32 was the most efficient MAO B inhibitor and acted as a competitive inhibitor on the two enzymes. Its K i values for MAO A and B were 11 and 0.15 μM, respectively. Structure-activity relationships suggest that these oxadiazinones should interact with a hydrophobic site and a nucleophilic site on MAO B for binding, while the functional group of the N-4 substituent should compete with the substrate for the active site of the enzyme.

Co-immobilized L-lactate oxidase and L-lactate dehydrogenase on a film mounted on oxygen electrode for highly sensitive L-lactate determination

Abstract Covalent immobilization of L-lactate oxidase (LOD) with L-lactate dehydrogenase (LDH) on a film tightly bound to an oxygen electrode, for rapid and sensitive L-lactate measurements, is described. Regeneration of L-lactate by substrate recycling provided an amplification of the sensor response, making it possible to decrease the detection limit of L-lactate from 10 μM to 20 nM. The apparent K m for L-lactate with the LOD-LDH coupled reaction was 1 μM, compared with 3 mM when utilizing only LOD. Linearity was obtained from 20 to 300 nM with both enzymes, whereas with LOD alone it was from 10 μM to 1 mM. Optimization of the biosensor was obtained with an increase in LOD and LDH film loading and low L-lactate concentration. The enzymes covalently bound to the film stabilized the biosensor (half life 8 weeks) for over 400 measurements. Low L-lactate excreted by E. coli bacteria metabolism can be assayed in turbid culture medium without pretreatment by the amplified L-lactate detection.

Assay of dehydrogenases with an O2-consuming biosensor

Coimmobilization of L-lactate oxidase (LOD) and L-lactate dehydrogenase (LDH) on a film bound to an oxygen electrode makes it possible, by recycling L-lactate and pyruvate, to lower their detection limit to 10 nM. With LOD immobilized alone and LDH in solution, easy LDH measurements were performed. The detection limit was 1 IUl-1. Similarly, this mixed biosensor, using an LDH sensitive to mercurials, was used for p-chloromercuribenzoate (PCMB) detection. Coimmobilization of LOD + LDH with an NAD(+)-dependent dehydrogenase allows the measurements of all dehydrogenases. NAD+ is recycled. The detection limit was 50 nM. Applications can be found in medicine, the food industry, and the environment.

Biosensors for heavy metal salt measurements with immobilized enzymes on a Clark electrode

Publisher Summary This chapter focuses on biosensors for heavy metal salt measurements with immobilized enzymes on a Clark electrode. Most immobilized enzymes are sensitive to 1 mM heavy metal salts. After immobilization at the surface of an affinity membrane (Ultrabind from Gelman), some enzymes are much more sensitive to lower concentrations of mercury salts. In a study described in the chapter, 50% inactivation (I50) was obtained with HgCl2 after 2 min incubation. Inactivation of the enzymes was measured with a Clark electrode whose sensitive tip was covered with the immobilized enzyme film. Addition of substrate induces oxygen consumption, which is inhibited by the heavy metal salt. For pyruvate oxidase, the sensitivity was further increased reaching 10 nM when the incubation with HgCl2 was performed in the absence of thiaminepyrophosphate-Mg. The biosensors are very easy to use, and the assay lasts less than 10 s. Applications can be found for solving pollution problems of water.