LOD of some acylcholinesterase sensors for OPs determination (AChE is the acetylcholinesterase and BuChE is the butyrylcholinesterase).
1. Introduction
The worldwide increasing use of organophosphorus (OP) pesticides which are powerful neurotoxins and the resulting environmental and public concerns (CDC, 2005) created a demand for the development of reliable, fast, sensitive, simple and low-costing methods for their quantification, appropriate for on-line and on-site measurements. The conventional chromatographic, spectroscopic and immunoassay techniques for OP compounds determination, despite of their accuracy and sensitivity, are not well suited to these tasks. In contrast, the electrochemical biosensors based methods fulfill all the mentioned requirements.
The biosensors are relatively new analytical devices developed taking advantage of the progress in the biotechnology and the material science, in particular, in association with the modern principles of transduction of the chemical information. They represent a variety of chemical sensors, transforming the concentration of the quantified substance into an analytically useful signal (Thévenot et al., 1999).
The electrochemical biosensors provide selective quantitative or semi-quantitative analytical information using a biological recognition element (enzymes, whole cells, organelles or particles, tissues, etc.), in direct spatial contact with an electrochemical transducer, converting the signal produced by the interaction between the bioreceptor and the analyte, into electrical one (Thévenot et al., 1999).
A great variety of electrochemical biosensors quantifying the organophosphorus pesticides have been designed over the last decades. This review gives a survey on the state of the art of organophosphorus compounds detection using enzyme- and bacterial-based electrochemical sensors.
The survey includes the presentation of the OP pesticides structure and biochemical action, as well as the sources of pollution and the regulatory norms.
The current chromatographic and immunoassay methods for OP analysis are briefly discussed. The emerging during the last decades electrochemical biosensors based techniques are presented as their alternative.
The analytical performances of the two main types of enzyme-based electrochemical sensors for OP determination (the organophosphorus hydrolase and the acylcholinesterases ones), involving respectively the direct enzyme transformation of the analyte, and the inhibition of the enzyme activity, are summarized.
The recent trends in the development and in the increasing application of bacterial sensor systems for OP analysis are revised.
The advantages and the limitations of the enzyme-based vs. the bacterial electrochemical sensors are discussed.
2. The organophosphorus pesticides
The chemical compounds including stable functional groups that contain the carbon-phosphorus bond or that are organic derivatives of inorganic phosphorus acids are known as organophosphorus (Quin, 2000). Most of them, with the following general structure (Corbett et al., 1984; Eto, 1974; Hassall, 1982):
are highly toxic and are used as chemical warfare agents and pesticides (insecticides, herbicides, fungicides, rodenticides, moluscocides, nematocides, and regulators of vegetal growth, among other).
According to their chemical constitution, the organophosphorus pesticides could be classified into several types (Gupta, 2006). Some representative structures are shown in Fig. 1.
The OPs structural variety is reflected in their physicochemical and biological properties (Corbett et al., 1984; Hassall, 1982; WHO, 1986). Data on individual OPs could be found in Dictionary of organophosphorus compounds (Edmundson, 1988), Handbook of pesticide toxicology (Hayes, 1991), The Pesticide manual: A world compendium (Worthing & Hance, 1991), at http://www.pesticideinfo.org/, etc.
The biochemical mode of action of the organophosphorus pesticides primarily involves the inhibition of the acetylcholinesterase occurring throughout the central and peripheral nervous system of vertebrates, through phosphorylation of the serine hydroxyl moiety of the enzyme active site, thus preventing the hydrolysis of the neurotransmitter acetylcholine, performed in an analogous manner (Corbett et al., 1984; Fukuto, 1990; Gupta, 2006; Matsumura, 1980), as shown in Fig. 2. The resulting acetylcholine accumulation at the nerve synapses disrupts the nerve impulses propagation.
Slow recovery of the acetylcholinesterase activity could be observed, because of the spontaneous hydrolysis of the phosphorylated enzyme (Fig. 2B). The nucleophilic attack of the phosphorylacetylcholinesterase by some reagents (hydroxylamine, oximes) leads to quicker enzyme reactivation. “Aging” consisting in loss of an alkyl group from an alkoxy group on a phosphoryl residue attached to the active-site serine causes irreversible enzyme inhibition (Fig. 2C).
OPs are among the most acutely toxic pesticides. They belong to the toxicity class I (highly toxic) or toxicity class II (moderately toxic), according to the EPA classification. Although less persistent than the organochlorine pesticides, their widespread usage poses risk to man and his environment. Pesticides pollution results from agricultural practices, from industrial waste or discharge, from seepage of buried toxic wastes, and from run-off during spraying (Larson et al., 1997; Majewski & Capel, 1995; Vighi & Funari, 1995). Pesticides production, distribution, use, exposure, environmental levels, and maximum permissible levels in drinking water and food are subject of regulations in accordance with the national and international legislations. The primary involved organizations are the US Environmental Protection Agency (EPA), the EU Commission, the World Health Organization (WHO), the Food and Agricultural Organization of the United Nations (FAO), and the Codex Alimentarius Commission.
The high acute toxicity of the OPs, rapid absorption by the organism, and fast degradation in the environment call for the development of adequate analytical tools for their “
Nowadays, the devices of choice for organophosphorus pesticides “
3. The electrochemical biosensors for OPs quantification
The electrochemical biosensors for OP pesticides analysis could be classed into two great groups according to the nature of the biological recognition element – enzymes or bacteria.
3.1. Enzyme electrochemical sensors
The function of the acylcholinesterases (acetylcholinesterase or butyrylcholinesterase) and phosphatases (acid or alkaline) electrochemical sensors is based on the ability of the OP compounds to inhibit these enzymes. The quantification is realized measuring the variation of the enzyme activity as a function of the organophosphorus pesticide concentration, applying electrochemical techniques. Thus, according to the transduction mode, the reported biosensors are mainly potentiometric or amperometric.
The potentiometric acylcholinesterase sensors involve the following reaction:
where R’ is an acetyl or butyryl moiety and ChE is the acylcholinesterase.
The pH change of the solution, resulting from the acid release during the enzyme catalyzed hydrolysis of the choline esters is recorded as a sensor response, the latter depending on the cholinesterase activity.
Another potentiometric system is that developed by Ghindilis (Ghindilis et al., 1996), based on mediatorless bioelectrocatalysis:
(ChO is the enzyme choline oxidase and HRP is the enzyme peroxidase)
The H2O2 electrocatalytical reduction causes a shift in the electrode potential. This tri-enzyme sensor allowed detecting 2x10-13 mol L-1 trichlorfon.
The amperometric acylcholinesterase sensors, providing in general faster response, as well as higher sensitivity and accuracy than the potentiometric do, are developed in two directions:
First generation ChE amperometric sensors
They exploit the bienzymatic processes described by Eq. 2 and Eq. 3. The current of H2O2 oxidation or O2 reduction, depending on the substrate concentration and the enzyme activity, is recorded as a sensor response. However, since the H2O2 oxidation is carried out at a potential of +0.60 V/SCE, many substances contained in biological liquids and submitted to an oxidation at the same potential (glutathione, ascorbates, urates, etc.) interfere, corrupting the determination. The output signal is influenced by the fluctuations in the oxygen concentration, too.
Second generation ChE amperometric sensors
They use synthetic substrates (thiocholine or indoxylacetate esters), transformed upon catalytic hydrolysis in products able to be easily oxidized, as for example:
However, R’-thiocholine is a subject of a spontaneous non-enzymatic hydrolysis. Although slight, it can produce an increase of the anodic current response. Thiocholine oxidation provoking a passivation of the platinum anodes, because of their interaction with the sulfur containing compounds (Nikol’skaya & Evtyugin, 1992) must be taken into consideration, too.
The process of direct thiocholine oxidation occurring at +0.80 V/SCE at conventional metal and graphite transducers (Martorell et al., 1994; Marty et al., 1992; Marty et al., 1993; Marty et al., 1995; Sužnjević et al., 1985) involves the transfer of one electron from the thiol and a dimerization of the intermediate to disulfide (Evtugyn et al., 1999, Liu et al., 2005). The high potential value however causes the appearance of a high background current, as well as electroactive compounds interferences.
Several types of electrodes providing a sensitive electrochemical detection of enzymatically generated thiocholine at low potential were reported, such as the ones chemically modified with phtalocyanines (Harlbert & Baldwin, 1985; Hart & Hartley, 1994; Skladal, 1991), Prussian blue (Ricci et al., 2004), tetracyanoquinodimethane (Kulys & D’Costa, 1991; Martorell et al., 1997) and ferrocene (Evtugyn et al., 1996). However, mediator addition also could provoke interferences.
The alternative route to achieve potential lowering avoiding electrode modification involves acylthiocholine enzymatic hydrolysis (Eq. 5), chemical reduction of the produced thiocholine in solution (Eq. 7), and electrochemical detection of the product of the homogeneous redox reaction (Eq. 8), as suggested by Neufeld (Neufeld et al., 2000) and Ovalle (Ovalle et al., 2009):
However, the reported sensitivity of the OPs (chlorofos) determination is lower in comparison to that, attained by direct thiocholine oxidation (Ovalle et al., 2009).
The exploited response-generating reaction in some acylcholinesterase sensors of second generation for OPs quantification is the electrochemical oxidation of the leucoindigo, produced upon enzymatic hydrolysis of indoxylacetate (Kulys, 1989):
The disadvantage of the method consists in the fact that the leucoindigo is exposed to a chemical, as well as to electrochemical oxidation involving O2, which complicates the formation of the analytical signal (Nikol’skaya & Evtyugin, 1992).
The phosphatases inhibition, although reversible (which avoid enzyme reactivation), is rarely applied in the electrochemical biosensors for OPs detection (Danzer & Schwedt, 1996; Mazzei et al., 1996).
The inhibition-based determinations are very sensitive, but indirect. Drawbacks of the method are also the lack of selectivity and the need, in some cases, of enzyme incubation and enzyme reactivation/regeneration. In addition, as shown by Gunaratna (Gunaratna & Wilson, 1990), the cholinesterase is very sensitive to its micro-environment and even small changes provoke significant lost of enzyme activity resulting in decreasing of the sensor sensitivity. An overview of the methods based on enzyme inhibition with emphasis on the non-ideal behavior of the enzyme inhibition-based biosensors and biosensing systems is presented by Luque de Castro (Luque de Castro & Herrera, 2003).
Direct OP pesticides analysis could be achieved applying organophosphorus hydrolase (OPH) electrochemical sensors (Anzai, 2006; Chough et al., 2002; Lei et al., 2007 ; Mulchandani et al., 2001a; Mulchandani et al., 2001b; Prieto-Simón et al., 2006; Rodriguez-Mozaz et al., 2004; Wang et al., 2003). The enzyme OPH demonstrates substrate specificity toward paraoxon, parathion, coumaphos, diazinon, dursban, methyl parathion, etc, and toward some chemical warfare agents (sarin, soman, tabun, VX, etc.) (Dumas et al., 1990; Munnecke, 1980). The detection of parathion is also possible using parathion hydrolase (PH) (Sacks et al., 2000). The enzymatically catalyzed OP substrates hydrolysis involves pH changes and generates electroactive products:
Thus, the detection could be performed in a single step, using potentiometric (pH sensitive) or amperometric transducers (Mulchandani et al., 2001a).
OPH-based systems allow the selective determination of the family of the OP compounds, in contrast to the enzyme inhibition based techniques, but the reported detection limit is higher (Mulchandani et al., 2006). An important drawback represents the complex, long-lasting, and expensive procedure for OPH or PH extraction and purification, performed in specialized microbiological laboratories (to note that these enzymes are not commercially available) (Prieto-Simón et al., 2006).
Some reviews summarize the performances of the enzyme electrochemical sensors for OP pesticides determination and the principles of their operation (Andreescu & Marty, 2006; Anzai, 2006; Jaffrezic-Renault, 2001; Mazzei et al., 1996; Mulchandani et al., 2001a; Noguer et al., 1999; Prieto-Simón et al., 2006; Rodriguez-Mozaz et al., 2004; Solé et al., 2003a; Solé et al., 2003b; Tran-Minh, 1985; Turdean et al., 2002). Selected relevant data, demonstrating the sensitivity of the enzyme sensors are given in Table 1 and Table 2.
The commune disadvantages of this group of biosensors are the instability of the response (due to enzyme leaking or deactivation), the observed interferences at high electrode potentials, the passivation of the electrode surface and the short life-time at ambient temperature.
Enzyme | Target | LOD | Reference |
AChE | paraoxon | 0.1 nM | Tran-Minh et al., 1990 |
AChE | malathion | 1 nM | Tran-Minh et al., 1990 |
AChE/BuChE | paraoxon | 2.8 ppb | Skladal, 1991 |
BuChE | diazinon | 2 ppb | Budnikov & Evtugyn, 1996 |
BuChE/ChO/HRP | chlorofos | 0.0002 nM | Ghindilis et al., 1996 |
AChE/BuChE | paraoxon | 0.08 ppb | Skladal et al., 1996 |
AChE | paraoxon | 0.5 ppb | Noguer et al., 1999 |
AChE/ChO | methyl parathion | 0.05 M | Lin et al., 2004 |
Enzyme | Target | LOD | Reference |
OPH | paraoxon | 90 nM | Mulchandani et al., 1999 |
OPH | methyl parathion | 70 nM | Mulchandani et al., 1999 |
OPH | paraoxon | 2 M | Mulchandani et al., 2001a |
OPH | methyl parathion | 2 M | Mulchandani et al., 2001a |
OPH | diazinon | 2 M | Mulchandani et al., 2001a |
OPH | parathion | 15 nM | Chough et al., 2002 |
OPH | paraoxon | 20 nM | Chough et al., 2002 |
OPH | paraoxon | 0.4 M | Lei et al., 2007 |
3.2. Bacterial electrochemical sensors
Bacteria-based electrochemical sensors are developed by coupling these microorganisms to electrochemical transducers. Bacteria offer several advantages over the isolated enzymes for biosensor application, as for example: lower cost, because of the elimination of the time-consuming and expensive processes of extraction of the intracellular enzymes and their purification; ability to catalyze sequential reactions involving multiple enzymes; resistance to pH and temperature changes, because of the retention of the enzymes in their natural environment; higher tolerance to toxic substances; enzyme activity recovery in nutrient medium (D’Souza, 1989).
The bacterial electrochemical sensors are less sensitive and less selective than the enzyme ones, and their response time is relatively long, because of the difusional constraints imposed by the bacterial cell wall. However, these drawbacks could be overcome, by genetic engineering and by cell permeabilizing (D’Souza, 1989) respectively, applying various techniques.
Only few bacterial electrochemical sensors for OP pesticides quantification have been developed until now. They include, as biological recognition element, genetically engineered
Ley (Lei et al., 2004) reports the construction of a hybrid biosensor for direct determination of OP pesticides using purified OPH for their initial hydrolysis and
The mentioned microbial and hybrid sensors for direct OP pesticides quantification display long term stability, good reproducibility and accuracy, and relatively short response time. However, the reached LOD is over the OP concentration in environmental samples and higher than that for acylcholinesterases inhibition-based sensors, immunoassays, and gas, liquid and thin layer chromatography (Mulchandani et al., 2006).
Recently, an electrochemical biosensor for OP pesticides trace level concentrations determination was developed and characterized (Stoytcheva et al., 2009). It integrates a hybrid biorecognition element consisting of immobilized
The conditions for maximal sensor response to choline are optimized according to the methodology of Design of Experiments. The analytical performances of the enzyme substrate determination in a wide concentration range (0.1 mol dm-3 - 20 mol dm-3 of acetylcholine) and different ACh activities are established. It is demonstrated that the biosensor ensures reproducible, accurate and reliable chlorofos quantification reaching a LOD of 1 nmol dm-3 and a sensitivity of 0.0252 A/p(mol dm-3) under optimal experimental conditions.
The biosensor response time is 200 s and the storage stability is tL50 = 49 days for the bacterial membrane at ambient temperature. The device is reusable, the bacterial membrane being not affected by OP. The biosensor was applied to chlorofos determination in contaminated milk.
The proposed approach combines the advantages of the bacterial sensors with those of the cholinesterases inhibition-based ones, namely: stable response and long life-time at ambient temperature, because of the conservation of the enzyme system of the bacteria in its natural environment; reproducible characteristics ensured controlling the bacterial charge and the bacterial activity; high sensitivity. In addition, it provides reliable, free of interferences measurement of the dissolved oxygen reduction current, the polymer membrane of the oxygen probe being permeable only for gases. The biosensor fabrication is simple and cost-effective, enzyme extraction and purification or genetic engineering being avoided.
The biosensor is suitable for general toxicity screening or for determining the concentration of isolated OP pollutants.
Some comparative data are presented in Table 3.
Microorganism | Target | LOD | Reference |
Recombinant P. coli | paraoxon | 2 M | Mulchandani et al., 1998 |
Recombinant P. coli | methyl parathion | 2 M | Mulchandani et al., 1998 |
Recombinant P. coli | diazinon | 5 M | Mulchandani et al., 1998 |
Recombinant Moraxella | methyl parathion | 1 M | Mulchandani et al., 2001c |
Recombinant Moraxella | paraoxon | 0.2 M | Mulchandani et al., 2001c |
Recombinant P. putida | paraoxon | 55 ppb | Lei et al., 2005 |
Recombinant P. putida | methyl paraoxon | 53 ppb | Lei et al., 2005 |
Recombinant P. putida | parathion | 58 ppb | Lei et al., 2005 |
Recombinant P. putida | fenitrothion | 277 ppb | Lei et al., 2006 |
Recombinant P. putida | EPN | 1.6 ppm | Lei et al., 2006 |
Recombinant Moraxella | paraoxon | 0.1 M | Mulchandani et al, 2006 |
Arthrobacter globiformis | chlorofos | 1 nM | Stoytcheva et al., 2009 |
4. Conclusion
Despite of the still limited application of the electrochemical biosensors for OPs quantification in real samples, their analytical potential is obvious. Thus, current efforts are axed on biosensors’ performance improvement, development of compact and portable or disposable devices for
References
- 1.
Andreescu S. Marty J. L. 2006 Twenty years research in cholinesterase biosensors:from basic research to practical applications. Biomol. Eng., 23,1 EOF 15 EOF ,1389-0344 - 2.
Anzai J. 2006 Use of biosensors for detecting organophosphorus agents . , 126, 12, (December, 2006) 1301-1308,0031-6903 EISSN: 1347-5231 - 3.
Aprea C. Colosio C. Mammone T. Minoia C. Maroni M. 2002 Biological monitoring of pesticide exposure: a review of analytical methods. J. Chromatogr. B, 769, 2, (April 2002) 191-219,1570-0232 1570 0232 - 4.
Budnikov H. V. Evtugyn G. A. 1996 Electrochemical biosensors for inhibitor determination: selectivity and sensitivity control . , 8, 8-9, (August-September, 1996) 817-820,1040-0397 ESSN: 1521-4109 - 5.
Campàs M. Prieto-Simón B. Marty J. L. 2009 A review of the use of genetically engineered enzymes in electrochemical biosensors . , 20, 1, (February 2009) 3-9,1084-9521 1084 9521 - 6.
CDC, Third national report on human exposure to environmental chemicals 2005 Centers for Disease Control and Prevention (CDC), Atlanta - 7.
Chough S. H. Mulchandani A. Mulchandani P. Chen W. Wang J. Rogers K. R. 2002 Organophosphorus hydrolase-based amperometric sensor: modulation of sensitivity and substrate selectivity . , 14, 4, (February, 2002) 273-276,1040-0397 EISSN: 1521-4109 - 8.
Corbett J. R. Wright K. Baillie A. C. 1984 The biochemical mode of action of pesticides , 2nd ed., Academic press,0-12187-860-0 13: 978-0-12-187860-3, London - 9.
Danzer T. Schwedt G. 1996 Chemometric methods for the development of a biosensor system and the evaluation of inhibition studies with solutions and mixtures of pesticides and heavy metals. Part I. Development of an enzyme electrodes system for pesticides and heavy metal screening using selected chemometric method s. 318, 3, (January, 1996) 275-286,0003-2670 0003 2670 - 10.
D’Souza S. F. 1989 Immobilized cells: techniques and applications. ., 29, 2, (June, 1989) 83-117,0046-8991 EISSN: 0973-7715 - 11.
Dumas D. P. Durst H. D. Landis W. G. Raushel F. M. Wild J. R. 1990 Inactivation of organophosphorus nerve agents by the phosphotriesterase from Pseudomonas diminuta. ., 227, 1, (February, 1990) 155-159,0003-9861 0003 9861 - 12.
Edmundson R. S. 1988 Dictionary of organophosphorus compounds , Chapman & Hall,100412257904 13: 978-0-412-25790-2, London - 13.
Eto M. 1974 Organophosphorus pesticides: organic and biological chemistry, CRS Press,100878190236 13: 978-0-87819-023-2, Cleveland - 14.
Evtugyn G. Budnikov H. Galyametdinov Yu. Suntsov E. 1996 Amperometric determination of thiocholine esters in the presence of butyrylcholinesterase. .,51 4 391 393 ,0044-4502 - 15.
Evtugyn G. Ivanov A. Gogol E. Marty J. L. Budnikov H. 1999 Amperometric flow-through biosensor for the determination of cholinesterase inhibitors . 385, 1-3, (April, 1999) 13-21,0003-2670 0003 2670 - 16.
Fukuto R. 1990 Mechanism of action of organophosphorus and carbamate insecticides . , 87, (July 1990)245 EOF -254,0091-6765 EISSN 15529924 - 17.
Ghindilis A. Morzunova H. Barmin A. Kurochkin I. 1996 Potentiometric biosensors for cholinesterase inhibitor analysis based on mediatorless bioelectrocatalysis. .,11 9 873 880 ,0956-5663 - 18.
Gunaratna C. Wilson G. 1990 Optimization of multienzyme flow reactors for determination of acetylcholine, ., 62, 4, (February, 1990) 402-407,0003-2700 EISSN: 1520-6882 - 19.
Gupta R. C. . Ed 2005 Toxicology of organophosphate & carbamate compounds, 1st ed., Elsevier Academic Press100120885239 13: 978-0-12-088523-7, London - 20.
Hayes W. J. 1991 Handbook of pesticide toxicology , Academic Press,100123341604 13: 978-0-12-334160-0, San Diego - 21.
Harlbert M. Baldwin R. 1985 Electrocatalytic and analytical response of cobalt phthalocyanine containing carbon paste electrodes toward sulfhydryl compounds. ., 57, 3, (March, 1985) 591-595,0003-2700 EISSN: 1520-6882 - 22.
Hart J. Hartley I. 1994 Voltammetric and amperometric studies of thiocholine at a screen-printed carbon electrode chemically modified with cobalt phthalocyanine: studies towards a pesticide sensor .119 2 259 265 ,0003-2654 - 23.
Hassall K. A. 1982 The chemistry of pesticides. Their metabolism, mode of action and uses in crop protection, Verlag Chemie,103527259694 13: 9783527259694, Weinheim, Deerfield Beach, Florida, Basel, 1982 - 24.
Jaffrezic-Renault N. 2001 New trends in biosensors for organophosphorus pesticides . , 1, 2, (July, 2001) 60-64,1424-8220 1424 8220 - 25.
Jeannot R. Dagnac T. 2006 In: . 3rd edition, Nollet L. (Ed.),841 889 , CRC Press, Boca Raton, London, New York - 26.
Kulys J. 1989 Amperometric enzyme electrodes in analytical chemistry , ., 335, 1, (January 1989) 86-91,0937-0633 EISSN: 1432-1130 - 27.
Kulys J. D’Costa E. J. 1991 Printed amperometric sensor based on TCNQ and cholinesterase . .,6 2 109 115 ,0956-5663 - 28.
Larson S. J. Capel P. D. Majewski M. S. 1997 Pesticides in surface waters: distribution, trends, and governing factors, CRC Press,101575040069 13: 978-1-57504-006-6 - 29.
Lee H. S. Kim Y. A. Chao Y. A. Lee Y. T. 2002 Oxidation of organophosphorus pesticides for the sensitive detection by a cholinesterase-based biosensor . , 46, 4, (January, 2002) 571-576,0045-6535 0045 6535 - 30.
Lei Y. Mulchandani P. Chen W. Wang J. Mulchandani A. 2004 Whole cell-enzyme hybrid amperometric biosensor for direct determination of organophosphorous nerve agents with p-nitrophenyl substituent. ., 85, 7, (March, 2004) 706-713,0006-3592 EISSN: 1097-0290. - 31.
Lei Y. Mulchandani P. Chen W. Mulchandani A. 2005 Direct determination of p-nitrophenyl substituent organophosphorus nerve agents using a recombinant Pseudomonas putida JS ., 53, 3, (February, 2005) 524-527,444 -modified Clark oxygen electrode.0021-8561 EISSN: 1520-5118 - 32.
Lei Y. Mulchandani P. Chen W. Mulchandani A. 2006 Biosensor for direct determination of fenitrothion and EPN using recombinant Pseudomonas putida JS444 with surface expressed organophosphorus hydrolase. 1. Modified Clark oxygen electrod e. 6, 4, (April, 2006) 466-472,1424-8220 1424 8220 - 33.
Lei C. Valenta M. Sapiralli K. P. Ackerman E. J. 2007 Biosensing paraoxon in simulated environmental samples by immobilized organophosphorus hydrolase in functionalized mesoporous silica. ., 36, 1, (January-February, 2007) 233-238,0047-2425 EISSN: 1537-2537 - 34.
Lin Y. H. Lu F. Wang J. 2004 Disposable carbon nanotube modified screen-printed biosensor for amperometric detection of organophosphorus pesticides and nerve agents . , 16, 1-2, (January, 2004) 145-149,1040-0397 EISSN: 1521-4109 - 35.
Liu G. Riechers S. Mellen M. Lin Y. 2005 Sensitive electrochemical detection of enzymatically generated thiocholine at carbon nanotube modified glassy carbon electrode . ., 7, 11, (November, 2005) 1163-1169,1388-2481 1388 2481 - 36.
Luque de Castro. M. D. Herrera M. C. 2003 Enzyme inhibition-based biosensors and biosensing systems: questionable analytical devices . 18, 2-3, (March, 2003) 279-294,0956-5663 0956 5663 - 37.
Majewski M. S. Capel P. D. 1995 Pesticides in the atmosphere: distribution, trends, and governing factors, CRC Press,101575040042 13: 978-1-57504-004-2 - 38.
Martorell D. Céspedes F. Martínez-Fàbregas E. Alegret S. 1994 Amperometric determination of pesticides using a biosensor based on a polishable graphie-epoxy biocomposite . , 290, 3, (May, 1994) 343-348,0003-2670 0003 2670 - 39.
Martorell D. Céspedes F. Martínez-Fàbregas E. Alegret S. 1997 Determination of organophosphorus and carbamate pesticides using a biosensor based on a polishable, 7,7,8,8-tetracyanoquino- dimethane- modified, graphite- epoxy biocompos ite. 337, 3, (January, 1997) 305-313,0003-2670 0003 2670 - 40.
Marty J. L. Mionetto N. Rouillon R. 1992 Entrapped enzymes in photocrosslinkable gel for enzyme electrodes . .,25 8 1389 1398 ,0003-2719 EISSN: 1532-236X - 41.
Marty J. L. Mionetto N. Noguer T. Ortega F. Roux C. 1993 Enzyme sensors for the detection of pesticides . .,8 6 273 280 ,0956-5663 - 42.
Marty J. L. Mionetto N. Lacorte S. Barceló D. 1995 Validation of an enzymatic biosensor with various liquid chromatographic techniques for determining organophosphorus pesticides and carbaryl in freeze-dried waters . , 311, 3, (August, 1995) 265-271,0003-2670 0003 2670 - 43.
Matsumura F. 1980 Toxicology of insecticides , Plenum Press,100306307871 13: 978-0-306-30787-4, New York - 44.
Mazzei F. Botré F. Botré C. 1996 Acid phosphatase/glucose oxidase-based biosensors for the determination of pesticides . 336, 1-3, (December, 1996) 67-75,0003-2670 0003 2670 - 45.
Mulchandani A. Mulchandani P. Kaneva I. Chen W. 1998 Biosensor for direct determination of organophosphate nerve agents using recombinant Escherichia coli with surface-expressed organophosphorus hydrolase. 1. Potentiometric microbial electro de. ., 70, 19, (October, 1998) 4140-4145,0003-2700 EISSN: 1520-6882 - 46.
Mulchandani A. Mulchandani P. Chen W. Wang J. Chen L. 1999 Amperometric thick-film strip electrodes for monitoring organophosphate nerve agents based on immobilized organophosphorus hydrolase. Anal. Chem., 71, 11, (June, 1999) 2246-2249,0003-2700 EISSN: 1520-6882 - 47.
Mulchandani A. Chen W. Mulchandani P. Wang J. Rogers K. R. 2001a Biosensors for direct determination of organophosphate pesticides. ., 16, 4-5, (June, 2001) 225-230,0956-5663 0956 5663 - 48.
Mulchandani P. Chen W. Mulchandani A. 2001b Flow injection amperometric enzyme biosensor for direct determination of organophosphate nerve agents. ., 35, 12, (June, 2001) 2562-2565,0001-3936 0013 936 X, EISSN: 1520-5851 - 49.
Mulchandani P. Chen W. Mulchandani A. Wang J. Chen L. 2001c Amperometric microbial biosensor for direct determination of organophosphate pesticides using recombinant microorganism with surface expressed organophosphorus hydrolase . ., 16, 7-8, (September, 2001) 433-437,0956-5663 0956 5663 - 50.
Mulchandani P. Chen W. Mulchandani A. 2006 Microbial biosensor for direct determination of nitrophenyl-substituted organophosphate nerve agents using genetically engineered Moraxella sp. 568, 1-2, (May, 2006) 217-221,0003-2670 0003 2670 - 51.
Munnecke D. M. 1980 Enzymatic detoxification of waste organophosphate pesticides. J. ., 28, 1, (January, 1980) 105-111,0021-8561 EISSN: 1520-5118 - 52.
Nikol’skaya E. B. Evtyugin G. A. 1992 Cholinesterases application in analytical chemistry. .,47 8 1358 1378 ,1061-9348 EISSN: 1608-3199 - 53.
Neufeld T. Eshkenazi I. Cohen E. Rishpon J. 2000 A micro flow injection electrochemical biosensor for organophosphorus pesticides. ., 15, 5-6, (August, 2000) 323-329,0956-5663 0956 5663 - 54.
Noguer T. Leca B. Jeanty G. Marty J. L. 1999 Biosensors based on enzyme inhibition: Detection of organophosphorus and carbamate insecticides and dithiocarbamate fungicides . .,3 3 171 178 ,0108-6900 X, EISSN: 1520-6521 - 55.
Ovalle M. Stoytcheva M. Zlatev R. Valdez B. 2009 Electrochemical study of rat brain acetylcholinesterase inhibition by chlorofos: kinetic aspects and analytical applications . ,DOI 10.1016/j.electacta.2009.09.008, (in press),0013-4686 0013 4686 - 56.
Periasamy A. P. Umasankar Y. Chen S. M. 2009 Nanomaterials-acetylcholinesterase enzyme matrices for organophosphorus pesticides electrochemical sensors: a review. , 2009, 9, (September, 2009) 4034-4055;1424-8220 1424 8220 - 57.
Prieto-Simón B. Campàs M. Andreescu S. Marty J. L. 2006 Trends in flow-based biosensing systems for pesticide assessment. , 6, 10, (October, 2006) 1161-1186,1424-8220 1424 8220 - 58.
Quin L. D. 2000 A guide to organophosphorus chemistry , Wiley-Interscience,100471318248 13: 978-0-471-31824-8 - 59.
Ricci F. Arduini F. Amine A. Moscone D. Palleschi G. 2004 Characterisation of Prussian blue modified screen-printed electrodes for thiol detection . ., 563, 2, (March, 2004) 229-237,0022-0728 0022 0728 - 60.
Richins R. Kaneva I. Mulchandani A. Chen W. 1997 Biodegradation of organophosphorus pesticides by surface-expressed organophosphorus hydrolase. ., 15, 10, (October, 1997) 984-987,1087-0156 EISSN: 1546-1696 - 61.
J. & Barceló, D.Rodriguez-Mozaz S. Marco M. P. Lopez de Alda. M. 2004 Biosensors for environmental applications: future development trends . .,76 4 723 752 ,0033-4545 EISSN: 1365-3075 - 62.
Sacks V. Eshkenazi I. Neufeld T. Dosoretz C. Rishpon J. 2000 Immobilized parathion hydrolase: An amperometric sensor for parathion. ., 72, 9, (May, 2000) 2055-2058,0003-2700 EISSN: 1520-6882 - 63.
Schlecht P. C. O’Connor P. F. Eds . 1994 , 4th ed., DHHS (NIOSH) Publication94 113 - 64.
Skladal P. 1991 Determination of organophosphate and carbamate pesticides using a cobalt phthalocyanine-modified carbon paste electrode and a cholinesterase enzyme membrane. 252, 1-2, (November, 1991) 11,0003-2670 0003 2670 - 65.
Skladal P. Fiala M. Krejcí J. 1996 Detection of pesticides in the environment using biosensors based on cholinesterases . ., 65, 1-4, 139-148,0306-7319 0306 7319 - 66.
Solé S. Merkoçi A. Alegret S. 2003a Determination of toxic substances based on enzyme inhibition. Part I. Electrochemical biosensors for the determination of pesticides using batch procedures . .,33 2 89 126 ,1040-8347 EISSN: 1547-6510 - 67.
Solé S. Merkoçi A. Alegret S. 2003b Determination of toxic substances based on enzyme inhibition. Part I. Electrochemical biosensors for the determination of pesticides using flow procedures. .,33 2 127 143 ,1040-8347 EISSN: 1547-6510 - 68.
Stoytcheva M. Zlatev R. Velkova Z. Valdez B. Ovalle M. Petkov L. 2009 Hybrid electrochemical biosensor for organophosphorus pesticides quantification . , 54, 6, (February, 2009) 1721-1727,0013-4686 0013 4686 - 69.
Sužnjević D. Ž. Veselinović D. S. Vukelić N. S. Pavlović D. Ž. Nikolić A. V. 1985 Investigation of the system butyrylthiocholineiodide-butyrocholinesterase by cyclovoltammetry and chronopotentiometry using inert working electrodes. .,50 2 83 88 ,0352-5139 - 70.
Thévenot D. R. Tóth K. Durst R. A. Wilson G. S. 1999 Electrochemical biosensors: recommended definitions and classification. .,71 12 2333 2348 ,0033-4545 EISSN: 1365-3075 - 71.
Tran-Minh C. 1985 Immobilized enzyme probes for determining inhibitors . .,7 41 75 ,100080332013 13: 978-0-08-033201-7 - 72.
Tran-Minh C. Pandey P. C. Kumaran S. 1990 Studies on acetylcholine sensor and its analytical application based on the inhibition of cholinesterase . .,5 6 461 471 ,0956-5663 - 73.
Turdean G. Popescu I. C. Oniciu L. 2002 Biocapteurs ampérométriques a cholinestérases pour la détermination des pesticides organophosphorés. ., 80, (March, 2002) 315-331,1480-3291 1480 3291 - 74.
Van Emon J. M. Ed . 2006 Immunoassay and other bioanalytical techniques , CRC Press,100849339421 13: 978-0-8493-3942-4, Boca Raton, London, New York - 75.
Vighi M. Funari E. 1995 Pesticide risk in groundwater , CRC Press,100873714393 13: 978-0-87371-439-6 - 76.
Wang J. Krause R. Block K. Musameh M. Mulchandani A. Schöning M. J. 2003 Flow injection amperometric detection of OP nerve agents based on an organophosphorus-hydrolase biosensor detector. 18, 2-3, (March, 2003) 255-260,0956-5663 0956 5663 - 77.
WHO/IPCS. 1986 Organophosphorus insecticides: a general introduction (Environmental health criteria Series63 ,109241542632 13: 978-92-4-154263-0, Geneva - 78.
Worthing C. R. Hance R. J. . Eds 1991 The pesticide manual: A world compendium, 9th ed., British Crop Protection,100948404426 13: 9780948404429, Surrey UK