Design and Optimization of Biosensor for Pyruvate Quantification

Recovery of phosphate, magnesium and ammonium from eutrophic water by struvite biomineralization through free and immobilized Bacillus cereus MRR2

The mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) is believed to have evolved from a trifunctional NADP-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase. It is unique in its absolute requirement for inorganic phosphate and magnesium ions to support dehydrogenase activity. To enable us to investigate the roles of these ions, a homology model of human NMDMC was constructed based on the structures of three homologous proteins. The model supports the hypothesis that the absolutely required Pi can bind in close proximity to the 2'-hydroxyl of NAD through interactions with Arg166 and Arg198. The characterization of mutants of Arg166, Asp190, and Arg198 show that Arg166 is primarily responsible for Pi binding, while Arg198 plays a secondary role, assisting in binding and properly orienting the ion in the cofactor binding site. Asp190 helps to properly position Arg166. Mutants of Asp133 suggest that the magnesium ion interacts with both Pi and the aspartate side chain and plays a role in positioning Pi and NAD. NMDMC uses Pi and magnesium to adapt an NADP binding site for NAD binding. This adaptation represents a novel variation of the classic Rossmann fold.

Formolase, a thiamine pyrophosphate (TPP)-dependent enzyme, catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. It has many important functions in the biosynthesis of carbon-based compounds and utilization of CO2. However, the enzyme has low activity and stability in the catalytic process, resulting in high cost in the applications. To improve the stability, formolase was immobilized onto magnetic nanoparticles, which were designed to have functional epoxy groups for covalently binding the enzyme. In the immobilization, effects of pH, temperature, and cofactor TPP on the immobilization were investigated and optimized. The results showed that the retention activity of immobilized formolase was highly related to TPP. In the presence of TPP, the specific activity of the immobilized formolase was 6.8 times higher than that without TPP. The optimal immobilization conditions were as follows: a temperature of 20 °C, a pH of 7.0, an immobilization time of 8 h, and an enzyme loading of 20 mg/g. Molecular docking was used to analyze the effect of TPP on the stabilization of the enzyme in the immobilization, which indicated that TTP could stabilize the enzyme structure during the immobilization. The stabilization effect of TPP could be a reference in the immobilization of other enzymes with TPP as the cofactor.

The amperometric biosensor was developed for determination of lactate concentration. As a bioselective element we used the enzyme lactate oxidase, coimmobilized with bovine serum albumin on the surface of amperometric transducer by glutaraldehyde covalent crosslinking. The platinum disc electrodes (Ø = 500 µm) served as transducers. The developed biosensor was highly sensitive to lactate, with a minimum limit of determination of lactate concentrations 3 µM and linear range 5 µM to 300-350 mM. It was shown that the proposed biosensor was characterized by high response replicability during several hours of non-stop measurement (the accuracy of measurement RSD = 3.4%) and satisfactory operational stability for several days. Optimal conditions for biosensor storage were analyzed and defined. One-month storage in the buffer solution at + 4 °C resulted in less than 50% loss of the biosensor activity. The impact of parameters of buffer solution (capacity and ionic strength) on the biosensor operation was analyzed. It was shown that the developed biosensor based on lactateoxidase used can successfully be used for lactate analysis in real biological liquids

Aim. To develop an electrochemical biosensor for the quantitative assessment of alanine aminotransferase (ALT) activity. Materials and Methods. Three-electrode amperometric scheme of detection with platinum disk working electrode covered with bioselective element based on pyruvate oxidase and photopolymer. Measurements were carried out with an applied potential of 0.6 V. Results. A laboratory prototype of an amperometric biosensor for ALT detection was developed. The immobilization method was selected, and the procedure was optimized for the type of bioselective material used. The composition of the working buffer was optimized, namely, the kind of buffer, buffer capacity, pH, content of coenzymes of the bioselective material (phosphate ions, magnesium ions, thiamine pyrophosphate), and the analyte (alanine, ketoglutarate, pyridoxal phosphate). The reproducibility of immobilization and reproducibility of responses of the developed biosensor, as well as its analytical characteristics (linear range, detection limit, response time, etc.), were analyzed. Conclusions. The developed biosensor was characterized by sufficiently good analytical parameters for its further optimization and testing when working with real blood serum samples.

Development of the enzyme-containing nanocomposites provides an excellent opportunity for the development of the sensitive and effective analytical devices – biosensors. In the present work, we used the nanocomposites that contained two enzymes (glucose oxidase and hexokinase), polymers, and gold nanoparticles (GNPs). Such nanostructure was expected to increase electron transfer in the bioselective element of biosensor and to improve the enzyme stability during the immobilization process. An amperometric biosensor based on the bienzyme system has been developed. The biosensor is sensitive to adenosine-5′-triphosphate (ATP) and glucose. The enzymes were immobilized onto the surface of a platinum disk electrode that was used as amperometric transducer. Three different methods of immobilization were investigated: cross-linking of the enzymes in the presence of bovine serum albumin, entrapment in a photo-cross-linkable modified polyvinyl alcohol (PVA-SbQ) matrix, and entrapment of the enzymes in a PVA/polyethylenimine matrix. The best results during the ATP determination were obtained with PVA-SbQ, and this immobilization method was modified by addition of 18-nm GNPs to the reacting mixture. The ATP detection could be achieved in all cases except for PVA/polyethylenimine, the best sensitivity and linear range being achieved by co-immobilizing glucose oxidase/hexokinase into the photo-cross-linked PVA-SbQ/GNP polymer matrix.

Changes in the mechanism of the reaction between phosphate and magnesium ions: Effect of initial concentration and contact time on removal of phosphate ions from aqueous media

Design of biosensors refers to the field of interdisciplinary research, therefore, it is necessary to develop a unified systems approach that is invariant to the physical nature of the used phenomena and processes to create an automated system for conceptual design of such elements. It is appropriate to choose the fundamentals of non-equilibrium thermodynamics as the basis for the development of this approach. This article discusses the energy-information model of diffusion phenomena, as the processes of diffusion is the general type of the processes that is occurring in bioselective elements. A number of physical effects are described by this model. In this paper authors continue researches in the field of automation of design of biosensors which was presented at the 6th International Conference on Information, Intelligence, Systems and Applications. The new information technology of functional and structural design of biosensors is described. It is based on the energy-information model of chains, invariant to the physical nature of the processes occurring in technical devices. And it uses the device parametric structural diagrams allowing algorithmization of the search and selection of new technical solutions with estimation of their performance.

Design of biosensors refers to the field of interdisciplinary research, therefore, it is necessary to develop a unified systems approach that is invariant to the physical nature of the used phenomena and processes to create an automated system for conceptual design of such elements. It is appropriate to choose the fundamentals of non-equilibrium thermodynamics as the basis for the development of this approach. They allow to obtain a complete system of transfer equations of the phenomena of different physical nature and other laws, without opening their molecular mechanism. This article discusses the energy-information model of diffusion phenomena, as the processes of diffusion is the general type of the processes that is occurring in bioselective elements. A number of physical effects are described by this model. They are used in the conceptual design of electrochemical biosensors.

In this work, an amperometric biosensor for highly sensitive and selective determination of pyruvate concentration was developed and optimized and the feasibility of it’s use for the analysis of alanine aminotransferase (ALT) activity was demonstrated. A platinum disk electrode was used as a transducer, and the bioselective element was based on pyruvate oxidase and PVA-SBQ photopolymer. The influence of solution parameters (buffer capacity, pH, ionic strength) was analyzed, the selectivity of the biosensor towards interferents and the reproducibility of responses (RSD = 12.66%) were studied. The main analytical characteristics of the biosensor were also analyzed (linear range of 10–500 μM pyruvate, sensitivity 66 nA/mM, limit of detection 1.57 μM). The obtained data indicate the prospects of using the developed biosensor for the determination of pyruvate in biological samples. Finally, a calibration curve of the response of the developed pyruvate-sensitive biosensor on the ALT content was created.

Today, the concentration of glutamate in the patient′s blood is an important indicator in medical diagnostics; therefore, it is necessary to have a simple, accurate, and fast tool to obtain the data. Here, a recently developed amperometric glutamate‐sensitive biosensor was optimized to improve its characteristics. The platinum disk electrode was used as a transducer. As a bioselective element we used the enzyme glutamate oxidase, covalently crosslinked with bovine serum albumin by glutaraldehyde. Circumstances of enzyme immobilization on the transducer‘s surface were optimized (enzyme and glutaraldehyde concentrations and immobilization duration). To test the ability of this biosensor to work in biological environments containing complex biological substances, the influence of the working solution was investigated (concentration of the working buffer, its temperature, presence of the protein in the analyzed sample). The linear range of biosensor was from 5 to 600 μM of glutamate and the sensitivity was 150–200 nA/mM. Measurements of glutamate concentrations in the blood plasma were performed by biosensor and glutamate dehydrogenase assay. The linear correlation between the methods was found in a range of 50.4–182.5 μM (R2=0.99). Thus, it has been shown that the developed biosensor makes it possible to measure the concentration of glutamate in blood plasma.

Phosphoprotein particles were isolated in their native state from the physiological fluid of the estuarine clam Rangia cuneata , and the characteristics of the mineral ion-protein complex which constitutes the native particle were investigated by using mineral ion binding and mineral ion exchange techniques. The particles are aspartic acid rich, highly phosphorylated proteins containing calcium, magnesium, and inorganic phosphate ions and covalently cross-linked via histidinoalanine residues. Twenty-nine percent of the amino acid residues are phosphorylated. In their native state, the particles contain a protected pool of calcium and inorganic phosphate ions and an exchangeable pool of calcium and magnesium ions. The Ca/PO4 ratio in the protected pool is about 2.5. The number of binding sites for the protected mineral is unknown, but on the average, the native particles contain about 0.2 inorganic phosphate ion per organic phosphate residue. There is 1.0 exchangeable metal ion binding site per organic phosphate residue, and there is probably a phosphoserine residue at each site. These sites bind calcium with an apparent binding constant (KCa) of 4 X 10(3) M-1 at 50% saturation under physiological conditions, and KCa/ KMg is about 1.6. In vivo, about 85% of the exchangeable sites are occupied. The total number of calcium ion binding sites (N) in the phosphoprotein particles is related to the number of organic phosphate residues (Po) and the number of bound inorganic phosphate ions (Pi) by the equation N = Po + 2. 5Pi . The phosphoprotein particles probably serve as both the transporter and reserve source of calcium ions for shell development.

N,N′‐bis{3,5‐di[(1‐pyrenylmethyl)carbamoyl]benzyl}pyridine‐2,6‐dicarbamide (compound 1) provides a pseudo‐tetrahedron cleft and multiple hydrogen bondings to form a 1:1 complex with phosphate (or pyrophosphate) ion in a highly selective manner, in comparison with other anions (F−, Cl−, Br−, SCN−, AcO−, NO3−, and ClO4−). Compound 1 bears four pyrene rings as the sensitive fluorescent readout unit for phosphate and pyrophosphate ions. The binding strength can be inferred from the emission intensity ratio of the pyrene monomer (λmax = 377 nm) to the excimer (λmax ˜ 480 nm). The enhanced multiple hydrogen bondings and ratiometric sensing mechanism render an opportunity for sensing pyrophosphate ion at micromolar concentration, even in water‐containing solution. The binding model is supported by fluorescence and 1H NMR studies.

The majority of biosensors for adenosine-5'-triphosphate (ATP) determination are based on cascades of enzymatic reactions; therefore, they are sensitive to glucose or glycerol (depending on the enzymatic system) as well as to ATP. The presence of unknown concentrations of these substances in the sample greatly complicates the determination of ATP. To overcome this disadvantage of known biosensors, we developed a biosensor system consisting of two biosensors: the first one is based on glucose oxidase and is intended for measuring glucose concentration, and the second one is based on glucose oxidase and hexokinase and is sensitive toward both glucose and ATP. Using glucose concentration measured by the first biosensor, we can analyze the total response to glucose and ATP obtained by the second biosensor. Platinum disc electrodes were used as amperometric transducers. The polyphenilenediamine membrane was deposited onto the surface of platinum electrodes to avoid the response to electroactive substances. The effect of glucose concentration on biosensor determination of ATP was studied. The reproducibility of biosensor responses to glucose and ATP during a day was tested (relative standard deviation, RSD, of responses to glucose was 3-6% and to ATP was 8-12%) as well as storage stability of the biosensors (no decrease of glucose responses and 43% drop of ATP responses during 50 days). The measurements of ATP and glucose in pharmaceutical vials (including mixtures of ATP and glucose) were carried out. It was shown that the developed biosensor system can be used for simultaneous analysis of glucose and ATP concentrations in water solutions.

Determination of total creatine kinase activity in blood serum using an amperometric biosensor based on glucose oxidase and hexokinase