Reactivation of an immobilized enzyme reactor for the determination of acetylcholinesterase inhibitors Flow injection determination of paraoxon

María, Cándido García de; Muñoz, Teresa Manzano; Townshend, Alan

doi:10.1016/0003-2670(94)80234-3

Cited by 32 publications

(12 citation statements)

References 15 publications

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“…The 2 μM lower detection limit (3 times the standard deviation of the response obtained for a blank) of the present microbial electrode for paraoxon is comparable to the OPH-based enzyme and microbial biosensors. , This detection limit, however, is 1−3 orders of magnitude higher than for AChE-based biosensors. − This will therefore limit the applicability of the present sensor for environmental monitoring. For any such application of the present biosensor, off-line sample preparation involving solvent extraction and sample concentration will be necessary.…”

Section: Resultsmentioning

confidence: 77%

“…Biosensing analytical devices, based on an acetylcholine esterase (AChE) inhibition test, using AChE-modified amperometric, − potentiometric, − or fiber optic − transducers have been reported. Although sensitive and ideal as single-use disposable sensors for environmental monitoring, biosensors based on AChE inhibition have limitations for application in on-line monitoring of detoxification process.…”

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confidence: 99%

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Biosensor for Direct Determination of Organophosphate Nerve Agents Using RecombinantEscherichia coliwith Surface-Expressed Organophosphorus Hydrolase. 1. Potentiometric Microbial Electrode

et al. 1998

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A potentiometric microbial biosensor for the direct measurement of organophosphate (OP) nerve agents was developed by modifying a pH electrode with an immobilized layer of Escherichia coli cells expressing organophosphorus hydrolase (OPH) on the cell surface. OPH catalyzes the hydrolysis of organophosporus pesticides to release protons, the concentration of which is proportional to the amount of hydrolyzed substrate. The sensor signal and response time were optimized with respect to the buffer pH, ionic concentration of buffer, temperature, and weight of cells immobilized using paraoxon as substrate. The best sensitivity and response time were obtained using a sensor constructed with 2.5 mg of cells and operating in pH 8.5, 1 mM HEPES buffer. Using these conditions, the biosensor was used to measure as low as 2 microM of paraoxon, methyl parathion, and diazinon. The biosensor had very good storage and multiple use stability. The use of cells with the metabolic enzyme expressed on cell surface as a biological transducer provides advantages of no resistances to mass transport of the analyte and product across the cell membrane and low cost due to elimination of enzyme purification, over the conventional microbial biosensors based on cells expressing enzyme intracellularly and enzyme-based sensors, respectively.

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Section: Resultsmentioning

confidence: 77%

mentioning

confidence: 99%

Biosensor for Direct Determination of Organophosphate Nerve Agents Using RecombinantEscherichia coliwith Surface-Expressed Organophosphorus Hydrolase. 1. Potentiometric Microbial Electrode

et al. 1998

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“…From the PMIRRAS data, the AChE secondary structure was reestablished 30 min after the dioxime injection following the inhibition by paraoxon. Spontaneous reactivation of the AChE after inhibition by paraoxon was reported earlier, where 100% of the enzyme activity was restored using dioxime as a reactivator. − However, no data were reported concerning the changes in the enzyme conformation. For the first time, we report in this paper the changes in the enzyme secondary structure after a reactivation following the paraoxon enzyme inhibition.…”

Section: Resultsmentioning

confidence: 85%

Polarization-Modulated FT-IR Spectroscopy Studies of Acetylcholinesterase Secondary Structure at the Air−Water Interface

1999

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The vibrational spectrum of acetylcholinesterase (AChE) at the air−water interface in its free form and bound to either its substrate, acetylthiocholine, or organophosphorus (OP) inhibitor has been studied by polarization modulation infrared reflection absorption spectroscopy (PMIRRAS). The shape and position of the amide I band was used to gauge the surface orientation of α-helices and β-sheets. The measured secondary structure content indicated that the enzyme did not unfold for the surface pressures used (0−30 mN/m). At low surface pressures, a strong amide I band indicated that the average tilt axis of the helices was aligned parallel to the air−water interface. Upon further compression, the α-helix component was significantly reduced, because the tilt axis of the helix relative to the water surface achieved a perpendicular orientation. PMIRRAS was also used to investigate the effect of phospholipids on molecular organization and orientation of AChE at the air−water interface. The enzyme was found to be fully inserted into the lipidic film during compression. The hydrolysis and inhibition were studied at the air−water interface. Band frequencies associated with acetylthiocholine binding to the enzyme active site and formation of the reaction products were observed. The OP inhibitor, paraoxon, was observed to unfold the enzyme at the air−water interface, because only high-frequency components associated with the extended conformation were observed upon compression. The secondary structure of the AChE was reestablished 30 min after a reactivator, trimethyl bis-(4-formylpyridinium bromide) dioxime, was injected beneath the paraoxon-inhibited AChE. For the first time, an in situ study of the protein conformation is reported using the PMIRRAS technique, and direct supporting evidence that the enzyme did not lose its native secondary structure upon spreading at the air−water interface is provided.

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“…Inhibitions and re-activation characteristics of a biosensor by the organophosphate pesticides were investigated [103,104]. Reactivation of an immobilized enzyme reactor was reported for the determination of acetylcholinesterase inhibitors in flow injection mode using 2-pyridinealdoxime methochloride [52,96].…”

Section: Reactivation Of the Inhibited Enzymementioning

confidence: 99%

Electrochemical Biosensors for the Detection of Pesticides~!2010-02-01~!2010-06-30~!2010-07-21~!

Mostafa¹

2010

TOELECJ

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Biosensors have been developed for the detection of pesticides using integrated enzymes, antibodies, cell and DNA-based biosensors. Enzymatic determination of pesticides is most often based on inhibition of the activity of selected enzymes such as cholinesterase, acid phosphatase, ascorbate oxidase, acetolactate synthase and aldehyde dehydrogenase. Enzymatic Biosensors were developed using various electrochemical signal transducers and different electrodes. Various immobilization protocols used for the formation of a biorecognition interface are also discussed: In addition, techniques of regeneration, single amplification, and miniaturization are evaluated for the development of immunosensor. Both batch and flow-injection analyses with enzyme biosensors are most intensively developed. It included that, in the future, compact, disposable and portable devices especially designed for in-field analysis with high sensitivity, selectivity; development of arrays and multiple sensors will continue another area of intensive research for biosensors.

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Reactivation of an immobilized enzyme reactor for the determination of acetylcholinesterase inhibitors Flow injection determination of paraoxon

Cited by 32 publications

References 15 publications

Biosensor for Direct Determination of Organophosphate Nerve Agents Using RecombinantEscherichia coliwith Surface-Expressed Organophosphorus Hydrolase. 1. Potentiometric Microbial Electrode

Biosensor for Direct Determination of Organophosphate Nerve Agents Using RecombinantEscherichia coliwith Surface-Expressed Organophosphorus Hydrolase. 1. Potentiometric Microbial Electrode

Polarization-Modulated FT-IR Spectroscopy Studies of Acetylcholinesterase Secondary Structure at the Air−Water Interface

Electrochemical Biosensors for the Detection of Pesticides~!2010-02-01~!2010-06-30~!2010-07-21~!

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