Canh Tran-Minh

Studies on acetylcholine sensor and its analytical application based on the inhibition of cholinesterase

Acetylcholine esterase electrodes, based on glass, Pd/PdO and Ir/IrO2 electrodes as pH sensor, using the immobilized acetylcholine esterase in acrylamide-methacrylamide hydrazides prepolymer are reported and compared. New data on the analysis of nicotine, fluoride ion, and some organophosphorus compounds are reported using the present AChE sensor based on the inhibition of the immobilized acetylcholine esterase. Reactivation of immobilized AChE after inhibition with reversible inhibitor, i.e. nicotine and fluoride ion is carried out using a mixture of working buffer and acetylcholine, whereas reactivation after inhibition with irreversible inhibitor, i.e. organophosphorus compounds is carried out using a mixture of acetylcholine and pyridine-2-aldoxime methiodide (PAM). The detection limits for the nicotine and fluoride ion are found to be 10(-5) M whereas for paraoxon, methyl parathion and malathion are found to be 10(-9) M and 10(-10) M.

Determination of organophosphorous and carbamate insecticides by flow injection analysis

A flow injection system, incorporating an acetylcholinesterase (AChE) single bead string reactor (SBSR), for the determination of some organophosphorous (azinphos-ethyl, azinphos-methyl, bromophos-methyl, dichlorovos, fenitrothion, malathion, paraoxon, parathion-ethyl and parathion-methyl) and carbamate insecticides (carbofuran and carbaryl) is presented. The detector is a simple pH electrode with a wall-jet entry. Variations in enzyme activity due to inhibition are measured from pH changes when the substrate (acetylcholine) is injected before and after the passage of the solution containing the insecticide. The percentage inhibition of enzyme activity is correlated to the insecticide concentration. Several parameters influencing the performance of the system are studied and discussed. The detection limits of the insecticides ranged from 0.5 to 275 ppb. The determination of these compounds was conducted in Hepes buffer and a synthetic sea water preparation. The enzyme reactor can be regenerated after inhibition with a dilute solution of 2-PAM and be reused for analysis. The immobilized enzyme did not lose any activity up to 12 weeks when stored at 4 degrees C.

Poly 3,4-ethylenedioxythiophene as an entrapment support for amperometric enzyme sensor

A conducting polymer of poly 3,4-ethylenedioxythiophene (PEDT) was used as a matrix for entrapment of enzymes onto a platinum electrode surface in order to construct amperometric biosensors. Glucose oxidase (GOD) was used as an example, and it was entrapped in the polymer during the electrochemical polymerization. Glucose in oxygenated solutions was tested by amperometric measurements at +650 mV (vs. SCE) in a batch system. The influence of several experimental parameters in the electropolymerization process was explored to optimize the analytical performance. The detection limit and sensitivity for this biosensor were 4×10−5 M and 15.2 mA M−1 cm−2, respectively. A linear range of response was found from 0.2 to 8 mM of glucose. The response time was 2–5 s. The stability of the electropolymerized films was evaluated in operational conditions. The glucose probe, stored in buffer at 4 °C when not in use, showed a residual activity of 40% after about 1 month. Glucose in synthetic serum was determined under flow injection conditions using an amperometric flow cell.

Whole-cell biosensor for direct determination of solvent vapours

A whole-cell biosensor has been designed to operate in the gas phase for determination of vapours of solvents. Methanol was chosen as an example of a volatile organic solvent to evaluate the algae sensor response. The use of whole cells as bioreceptors allows detection of those compounds in the gas phase without preliminary trapping of the analytes in a solution. In addition, near real-time monitoring is possible by the measurement of the algae photosynthetic activity with an oxygen electrode.

Determination of penicillin-V in standard solution and in fermentation broth by flow-injection analysis using fast-responding enzyme glass electrodes in different detection cells

An enzyme immobilization technique to measure penicillin concentration was developed. Penicillinase is immobilized by cross-linking with a very fine film of glutaraldehyde, which is sprayed on to the sensitive ends of two different pH glass electrodes. An extremely short response time of less than 10 s is guaranteed with electrodes made by this technique when measurements of penicillin are conducted in batch. The electrodes show good stability for 12 days when the sensor remains at room temperature. As the response time of the sensor is very short, an immediate determination of penicillin-V is possible by incorporating this enzyme electrode in a continuous-flow system, thereby dispensing with the need for an on-line enzyme reactor. The penicillin concentration is calculated as the difference in the potentiometric signal obtained as a peak when the sample flows past the sensor. Sample throughputs up to a maximum of 200 h−1 can be achieved. Samples are injected into a carrier stream consisting of a buffer of constant pH. Three different configurations of detection cells were designed and tested with the penicillinase electrode. First the performance of a magnetically stirred flow cell was studied. Subsequently a modified flow-through cell was tested, where the sensor was placed perpendicular to the direction of flow. A wall-jet cell was used to test the electrode where the flow direction coincided with the electrode axis. Finally, the magnetically stirred flow cell was used to detect penicillin-V in fermentation broth samples.

Whole-cell biosensor for determination of volatile organic compounds in the form of aerosols

A biosensor using Chlorella microalgae immobilized on the membrane of an oxygen electrode has been designed to operate in the gas phase for determination of volatile organic compounds in the form of aerosols. Perchloroethylene (tetrachloroethylene) is used as an example to produce aerosols and vapours since it is toxic to the people working in dry-cleaning atmospheres and municipal treatment plants. A home-made controlled atmosphere chamber is constructed for perchloroethylene detection. Monitoring of perchloroethylene is achieved by the measurement of oxygen production during the algae photosynthetic process. The use of whole cells as bioreceptors allows the detection of a toxic aerosol in the gas phase without preliminary trapping of the analyte in a solution.

Rapid measurement of penicillin contained in complex media using enzyme-loaded glass electrodes

Abstract An enzyme electrode based on a glass electrode for measuring penicillin-V concentration is described. In preparing this electrode, penicillinase is immobilized by cross-linking with a very fine film of glutaraldehyde, which is sprayed directly over the sensitive end of a pH glass electrode. The penicillin sensor was used for the determination of penicillin-V concentrations in phosphate buffer, fermentation broth and dairy milk, the ranges of concentrations detected being 1.5–15, 2.5–15 and 2–12 mM, respectively. The linearity, stability, sensitivity, reversibility and pH and buffer strength dependence of the electrode were examined and are discussed. An extremely short response time of less than 10 s is obtained with electrodes made in this manner.

Polycyclic aromatic hydrocarbons (PAHs) biodegradation in the soil of a former gasworks site : selection and study of PAHs-degrading microorganisms

Abstract Natural PAHs biodegradation was shown in the soil of a former gasworks site and the influence on biodegradation efficiency of sawdust addition studied. Cultures enriched by serial transfer through a mineral medium containing either fluorene, phenanthrene, anthracene, fluoranthene, or pyrene were used to obtain several bacterial communities. The obtained consortia were able to grow on different individual PAHs as their sole carbon source and consisted of a mixing of gram-negative and gram-positive bacterial strains. The reintroduction of the bacterial communities into sterilized polluted soil microcosms was investigated by following the PAHs and the microbial levels in the soil.

Biosensors in flow-injection systems for biomedical analysis, process and environmental monitoring.

This paper presents the construction of various biosensors using thin‐film layers incorporated in flow injection devices, providing automated systems for biomedical analysis, process and environmental monitoring. A urease sensor has been developed in conjunction with a flow injection system for the automatic determination of urea. Use of the spraying immobilization technique gives rise to a response time of a few seconds, which allows sample throughputs up to 200 h−1. With a penicillin biosensor adapted in an appropriate cell detection, on‐line measurements of penicillin V in the fermentation broth are achieved during the whole fermentation process; the results are compared with the HPLC method. Linearity, sensitivity and reproducibility of the biosensor are studied with regards to sample dilution in a stirred flow detection cell to provide optimal operating conditions. Measurements without any change in parameters are obtained during the whole fermentation process. Acetylcholinesterase sensors have been used in batch systems for the determination of pesticides, but they require large amounts of substrate. When those enzyme sensors are combined with flow injection systems, only small volumes (100 μl) of substrate are injected into the carrier stream and an automated system can be obtained for continuous control of water quality.

Synthesis of benzoates by enzymatic catalysis in heterogeneous medium

The enzyme-catalyzed synthesis of methyl and 1-butyl benzoates is reported. Direct esterification is performed with good yields by lipase from Candida rugosa suspended in a hexane/toluene mixture. Influence of substrates concentration, water content and toluene ratio are examined. Methanol and 1-butanol are lipase-inhibitors: they interact with the water adsorbed on lipase and on the enzyme as well. Increasing benzoic acid concentration in the system partially eliminates the alcohol inhibition. Water content greatly affects reaction kinetics and must be optimized to both activate the lipase and reach good conversion yields. Toluene, used to solubilize benzoic acid, negatively acts on reaction kinetics, mostly by increasing the partition of benzoic acid for the organic phase. The kinetic modelisation of 1-butyl benzoate synthesis agrees with a ping-pong Bi–Bi mechanism with 1-butanol inhibition. Apparent reaction coefficients are then determined.