A. V. Eremenko

Biosensor detection of neuropathy target esterase in whole blood as a biomarker of exposure to neuropathic organophosphorus compounds.

Abstract Neuropathy target esterase (NTE) is the target protein for neuropathic organophosphorus (OP) compounds that produce OP compound-induced delayed neurotoxicity (OPIDN). Inhibition/aging of brain NTE within hours of exposure predicts the potential for development of OPIDN in susceptible animal models. Lymphocyte NTE has also found limited use as a biomarker of human exposure to neuropathic OP compounds. Recently, a highly sensitive biosensor was developed for NTE activity using a tyrosinase carbon-paste electrode for amperometric detection of phenol produced by hydrolysis of the substrate, phenyl valerate. The I50 (20 min at 37°C) for N,N′-di-2-propylphosphorodiamid ofluoridate (mipafox) against hen lymphocyte NTE was 6.94 ± 0.28 μM amperometrically and 6.02 ± 0.71 μM colorimetrically. For O,O-di-1-propyl O-2,2-dichlorvinyl phosphate (PrDChVP), the I50 against hen brain NTE was 39 ±8 nM amperometrically and 42 ±2 nM colorimetrically. The biosensor enables NTE to be assayed in whole blood, whereas this cannot be done with the usual colorimetric method. Amperometrically, I50 values for PrDChVP against hen and human blood NTE were 66 ±3 and 70 ± 14nM, respectively. To study the possibility of using blood NTE inhibition as a biochemical marker of neuropathic OP compound exposure, NTE activities in brain and lymphocytes as well in brain and blood were measured 24 h after dosing hens with PrDChVP. Brain, lymphocyte, and blood NTE were inhibited in a dose-responsive manner, and NTE inhibition was highy correlated between brain and lymphocyte (r=.994) and between brain and blood (r=.997). The results suggest that the biosensor NTE assay for whole blood could serve as a biomarker of exposure to neuropathic OP compounds as well as a predictor of OPIDN and an adjunct to its early diagnosis.

Biosensor assay of neuropathy target esterase in whole blood as a new approach to OPIDN risk assessment: review of progress.

Organophosphates (OPs) that inhibit neuropathy target esterase (NTE) with subsequent ageing can produce OP-induced delayed neuropathy (OPIDN). NTE inhibition in lymphocytes can be used as a biomarker of exposure to neuropathic OPs. An electrochemical method was developed to assay NTE in whole blood. The high sensitivity of the tyrosinase carbon-paste biosensors for the phenol produced by hydrolysis of the substrate, phenyl valerate, allowed NTE activity to be measured in diluted samples of whole blood, which cannot be done using the standard colorimetric assay. The biosensor was used to establish correlations of NTE inhibitions in blood with that in lymphocytes and brain after dosing hens with a neuropathic OP. The results of further studies demonstrated that whole blood NTE is a reliable biomarker of neuropathic OPs for up to 96 hours after exposure. These validation results suggest that the biosensor NTE assay for whole blood could be developed to measure human exposure to neuropathic OPs as a predictor of OPIDN. The small blood volume required (100 μL), simplicity of sample preparation and rapid analysis times indicate that the biosensor should be useful in biomonitoring and epidemiological studies. The present paper is an overview of our previous and ongoing work in this area. Human & Experimental Toxicology (2007) 26, 273-282

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.

Hydrogen peroxide-sensitive amperometric sensor based on manganese dioxide nanoparticles

A method for the synthesis of manganese dioxide nanoparticles in reverse micelles is developed and optimized. Nanoparticles are characterized by transmission electron microscopy. An amperometric sensor to detect hydrogen peroxide is developed based on thin films of the synthesized manganese dioxide nanoparticles. The possibility of its use for the electrochemical detection of hydrogen peroxide and its operational and longterm stability are examined. The effect of measuring the response potential, pH, and the ionic strength of solution, as well as of interfering compounds, on the results of hydrogen peroxide measurements are studied. The dependence of response values on the hydrogen peroxide concentration is obtained. The linear range of this dependence is in the interval from 7.8 × 10−8 to 7.8 × 10−4 M. The detection limit is 7.8 × 10−8 M (3σ), and the sensitivity is 922 mA/(M · cm2). The possibility of using the proposed hydrogen-peroxide-sensitive sensor for producing more complex enzyme amperometric sensors based on oxide reductases is exemplified by a choline oxidase biosensor.

Blood esterases as a complex biomarker for exposure to organophosphorus compounds

The growing threat of international terrorism brings with it new scenarios for disaster. For example, in the case of toxic organophosphorus compounds (OPs), it possible for terrorists to use known agents or inadvertently to produce highly toxic OPs of unknown structure as the result of attacks on chemical plants or stockpiles of pesticides and other chemicals. Defending against such agents requires rapid, sensitive, and specific detection of them and their biological effects. Thus, the development of biomarkers of human exposures to OPs is a vital component of the system of prediction and early diagnosis of induced diseases. The phosphylating properties of OPs lead to their differential interactions with various serine esterases. These enzymes include primary targets, e.g., acetylcholinesterase (AChE, acute toxicity) and neuropathy target esterase (NTE, delayed neuropathy, OPIDN); as well as secondary targets, e.g., butyrylcholinesterase (BChE) and carboxylesterase (CaE), which act as scavengers of OPs. The set of activities of these esterases as well as that of paraoxonase (PON1), which can hydrolyze and detoxify OPs, constitutes the “esterase status” of an organism that largely determines indi-vidual sensitivity to OPs and that may be used as a complex biomarker of exposure. This complex biomarker is more effective and informative than the standard determination of erythrocyte AChE and total blood cholinesterases. In particular, it assists with distinguishing between acute and delayed neurotoxicity induced by OPs, as we showed in experiments on acute exposure of hens to a neuropathic compound, O,O-dipropyl-O-dichlorovinyl phosphate. In addition, measuring decreased activities of BChE and CaE, which are often more sensitive biomarkers of OP exposure, allows us to reveal exposure to low doses, as demonstrated by treating mice with low doses of phosphorylated oximes. The aim of the ISTC Project summarized here is to develop a smart biosensor system for simultaneous analysis of a set of blood esterases including AChE, BChE, NTE, CaE, and PON1. The speed, sensitivity, and integrated approach of the method will allow hazards to be assessed and appropriate interventions to be recommended before overt toxic damage has occurred.

Nanostructured polyelectrolyte films for engineering highly sensitive tyrosinase biosensors: Specifics of enzyme-polyelectrolyte structures

The correlation was studied between the activity of tyrosinase electrodes engineered by layer-by-layer deposition technology and the number of PDDA/tyrosinase layers (where PDDA stands for poly(diallyldimethylammonium chloride)). The last deposited layer is the active one regardless of the number of layers. Atomic force microscopy (AFM) characterization of the topology of (PDDA/tyrosinase)1 and (PDDA/tyrosinase)2 films showed that these films have identical structures. The operational stability of tyrosinase biosensors coated with (PDDA/tyrosinase)1 films was examined. Various methods for stabilizing tyrosinase biosensors were tested. Crosslinking with glutaraldehyde (GA) improved 2.5-fold the operational stability of (PDDA/tyrosinase)1 films.

Opioid peptides determination by tyrosinase-modified oxygen electrode

Abstract Opioid peptides morphiceptin (Tyr-Pro-Phe-Pro-NH2), [D-Ala2, D-Leu5]-enkephalin (Tyr-D-Ala-Gly-Phe-D-Leu, DADLE) and [D-Thr2]-Leu-enkephalin-Thr (Tyr-D-Thr-Gly-Phe-Leu-Thr, DTLET) containing tyrosine has been studied in the reaction with mushroom tyrosinase immobilized on Clark-type oxygen electrode. A 4–6-times higher response is observed for tyrosine compared to peptides. The detection limit of the tyrosinase-modified electrode for DADLE, morphiceptin and DTLET was 6 μM, 9 μM and 16 μM, respectively.

Multi-strip assay and multimodal biosensors for environmental and medical monitoring of neurotoxicants

The present report describes two types of biosensor systems. The first is for direct monitoring of neurotoxicants. The second is for monitoring individuals exposed to cholinergic and/or neuropathic organophosphorus compounds (OPs) by analyzing activities of their blood esterases. Both systems are rapid, simple, and sensitive. Assemblies of synthetic polymers, biomolecules, nanoparticles, and electrochemical transducers allow the biosensor systems to be used at the point of care and for field measurements in environmental, medical toxicology, veterinary, and antiterrorist applications. The selective monitoring of anticholinesterase compounds is based on a simple analytical method: the residual cholinesterase (ChE) activity assay after incubation with inhibitors. The specially developed design includes “programmable” strips and an automatic flow-injection amperometric analyzer for ChE activity analysis based on biosensor technology for choline detection. Capabilities include estimating the general toxicity of a sample as well as carrying out selective quantitative and qualitative assays of OPs, and other antiChE agents in mixtures. Combining layer-by-layer electrostatic assembly of enzymes and polyelectrolytes with extremely sensitive amperometric detection of hydrogen peroxide based on carbon electrodes coated by nanoparticles of MnO2 yields a simple biosensor device capable of sensitive (< 0.1 pM) detection of neurotoxicants in 20 minutes. A new method of simultaneous quantitative determination of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in mixtures is also described in the report; the method is fast and precise. Average error of determination of enzyme activity is 8%. Highly sensitive tyrosinase and choline oxidase biosensors based on nanostructured polyelectrolyte films were developed for these purposes. The methodology of neuropathy target esterase (NTE) activity assay in whole human blood was realized using an amperometric analyzer coupled with a tyrosinase biosensor for phenol detection. These analytical approaches were developed to serve as a monitoring system for individuals exposed to cholinergic and/or neuropathic OPs. Thus, the technologies described here not only have enormous potential for use in responding to terrorist chemical threats, but they also have many civilian applications, which would likely be the larger market sector.

BIOSENSORS AND NANOTECHNOLOGICAL IMMUNOCHIPS FOR THE DETECTION AND MONITORING OF CHEMICAL AND BIOLOGICAL AGENTS

The elaboration of highly sensitive and express methods for quantitative and qualitative detection and monitoring of chemical warfare agents (CWA), organophosphate and carbamate pesticides, compounds with delayed neurotoxicity, and pathogenic microorganisms and viruses is discussed. The application of potentiometric and amperometric biosensors, automatic biosensors discriminating the neurotoxins of different classes, is performed. The information about biosensors detecting the compounds with delayed neurotoxicity through the evaluation of “neurotoxic esterase” activity in the blood is presented. The use of immunochip technology for the detection of pathogenic microorganisms and viruses is demonstrated. The enzymatic methods of destruction of organophosphorus neurotoxins are considered as the base of new defense technology

[Tyrosine hydroxylase activity in two-dimensional monomolecular films].

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.