Microchimica Acta (v.170, #3-4)

The Fourth International Workshop on Biosensors for Food Safety and Environmental Monitoring by Aziz Amine; Chris M. A. Brett; Giuseppe Palleschi (191-192).

Biosensors based on cholinesterase inhibition for insecticides, nerve agents and aflatoxin B1 detection (review) by Fabiana Arduini; Aziz Amine; Danila Moscone; Giuseppe Palleschi (193-214).
The present review reports the research carried out during last 9 years on biosensors based on cholinesterase inhibition for nerve agents, organophosphorus and carbammic insecticides, and aflatoxin B1 detection. Relative applications in environmental and food areas are also reported. Special attention is paid to the optimization of parameters such as enzyme immobilization, substrate concentration, and incubation time in the case of reversible inhibition by aflatoxin B1 or irreversible inhibition by organophosphorus and carbamic insecticides, and nerve agents in order to optimize and improve the analytical performances of the biosensor. Evaluation of selectivity of the system is also discussed.
Keywords: Biosensors; Inhibition; Insecticides; Cholinesterase; Nerve agents; Aflatoxin

Biosensors for effective environmental and agrifood protection and commercialization: from research to market by Viviana Scognamiglio; Gianni Pezzotti; Ittalo Pezzotti; Juan Cano; Katia Buonasera; Daniela Giannini; Maria Teresa Giardi (215-225).
Biosensors are projected to find many applications due to their high selectivity and sensitivity, rapid reaction, economy and ease of handling in field measurements. Even though biosensors for a wide range of environmental pollutants have been extensively reported in the literature, the decision to develop a suitable biosensing system that can be approved by a regulatory perspective for environmental applications is fraught with technical issues. These issues mainly concern the biological recognition element, the physico-chemical transducer and the interfaces between the biological and the physical components, but also aspects of fluidics, electronics, and software for data processing. This article reviews methods together with a process to move biosensor technology from research laboratories to market, focusing as a case in point on challenges and possible opportunities in the development of photosynthetic-based biosensors for environmental applications.
Keywords: Biosensor platform; Electro-optical transduction systems; Analytical methods; Photosynthetic organisms; Pollutants; Environmental monitoring; In field analyses; Biosensor market

We report on the integration of micro and nanostructured materials in biosensing devices to obtain analytical nano-bio-systems with improved performance. The simplest case of electrochemical biosensing presented here is based on graphite microparticles to obtain a graphite-epoxy composite. The integration of the bioreceptor within the composite provides an analytical biosystem with improved performances for biosensing. The further addition of gold nanoparticles results in an analytical nano-bio-system which allows an improved strategy for the immobilization of the bioreceptor. And finally, the integration of magnetic beads to graphite-epoxy composite magneto electrodes provides further advantages in terms of separation of the analyte from complex matrix and enhanced biological reactions, resulting in analytical magneto-nano-bio-systems. The application of these systems mainly in food safety and environmental monitoring are reported. The benefits of the integration of these materials to obtain improved analytical systems are discussed and represent an example of converging technologies.
Keywords: Converging technologies; Immunosensor; DNA biosensors; Pathogenic bacteria; Pesticides; Antibiotic residues; Allergens; Magnetic beads; Gold nanoparticles; Graphite microparticles

A disposable biosensor for the determination of alpha-amylase in human saliva by Mika Mahosenaho; Felice Caprio; Laura Micheli; Adama M. Sesay; Giuseppe Palleschi; Vesa Virtanen (243-249).
A disposable tri-enzymatic biosensor is presented for the determination of α-amylase in human saliva. It is based on the quantity of maltose generated by hydrolysis of maltopentose in the presence of salivary α-amylase. The biosensor is fabricated by co-immobilization of the enzymes α-glucosidase, glucose oxidase, and mutarotase on screen-printed electrodes modified with Prussian Blue. The assay can be performed with a “drop” of sample, this allowing for ease and simplicity. A linear relationship is found for the range from 5 to 250 units per mL, with an LOD of 5 units per mL. The biosensor is stable for at least one month and over this time retains 80% of its original activity. The system was then evaluated for matrix effects of human saliva and compared to a spectrometric method using a commercially available kit.
Keywords: Biosensor; Alpha amylase; Biomarker; Saliva; Stress

Biosensor for determination of carboxylic acids in wines based on the inhibition of sarcosine oxidase by Jiri Zeravik; Karel Lacina; Milan Jilek; Jiri Vlcek; Petr Skládal (251-256).
The flow-through amperometric biosensor is presented for determination of carboxylic acids. It is based on two sensor layers that are deposited on a platinum electrode. The inner layer serves to eliminate interferences by limiting diffusion of electrochemically active substances such as ascorbic acid. This layer is electro-polymerized using an equimolar mixture of o-phenylenediamine and resorcinol. The outer layer is prepared by cross-linking the enzyme sarcosine oxidase and bovine serum albumin using glutaraldehyde. The formation of enzymatically produced hydrogen peroxide is monitored at 600 mV vs. an Ag/AgCl reference electrode. The addition of carboxylic acids causes competitive inhibition of the enzyme and a decrease in signal. The assay was optimized for determination of carboxylic acids in wine samples. Following 10-fold dilution, most samples contain 1–10 mM individual carboxylic acids and thus a 5 mM concentration of sarcosine was chosen as being optimal for competition. In case of real samples, the biosensor measures the sum of all carboxylic acids, which serves as a parameter describing the quality of wines. Results from testing several wine samples are reported.
Keywords: Biosensor; Sarcosine oxidase; Carboxylic acid; Wine

Biosensors with novel modified electrode structures for glucose determination have been developed by using different combinations of multiwalled carbon nanotubes (CNTs) and polyazine redox polymer, poly(neutral red) or poly(brilliant cresyl blue) on glassy carbon electrodes (GCE). CNT films were formed using functionalised CNTs, covalently immobilised by crosslinking in a chitosan matrix, and the azine dyes were electropolymerised directly on GCE or on top of the GCE modified with CNTs. Glucose oxidase (GOx) was immobilised by crosslinking with glutaraldehyde on top of the GCE modified with CNT, poly(azines), or combinations of poly(azines) and CNT. An assembly with enzyme/nanotube mixture immobilised on top of polyazine films was also studied. The mechanism of functioning was investigated in the presence and absence of oxygen and also by using bienzymatic devices containing glucose oxidase and catalase. Of the combinations studied, the best performance was obtained with a PBCB/CNT/GOx biosensor at a potential of −0.3 V vs. SCE with a detection limit of 11 μmol L−1.
Keywords: Functionalised carbon nanotubes; Chitosan matrix; Azine dyes; Redox polymer; Glucose oxidase; Catalase

A BDD electrode modified with polyaniline (PANI) film doped with polyvinyl sulphonate (PVS), containing in-situ deposited tyrosinase (Tyr) enzyme was prepared and characterized. The PANI-PVS film was electrochemically polymerized onto the BDD surface by cyclic voltammetry (CV) at 50 mV.s−1 (versus Ag/AgCl). An increase in current density with increasing number of cycles was an indication of the polymer growth. The morphologies of the composite films were characterized by scanning electron microscopy (SEM). Electrochemical characterization confirmed the successful doping of the PANI film by PVS and that the template PVS directed the electron transfer at the biosensor interface. SEM provided evidence of the influence of the synthesis medium on the morphology and surface area of the composite film. Synthesis from HCl produced homogenous, finely granular thin film with increased surface area which is thought to be responsible for the increased redox currents measured. PANI/PVS served both as an efficient mediator and biocompatible enzyme immobilisation platform for the substrate L-tyrosine at low concentrations with apparent Michaelis-Menton ( $$ {hbox{K}}_{ m{m}}^{ m{app}} $$ ) 1 × 10−2 µM.
Keywords: Biosensor; Boron-doped diamond; Polyaniline composite film; Tyrosinase

DNA hybridization detection by electrochemical impedance spectroscopy using interdigitated gold nanoelectrodes by Alessandra Bonanni; Irene Fernández-Cuesta; Xavier Borrisé; Francesc Pérez-Murano; Salvador Alegret; Manel del Valle (275-281).
A DNA biosensor is presented that is based on gold interdigitated nanoelectrodes of 100 nm width and 250 nm pitch. A single-strand oligonucleotide acts as the capture probe. Because of its nanometer dimensions, the device shows improved sensitivity when compared to similar systems. This encouraged us to perform a direct and unlabelled detection. After incubation with the DNA target, the impedance spectrum was recorded between 1 kHz and 10 MHz to obtain the net capacitance change. The use of a biotinylated DNA signalling probe permitted the integration of an amplification stage in a sandwich format that employs streptavidin-modified gold nanoparticles. The strategy was preliminarily tested by detecting the breast cancer related BRCA1 gene, where the noncomplementary, wild and mutated forms were easily differentiated at a concentration level of 3 μM (corresponding to a 30 pmol quantity).
Keywords: Nanoelectrodes; Interdigitated electrodes; Impedimetric genosensor; Gold nanoparticle amplification; BRCA1 gene

Real-time detection and quantification of food-borne bacteria draws increasing interest for evaluation of food quality and safety. Since living cells invariably contain adenosine triphosphate (ATP), the detection of bacterial ATP presents a fascinating method to determine its presence in food. Care must be taken however, to remove food-derived extracellular ATP, which will interfere with detection by ATP-luminescence. We developed a microfluidic and dielectrophoretic (DEP) device for intracellular ATP detection, which captures microorganisms by DEP force and washes extracellular ATP away. The yield of capture by DEP force at a 10 μL∙min−1 flow rate used in experiments was 87.7%. At constant ATP level the electrical sensor responded in proportion to the bacterial concentration. With a constant bacterial concentration and varying ATP, the signal did not change. These results show that the device can remove the extracellular ATP contribution from food to be sampled.
Keywords: Real-time detection; ATP(adenosine triphosphate); Luminescence; Dielectrophoresis; Microfluidics

Myeloperoxidase-mediated oxidation of organophosphorus pesticides as a pre-step in their determination by AChE based bioanalytical methods by Tamara Lazarević Pašti; Tatjana Momić; Antonije Onjia; Ljubodrag Vujisić; Vesna Vasić (289-297).
In order to improve the sensitivity of assays for inhibitors of the enzyme acetylcholine esterase (AChE), an effective method was developed for the conversion of the organophosphate pesticides (OPs) diazinon, malathion, chlorpyrifos, azinphos-methyl and phorate into more toxic inhibitors. This was accomplished by converting them from the thio form into their oxo form using the enzyme myeloperoxidase. The oxo forms, which are the only products of conversion, were determined by AChE bioassays, using either the free enzyme, or a flow injection analysis manifold with immobilized AChE and spectrophotometric detection. All modified OPs exhibited inhibitory power at ppb levels and within 10 min. The method is considered to represent an excellent means for improving the sensitivity of assays for determination of OPs.
Keywords: Organophosphate pesticides; Myeloperoxidase; Oxidation; Bioanalytics; Acethylcholinesterase

A novel approach to the electrochemical determination of heavy metals in tap water using anodic stripping voltammetry was developed using screen-printed electrodes modified with gold films. After optimisation of the experimental conditions, the screen-printed electrodes modified with gold films displayed excellent linear behaviour in the examined concentration range from 2 to 16 µg L-1 mercury and lead in 50 mM HCl with a detection limit of 1.5 µg L-1 and 0.5 µg L-1 for mercury and lead, respectively. In order to decrease the working range down to less than 1 µg L-1, a preconcentration step based on the use of magnetic particles modified with thiols was introduced into the protocol. Applying optimum binding conditions, the assay using screen-printed electrodes modified with gold films displayed excellent linear behaviour in the concentration range 0.1 to 0.8 µg L-1 in 50 mM HCl. The detection limit after a 120 s deposition time for mercury and lead were 0.08 µg L-1 and 0.02 µg L-1, respectively. The method has been applied to the determination of mercury and lead traces in tap water
Keywords: Screen-printed electrodes; Square-wave anodic voltammetry; Gold film; Heavy metals; Magnetic particles; Thiols

Shuttle-like copper oxide (CuO) was prepared by a hydrothermal decomposition process. The resulting material was characterized by scanning electron microscopy and X-ray diffraction. It was then immobilized on the surface of a glassy carbon electrode modified with a film of poly(thionine). A pair of well-defined and reversible redox peaks for Hg(II) was observed with the resulting electrode in pH 7.0 solutions. The anodic and cathodic peak potentials occurred at 0.260 V and 0.220 V (vs. Ag/AgCl), respectively. The modified electrode displayed excellent amperometric response to Hg(II), with a linear range from 40 nM to 5.0 mM and a detection limit of 8.5 nM at a signal-to-noise ratio of 3. The sensor exhibited high selectivity and reproducibility and was successfully applied to the determination of Hg(II) in water samples.
Keywords: CuO nanoshuttle; Hg2+ ; Poly(thionine); Sensor

Newly synthesized poly(aryleneethynylene)s carrying substituents such as leucine, glycine and methionine have been prepared and tested for their ability to bind Hg(II) ions. Binding resulted in strong quenching of the fluorescence emission of all the compounds tested. A comparative study of the quenching efficiency by Hg(II) was then carried out in a flow injection system. Quenching efficiency was amplified in the polymeric material because of the so-called “wire effect” that is observed with organic conducting polymers. Under the experimental conditions used, detection limits are in the 50–100 ppb range. The selectivity for Hg(II) is remarkable in that a series of other divalent cations did not give any measurable effect, a slight response being observed for methylmercury only. The materials hold promise for selective determination of Hg(II) ion.
Keywords: Conducting polymers; Hg(II); Poly(aryleneethynylene)s; Optical detection; Fluorescence; Sensors; Aminoacidic based sensors

Application of screen-printed microband biosensors incorporated with cells to monitor metabolic effects of potential environmental toxins by Roy M. Pemberton; Frankie J. Rawson; Jinsheng Xu; Robin Pittson; Guido A. Drago; John Griffiths; Simon K. Jackson; John P. Hart (321-330).
Microband biosensors were fabricated from a screen-printed water-based carbon ink containing cobalt phthalocyanine redox mediator and glucose oxidase or lactate oxidase enzyme. The microbiosensors were characterised for their ability to monitor ferrocyanide and H2O2 in phosphate buffer solution: sigmoidal cyclic voltammograms, high current density values and steady-state amperometric responses confirmed the existence of radial-diffusion-limiting microelectrode behaviour. The lactate microband biosensors were then used, in conjunction with a screen-printed Ag/AgCl reference and platinum counter electrode, to monitor lactate levels in culture medium, with a linear range of 0.5–5 mM, sensitivity of 20 nA.mM−1, and dynamic range up to >9 mM. The lactate microband biosensors could operate continuously in culture medium over extended times (up to 24 h) at 37 °C. These biosensors were then applied to detect changes in lactate release from cultured cells in response to toxic challenge: m-dinitrobenzene (500 μM) caused a reduction in lactate production by high-passage number HepG2 single cells; D-galactosamine (20 mM) induced release of lactate by HepG2 spheroid cultures. This novel use of microband biosensors in cell culture has the potential for further application in toxicity monitoring, in both environmental and pharmaceutical areas.
Keywords: Microband electrode biosensor; HepG2 cell culture; Continuous monitoring; Toxicity

Studies towards an amperometric phosphate ion biosensor for urine and water analysis by Lucy Gilbert; Simon Browning; Andrew T. A. Jenkins; John P. Hart (331-336).
An amperometric biosensor for phosphate ion is described that is based on a cobalt phthalocyanine modified screen-printed carbon electrode (CoPC-SPCE). The biosensor operation is based on the enzyme pyruvate oxidase (PyOd) which catalyses the oxidative decarboxylation of pyruvate, in the presence of inorganic phosphate and O2, to acetyl phosphate, hydrogen peroxide (H2O2) and CO2. The transducer allows the electrocatalytic oxidation of H2O2 in order to generate the analytical signal. The enzyme was immobilised onto the CoPC-SPCE using a sandwich format. The inner membrane was formed in situ by depositing an acetone solution containing cellulose acetate first onto the transducer surface. The enzyme and cofactors were then deposited onto this layer and allowed to dry; finally a second aliquot of the cellulose acetate solution was deposited onto the enzyme layer and allowed to dry. The biosensor was characterised by amperometry in stirred solution to produce current-voltage curves and for calibration studies. From these it was deduced that a reliable electrocatalytic response was obtained for phosphate ion; an operating potential of +0.4 V was selected for the analysis of urine samples. The precision of the response for urine analysis and recovery data for potable water suggests that the biosensor could have applications in clinical and environmental monitoring.
Keywords: Biosensor; Phosphate; Amperometric; Urine; Tap water

Formaldehyde-sensitive conductometric sensors based on commercial and recombinant formaldehyde dehydrogenase by Yaroslav I. Korpan; Olexandr O. Soldatkin; Olga F. Sosovska; Halyna M. Klepach; Elisabeth Csöregi; Francis Vocanson; Nicole Jaffrezic-Renault; Mykhailo V. Gonchar (337-344).
Novel formaldehyde-sensitive conductometric biosensors have been developed that are based on commercial bacterial formaldehyde dehydrogenase (FDH) from Pseudomonas putida and recombinant formaldehyde dehydrogenase (rFDH) from the yeast Hansenula polymorpha as the bio-recognition elements. The bio-recognition membranes have mono-layer architecture and consist of enzyme cross-linked with albumin and of the cofactors NAD (for FDH-based sensor) or NAD and glutathione (for rFDH-based sensor). This architecture of the biosensor allows the determination of formaldehyde without adding NAD and glutathione to the analyzed sample at every analysis and conducting measurements on the same transducer without cofactors regeneration since the bio-membrane contains it at high concentration (100 mM for NAD and 20 mM for glutathione). The response is linear in the range from 10 to 200 mM of formaldehyde concentration depending on the enzyme used. The dependence of the biosensor output signals on pH and buffer concentration as well as operational/storage stability and selectivity/specificity of the developed conductometric biosensors have been investigated. The relative standard deviation of the intra-sensor response did not exceed 4% and 10% for rFDH- and FDH-based sensors, respectively. The relative standard deviation of the inter-sensor response constituted 20% for both dehydrogenases used. The biosensors have been validated for formaldehyde detection in some real samples of pharmaceutical (Formidron), disinfectant (Descoton forte) and an industrial product (Formalin). A good correlation does exist between the concentration values measured by the conductometric biosensor developed in this work, an enzymatic method, amperometric biosensors developed earlier, and standard analytical methods of formaldehyde determination.
Keywords: Thin-film interdigitated gold planar electrodes; Conductometric biosensor; Formaldehyde dehydrogenase; Formaldehyde detection