Detection of bacteria viruses parasites and fungi bioterrorism prevention NATO science for peace and security series a chemistry and biology

Detection of Bacteria, Viruses, Parasites and Fungi NATO Science for Peace and Security Series This Series presents the results of scientific meetings supported under the NATO Programme: Science for Peace and Security (SPS) The NATO SPS Programme supports meetings in the following Key Priority areas: (1) Defence Against Terrorism; (2) Countering other Threats to Security and (3) NATO, Partner and Mediterranean Dialogue Country Priorities The types of meeting supported are generally “Advanced Study Institutes” and “Advanced Research Workshops” The NATO SPS Series collects together the results of these meetings The meetings are coorganized by scientists from NATO countries and scientists from NATO’s “Partner” or “Mediterranean Dialogue” countries The observations and recommendations made at the meetings, as well as the contents of the volumes in the Series, reflect those of participants and contributors only; they should not necessarily be regarded as reflecting NATO views or policy Advanced Study Institutes (ASI) are high-level tutorial courses intended to convey the latest developments in a subject to an advanced-level audience Advanced Research Workshops (ARW) are expert meetings where an intense but informal exchange of views at the frontiers of a subject aims at identifying directions for future action Following a transformation of the programme in 2006 the Series has been re-named and re-organised Recent volumes on topics not related to security, which result from meetings supported under the programme earlier, may be found in the NATO Science Series The Series is published by IOS Press, Amsterdam, and Springer, Dordrecht, in conjunction with the NATO Public Diplomacy Division Sub-Series A B C D E Chemistry and Biology Physics and Biophysics Environmental Security Information and Communication Security Human and Societal Dynamics http://www.nato.int/science http://www.springer.com http://www.iospress.nl Series A: Chemistry and Biology Springer Springer Springer IOS Press IOS Press Detection of Bacteria, Viruses, Parasites and Fungi Bioterrorism Prevention edited by Mariapia Viola Magni Università degli Studi di Perugia Italy Published in Cooperation with NATO Public Diplomacy Division Proceedings of the NATO Advanced Research Workshop on Detection of Bacteria, Viruses, Parasites and Fungi Perugia, Italy November 18-21, 2008 Library of Congress Control Number: 2010927631 ISBN 978-90-481-8543-6 (PB) ISBN 978-90-481-8542-9 (HB) ISBN 978-90-481-8544-3 (e-Book) Published by Springer, P.O Box 17, 3300 AA Dordrecht, The Netherlands www.springer.com Printed on acid-free paper All Rights Reserved © Springer Science + Business Media B.V 2010 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work Contents Part Iâ•… Bioterrorism Prevention Bioterrorism: A Potential Weapon for Terrorist Attacks Through Food and Water Contamination: Evolution of Our Understanding of the Use of Chemical and Bacteriological Weapons Vincenzo Costigliola and Franco Quagliata Detection and Quantification of Bacteria and Fungi Using Solid-Phase Cytometry Lies ME Vanhee, Eva D’Haese, Ils Cools, Hans J Nelis, and Tom Coenye Vulnerability Assessment and Mapping of Akounq Groundwater Body, Armenia Vahram Vardanyan and Artashes Aginian 25 43 Part IIâ•… Bacteria Diagnostic Tests Methods for Detection of Shiga-Toxin Producing Escherichia coli (STEC) Jordan Madic Genetic Diversity of Enterococci in Bryndza Cheese Roman Dušinský, Anna Belicová, Libor Ebringer, Dušan Jurkovič, Lívia Križková, Mária Mikulášová, and Juraj Krajčovič 53 87 Salmonella Infections 125 Constantine Arvanitakis Molecular Diagnostics of Staphylococcus aureus 139 Jiří Doškař, Roman Pantůček, Vladislava Růžičková, and Ivo Sedláček v vi Contents Molecular Methods to Detect Bacillus cereus and Bacillus thuringiensis in Foods 185 Marisa Manzano, Lucilla Iacumin, Cristina Giusto, and Giuseppe Comi Control of Listeria monocytogenes in San Daniele Dry Cured Ham by Different Technologies: Reduction of L Monocytogenes in Dry Cured Ham 211 Lucilla Iacumin, Marisa Manzano, Milena Osualdini, Carlo Cantoni, and Giuseppe Comi Part IIIâ•… Viral Infections Epidemiology and Diagnosis of Hepatitis B in the Mediterranean Region and Elsewhere 239 Nurdan Tưzün, Ưzdal Ersoy, Fatih Eren, and Veysel Tahan Hepatitis E Virus (HEV) – An Emerging Viral Pathogen 261 Avrelija Cencič and Walter Chingwaru Dengue Virus Diagnostics 275 Evgeni Eltzov, Danit Atias, Levi Gheber, and Robert S Marks Part IVâ•… Parasites and Fungi Infectious Forms of Parasites in Food: Man Embedded in Ecosystems 299 Eduardo Dei-Cas, Cécile-Marie Aliouat, Gabriela Certad, Colette Creusy, and Karine Guyot Unusual Developmental Pattern of Expression of Enzymes Involved in DNA Biosynthesis in Trichinella spiralis and Trichinella pseudospiralis 333 Magdalena Dąbrowska, Barbara Gołos, Elżbieta Wałajtys-Rode, Patrycja Wińska, Joanna Cieśla, Zbigniew Zieliński, Elżbieta Jagielska, and Wojciech Rode Immunodulation and Helminths: Towards New Strategies for Treatment of Immune-Mediated Diseases? 357 Fabrizio Bruschi, Lorena Chiumiento, and Gianfranco Del Prete Contents vii Parasites and Oncogenesi with a Special Reference to Gastro-Intestinal Neoplasia Induced by Cryptosporidium parvum 381 C Creusy, G Certad, K Guyot, and E Dei-Cas Smart Biosensors for Determination of Mycotoxines 389 Lyubov Yotova, Ivo Grabchev, Rossica Betcheva, and Dessislava Marinkova Introduction This publication represents the result of the fruitful workshop organised with the aim to attract the attention on the possibility of bio terrorism attack, with the support of NATO funds In the last years the attention was strongly concentrated on the terrorism view similar to “military type attacks:” bomb on the trains, kamikazes, airplanes etc As consequence many devices studied are directed to prevent these attacks such as the control of the passengers before the flight For the people terrorism is therefore equivalent to bomb or similar and nobody think that there is also other possible and sophisticated means that can be used by the terrorist In 1995 Sarin gas in the Tokio subway killed 12 people and affected 5,000 persons In the USA anthrax was sent by mail to many federal offices These events and other cases attract the attention on these possible terrorist attacks and the first recommendations for preventing theses events were\elaborated in the United State and in Europe The possible agents and the modality that can be used for the diffusion are analysed and food and water are considered the principal and more favourable way The story and the principal decision about this were reported in the first article of this collection which introduces the concept of bio-terrorism Then the attention was concentrated on the possible and more diffuse agents which may contaminate the food, the water and the environment and the possible methods which may be sufficient precise, rapid and easy to use on a large number of person and samples Dr Coenye elaborated a fast method consisting in solid-phase cytometry which permits to analyse many samples and gives the possibility to visualise few bacteria or other kind of cells also if they are in small number The analysis was made on air samples, on water and food samples As regards the water possible contamination of the source in relation to the particular structure of the soil was suggested by Vardanyan and Aginian Then the study was divided in four sectors of possible contaminant: bacteria especially the more diffuse, virus, parasites and pollutants Of each the possible way of diffusion, the consequent diseases and the more modern and safe methods used for diagnosis were presented and discussed in view of the results obtained ix 400 L Yotova et al attachment site General single-step reaction used widely to create derivatized conducting polythiophenes is presented, we outline the two-step synthesis of a representative biotinylated (B) polythiophene copolymer, B-PUMT, where the biotin is connected by an ester linkage to the pendant CH2OH group on the thiophene polymer backbone (Marx et€al 1994) These biotin ligands recognize and bind each of the four subunits of the tetrameric proteins streptavidin and avidin with affinity constants approaching those of covalent bonds, around 1015 per M (Wilchek and Bayer 1989) These two proteins have been widely used to covalently derivatives biological elements can be immobilized upon the biotinylated polymer chain, following prior immobilization of the polymer upon the surface of the chosen platform (Knoph et€al 2007b) During the last several years, optical biosensors gradually are involved in aflatoxins analyses They are based on optical fibers or surface plasmon resonance Biosensors are devices, ideally small and portable, that allows the selective measurement of chemical and biochemical analytes They consist of two components: the transducer and the chemical recognition element Chemical recognition is accomplished by exploiting the natural selectivity of biochemical species such as enzymes, antibodies, chemoreceptors and nucleic acids In the presence of the analyte, these agents, immobilized at the surface of the transducer, cause change in measurable property in the local environment The transducer converts the biochemical signal in electronic signal Transducers may measure electrochemical, optical, thermal or adsorption processes that change in the presence of analyte Polyamidoamine Dendrimers as Potential Substances for Obtaining Biosensors The rapid detection of organic and biological compounds in the environment and in medicine is of a particular interest for identifying different biologically active substances of metabolic or industrial origin Fluorescence as a signal for identifying the presence and quality of different compounds is widely used For this purpose a number of appropriate sensors has been developed using fluorophores with different chemical structures Among them polymer sensors are considered as quite promising Synthesis of linear polymers has as a result obtaining of final products with heterogeneity that could influence the optical performance of the polymer sensor Poly (amidoamine) dendrimers (PAMAM) are new interesting class of star polymers They have mono dispersive, well defined and developed three-dimensional structures comprising functional groups at high concentration In its core they have amidic groups Structurally modified PAMAM dendrimers with 1,8-naphthalimide derivatives are fluorescent dendrimers and they can be applied as effective and selective sensors for different metal cations and protons in organic solvents This property can be transferred to other polymer matrixes (for example textiles) In this study we present our first results about the possibility for the incorporation of bio-receptors into some new PAMAM dendrimers Dendrimers under study contain peripherally bonded 1,8-naphthalimide derivatives (Figs.€1 and 2) Smart Biosensors for Determination of Mycotoxines A A N O A NH O O N O O N O O HN O O NH O N HN O O O HN O HN O O O O O NH N O O O O NH N A O O O N A O O O N O A O NH N NH N O N O O HN N A A O NH O NH N O NH N O O O NH N HN N HN N HN O O N A HN O NH N O HN N N A NH O NH N O O O N O HN O N A O N O A A N HN O HN N 401 O O O N O O HN O NH N N A O A A Fig.€1â•… Chemical structure of dendrimer Fig.€2â•… Picture of dendrimer membrane with covalently immobilized fluorescent dye and enzyme 402 L Yotova et al These dendrimers have been additionally modified with biologically active molecules, which can react with the available amidic functional groups Result from the first photophysical and biochemical investigations of the new structurally modified dendrimers are discussed The comparison between the bio-modified PAMAM dendrimers and some linear polymers is given (Grabchev et€al 2007a) Photophysical and Biological Properties of Fluorescent PAMAM Dendrimer Dendrimers are a relatively new category of star-shaped polymers They are monodisperse, perfectly branched well-defined polymers, possessing a number of functional end groups that allow different modification of the dendrimers A great deal attention has been paid to this class of macromolecules owing to their new form of structure organization, which combines the properties of low, and high molecular weight compounds In this study we present the result on the preparation of fluorescence PAMAM dendrimer –acetyl cellulose membrane by spin coating method Fluorescent PAMAM contained chemically bonded fluorescent dye Glucose oxidase widely applied in clinical diagnostic was used as a model enzyme widely applied in order to check the possibility to use the membrane as a biosensor The enzyme was covalently immobilized on the dendrimer membrane by preliminary oxidation of carbohydrate residues of the glucose oxidase with periodic acid The chemical modification of PAMAM was supposed to occur in the dendrimer core where the enzyme can react with amidic functional groups Thus the model enzyme and the immobilized dye form a stable conjugate with a high activity Lipoxygenase, peroxidase and aflatoxine antibodies were covalently immobilized Membrane obtained was 40 m thick and several photophysical properties including the photostability were investigated Fluorescence intensity was studied as well and the effect of Cu2+ and Co2+ cations in water solution was measured Based on preliminary photophysical end biological investigations a conclusion can be taken that the dendrimer membrane possesses promising properties for applying in biosensor and immunosensor constructions with fluorescent detection (Grabchev et€al 2007b) (Fig.€3) Magnetic Particles Magnetic nanoparticles modified with organic molecules have been widely used for biotechnological and biomedical applications because their properties can be magnetically controlled by applying an external magnetic field (Osaka et€ al 2006a) They offer a high potential for numerous biomedical applications, such as cell separation (Safarik and Safarikova 2002), purification of nucleic acid (Berensmeier 2006), hyperthermia (Lao and Ramanujan 2004) and immunosensors (Sheng-Fu Wang and Tan Yu-Mei 2007) Smart Biosensors for Determination of Mycotoxines 403 Normalized Intensity 1,0 0,8 0,6 E F 0,4 0,2 0,0 350 400 450 500 550 600 650 Wavelength / nm Fig.€3â•… Normalized excitation and fluorescence spectra of membrane Magnetic nanoparticles as immobilization materials have the following advantages: (1) more specific surface area obtained for the binding of larger amounts of biomolecules; (2) lower mass transfer resistance; (3) selective separation of the immobilized biomolecules from a reaction mixture on application of a magnetic field Among these materials, Fe3O4 magnetic nanoparticles are the most commonly studied Fe3O4 magnetic nanoparticles have good biocompatibility, strong superparamagnetism, low toxicity, and an easy preparation process, and their use in biosensors has already shown attractive prospects (Sheng-Fu Wang and Tan Yu-Mei 2007) Horseradish peroxidase (HRP) is an important peroxidase that contains heme, which is the protein active site with the resting state of the heme iron, Fe(III), as prosthetic group It can catalyze the H2O2 dependent one-electron oxidation of a great variety of substrates, and has been commonly employed to construct H2O2 biosensors (Hai-Li Zhang et€ al 2008) Peroxidase is the most frequently used enzyme for the construction of immunosensors Successful immobilization of horseradish peroxidase on modified magnetic particles and their employment in the amperometric biosensors are mentioned in (Hai-Li Zhang et€ al 2008; Yu et€ al 2006) The objectives of this our study were to investigate the properties of immobilized horseradish peroxidase on magnetic particle coated with copolymer of acrylamide and acrylonitrile, used furder in fibre optic smart biosensor constructions with simultaneously immobilized different antibodies for aflatoxines (Fig.€4) Detection of Foodborne Pathogens Using Optical Biosensors Biosensors use a combination of biological receptors and physical or chemical transducers, which represent a new and unique technology with great potential to meet the need for rapid detection of low levels of biomolecules (Baeumner 2003; Bhunia and Lathrop 2003; Ivnitski et€ al 1999; Vo-Dinh et€ al 2001) Optical sensors measure a parameter of the reaction between a receptor and an analyte 404 L Yotova et al a Fibre optic Fibre optic b Compound (II) H2O2 Compound (I) H2O magnetic particle-HRP-anti-AFB1 mycotoxin Fig.€4â•… The future immunosensor with fibre optic (a) and reaction with mycotoxin (b) as a quantifiable optical signal Optical biosensors offer the advantages of noninvasive, nondestructive, continuous, and simultaneous multianalyte detection (Taitt et€al 2005) Optical systems not interfere with metabolism, and thus in€vivo measurements are possible In this chapter, some applications of the following optical sensing principles in foodborne pathogen detection are discussed: (1) SPR, (2) resonance mirror (RM), (3) fiber-optic biosensor, (4) array biosensor, (5) Raman spectroscopy, and (6) light-addressable potentiometric sensor (LAPS) (Knoph et€al 2007c) Biosensors Based on Surface Plasmon Resonance (SPR) The surface plasmon resonance is a measurement of the changes in the mass concentration that appear on the surface of the sensor and are due to the molecule binding At specific wavelength and angle of incidence the free electrons in the thin metal film of the sensor surface will absorb the light and the intensity of the reflected light will decrease The angle of incidence in this process changes as mass concentration of the sensor surface changes The variations in the mass concentration measured by the sensor are due to the binding and dissociation of interacting molecules In this case those molecules are between the immobilised agent (the mycotoxin) and a specific antibody added to the sample There are immunochemical biosensors applied for mycotoxin assays Gaag at all developed a method based on surface plasmon resonance for measuring of four different mycotoxins – aflatoxin B1, zearalenone, ochratoxin A and fumarotoxin B1 (Paepens et€al 2004) These systems utilize surface Plasmon resonance (SPR) and the Resonant Mirror, respectively and have proved especially convenient for the study of affinity Smart Biosensors for Determination of Mycotoxines 405 kinetic constants and screening of binding partners without the need for labeled molecules Surface Plasmon resonance occurs when plane – polarized light is internally reflected at an interface between media of different refractive indices, separated by thin metal film Light energy is absorbed by the metal film but is at a maximum at a specific input angle Typically it is possible to follow the binding of a macromolecular antigen to a surface immobilized antibody without need a label (Maragos 2002) Daly et€ al (2000) gives an example for aflatoxine determination by a specific antibody too Sensor systems based on SPR now are commercially available (Nagata and Handa 2000) Some of the latest investigations show that on the base of smart polymers with simultaneously immobilized biorecognition molecules as enzymes, antibodies, nucleic acids with fluorescent dyes can be constructed biosensors by means of SPR measurements In this method the fixed concentration of the mycotoxin specific antibodies are mixed with a sample containing some concentration of the mycotoxin Then the mixture is passed over the sensor surface on which the mycotoxin is immobilised The surface plasmon resonance has a number of benefits –â•fi –â•fi –â•fi –â•fi –â•fi A very small volume of the sample is needed The metal chip can be reused The kinetics of the reaction antibody – antigen can be detected A number of analytes can be detected The method is easy to use SPR was developed for several mycotoxins Different mycotoxins have been found simultaneously within a period of 25 with a detection limit for aflatoxin В1, zearelenon, ochratoxin A, fumonisin В1 and deoxinivalenol respectively – 0.2, 0.01, 0.1, 50, 0.5 mg L−1 (Zheng et€al 2005) Fiber Optic Immunosensor The Fiber optic immunosensors are the second major generation of biosensors Many of the biosensors are a mini version of the classical spectrophotometry The most frequently used is the fiber optic chain or membrane, covered with a biological element The optical biosensors are only optical sensors where the element to be detected is of biological origin This provides greater specificity for the analyte An evanescent wave is generated in the space between the optical fiber and the outside lower refractive index material Fluorescent molecules in this region can absorb energy from the evanescent wave and fluoresce A portion of the fluorescence will be coupled back into the fiber and can be detected By immobilizing antibodies to the surface of an optical fiber, fluorescent interference from the bulk solution is almost completely eliminated The signal generated in the assay corresponds 406 L Yotova et al to the toxin concentration but varies depending on the assay format (Eaton and Groopman 1994) Studies of the detection of fumonisin B1 by using a fiber-optic immunosensor showed that the sensor could detect fumonisin B1 in a quantitative range of 10–1,000 ng mL−1 with a limit of detection of 10 ng mL−1 Benefits of the Method –â•fi –â•fi –â•fi –â•fi High specificity Ease of miniaturization Real-time monitoring Adaptability for remote sensing However, the method may have limitations in sensitivity Using immunoaffinity column clean up can enhance the sensitivity Additionally, solvents may affect the accuracy of the method because they can change the refractive index of a medium (Zheng and Richard 2006) Fibre Optic Oxygen Enzyme Biosensor The purpose of this study was to design biosensors on the basis of covalently immobilised lipoxygenase and oxygen electrodes The measurements performed with the two types of biosensors show that the linear range of this type of enzyme electrodes using natural oxygen mediator manifest a wide range of measurement The immobilised enzyme showed high operative stability, which makes the measurements easily reproducible Both electrodes have very good correlation coefficients and a small standard deviation The performed investigations prove that they can be used for measuring real food (Yotova and Marinkova 2008) Lipoxygenase is iron that contains dioxygenase and is found in plants and mammals, also produced by some microorganisms It has been established that the lipoxygenase from soybeans can catalyse the oxidation of a wide range of endogenous chemicals (Kulkarni 1996) For the first time bioactivation of aflatoxin B1 from soybeans lipoxygenase was demonstrated by Liu and Massey (Liu and Massey 1992) Datta and Kulkarni have also investigated it, but in the presence of linoleic acid which is the natural substrate of the enzyme (Datta and Kulkarni 1994) Obtaining Polymer Membrane Powder copolymer of acrylonitrile and acrylamide (200 mg) was diluted in mL of dimethylformamide and the mixture was poured in petri and kept there until Smart Biosensors for Determination of Mycotoxines 407 dimethylformamide evaporated completely The obtained membrane was activated with 50 mL phosphate buffer рН = and 12.5 mL formaldehyde for h at t = 45°С in a closed container being constantly stirred The activated membrane was carefully washed with distilled water until there was no formaldehyde Enzyme Immobilization Procedure The immobilization was performed with 1% solution of microbial lipoxygenase [E.C 1.13.11], from Pseudomonas sp., produced by Biovet, Peshtera, Bulgaria stirring it constantly in a closed container at t = 25°C for 21 h in phosphate buffers with рН = 5, and and acetate buffer рН = After the completion of the procedure, the carrier granules were washed many times in buchner funnel with distilled water and М NaCl until there was no absorption at l = 280 nm Measurements of the Kinetics of the Enzyme Reaction The measurements were performed spectrophotometrically at l = 234 nm using substrate solutions with different concentrations as well as by accounting for the O2 consumption in the enzyme reaction The latter procedure was performed with an electrode for O2 An optical sensor was constructed on the immobilised enzyme based on spectrophotometer with optical fibres and probe for oxygen measurement AvaSoft – Oxy, AvaSpec US B1 (Avantes – the Netherlands) Using substrate solutions in the range 0.4–3.5 mМ the dependency of the speed of the reaction on the concentration of the substrate (linoleic acid) was tracked The microbial lipoxygenase was purified using ultrafiltration In this purification the specific activity of the enzyme preparation increased three times compared to the commercial preparation 2.214 Е mg−1 (U mg−1) and 860 E mg−1, respectively The microbial lipoxygenase was immobilised on polymer membrane, which was done according to the procedure in the methodological part Examination of the obtained conjugate for protein contents was performed spectrophotometricaly in the way it was done for the enzyme immobilised on polymer granules The relative activity of the immobilised enzyme is 65% and the protein contents 5.4 mg protein/g dry carrier In the Table€ the main characteristics of free and immobilised at рН = 5, microbial lipoxygenase are shown The stability of the microbial lipoxygenase immobilised on polymer carrier was determined by performing measurements of the activity with the same carrier with covalently bound enzyme for a period of 20 days (Fig.€5) As can be seen from the figure, the enzyme preserved its activity almost 100% for the whole period of investigation The biosensors were constructed with optical sensor for oxygen based on optical fibre Аvaspec –Oxy (Avantes, Holland) The membrane with immobilised enzyme was attached to the electrodes with a dialysis membrane and a pipette tip 408 L Yotova et al Table€1â•… Catalytic properties of lipoxygenase from Penicillium sp Amount of bound enzyme, mg g−1 dry Residual pН carrier activity (%) optimum Enzyme Residual activity (%) Free Immobilized on granulated carrier Immobilized on membrane t (°C) Optimum Km [M] – 2.9 – 94 8.3 7.5 35 35 × 10−6 × 10−6 5.4 65 7.5 35 – 100 50 0 10 15 20 t (days) Fig.€5â•… Stability of microbial lipoxygenase immobilized on granulated polymer carrier in time 100 90 80 O 2% 70 60 50 40 30 20 10 0,0 0,1 0,2 0,3 0,4 0,5 Aflatoxin B1 àM Fig.6õ Standard curve for determination of Aflatoxin B1 using biosensor AvaSpec – Oxy In a measurement cell 20 mL of 0.1 Tris-buffer with рН 7.2 the respective sensor was incubated for with aflatoxin B1 in concentration range of 0.0 5–0.5 µM previously diluted in dimethyl sulphoxide After of incubation at 37°С 0.1 mL linoleic acid was added to the samples so that the final concentration of the sample was µM Based on the measurements, standard curves were constructed for the optical enzyme electrode (Fig.€6) Smart Biosensors for Determination of Mycotoxines Table€2â•… Statistical processing of standard curves Correlation Electrode coefficient Standard deviation AvaSpec – Oxy 0.09978 0.02014 409 Kohren criteria 0.1200 The obtained standard curves were statistically processed and are shown in Table€2 above The performed investigations prove that they can be used for measuring real food Application of Magnetic Particles in the Design of Biosensors Nano particles are an attractive target due to their unique characteristics Especially the magnetic nano particals are of interest not only because of their properties, but also because of their potential application in many fields as density particles, magnetic fluids and biomedical material for highly sensitive immune analyses (Osaka et€al 2006b) An immunological method has been developed based on AFB1 antibodyfunctionalised composite magnetic nano particles based on Fe3O4/SiO2 For this purpose anti-AFB1 is immobilized covalently on the surface of the nucleus Te selectivity, reproducibility and stability of the immunological system are within the allowable error Compared to the conventional ELISA the proposed immunosystem is simple and rapid (Li Wang and Xian-Xue Gan 2009) Magnetic particles coated with copolymer of acrylamide and acrylonitrile has been prepared Those particles were used as a matrix for a covalent binding of peroxidase The periodic-oxidized enzyme was bound to the matrix by immobilization procedure at 4°C for 18 h The immobilized enzyme showed relative activity of 86% The following results were obtained for pH and optimum temperature of the immobilized enzyme – 7.0°C and 30°C, respectively The analysis of the kinetic parameters of the immobilized enzyme showed values of Vmax − 0.0517 M × 10−6 s−1 and Km − 2.3 × 10−4 M (Yotova et€al 2008) The copolymerization of acrylonitrile and acrylamide occurs by a free radical mechanism with an initial stage of initiation resulting in free radicals FeSO4, NaS2O5 and (NH4)2S2O8 are used as initiators The following reagents were added to obtain copolymer containing 20% acrylamide: H2O-174 mL, acrylonitrile-12 g, acrylamide-3 g, NaS2O5 − 0.125 g, FeSO4 − 0.125 g, (NH4)2S2O8 − 0.125 g Copolymerization takes h at room temperature under constant stirring The copolymer is filtered and profusely flushed with water in buchner funnel, and finally with methanol It is dried in a vacuum oven at 50°С The dried polymer is dissolved in dimethylformamide and then using a syringe it is poured drop by drop in a solution of ethanol and water (1:4) and surfactant The spherical particles thus formed have an average diameter of ~1 mm The water contents of the carrier thus formed were determined to be – 80% The spherical particles with magnetic properties were obtained by producing a 10% solution of copolymer containing 5% magnetite 410 L Yotova et al Horseradish peroxidase (HRP, EC 1.11.17) was obtained from Fluka The oxidation of carbohydrate moieties of enzyme with periodic acid (0.04 mM in 0.05 mM acetate buffer, pH 5.0) was performed according to Zaborsky and Ogletree (1974) The unreacted periodic acid was removed with 0.025-ethylene glycol The oxidized enzymes were dialyzed against 50 mM phosphate buffer with pH 6.0 for 18 h The immobilization of HRP was performed in the following manner: 20 mL of oxidized dialytic solution of peroxidase was added to 1.0 g of absolutely dry particles The process was implemented by constant stirring with a magnetic stirrer for 18 h at t = 4°С, in dark The copolymer obtained according to the methodological part has a spherical shape with magnetic properties and average diameter of 0.6–0.8 mm (Fig.€7) The magnetic particles have a high magnetic stability, which is a necessary condition to obtain the desired shape and size relevant to the requirements of the experiment The mechanical robustness of the particles was preserved throughout the process The magnetic particles coated with copolymer were used as a matrix for covalent binding of the peroxidase The results from the investigations on the activity of the free and immobilized peroxidase at рН = are shown in Table€3 It can be seen from the table that the enzyme preserves its high relative activity The pH optima of free and immobilized peroxides were performed following the methodological part and are given in Fig.€8 Fig.€ 7â•… Picture of received magnetic particle Table€3â•… Investigation on the activity of the free and immobilized enzyme Specific activity Amount of bound protein Enzyme U mg−1 Relative activity % mg g−1 dry carrier Soluble enzyme Immobilized enzyme 210 181 – 86 – 0.613 Smart Biosensors for Determination of Mycotoxines 411 110 100 Residual activity (%) 90 80 70 60 50 40 30 Soluble HRP Immobilized HRP 20 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 pH Fig.€8â•… Effect of pH on the activity of soluble and immobilized peroxidase When examining the effects related to the рН optimum the effect of the matrices on the enzyme should be considered In this case the рН optimum increases to 7.0 This change occurs due to the irregular distribution of the protons caused by the positive or negative loads of the employed carrier as well as the presence of diffusion limitations In this case it can be assumed that the magnetic particles affect the enzyme molecules The experiments on the temperature optimum also showed a shift of the optimum of the immobilized enzyme at 30°С (Fig.€9) The immobilization of enzymes to different carriers usually leads to a change in their kinetic behaviour characterized by different constants, the most important being the Michaelis constant (Km) Table€ shows a comparison between the kinetic parameters of soluble and immobilized peroxidase and on magnetic particles In an investigation similar to ours, the following values for Km of free and immobilized peroxidise, respectively 5.5 and 3.6 mM were obtained in immobilization on chitosan The results from this investigation show that the immobilized enzyme has a greater affinity to the substrate compared to the free enzyme (Mohamed et€al 2008) Magnetic particles coated with copolymer of acrylamide and acrylonitriles were successfully obtained The peroxidase immobilized on these particles preserves its high relative activity It was established that the catalytic parameters of the immobilized peroxidase change as follows: topt − 30°C, pHopt − и Km − 2.3 × 10−4 Based on the results obtained from the covalent immobilization of peroxidase to magnetic particles coated with copolymer of acrylamide and acrylonitrile we can outline the steps in construction of a smart biosensor for mycotoxins 412 L Yotova et al 110 100 Residual activity (%) 90 80 70 60 50 40 30 Immobilized HRP Soluble HRP 20 10 25 30 35 40 45 T (°C) Fig.€9â•… Effect of temperature on soluble and immobilized peroxide Table€4â•… Kinetic parameters of soluble and immobilized peroxidase R2 Enzyme Кm [М] Vm [M × 10−6 s−1] Soluble enzyme Immobilized enzyme 1.19 × 10−3 2.3 × 10−4 – 0.0517 – 0.99885 Acknowledgmentâ•… This review is supported for publication from NATO Scientific Committee and our research investigation was financial supported by project 322, National Fund “Scientific Investigations” of Republic of Bulgaria References Baeumner AJ (2003) Biosensors for environmental pollutants and food contaminants Anal Bioanal Chem 377:434–445 Berensmeier S (2006) Magnetic particles for the separation and purification of nucleic acids Appl Microbiol Biotechnol 73:495–504 Bhunia AK, Lathrop A (2003) Pathogen detection, food-borne McGraw-Hill yearbook of science and technology McGraw-Hill, New York, pp 320–323 Daly S, Keating G, Dillon P (2000) Development of surface plasmon resonance-based immunoassay for aflatoxin B (1) J Agric Food Chem 48:5097–5104 Datta K, Kulkarni A (1994) Oxidative metabolism of aflatoxin B1 by lipoxygenase purified from human term placenta and intrauterine conceptual tissues Teratology 50:311–317 Eaton D, Groopman JD (1994) The toxicology of aflatoxins Academic, New York, pp 383–426 Grabchev I, Betcheva R, Yotova L (2007a) Polyamidoamine dendrimers as potential substances for obtaining biosensors COST Action 868- Biotechnical fictionalization of renewable polymeric materials, Sitges, Spain, 16–17 April 2007 Smart Biosensors for Determination of Mycotoxines 413 Grabchev I, Betcheva R, Yotova L (2007b) Photophysical and biological properties of fluorescent PAMAM dendrimer Biotechnical fictionalization of renewable polymeric materials, Graz, Austria, 12–14 September 2007 Hai-Li Zhang, Guo-Song Lai, Han De-Yan, Yu Ai-Min (2008) An amperometric hydrogen peroxide biosensor based on horseradish peroxidase on an electrode modified with magnetic dextran microspheres Anal Bioanal Chem 390:971–977 Ivnitski D, Abdel-Hamid I, Atanasov P, Wilkins E (1999) Biosensors for detection of pathogenic bacteria Biosens Bioelectron 14:599–624 Karova E (1998) Microbiology Academic Publishing house, Plovdiv Knoph GK, Bassi AS, Geng T, Bhunia A (2007a) Optical biosensors in foodborne pathogen detection, foodborne pathogens In: Knopf GK, Bassi AS (eds) Smart biosensor techonology CRC Press/Taylor and Francis group, Boca Raton, FL, pp 506–507 Knoph GK, Bassi AS, Marx KA (2007b) Toward understanding the intelligent properties of biological macromolecule-implifications for their design into biosensors In: Knopf GK, Bassi AS (eds) Smart biosensor techonology CRC Press/Taylor and Francis group, Boca Raton, FL 4–6 Knoph GK, Bassi AS, Geng T, Bhunia A (2007c) Optical biosensors in foodborne pathogen detection, detection of foodborne pathogens using optical biosensors In: Knopf GK, Bassi AS (eds) Smart biosensor techonology CRC Press/Taylor and Francis group, Boca Raton, FL 507 Kulkarni AP (1996) Role of xenobiotic metabolism in developmental toxicity In: Hood RD (ed) Handbook of developmental toxicology CRC Press, Boca Raton, FL, pp 383–421 Lao LL, Ramanujan RV (2004) Magnetic and hydrogel composite materials for hyperthermia applications J Mater Sci Mater Med 15:1061–1064 Larinov OG, Pisareva O Yu (1997) Structure of chemical compounds methods of analysis and process control Pharmaceutical 31(7):49–53 Li Wang, Xian-Xue Gan (2009) Biomolecule-functionalized magnetic nanoparticles for flowthrough quartz crystal microbalance immunoassay of aflatoxin B1 Bioprocess Biosyst Eng 32:109–116 Liu L, Massey T (1992) Bioactivation of aflatoxin B1 by lipoxygenases, prostaglandin H synthase and cytochrome P450 monooxygenase in guinea-pig tissues Carcinogenesis 13:533–539 Maragos CM (2002) Novel assays and sensor platforms for the detection of aflatoxins Adv Exp, Med Biol 504:84–93 Marx KA, Samuelson LA, Kamath M, Lim JO, Sengupta S, Kaplan D, Kumar J, Tripathy SK (1994) Inteligent biomaterials based on Langmuir-Blodgett monolayer films In: Birge BR (ed) Molecular and biomolecular electronics Advanced in chemical series, Vol 240 American Chemical Society Books, Washington, DC, pp 395–412 Maxwell CK, Diaz-Lano LG, Smith TK (2003) Mycotoxins in pet food Agric Food Chem 54:9623–9635 Mohamed SA, Aly SA, Mohamed TM, Salah HA (2008) Immobilization of horseradish peroxidase on nonwoven polyester fabric coated with chitosan Appl Biochem Biotechnol 144:169–179 Murphy PA, Suzanne Hendich PHD, Landgren C, Cory M (2006) Food mycotoxins: an update Appl Biochem Biotechnol Hypotheses Food Sci 71(5):51–55 Nagata K, Handa H (2000) Real-time analysis of biomolecular interactions – applications of biacore, 1st edn Springer, Tokyo Osaka T, Matsunaga T, Nakanishi T, Arakaki A, Niwa D, Iida H (2006a) Synthesis of magnetic nanoparticles and their application to bioassays Anal Bioanal Chem 384:593–600 Osaka T, Matsunaga T, Nakanishi T, Arakaki A, Niwa D (2006b) Synthesis of magnetic nanoparticles and their application to bioassays Anal Bioanal Chem 384:593–600 Paepens C, De Saeger S, Sibanda L, Barna-Vetro I, Van Hove F, Van Peteghem C (2004) A flow-through enzyme immunoassay for the screening of fumonisins in maize Analytica Chimica Acta 523:229–235 Rudolf K, Alexandra M (2007) Mycotoxin analysis: state if the art and future trends Anal Bioanal Chem 387:145–148 Safarik I, Safarikova M (2002) Magnetic nanoparticles and biosciences Monatshefte fűr Chemie 133:737–759 414 L Yotova et al Santacroce MP, Conversano MC, Casalano E, Lai O, Zizzadoro C, Centoducati G, Crescenzo G (2007) Aflatoxins in aquatic species: metabolism, toxicity Rev Fish Biol Fisheries 18:99–130 Sheng-Fu Wang, Tan Yu-Mei (2007) A novel amperometric immunosensor based on Fe3O4 magnetic nanoparticles/chitosan composite film for determination of ferritin Anal Bioanal Chem 387:703–708 Taitt CR, Anderson GP, Ligler FS (2005) Evanescent wave fluorescence biosensors Biosens Bioelectron 20:2470–2487 Tcvetkova T, Danev S (2000) Analytical principles and procedures in the clinical laboratory Biomedical and Life Sciences 9:15–20 Valkov P (ed) (2003) Chemical food pollutants Academic Publishing House, Plovdiv Vlahov S (ed) (2006) Microbiology Academic Publishing House, Plovdiv Vlahov S, Ivanon A (eds) (1996) General microbiology Academic Publishing House, Plovdiv Vo-Dinh T, Cullum BM, Stokes DL (2001) Nanosensors and biochips: frontier in biomolecular diagnostic Sens Actuators B Chem 74:2–11 Waldo GS (2003) Genetic screens and directed evolution for protein stability Curr Opinion Chem Biol 7:33–38 Wilchek M, Bayer EA (1989) Avidin-biotin technology ten years on: has it lived up to its expectations? Trends Biochem Sci 14:408–412 Yotova L, Marinkova D (2008) Application of immobilized lipoxygenase in the biosensors design for aflatoxin B1 BioPS’08 November 4–5, III.37–III.46 Yotova L, Ivanov T, Mironova V, Chuchuranova V (2008) Investigation of the properties of immobilized horseradish peroxidase on magnetic particles BioPS’08, November 4–5, III.29–III.36 Yu D, Blankert B, Bodoki E, Bollo S, Vire J, Sandulesku R, Nomura A, Kauffman J (2006) Amperometric biosensor based on horseradish peroxidase-immobilized magnetic particles Sens Actuators B Chem 113:749–754 Zaborsky O, Ogletree R (1974) Immobilization of enzymes on porous silica supports J Biochem Biophys Res Cooun 61:210–216 Zheng MZ, Richard JL (2006) A review of rapid methods for the analysis of mycotoxins Mycopathologia 161:261–273 Zheng Z, Humprey CW, King RS, Richard JL (2005) Validation of an ELISA test kit for the detection of total aflatoxins in grain and grain products Mycopathologia 159:255–263 ... Chemical Weapon Convention V Costigliola (*) and F Quagliata European Medical Association, Brussels, Belgium M.V Magni (ed.), Detection of Bacteria, Viruses, Parasites and Fungi, NATO Science for Peace. .. Vulnerability Assessment and Mapping of Akounq Groundwater Body, Armenia Vahram Vardanyan and Artashes Aginian 25 43 Part IIâ•… Bacteria Diagnostic Tests Methods for Detection of Shiga-Toxin.. .Detection of Bacteria, Viruses, Parasites and Fungi NATO Science for Peace and Security Series This Series presents the results of scientific meetings supported under the NATO Programme: Science