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The characterization of an economic and ease-to-use carbon paste acetylcholinesterase (AChE) based biosensor to determine the concentration of pesticides Paraoxon and Dichlorvos is discussed. AChE hydrolyses acetylthiocholine (ATCh) in thiocoline (TC) and acetic acid (AA). When AChE is immobilized into a paste carbon working electrode kept at +410 mV vs. Ag/AgCl electrode, the enzyme reaction rate using acetylthiocholine chloride (ATCl) as substrate is monitored as a current intensity. Because Paraoxon and Dichlorvos inhibit the AChE reaction, the decrease of the current intensity, at fixed ATCl concentration, is a measure of their concentration. Linear calibration curves for Paraoxon and Dichlorvos determination have been obtained. The detection limits resulted to be 0.86 ppb and 4.2 ppb for Paraoxon and Dichlorvos, respectively, while the extension of the linear range was up 23 ppb for the former pesticide and up to 33 ppb for the latter. Because the inhibited enzyme can be reactivated when immediately treated with an oxime, the biosensor reactivation has been studied when 1,1'-trimethylene bis 4-formylpyridinium bromide dioxime (TMB-4) and pyridine 2-aldoxime methiodide (2-PAM) were used. TMB-4 resulted more effective. The comparison with the behavior of similar AChE based biosensors is also presented.

The potential of Raman spectroscopy with excitation in the visible as a tool for quantitative determina- tion of single components in food industry products was investigated by focusing the attention on glu- cose content in commercial sport drinks. At this aim, micro-Raman spectra in the 600-1600 cm À1 wavenumber shift region of four sport drinks were recorded, showing well defined and separated vibra- tional fingerprints of the various contained sugars (glucose, fructose and sucrose). By profiting of the spectral separation of some peculiar peaks, glucose content was quantified by using a multivariate statis- tical analysis based on the interval Partial Least Square (iPLS) approach. The iPLS model needed for data analysis procedure was built by using glucose aqueous solutions at known sugar concentrations as cali- bration data. This model was then applied to sport drink spectra and gave predicted glucose concentra- tions in good agreement with the values obtained by using a biochemical assay. These results represent a significant step towards the development of a fast and simple method for the on-line glucose quantifica- tion in products of food and beverage industry.

A hollow-fiber enzyme reactor, operating under isothermal and nonisothermal conditions, was built employing a polypropylene hollow fiber onto which beta-galactosidase was immobilized. Hexamethylenediamine and glutaraldehyde were used as spacer and coupling agent, respectively. Glucose production was studied as a function of temperature, substrate concentration, and size of the transmembrane temperature gradient. The actual average temperature differences across the polypropylene fiber, to which reference was done to evaluate the effect of the nonisothermal conditions, were calculated by means of a mathematical approach, which made it possible to know, using computer simulation, the radial and axial temperature profiles inside the bioreactor and across the membrane. Percent activity increases, proportional to the size of the temperature gradients, were found when the enzyme activities under nonisothermal conditions were compared to those measured under comparable isothermal conditions. Percent reductions of the production times, proportional to the applied temperature gradients, were also calculated. The advantage of employing nonisothermal bioreactors in biotechnological industrial process was discussed.

The effects of low intensity, low frequency magnetic fields (MFs) on catalytic activity of the calcium dependent protease calpain was determined following the enzyme activation both in “in vitro” and “in vivo” conditions. We have observed that a 0.3 mT MF induces a significant increase in the requirement of the protease for this metal ion. This change is detectable at low [Ca2+] and disappears when the level of Ca2+ is raised to saturating amounts. The observed effects are not due to transient MF− induced conformational changes occurring in calpain, but to direct effects of the MF on Ca2+ ions, which become less available for the binding sites present in calpain. Altogether, these results indicate that exposure to low intensity, low frequency MFs alters the intracellular Ca2+ “availability,” thereby modifying the related cell response. Bioelectromagnetics 27:43–50, 2006. © 2005 Wiley-Liss, Inc.

The dynamic and steady-state responses as well as the response times of a glucose biosensor have been studied under isothermal and non-isothermal conditions as a function of analyte concentration. The presence of a temperature gradient across the catalytic membrane system improved the biosensor characteristics, because the dynamic and steady-state responses increased and the response times decreased under non-isothermal conditions. For example, a macroscopic temperature difference of 20 degrees C applied across the catalytic membrane system increases the biosensor sensitivity of 70% and reduces of 50% its response time. The dependence of the observed effects on the magnitude of the temperature difference applied has been correlated with the substrate (and products) transport across the catalytic membrane system due to the process of thermodialysis.

The effect of tamoxifen (TAM) on the black goby Gobius niger exposed to different contaminants was analyzed by investigating the response of a set of biomarkers involved in reproduction and cell recovery. While the effects of TAM are well known in mammalian breast and endometrial cells (with severe consequences on physiology), few contrasting data are available for fish. In this work the expression of vitellogenin (vtg), estrogen receptor alpha (ERalpha), cytochrome P4501A1 (CYP1A1), and heat shock protein 70 (HSP70) genes was evaluated in fish treated with TAM alone or co-injected with nonylphenol, estradiol, and beta-naphtoflavone. The induction of vtg observed in male fish treated with estrogens was significantly lowered by the co-injection of TAM. ERalpha and HSP70 gene expressions were significantly upregulated in all experimental groups; however, TAM treatment did not change CYP1A1 gene expression. Together these data confirm the mixed estrogenic/anti-estrogenic action of TAM, indicating its interference in fish reproduction and physiology.

A fluidized bed reactor, filled with laccase-based beads, has been employed to bioremediate aqueous solutions polluted by endocrine disruptors belonging to the alkylphenols (APs) class. In particular Octylphenol and Nonylphenol have been studied. The catalytic activity of free and immobilized laccase from Trametes versicolor has been characterized as a function of pH, temperature and substrate concentration in the reaction medium. In view of practical applications for each substrate concentration the removal efficiency (RE), the time to halve the initial concentration (tau(50)), and the t(c-0), i.e. the time to reach complete pollutant removal, have been calculated. The immobilized laccase exhibited a lower affinity for octylphenol (K-m = 1.11 mM) than for Nonylphenol (K-m = 0.72 mM), but all the other parameters of applicative interest resulted more significant for octylphenol. For example, the times to reach the complete removal of octylphenol compared to those for nonylphenol at the same concentration is shorter of about 15% (at low concentrations) up to 40% (at high concentrations). The study of cell proliferation with MPP89 cells, a human mesothelioma cell line, and the assay with the YES test indicated the loss of estrogenic activity of the APs solutions after laccase treatment. (C) 2013 Elsevier B.V. All rights reserved.

The biodegradation of waters polluted by some bisphenols, endowed with endocrine activity, has been studied by means of laccase or tyrosinase immobilized on polyacrylonitrile (PAN) beads. Bisphenol A (BPA), Bisphenol B (BPB), Bisphenol F (BPF) and Tetrachlorobisphenol A (TCBPA) have been used. The laccase-PAN beads system has been characterized as a function of pH, temperature and substrate concentration. The biochemical parameters so obtained have been compared with those of the free enzyme to evidence the modification induced by the immobilization process. Once characterized, the laccase-PAN beads have been employed in a fluidized bed reactor to determine for each of the four bisphenols the degradation rate constant (k); the τ50, i.e., the time to obtain the 50% of degradation, and the removal efficiency (RE90) after 90 min of enzyme treatment. The same parameters have been measured for each of the four pollutants with the same fluidized bed bioreactor loaded with tyrosinase-PAN beads. The internal comparison, i.e., in each of the two catalytic systems, has shown that both enzymes exhibit a removal efficiency in the following order BPF>BPA>BPB>TCBPA. The external comparison, i.e., the comparison between the two catalytic system, has shown that the catalytic power of laccase were higher than that of tyrosinase. The operational stability of both catalytic systems resulted excellent, since they maintained more than 80% of the initial activity after 30 days of work. © 2010 Springer Science+Business Media B.V.

Octylphenol (OP) is an endocrine-disrupting chemical that accumulates in various organs. It has also been shown to exert noxious effects on the central nervous system. In the present study, we measured in Sprague–Dawley rats the degree of OP accumulation in different areas of the brain and investigated the effect of OP in pain modulation. Two groups of male Sprague–Dawley rats were treated for 20 days with 50 mg/kg BW/day of OP (group 1) or vehicle (group 2). At the end of the treatment, the formalin test was performed to evaluate the effect of OP exposure on pain. Soon after, rats were sacrificed, and the accumulation of OP in the cerebral cortex, hippocampus, hypothalamus, cerebellum, thalamus, striatum, mesencephalus and ventral hindbrain was measured by HPLC analysis. The results showed a greater accumulation of OP in the cerebral cortex compared to all the other areas; there was also more accumulation in the cerebellum compared to the mesencephalus and thalamus. No accumulation was found in the striatum. These results suggest that there is a preferential accumulation of OP in different areas of the brain with consequences to neural behaviour. On the contrary, experiments on facial grooming did not show significant effects of OP on pain.

Recently, aqueous solutions polluted by BPA have been bioremediated by us using laccase immobilized on hydrophobic membranes in non-isothermal bioreactors. BPA degradation was checked using analytical methods. To assess in vitro the occurred bioremediation, the proliferation and viability indexes of MCF-7 cells incubated in the presence of aqueous solutions of BPA, or of enzyme-treated BPA solutions, have been measured as a function of the initial BPA concentration.The results demonstrated that: i) at each initial BPA concentration used, both the proliferation and viability indexes are a function of the duration of enzyme treatment; ii) proliferation and viability are uncoupled biological processes with respect to BPA enzyme treatment.Non-isothermal bioreactors are a useful tool for the bioremediation of aqueous solutions polluted by BPA, which is an example of an endocrine disruptor that belongs to the alkyl phenol family. Keywords: MCF-7, BPA, Bioremediation, Proliferation index, Viability index

Different tyrosinase carbon paste modified electrodes to determine bisphenol A (BPA) concentration in aqueous solutions have been constructed. Variables examined were in the carbon paste composition and in particular: (i) the immobilized enzyme amount; (ii) the carbon type (powder, single or multi-walled nanotubes); (iii) the nature of the pasting oil (mineral oil, hexadecane and dodecane). For each biosensor type the amperometric response was evaluated with reference to the linear range and sensitivity. Constant reference has been made to the amperometric signals obtained, under the same experimental conditions, towards the catechol, a specific phenolic substrate for tyrosinase. The most efficient biosensors were those constructed by using the following composition for the carbon paste: 10% of tyrosinase, 45% of single wall carbon nanotubes (SWCN) and 45% of mineral oil. This biosensor formulation displayed the following electrochemical characteristics: a sensitivity equal to 138 μA/mM, LOD of 0.02 μM (based on three times the S/N ratio), linear range of 0.1–12 μM and response time of 6 min. This experimental work represents a first attempt at construction of a new carbon nanotube-tyrosinase based biosensor able to determine the concentration of BPA, one of the most ubiquitous and hazardous endocrine disruptors which can pollute the drinking and surface water, as well as many products of the food chain.

By using soluble and insoluble glucose oxidase, the changes in intrinsic emission fluorescence in the visible spectral region were studied as a function of glucose concentration. Insoluble glucose oxidase (GOD) was obtained by entrapment in a gelatine membrane or by covalent attachment on an agarose membrane grafted with hexamethylendiamine. The intensity of the fluorescence emission peak at 520 nm or the value of the integral fluorescence area from 480 to 580 nm were taken as physical parameters representative of the glucose concentration during the enzyme reaction. By using these parameters, linear calibration curves for glucose concentration were obtained. The extension of the calibration curve and the sensitivity of the adopted systems were found to be dependent on the enzyme state (free or immobilized) and on the immobilization method. In particular, it was found that the extent of the linear range of the calibration curves is increased of one order of magnitude when the glucose oxidase is immobilized, while the sensitivity of the measure is decreased of one order of magnitude by the immobilization process. Measures carried out by using the integral fluorescence area resulted more sensitive than those obtained with the peak size. Useful indications for the construction of optical fibre-based sensors were drawn from the reported results. (C) 2004 Elsevier B.V. All rights reserved.

The influence of an extremely low frequency (ELF) magnetic field (50 Hz and 1 mT, EMF) on the activity of a soluble and insoluble horseradish peroxidase (E.C. 1.11.17) has been studied as a function of time. Insoluble derivatives were obtained by enzyme entrapment into two different gelatin membranes or by covalent attachment of the enzyme on two nylon membranes, differently preactivated. Results have shown that the field affects the inactivation rate of the soluble enzyme, while no effects are observed with insoluble derivatives. Since in vivo enzymes are immobilised into the biomembrane bilayer or entrapped into the cytoplasmic mixture, one might speculate that our experimental conditions do not reflect the catalytic activity of the enzymes in vivo.

This work studies protease concentration decrease in aqueous solutions in contact with a modified polyethersulphone graft membrane onto which antiproteases were immobilized. As a model of protease/antiprotease interaction, elastase and α1-antitrypsin were used. Experiments were carried out either under fixed amounts of immobilized antiproteases and variable protease concentration or under fixed protease concentration and variable amounts of immobilized antiproteases. In both cases, active protease concentrations decreased with increase in contact time with the membrane. Experimental conditions under which active elastase concentration becomes zero were also found. Occurrence of the same phenomenology has also been ascertained with protease solutions obtained from human blood neutrophils. The membrane activated with α1-antitrypsin showed differential inhibitory power on elastase and cathepsin G. This technology could open new perspectives in manufacturing new membranes to be used in hemodialysis and extracorporeal circulation when elastase is released.

The tryptophanyl emission decay of the mesophilic beta-galactosidase from Aspergillus oryzae free in buffer and entrapped in agarose gel is investigated as a function of temperature and compared to that of the hyperthermophilic enzyme from Sulfolobus solfataricus. Both enzymes are tetrameric proteins with a large number of tryptophanyl residues, so the fluorescence emission can provide information on the conformational dynamics of the overall protein structure rather than that of the local environment. The tryptophanyl emission decays are best fitted by bimodal Lorentzian distributions. The long-lived component is ascribed to close, deeply buried tryptophanyl residues with reduced mobility; the short-lived one arises from tryptophanyl residues located in more flexible external regions of each subunit, some of which are involved in forming the catalytic site. The center of both lifetime distribution components at each temperature increases when going from the free in solution mesophilic enzyme to the gel-entrapped and hyperthermophilic enzyme, thus indicating that confinement of the mesophilic enzyme in the agarose gel limits the freedom of the polypeptide chain. A more complex dependence is observed for the distribution widths. Computer modeling techniques are used to recognize that the catalytic sites are similar for the mesophilic and hyperthermophilic beta-galactosidases. The effect due to gel entrapment is considered in dynamic simulations by imposing harmonic restraints to solvent-exposed atoms of the protein with the exclusion of those around the active site. The temperature dependence of the tryptophanyl fluorescence emission decay and the dynamic simulation confirm that more rigid structures, as in the case of the immobilized and/or hyperthermophilic enzyme, require higher temperatures to achieve the requisite conformational dynamics for an effective catalytic action and strongly suggest a link between conformational rigidity and enhanced thermal stability.

The effect of methanol on the kinetically controlled synthesis of cephalexin by free and immobilized penicillin G acylase (PGA) was investigated. Catalytic and hydrophobic membranes were obtained by chemical grafting, activation, and PGA immobilization on hydrophobic nylon supports. Butyl methacrylate (BMA) was used as graft monomer. Increasing concentrations of methanol were found to cause a greater deleterious effect on the activity of free than on that of the immobilized enzyme. Methanol, however, improved the kinetic stability of cephalexin synthesized by free PGA, resulting in higher maximum yields. By contrast, immobilized PGA reached 100% yields even in the absence of the cosolvent. Cephalexin synthesis by the catalytic membrane was also performed in a non-isothermal bioreactor. Under these conditions, a 94% increase of the synthetic activity and complete conversion of the limiting substrate to cephalexin were obtained. The addition of methanol reduced the non-isothermal activity increase. The physical cause responsible for the non-isothermal behavior of the hydrophobic catalytic membrane was identified in the process of thermodialysis.

The behavior of three different catalytic membranes, obtained by immobilizing urease on nylon sheets chemically grafted with methyl methacrylate, was studied in a bioreactor operating under isothermal and non-isothermal conditions. Membrane activation was carried out by condensation or acyl azide reaction, and spacers of different lengths, such as hexamethylendiamine or hydrazine, were used. Under isothermal conditions, the activities of the catalytic membranes and soluble urease were characterized as a function of pH, temperature, and urea concentration. Both enzyme forms showed the same optimum pH, whereas the optimum temperature was lower for the immobilized enzymes. The spacer length appeared to determine broader pH- and temperature-activity profiles for the urease derivatives. The apparent K(m) values of the insoluble urease were dependent on membrane type and were higher than those of the soluble counterpart, thus indicating an affinity loss for urea. Under non-isothermal conditions, all membranes exhibited an increase of percentage activity proportional to the applied temperature difference and decreasing with the increase of urea concentrations. A decrease of the apparent K(m) was also observed. These results suggest that substrate diffusion limitations due to the immobilization process can be overcome in the presence of temperature gradients. In addition, the remarkable reduction of the production times supports the use of non-isothermal bioreactors for the treatment of urea-polluted waste waters.

The behaviour of five different hydrophobic β-galactosidase derivatives, obtained by grafting different amount of butylmethacrylate (BMA) on planar nylon membranes, has been studied under isothermal and non-isothermal conditions. Under isothermal conditions the effect of the grafting percentage on the enzyme activity has been studied as a function of pH, temperature and substrate concentration. Independently from the parameters under observation, the yield of the catalytic process reaches the maximum value at a grafting percentage value equal to 21%. The apparent K m values result linearly increasing with the increase of the grafting percentage, while the apparent V max exhibits a maximum value. Under non-isothermal conditions, a decrease of the apparent K m values and increase of the apparent V max has been found in respect to the same values obtained under isothermal conditions. The percentage activity increases induced by the presence of a temperature gradient have been found to decrease with the increase of the percentage of graft BMA. A parameter correlating the percentage increase of enzyme activity under non-isothermal conditions with the hydrophobicity of the catalytic membrane has also been identified. This parameter is the ratio between thermoosmotic and hydraulic permeability. Results have been discussed in terms of reduction of diffusion limitations for substrate and products movement towards or away from the catalytic site by the process of thermodialysis. The usefulness of using non-isothermal bioreactors in industrial biotechnological processes has been confirmed.

The results obtained with a glucose biosensor operating under non-isothermal conditions are presented and discussed. Glucose oxidase, immobilized onto Nylon membranes, was used as biological element. An amperometric two electrodes system was employed to measure the anodic current produced by oxidation of hydrogen peroxide. Non-isothermal conditions were characterized in terms of the temperature difference, ΔT = Tw-Tc, and of the average temperature of the system, Tav = (Tw+Tc)/ 2, Tw and Tc being the temperature in the warm and cold half-cells constituting the biosensor. Comparison between the functioning of the biosensor under isothermal and non-isothermal conditions was performed. It was found that, under non-isothermal conditions, the dynamic response and sensitivity increased, while the response times and the detection limit decreased, if comparison was done with the same parameters measured under isothermal conditions. The increase of the dynamic response was found to be proportional to the applied temperature gradient. The results obtained with a glucose biosensor operating under non-isothermal conditions are presented and discussed. Glucose oxidase, immobilized onto Nylon membranes, was used as biological element. An amperometric two electrodes system was employed to measure the anodic current produced by oxidation of hydrogen peroxide. Non-isothermal conditions were characterized in terms of the temperature difference, ΔT = Tw-Tc, and of the average temperature of the system, Tav = (Tw+Tc)/ 2, Tw and Tc being the temperature in the warm and cold half-cells constituting the biosensor. Comparison between the functioning of the biosensor under isothermal and non-isothermal conditions was performed. It was found that, under non-isothermal conditions, the dynamic response and sensitivity increased, while the response times and the detection limit decreased, if comparison was done with the same parameters measured under isothermal conditions. The increase of the dynamic response was found to be proportional to the applied temperature gradient.