Design and characterization of a lactate biosensor based on immobilized lactate oxidase onto gold surfaces

The design and characterization of a lactate biosensor and its application to the determination of this analyte in wine and beer are described. The biosensor is developed through the immobilization of lactate oxidase (LOx) using two different strategies including direct adsorption and covalent binding. The characterization of the resulting lactate oxidase monolayers was performed in aqueous phosphate buffer solutions using atomic force microscopy (AFM) and quartz crystal microbalance (QCM) techniques. In presence of lactate and using hydroxymethylferrocene as a redox mediator, biosensors obtained by either direct adsorption or by covalent binding exhibit a clear electrocatalytic activity, and lactate could be determined amperometrically at 300 mV versus SSCE. Results obtained under these conditions give a linear current response versus lactate concentration up to 0.3 mM, with a detection limit of 10 μM of lactate and a sensitivity of 0.77 ± 0.08 μA mM⁻¹. Finally, biosensors were applied to the determination of lactate in wine and beer. The results obtained are in good agreement with those obtained by a well-established enzymatic-spectrophotometric assay kit.     

Bacterial spores as platforms for bioanalytical and biomedical applications

Genetically engineered bacteria-based sensing systems have been employed in a variety of analyses because of their selectivity, sensitivity, and ease of use. These systems, however, have found limited applications in the field because of the inability of bacteria to survive long term, especially under extreme environmental conditions. In nature, certain bacteria, such as those from Clostridium and Bacillus genera, when exposed to threatening environmental conditions are capable of cocooning themselves into a vegetative state known as spores. To overcome the aforementioned limitation of bacterial sensing systems, the use of microorganisms capable of sporulation has recently been proposed. The ability of spores to endow bacteria-based sensing systems with long lives, along with their ability to cycle between the vegetative spore state and the germinated living cell, contributes to their attractiveness as vehicles for cell-based biosensors. An additional application where spores have shown promise is in surface display systems. In that regard, spores expressing certain enzymes, proteins, or peptides on their surface have been presented as a stable, simple, and safe new tool for the biospecific recognition of target analytes, the biocatalytic production of chemicals, and the delivery of biomolecules of pharmaceutical relevance. This review focuses on the application of spores as a packaging method for whole-cell biosensors, surface display of recombinant proteins on spores for bioanalytical and biotechnological applications, and the use of spores as vehicles for vaccines and therapeutic agents.     

Novel electron transfer mediators based on dichloroindophenol derivatives for lactate oxidase

Novel indophenol derivatives were synthesized and characterized electrochemically with respect to their abilities to act as electron-transfer mediators for lactate oxidase. These compounds showed suitable redox potentials and high second-order rate constants, k_med, in solution for electron-transfer from the reduced enzyme. A lactate sensor using these derivatives demonstrated high sensitivity and good substrate selectivity. This sensor could also achieve excellent durability which retained more than 70% residual activity and good linearity in the range from 0 to 16 mM lactate concentration even after 10 days.     

Fibre optic fluorometric enzyme sensors for hydrogen peroxide and lactate,based on horseradish peroxidase and lactate oxidase

An optical biosensor for the determination of hydrogen peroxide based on immobilized horseradish peroxidase is described. The fluorescence of the dimeric product of the enzyme catalysed oxidation of homovanillic acid is utilized to determine the concentration of H₂O₂. The membrane-bound enzyme is attached to a bifurcated fibre bundle permitting excitation and detection of the fluorescence by a fluorometer. The response of the sensor is linear from 1 to 130 M hydrogen peroxide; the coefficient of variation is 3%. The sensor is stable for more than 10 weeks. The operating pH for maximal sensor response is 8.15. This allows the sensor to be used in combination with oxidase reactions producing hydrogen peroxide, as is demonstrated with a co-immobilized lactate oxidase-horseradish peroxidase optode for the determination of L-lactate. The fluorescence intensity of this sensor depends linearly on the concentration of lactate between 3 and 200 M and a throughput of 10 samples per hour is possible. The precision is in the same range as that of the monoenzyme optode. The lifetime of the bienzyme sensor for lactate is considerably shorter than that of the peroxidase sensor; it is limited by the stability of the immobilized lactate oxidase enzyme. The sensor has been applied to the determination of lactate in control serum.     

A sensitive biosensor for lactate based on layer-by-layer assembling MnO2 nanoparticles and lactate oxidase on ion-sensitive field-effect transistors

A sensitive enzyme-based FET biosensor for lactate has been obtained by introducing MnO2 nanoparticles at the gate surface via a layer-by-layer assembling method.     

Amperometric tetrathiafulvalene-mediated lactate electrode using lactate oxidase absorbed on carbon foil

The features of a new sensor for determining l-lactate are reported. The enzyme lactate oxidase and the mediator, tetrathiafulvalene (TTF), are absorbed on carbon foil disks previously bonded onto the ends of glass tubes. Linear calibration graphs were obtained in the range 10^?4?10^?3 M with physiological phosphate buffer (pH 7.35) and at 30°C with a response time of a few seconds. Calibration graphs in the range 10^?3?10^?2 M were also obtained and the difference in response times between these two ranges were investigated. The results are promising for assembling disposable lactate sensors for in vitro or for in or ex vivo measurements.     

Biosensors based on thermistors and semiconductors and their bioanalytical applications

Calorimetry is a technique with general applicability in bioanalysis. The enzyme thermistor (ET) is a simple flow calorimeter primarily intended for rapid metabolite assays. The reaction heat in a small column containing immobilized enzyme is measured as a temperature change of the effluent of the column. The temperature change is linear versus substrate concentrations over wide ranges from 1 μM to several hundred mM, depending on the actual enzyme reaction. Up to 60 samples per hour can be analysed. A large number of metabolite assays of clinical as well as biotechnological interest have been studied. The ET assay can easily be automated and is well suited for monitoring and control of biotechnological processes due to its high operational stability. For monitoring molecules larger than enzyme substrates, a thermometric enzyme immunoassay (TELISA) has been developed and automated.Hydrogen- and ammonia-sensitive semiconductors of Pd-MOS type combine with enzymes and cells into highly sensitive biosensors. The H₂ sensor is also useful for direct monitoring of biological processes producing H₂. Addition of another layer of a catalytic metal such as Ir on the MOS device results in enhanced NH₃ sensitivity. Highly-sensitive methods for the determination of, e.g., urea and creatinine have been developed. A common feature of these sensors is that the detection is made in the gas phase, while the reaction is carried out in the aqueous phase, which facilitates work with crude samples.     

A voltammetric study of interdomain electron transfer within sulfite oxidase

Protein film voltammetry of chicken liver sulfite oxidase (SO) bound at the pyrolytic graphite "edge" or modified gold electrodes shows that catalytic electron transport is controlled by the inherent electrochemical characteristics of the heme b domain and conformational changes that allow intramolecular electron transfer with the molybdenum active site. In the absence of sulfite, a single nonturnover electrochemical signal is observed at +90 mV (vs SHE) that is assigned to heme b. In the presence of sulfite, this signal transforms into a catalytic wave at similar potential. The shape and negligible pH dependence of this wave indicate that catalytic turnover is controlled by the one-electron transfers through heme b. The smaller turnover numbers obtained in this experiment (k(cat) approximately 2-4 s(-1), as compared to 100 s(-1) in solution) suggest that only a small fraction of SO is bound at the electrode in a manner that permits the conformational change necessary for fast interdomain electron transfer.     

Thick-film voltammetric sensors based on ruthenium dioxide

Commercial resistive pastes are applied on alumina substrates by planar thick-film technology. The electrochemical properties of these films are studied in voltammetric experiments. Results indicate that thick-film RuO₂ electrodes offer considerable advantages over classically manufactured electrodes. The preparation of these electrodes is straightforward and easy. They behave well with redox systems, have small capacitive currents and are robust.     

Signal amplification by substrate recycling on polyaniline/lactate oxidase/lactate dehydrogenase bienzyme electrodes

The bienzyme electrodes were fabricated by coimmobilization of lactate oxidase (LOD) and lactate dehydrogenase (LDH) onto electrochemically prepared polyaniline (PANI) films. These PANI/LOD/LDH bienzyme electrodes were shown to provide signal amplification by substrate recycling, making it possible to detect l-lactate at lower concentrations (0.1-1 mM). The PANI/LOD/LDH bienzyme electrodes were found to be stable for about 21 d at 4–10°C.     

Nanomechanical properties of globular proteins: lactate oxidase

We report on the study of the nanomechanical properties of a lactate oxidase (LOx) monolayer immobilized on gold substrates by atomic force microscopy techniques operating under buffer conditions. Topographical contact mode imaging evidenced the protein deformation under the applied tip load. We performed approaching force curves with both stiff and soft cantilevers by imposing maximum loads of 1.6 nN and 400 pN, respectively. We found that the experimental data were well fitted by the Hertz model for a conical indenter. The use of two types of cantilevers allowed us to check further the consistency of the applicability of the Hertz model to the experimental data. After analyzing 180 curves, we obtained an average value of Young's modulus for the LOx layer in the 0.5-0.8 GPa range. These results agreed with those obtained for LOx submonolayer deposits on mica substrates, which allows discarding any important contribution from the underlying substrate on the measured properties. This range of values is closer to those obtained by other techniques on other globular proteins in comparison with those reported in previous AFM studies on similar systems. We found that for our experimental conditions the force curves can be, in principle, well fitted by the Hertz model for both conical and spherical indenter geometries. However, as the Young's modulus obtained for both geometries can differ appreciably, it becomes necessary to assess which indenter geometry is more adequate to explain the experimental data. For such purpose a systematic study of the indentation versus applied force curves obtained from both fittings for all the experimental curves was done.     

Lactate biosensor based on a bionanocomposite composed of titanium oxide nanoparticles, photocatalytically reduced graphene, and lactate oxidase

We have developed a lactate biosensor based on a bionanocomposite (BNC) composed of titanium dioxide nanoparticles (TiO₂-NPs), photocatalytically reduced graphene, and lactate oxidase. Graphene oxide was photochemically reduced (without using any chemical reagents) in the presence of TiO₂-NPs to give graphene nanosheets that were characterized by atomic force microscopy, Raman and X-ray photoelectron spectroscopy. The results show the nanosheets to possess few oxygen functionalities only and to be decorated with TiO₂-NPs. These nanosheets typically are at least 1 μm long and have a thickness of 4.2 nm. A BNC was obtained by mixing lactate oxidase with the nanosheets and immobilized on the surface of a glassy carbon electrode. The resulting biosensor was applied to the determination of lactate. Compared to a sensor without TiO₂-NPs, the sensor exhibits higher sensitivity (6.0 μA mM^?1), a better detection limit (0.6 μM), a wider linear response (2.0 μM to 0.40 mM), and better reproducibility (3.2 %).

Adlayers based on molecular platforms of trioxatriangulenium

The platform approach for preparation of molecular adlayers with freestanding functional groups was extended to systems on the basis of the trioxatriangulenium ion. Self-assembled monolayers of these compounds were prepared on Au(111) surfaces and characterized by scanning tunneling microscopy, revealing highly-ordered structures.     

乳酸胆固醇酯的合成与表征

乳酸酯广泛应用于化妆品、药剂、乳化剂等方面。胆固醇（胆甾醇）酯是一种具有液晶相的化合物,其在一定条件下,会随温度、磁场、电场、机械应力、气体浓度变化,而发生色彩的变化,可用于制作液晶温度计、气敏元件、电子元件、变色物质等,还可用于无损伤探伤、微波测量、治病诊断、定向反应等化学、化工、冶金、医学等领域。     

A lactate electrode with lactate oxidase immobilized on nylon net for blood serum samples in flow systems

Commercially available lactate oxidase from Mycobacterium smegmatis is immobilized on a nylon net which is fixed on an oxygen probe to provide a simple l-lactate sensor. A citrate buffer, pH 6.0, is the only reagent required. The high activity of the enzyme obtained with this immobilization process permits the use of only 20–100 μl of plasma; diluted with citrate buffer to 2 ml, the sample is pumped through a flow cell. The high dilution offsets inhibitory effects of some anions present in blood such as oxalate, hydrogencarbonate and chloride. The response of the sensor is linear over the range 0.2– 2 × 10^?4 M l-acetate. The lifetime is about two months. Effects of pH, temperature and different buffers are described and results on serum samples are reported.     

Differential pulse voltammetric studies on the effects of Al（Ⅲ） on the lactate dehydrogenase activity

In this paper, differential pulse voltammetry (DPV) was applied to study the effects of aluminum Al(Ⅲ) on the lactate dehydrogenase (LDH) activity. Michaelis–Menten constant (KmN ADH) and maximum velocity (vmax) in the enzyme promoting catalytic reaction of "pyruvate(Pyr) NADH H LDDHlactate NAD " under different conditions by monitoring DPV reduction current of NAD were reported.     

Synthesis, characterization and cyclic voltammetric studies of ruthenium oximates

Summary Using 1,2-naphthoquinone-1-oxime (HL) as the principal ligand, four mixed-ligand ruthenium oximate complexes - namely [Ru(bipy)₂(L)]ClO₄, [Ru(pap)₂(L)]-ClO₄, [Ru(bipy)(L)₂] and [Ru(PPh₃)₂(L)₂], where bipy = 2,2′-bipyridine and pap = 2-(phenylazo)pyridine- have been synthesized and characterized. In all these complexes, Ru exists in the +2 state. They are diamagnetic and, in solution, show several intense metal-to-ligand charge transfer (MLCT) transitions in the vis. region. In MeCN solution, all four complexes show a reversible Ru^II-Ru^III oxidation on the positive side of a standard calomel electrode (s.c.e.), the potential of which varies with the compositions of the complexes. Reductions of the coordinated co-ligands (bipy or pap) are also observed.