Bioelectrocatalytic application of titania nanotube array for molecule detection

A bioelectrocatalysis system based on titania nanotube electrode has been developed for the quantitative detection application. Highly ordered titania nanotube array with inner diameter of 60 nm and total length of 540 nm was formed by anodizing titanium foils. The functionalization modification was achieved by embedding glucose oxidases inside tubule channels and electropolymerizing pyrrole for interfacial immobilization. Morphology and microstructure characterization, electrochemical properties and bioelectrocatalytic reactivities of this composite were fully investigated. The direct detection of hydrogen peroxide by electrocatalytic reduction reaction was fulfilled on pure titania nanotube array with a detection limit up to 2.0 × 10⁻⁴ mM. A biosensor based on the glucose oxidase–titania/titanium electrode was constructed for amperometric detection and quantitative determination of glucose in a phosphate buffer solution (pH 6.8) under a potentiostatic condition (−0.4 V versus SCE). The resulting glucose biosensor showed an excellent performance with a response time below 5.6 s and a detection limit of 2.0 × 10⁻³ mM. The corresponding detection sensitivity was 45.5 μA mM⁻¹ cm⁻². A good operational reliability was also achieved with relative standard deviations below 3.0%. This novel biosensor exhibited quite high response sensitivity and low detection limit for potential applications.     

Glucose biosensor based on nano-SiO2 and "unprotected" Pt nanoclusters

The “unprotected” Pt nanoclusters (average size 2 nm) mixed with the nanoscale SiO₂ particles (average size 13 nm) were used as a glucose oxidase immobilization carrier to fabricate the amperometric glucose biosensor. The bioactivity of glucose oxidase (GOx) immobilized on the composite was maintained and the as-prepared biosensor demonstrated high sensitivity (3.85 μA mM⁻¹) and good stability in glucose solution. The Pt–SiO₂ biosensor showed a detection limit of 1.5 μM with a linear range from 0.27 to 4.08 mM. In addition, the biosensor can be operated under wide pH range (pH 4.9–7.5) without great changes in its sensitivity. Cyclic voltammetry measurements showed a mixed controlled electrode reaction.     

Pigment content and molar extinction coefficients of photochemical reaction centers from Rhodopseudomonas spheroides

Reaction center particles isolated from carotenoidless mutant Rhodopseudomonas spheroides were studied with the aim of determining the pigment composition and the molar extinction coefficients.

Two independent sets of measurements using a variety of methods show that a sample with A_{800 nm} = 1.00 contains 20.8 ± 0.8 μM tetrapyrrole and that the ratio of bacteriochlorophyll to bacteriopheophytin is 2:1.

Measurements were made of the absorption changes attending the oxidation of cytochrome c coupled to reduction of the photooxidized primary electron donor in reaction centers, using laser flash excitation. The ratio of the absorption change at 865 nm (due to the bleaching of P870) to that at 550 nm (oxidation of cytochrome) was found to be 5.77.

These results, combined with other data, yield a pigment composition of 4 bacteriochlorophyll and 2 bacteriopheophytin molecules in a reaction center. Based on this choice, extinction coefficients are determined for the 802- and 865-nm bands: _{802 nm} = 288 (± 14) mM⁻¹ · cm⁻¹ and _{865 nm} = 128 (± 6) mM⁻¹ · cm⁻¹. For reversible bleaching of the 865-nm band, Δ^{red - ox}_865nm = 112 (± 6) mM⁻¹ · cm⁻¹ (referred to the molarity of reaction centers). Earlier reported values of photochemical quantum efficiency are recomputed, and the revised values are shown to be compatible with those obtained from measurements of fluorescence transients.     

Effects of ultraviolet-B radiation on common bean (Phaseolus vulgaris L.) plants grown under nitrogen deficiency

This work reports on the significance of UV-B absorbing compounds and DNA photorepair in protecting bean plants from UV-B radiation under nitrogen restriction. Bean plants grown in sterile vermiculite and irrigated periodically with a nutrient solution containing 12 or 1 mM of nitrate were irradiated with 22 μW cm⁻² of UV-B, 4 h daily during 10 days after the first trifoliate leaf was developed. This intensity was equivalent to 3.2 kJ m⁻² per day, approximately. PAR fluence rate was 350 ± 50 μmol quanta m⁻² s⁻¹. Control plants did not receive UV-B irradiation. Leaf expansion was negatively affected by both nitrate restriction and UV-B irradiation. This decrease was paralleled by a significant increase in starch, which was exacerbated by the combined action of both factors. Combined action of low nitrogen and UV-B also negatively affected the CO₂ assimilation rate and the stomatal conductance. Formation of UV-B absorbing compounds was significantly increased by both UV-B irradiation and nitrogen restriction and this increase was exacerbated by the combination of both factors. No significant increase in dimer formation was detected in irradiated plants at the UV-B dose used. Significant dimer formation was only obtained by using very high UV-B intensities. This suggests that under an irradiation level of 22 μW cm⁻² of UV-B, which is close to natural conditions, protective mechanisms such as pigment screening and DNA photorepair were probably sufficient to prevent any dimer formation in leaves.     

Layer-by-layer self-assembly aluminum Keggin ions/Prussian blue nanoparticles ultrathin films towards multifunctional sensing applications

In this study we described the nanocomposites films of specially synthesized inorganic Prussian blue (PB) nanoparticles and polyoxocation Al₁₃ Keggin ions that possess the excellent sensing activities. Film fabrication using layer-by-layer (LBL) self-assembly technique was followed by electrochemical characterization. The assembled multilayer Al₁₃/PB films as sensor devices for both the catalytic reduction of H₂O₂ and detecting the change of relative humidity were also investigated. The sensitivity of the biosensor was 0.886 mA cm⁻² mM⁻¹, and about two orders of magnitude change in resistance was observed as the relative humidity increasing from 5 to 95%. Both sensors exhibited good reproducibility, wide linear range. The performance and multifunctional abilities of these nanocomposites promise potential applications in biosensors, environmental controlling system and biomedical devices.     

Amperometric glucose biosensor based on self-assembly hydrophobin with high efficiency of enzyme utilization

Hydrophobins are a family of natural self-assembling proteins with high biocompability, which are apt to form strong and ordered assembly onto many kinds of surfaces. These physical-chemical and biological properties make hydrophobins suitable for surface modification and biomolecule immobilization purposes. A class II hydrophobin HFBI was used as enzyme immobilization matrix on platinum electrode to construct amperometric glucose biosensor. Permeability of HFBI self-assembling film was optimized by selecting the proper HFBI concentration for electrode modification, in order to allow H₂O₂ permeating while prevent interfering compounds accessing. HFBI self-assembly and glucose oxidase (GOx) immobilization was monitored by quartz crystal microbalance (QCM), and characterization of the modified electrode surface was obtained by scanning electron microscope (SEM). The resulting glucose biosensors showed rapid response time within 6 s, limits of detection of 0.09 mM glucose (signal-to-noise ratio = 3), wide linear range from 0.5 to 20 mM, high sensitivity of 4.214 × 10⁻³ A M⁻¹ cm⁻², also well selectivity, reproducibility and lifetime. The all-protein modified biosensor exhibited especially high efficiency of enzyme utilization, producing at most 712 μA responsive current for per unit activity of GOx. This work provided a promising new immobilization matrix with high biocompatibility and adequate electroactivity for further research in biosensing and other surface functionalizing.     

Human fibroblast culture on a crosslinked dermal porcine collagen matrix

The use of a novel porcine-derived collagen biomaterial as a dermal tissue engineering matrix was examined. The matrix is derived from porcine dermis, and is processed to retain the native collagen (Type 1) and elastin structure. Human primary fibroblasts were cultured on the matrix to examine its potential for creating a dermal replacement. Attachment of fibroblasts on the collagen was compared to tissue culture plastic and PET membranes. Cell proliferation was assessed using the MTT assay and DAPI staining. For seeding densities of 5×10⁴ and 1×10⁵ cells cm⁻², PET and plastic demonstrated >95% attachment of seeded numbers after 3 h. The collagen matrix reached levels >80% after 3–4 h with no influence of the seeding density. Matrix samples with perforating pores of 40 μm diameter were also studied. After 216 h culture in static culture, with media replacement every 3 days, the final cell numbers reached 2.1×10⁵ (perforated) and 2.0×10⁵ cells cm⁻² (unperforated). In comparison fibroblast culture in a perfusion bioreactor, with continuous media replacement, reached 2.3×10⁵ (unperforated) and 2.5×10⁵ cells cm⁻² (perforated) after 216 h.     

The use of different eye regions in the mantis shrimp Hemisquilla californiensis Stephenson, 1967 (Crustacea: Stomatopoda) for detecting objects

A behavioral assay was used to assess the ability of the stomatopod Hemisquilla californiensis to perceive and respond to a moving target under different wavelengths and intensities of light illumination. Subjects responded to targets rotating horizontally across their visual field by a brief startle response of their eyes or antennules but did not track the targets. Under white light responses were elicited down to a light intensity of 0.9 μW cm^− 2. Responses were seen in blue light at intensities as low as 0.5 μW cm^− 2, and in green light down to 1.0 μW cm^− 2. The animals were less sensitive to red light, with no responses seen at intensities below 3.0 μW cm^− 2. Subjects did not respond to the targets at all under infrared light. This response pattern mirrors the computed sensitivity spectrum of ommatidia in the species' peripheral hemispheres but not that in most of the central bands. We conclude that this species uses the monochromatic vision in the peripheral hemispheres of its eyes to recognize objects and that the sharply tuned color receptors of the central band serve to add supplemental information if light conditions allow.     

Potassium selective chemically modified field effect transistors based on AlGaN/GaN two-dimensional electron gas heterostructures

We investigate the use of the AlGaN/GaN high electron mobility transistor (HEMT) as a novel transducer for the development of ion-selective chemically modified HEMT sensors (ChemHEMTs). For this, polyvinyl chloride (PVC) membrane doped with ion-selective ionophores is deposited onto the area of the gate for the chemical recognition step, while the AlGaN/GaN HEMT is used as the transducer. In particular, the use of a valinocycin doped membrane with thickness of 50 μm generates a sensor with excellent analytical characteristics for the monitoring of K⁺. The K⁺-ChemHEMT has sensitivity of 52.4 mV/pK⁺in the linear range of 10⁻⁵ to 10⁻² M, while the detection limit is in the order of 3.1 × 10⁻⁶ M. Also, the sensor shows selectivity similar to valinomycin-based ISEs, while the signal stability over time and the measurement to measurement reproducibility are very good.     

Morphological and physiological responses of canola (Brassica napus) siliquas and seeds to UVB and CO2 under controlled environment conditions

Combined effects of UVB radiation and CO₂ concentration on plant reproductive parts have received little attention. We studied morphological and physiological responses of siliquas and seeds of canola (Brassica napus L. cv. 46A65) to UVB and CO₂ under four controlled experimental conditions: UVB radiation (4.2 kJ m⁻² d⁻¹) with ambient level of CO₂ (370 μmol mol⁻¹) (control); UVB radiation (4.2 kJ m⁻² d⁻¹) with elevated level of CO₂ (740 μmol mol⁻¹); no UVB radiation (0 kJ m⁻² d⁻¹) with ambient level of CO₂ (370 μmol mol⁻¹); and no UVB radiation (0 kJ m⁻² d⁻¹) with elevated level of CO₂ (740 μmol mol⁻¹). UVB radiation affected the outer appearance of siliquas, such as colour, as well as their anatomical structures. At both CO₂ levels, the UVB radiation of 4.2 kJ m⁻² d⁻¹ reduced the size of seeds, which had different surface patterns than those from no UVB radiation. At both CO₂ levels, 4.2 kJ m⁻² d⁻¹ of UVB decreased net CO₂ assimilation (A_N) and water use efficiency (WUE), but had no effect on transpiration (E). Elevated CO₂ increased A_N and WUE, but decreased E, under both UVB conditions. At both CO₂ levels, the UVB radiation of 4.2 kJ m⁻² d⁻¹ decreased chlorophyll fluorescence, total chlorophyll (Chl), Chl a and Chl b, but had no effect on the ratio of Chl a/b and the concentration of UV-screening pigments. Elevated CO₂ increased total Chl and the concentration of UV-screening pigments under 4.2 kJ m⁻² d⁻¹ of UVB radiation. Neither UVB nor CO₂ affected wax content of siliqua surface. Many significant relationships were found between the above-mentioned parameters. This study revealed that UVB radiation exerts an adverse effect on canola siliquas and seeds, and some of the detrimental effects of UVB on these reproductive parts can partially be mitigated by CO₂.     

Effect of leaf age on CAM activity in Littorella uniflora

In this study the effect of ontogenetic drift on crassulacean acid metabolism (CAM) was investigated in the aquatic CAM-isoetid Littorella uniflora. The results of this study strengthen the general hypothesis of CAM being a carbon-conserving mechanism in aquatic plants, because high-CAM capacity (45–183 μequiv. g⁻¹ FW) was present in all leaves of L. uniflora irrespective of age. Since possession of CAM in aquatic plants allows CO₂ uptake throughout the light/dark cycle, presence of CAM in all leaves influences the carbon balance of L. uniflora positively. On average for all lakes, different leaf classes accounted for 11–36% of the total dark CO₂ uptake by the individual plant.

The capacity for both CAM and photosynthesis declined with increasing leaf age, and was in the oldest leaves only 25–53% of the capacity in the youngest. The photosynthetic capacity was estimated to be sufficiently high to ensure refixation of the CO₂ released from malate during decarboxylation in the daytime. In line with this, a linear coupling between CAM capacity and photosynthetic capacity was found. Parallel to the change in photosynthetic capacity, an age-related change in total ribulose-bisphosphate carboxylase/oxygenase (rubisco) activity from 732 μmol C g⁻¹ DW h⁻¹ in the youngest leaves to 346 μmol C g⁻¹ DW h⁻¹ in the oldest was observed. In contrast, no significant change in phosphoenolpyruvate carboxylase (PEPcase) activity with leaf age was observed (means ranged between 46 and 156 μmol C g⁻¹ DW h⁻¹).     

Enhancement of growth and gymnodimine production by the marine dinoflagellate, Karenia selliformis

Because of its novel bioactive properties the production of gymnodimine for use as a pharmaceutical precursor has aroused interest. The dinoflagellate, Karenia selliformis produces gymnodimine when grown in bulk culture using GP + selenium medium but the growth rates (μ) and levels of gymnodimine are low (μ, 0.05 days⁻¹; gymnodimine 250 μg L⁻¹ max). We describe the effects of organic acid additions (acetate, glycolate, alanine and glutamate additions and combinations of these) in enhancing growth and gymnodimine production in axenic cultures. The most effective organic acid combinations in decreasing order were: glycolate/alanine > acetate > glycolate. Glycolate/alanine optimised gymnodimine production by prolonging growth (maximum cell yield, 1.76 × 10⁵ cells mL⁻¹; gymnodimine, 1260 μg L⁻¹; growth rate (μ), 0.2 days⁻¹) compared to the control (growth maximum cell yield, 7.8 × 10⁴ cells mL⁻¹; gymnodimine, 780 μg L⁻¹; μ, 0.17 days⁻¹). Acetate enhanced gymnodimine by stimulating growth rate (μ, 0.23 days⁻¹) and the large concentration of gymnodimine per cell (16 pg cell⁻¹ cf. 9.8 pg cell⁻¹ for the control) suggests a role for this compound in gymnodimine biosynthesis. Amending culture media with Mn²⁺ additions resulted in slightly decreased growth in control cultures and increased the gymnodimine while in glycolate/alanine cultures growth was stimulated but gymnodimine production decreased. The results suggest that the organic acid can enhance gymnodimine production by either enhancing growth maximum or the biosynthetic pathway.     

The non-specific inhibition of enzymes by environmental pollutants: a study of a model system towards the development of electrochemical biosensor arrays

Previous research has shown that lactate dehydrogenase (LDH) was competitively inhibited by pentachlorophenol (PCP) and a modified assay produced a detection limit of 1 μM (270 μg l⁻¹). This work used spectrophotometric rate-determination but in order to move towards biosensor development the selected detection method was electrochemical. The linkage of LDH to lactate oxidase (LOD) provided the electroactive species, hydrogen peroxide. This could be monitored using a screen-printed carbon electrode (SPCE) incorporating the mediator, cobalt phthalocyanine, at a potential of +300 mV (vs. Ag/AgCl). A linked LDH/LOD system was optimised with respect to inhibition by PCP. It was found that the SPCE support material, PVC, acted to reduce inhibition, possibly by combining with PCP. A cellulose acetate membrane removed this effect. Inhibition of the system was greatest at enzyme activities of 5 U ml⁻¹ LDH and 0.8 U ml⁻¹ LOD in reactions containing 246 μM pyruvate and 7.5 μM NADPH. PCP detection limits were an EC₁₀ of 800 nM (213 μg l⁻¹) and a minimum inhibition detectable (MID) limit of 650 nM (173 μg l⁻¹). The inclusion of a third enzyme, glucose dehydrogenase (GDH), provided cofactor recycling to enable low concentrations of NADPH to be incorporated within the assay. NADPH was reduced from 7.5 to 2 μM. PCP detection limits were obtained for an assay containing 5 U ml⁻¹ LDH, 0.8 U ml⁻¹ LOD and 0.1 U ml⁻¹ GDH with 246 μM pyruvate, 400 mM glucose and 2 μM NADPH. The EC₁₀ limit was 150 nM (39.9 μg l⁻¹) and the MID was 100 nM (26.6 μg l⁻¹). The design of the inhibition assays discussed has significance as a model for other enzymes and moves forward the possibility of an electrochemical biosensor array for pollution monitoring.     

Contribution of the heme propionate groups to the electron transfer and electrostatic properties of myoglobin

The role of the heme propionate groups in determining the electron transfer and electrostatic properties of myoglobin have been studied by thermodynamic, kinetic, and spectroscopic studies of horse heart myoglobin in which the heme propionate groups are esterified. Spectroelectrochemical analysis has established that the E_m,7 of dimethylester heme-substituted Mb (DME-Mb) (E_m,7 = 100.2(2) mV vs. NHE (Normal Hydrogen Electrode) (25 °C) is increased  40 mV relative to that of the native protein with ΔH° = −12.9(2) kcal/mol and ΔS° = −51.0(8) cal/mol/deg (pH 7.0, μ = 0.1 M (phosphate)). The second order rate constant for reduction of DME-metMb by Fe(EDTA)²⁻ is increased  > 400-fold relative to that for reduction of native metMb to a value of 1.34(2) × 10³ M⁻¹ s⁻¹ with ΔS^‡ = −13(1) cal/mol/deg and ΔH^‡ = 9.2(3) (pH 7.0, μ = 0.1 M (phosphate)). Analysis of the pH dependences of the reduction potential and rate constant for reduction by Fe(EDTA)²⁻ demonstrates that heme propionate esterification introduces significant changes into the electrostatic interactions in myoglobin. These changes are also manifested by differences in the pH dependences of the ¹H NMR spectra of native and DME-metMb that reveal shifts in pK_a values for specific His residues as the result of heme propionate esterification. In sum, the current results establish that heme propionate esterification not only affects the electron transfer properties of myoglobin but also influences the titration behavior of specific His residues.     

CYP450 biosensors based on gold chips for antiepileptic drugs determination

Three-electrode configuration chips containing a Pt, Au and a screen-printed Ag/AgCl as counter, working and reference electrode, respectively, have been developed. Selective determination of Phenobarbital (PB) has been carried out by Cytochrome P450 2B4 (CYP450) immobilization into a polypyrrole matrix onto the gold working electrode. Chronoamperometric experiments show a PB diffusion coefficient of 2.42 × 10⁻⁶ cm² s⁻¹, a reproducibility and repeatability in terms of residual standard deviation (RSD) of 13% and 5.51%, respectively, and a limit of detection (LOD) of 0.289 μmol dm⁻³ ( = β = 0.05) for the developed CYP450-biosensor chip. Its performance has been showed by the determination of PB in pharmaceutical drugs. HPLC has been used as reference technique.     

Growth and photosynthesis of Hydrocotyle ranunculoides L. fil. in Central Europe

Floating Pennywort (Hydrocotyle ranunculoides L. fil.) is a worldwide distributed aquatic plant. The species is native to North America and quite common also in Central and South America. In Europe, Japan and Australia it is known as an alien plant, sometimes causing serious problems for affected ecosystems and human use of water bodies. Starting from Western Europe with an eastwards directed spread, Floating Pennywort was recorded in Germany in 2004 for the first time. Since then, the species spread out and got established in western parts of Central Europe. For a definite prediction of the potential of a further spread, data about biology, in particular growth and photosynthesis are needed. Here, regeneration capacity, growth at different nutrient availabilities and photosynthesis of H. ranunculoides were investigated. In addition biomass samples were taken in the field. Results show an enormous regeneration capacity (e.g., by forming new shoots from small shoot fragments), increasing growth rates under increasing nutrient availability and a maximum increase of biomass reaching 0.132±0.008 g g⁻¹ dw d⁻¹. Dense populations of H. ranunculoides growing in ponds and oxbows were found at high nutrient content of the substrate, the biomass reaching there up to 532.4±14.2 g dw m⁻². Gas exchange analysis showed a physiological optimum of H. ranunculoides CO₂ uptake at temperatures between 25 and 35 °C and high photon flux densities (PPFD) above 800 μmol photons m⁻² s⁻¹. In comparison, native Hydrocotyle vulgaris showed an optimum of net photosynthesis at 20–30 °C and a light saturation of CO₂ gas exchange at 350 μmol photons m⁻² s⁻¹.     

Influence of the alkyl-substituted silane precursor on sol–gel encapsulated lipase activity

Lipase from Candida rugosa was encapsulated within a chemically inert sol–gel support prepared by polycondensation of three precursor types (tetraethoxysilane (TEOS), methyltrimethoxysilane (MTMS) and polydimethylsilane (PDMS)) in the presence and absence of polyethylene glycol (PEG) and polyvinyl alcohol (PVA) as additives. Silica and their derivatives were characterised with regard to mean pore diameter, specific surface area, pore size distribution (BET method), weight loss upon heating thermogramivemetric analysis (TGA), chemical composition Fourier transform infrared spectroscopy (FT-IR), and catalytic activities. Immobilisation yields based on the recovered lipase activity vary from 3.02 to 31.98% and the highest efficiency was attained when lipase was encapsulated using TEOS in the presence of the PEG. Further information was obtained by testing the derivatives in esterification reactions and a different reactivity profile was found. Better performance was obtained with derivatives containing lipase encapsulated within gels prepared with MTMS as precursor in the presence of PEG. This lipase preparation exhibits increased esterification activity (155 μmol g⁻¹ min⁻¹), up to of three times greater than that prepared with TEOS (52 μmol g⁻¹ min⁻¹), and almost twice that prepared with MTMS/PDMS (89 μmol g⁻¹ min⁻¹) as precursors.     

Fabrication of sucrose biosensor based on single mode planar optical waveguide using co-immobilized plant invertase and GOD

In present studies, the new optical sensing platform based on optical planar waveguide (OPWG) for sucrose estimation was reported. An evanescent-wave biosensor was designed by using novel agarose–guar gum (AG) biopolymer composite sol–gel with entrapped enzymes (acid invertase (INV) and glucose oxidase (GOD)). Partially purified watermelon invertase isolated from Citrullus vulgaris fruit (specific activity 832 units mg⁻¹) in combination with GOD was physically entrapped in AG sol–gel and cladded on the surface of optical planar waveguide. Na⁺–K⁺ ion-exchanged glass optical waveguides were prepared and employed for the fabrication of sucrose biosensor. By addressing the enzyme modified waveguide structure with, the optogeometric properties of adsorbed enzyme layer (12 μm) at the sensor solid–liquid interface were studied. The OPWG sensor with short response time (110 s) was characterized using the 0.2 M acetate buffer, pH 5.5. The fabricated sucrose sensor showed concentration dependent linear response in the range 1 × 10⁻¹⁰ to 1 × 10⁻⁶ M of sucrose. Lower limit of detection of this novel AG–INV–GOD cladded OPWG sensor was found to be 2.5 × 10⁻¹¹ M sucrose, which indicates that the developed biosensor has higher sensitivity towards sucrose as compared to earlier reported sensors using various transducer systems. Biochips when stored at room temperature, showed high stability for 81 days with 80% retention of original sensitivity. These sucrose sensing biochips showed good operational efficiency for 10 cycles. The proper confinement of acid invertase and glucose oxidase in hydrogel composite was confirmed by scanning electron microscopy (SEM) images. The constructed OPWG sensor is versatile, easy to fabricate and can be used for sucrose measurements with very high sensitivity.     

A study in UF-membrane reactor on activity and stability of nitrile hydratase from Microbacterium imperiale CBS 498-74 resting cells for propionamide production

The bioconversion of propionitrile to propionamide was catalysed by nitrile hydratase (NHase) using resting cells of Microbacterium imperiale CBS 498-74 (formerly, Brevibacterium imperiale). This microorganism, cultivated in a shake flask, at 28 °C, presented a specific NHase activity of 34.4 U mg_DCW⁻¹ (dry cell weight). The kinetic parameters, K_m and V_max, tested in 50 mM sodium phosphate buffer, pH 7.0, in the propionitrile bioconversion was evaluated in batch reactor at 10 °C and resulted 21.6 mM and 11.04 μmol min⁻¹ mg_DCW⁻¹, respectively. The measured apparent activation energy, 25.54 kJ mol⁻¹, indicated a partial control by mass transport, more likely through the cell wall.

UF-membrane reactors were used for kinetic characterisation of the NHase catalysed reaction. The time dependence of enzyme deactivation on reaction temperature (from 5 to 25 °C), on substrate concentrations (from 100 to 800 mM), and on resting cell loading (from 1.5 to 200 μg  ml⁻¹) indicated: lower diffusional control (E_a=37.73 kJ mol⁻¹); and NHase irreversible damage caused by high substrate concentration. Finally, it is noteworthy that in an integral reactor continuously operating for 30 h, at 10 °C, 100% conversion of propionitrile (200 mM) was attained using 200 μg  ml⁻¹ of resting cells, with a maximum volumetric productivity of 0.5 g l⁻¹ h⁻¹.     

Enzyme-mediated amperometric biosensors prepared with the Layer-by-Layer (LbL) adsorption technique

Glucose oxidase (GOD) has been immobilized in Layer-by-Layer (LbL) films, adsorbed alternately with poly(allylamine) hydrochloride (PAH) layers, onto an ITO substrate modified with a Prussian Blue (PB) layer. The ITO/PB/GOD-PAH heterostructures were tested in amperometric glucose biosensors, with a high sensitivity of 16 μA mmol⁻¹ l cm^-2 and a limit of detection of 0.20 mmol l⁻¹ being achieved. This high sensitivity is attributed to the ultrathin nature of the film in addition to the low operating potentials that could be used due to the efficient catalysis of H₂O₂ produced in the enzymatic reaction in the presence of Prussian Blue. The biosensors are highly selective to glucose, as demonstrated by the lack of interference from possible interferents such as ascorbic and uric acids and acetominophen. The stability of the biosensors was checked by observing an almost constant sensitivity for a period of approximately 20 days, thus indicating a stable adsorption of GOD.