MerR-fluorescent protein chimera biosensor for fast and sensitive detection of Hg2+ in drinking water.

Mercury ion (Hg2+ ) is a universal pollutant and its detection is crucial for public healthcare. In this study, we developed a novel fluorescent biosensor by construction of a protein fusion between the mercury-sensing transcription factor MerR and enhanced yellow fluorescent protein (EYFP). Hg2+ -induced conformational change of MerR was transduced into fluorescence signal. Fluorescence intensity of the biosensor protein decreased with increasing concentrations of Hg2+ and a linear response was obtained in the range of 0.5-40 nM. The limit of detection was 0.5 nM, which was much lower than the maximum allowed level in water. The biosensor specificity was highly dependent on type and concentration of metal ion. The biosensor exhibited high specificity in a mixture of metal ions at 0.5 nM concentration. However, the interference effect was more pronounced at 40 nM concentration of metal ions. The measurement was completed in less than 1 Min with no need for sample preparation or preincubation steps. The biosensor achieved accurate and reliable detection in the spiked drinking water sample, as validated by the inductively coupled plasma optical emission spectrometry.

Development of genetically encoded fluorescent protein constructs of hyperthermophilic maltose-binding protein.

Circularly permuted green fluorescent protein (cGFP) was inserted into the hyperthermophilic maltose binding protein at two different locations. cGFP was inserted between amino acid residues 206 and 207, or fused to the N-terminal of maltose binding protein from Thermotoga maritima. The cloned DNA constructs were expressed in Escherichia coli cells, and purified by metal chelate affinity chromatography. Conformational change upon ligand binding was monitored by the increase in fluorescence intensity. Both of the fusion proteins developed significant fluorescence change at 0.5 mM maltose concentration, whereas their maltose binding affinities and optimum incubation times were different. Fluorescent biosensors based on mesophilic maltose binding proteins have been described in the literature, but there is a growing interest in biosensors based on thermostable proteins. Therefore, the developed protein constructs could be models for thermophilic protein-based fluorescent biosensors.

The covalent bioconjugate of multiwalled carbon nanotube and amino-modified linearized plasmid DNA for gene delivery.

Carbon nanotubes (CNTs) are allotropes of carbon, which have unique physical, mechanical, and electronic properties. Among various biomedical applications, CNTs also attract interest as nonviral gene delivery systems. Functionalization of CNTs with cationic groups enables delivery of negatively charged DNA into cells. In contrast to this well-known strategy for DNA delivery, our approach included the covalent attachment of linearized plasmid DNA to carboxylated multiwalled CNTs (MWCNTs). Carboxyl groups were introduced onto MWCNTs by oxidative treatment, and then the carboxyl groups were activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The whole pQE-70 vector including the gene encoding green fluorescent protein (GFP) was subjected to polymerase chain reaction (PCR) using the modified nucleotide N6-(6-Amino)hexyl-2'-deoxyadenosine-5'-triphosphate. Hence, free amino groups were introduced onto the linearized plasmid. Covalent bonding between the amino-modified plasmid DNA and the carboxylated MWCNTs was achieved via EDC chemistry. The resulting bioconjugate was successfully transformed into chemically competent Escherichia coli cells, without necessity of a heat-shock step at 42°C. The presence of Ca(2+) in transformation medium was required to neutralize the electrostatic repulsion between DNA and negatively charged outer layer of E. coli. The transformants, which were able to express GFP were inspected manually on ampicillin agar plates. Our study represents a novelty with respect to other noncovalent CNT gene delivery systems. Considering the interest for delivery of linear DNA fragments, our study could give insights into further studies.

Development of a novel fluorescent protein construct by genetically fusing green fluorescent protein to the N-terminal of aspartate dehydrogenase.

We developed a fluorescent protein construct by genetically fusing green fluorescent protein (GFP) to aspartate dehydrogenase from Thermotoga maritima. The fusion protein was cloned, heterologously expressed in Escherichia coli cells, and purified by Ni-chelate affinity chromatography. It was then introduced into a measurement cuvette to monitor its fluorescence signal. Aspartate dehydrogenase functioned as the biorecognition element, and aspartate-induced conformational change was converted to a fluorescence signal by GFP. The recombinant protein responded to l-aspartate (l-Asp) linearly within the concentration range of 1-50 mM, and it was capable of giving a fluorescence signal in 1 Min. Although a linear response was also observed for l-Glu, the fluorescence signal was 2.7 times lower than that observed for l-Asp. In the present study, we describe two novelties: development of a genetically encoded fluorescent protein construct for monitoring of l-Asp in vitro, and employment of aspartate dehydrogenase scaffold as a biorecognition element. A few genetically encoded amino-acid biosensors have been described in the literature, but to our knowledge, a protein has not been constructed solely for determination of l-Asp. Periplasmic ligand binding proteins offer high binding affinity in the micromolar range, and they are frequently used as biorecognition elements. Instead of choosing a periplasmic l-Asp binding protein, we attempted to use the substrate specificity of aspartate dehydrogenase enzyme.

Directed evolution of (ßα)(8)-barrel enzymes: establishing phosphoribosylanthranilate isomerisation activity on the scaffold of the tryptophan synthase α-subunit.

Phosphoribosylanthranilate (PRA) isomerase (TrpF) and tryptophan synthase α-subunit (TrpA) are (ßα)(8)-barrel enzymes that are involved in the biosynthesis of tryptophan. They contain a conserved phosphate binding site, which indicates a common evolutionary origin. In order to experimentally back this hypothesis, we have established TrpF activity on the scaffold of TrpA from Salmonella typhimurium using protein engineering. Based on the superposition of crystal structures with bound ligands, two residues in the active site of TrpA were replaced with catalytic residues from TrpF using site-directed mutagenesis. This TrpA variant as well as wild-type TrpA were each subjected to random mutagenesis using error-prone PCR. The two resulting trpA gene libraries were used to transform an auxotrophic Escherichia coli trpF deletion strain, and TrpA variants with PRA isomerisation activity were isolated by in vivo complementation. The amino acid substitutions of the selected TrpA variants were recombined by DNA shuffling, again followed by complementation in vivo. Several TrpA variants were produced in E. coli and purified, and their catalytic TrpF activities were determined in vitro by steady-state enzyme kinetics. Our results support that TrpA and TrpF have evolved by gene duplication and diversification from each other or a common predecessor, and provide insights into the minimum requirements for the catalysis of PRA isomerisation.

Modification of lysozyme with oleoyl chloride for broadening the antimicrobial specificity.

Lysozyme from egg white was modified by covalent attachment of an oleyl group to the free amino groups of lysozyme. The aim of the chemical modification was to develop an effective antimicrobial lysozyme derivative against both gram-negative and gram-positive bacteria. Lysozyme with various degrees of modification was obtained by changing oleoyl chloride/lysozyme mass ratio. Lysozyme derivatives evidently exhibited an antimicrobial effect against Escherichia coli (ATCC 29998). The modification slightly changed the antimicrobial effect of lysozyme derivative against Staphylococcus aureus (ATCC 121002). Since there was a positive correlation between the modification degree and the antimicrobial effect against E. coli, it was concluded that the change in antimicrobial behavior was due to an increase in hydrophobicity of the enzyme molecule enabling it to penetrate through the bacterial membrane of E. coli. It was also shown that oleoyl chloride with an MIC value of 10 mg/mL was effective against both E. coli and S. aureus.

Construction and comparison of Trametes versicolor laccase biosensors capable of detecting xenobiotics.

Amperometric biosensors using laccase from Trametes versicolor as a bioelement were developed for 2,4-dichloro phenoxy acetic acid (2,4-D). Laccase enzyme was immobilized by gelatin and glutaraldehyde onto a Clark oxygen probe and screen printed electrodes (SPEs). Amperometric and chronoamperometric measurements were carried out with the biosensors. First, the effect of laccase activity on the biosensor performances was investigated for both biosensors, and then optimum pH and temperature and also thermal stability of the biosensors were tested. In addition, the detection ranges of some phenolic compounds were obtained by the help of calibration graphs of them. In repeatability studies, variation coefficients and standard deviations for both biosensors were also calculated by the studies done for this purposes. Finally, the biosensors were applied to the determination of 2,4-D in a real herbicide sample.

Maltose biosensing based on co-immobilization of alpha-glucosidase and pyranose oxidase.

A new bi-enzymatic system was designed by co-immobilization of alpha-glucosidase (AG) and pyranose oxidase (PyOx) for maltose analysis. The immobilization was carried out by cross-linking enzyme mixture, chitosan (CHIT) and carbon nanotube (CNT) via glutaraldehyde. The structure of biosensor including enzyme, CHIT, glutaraldehyde and CNT amount together with operational conditions like pH, temperature and applied potential were optimized. Then analytical characterization was performed. A fast linear response of the biosensor was observed for maltose in the concentration range from 0.25 to 2.0 mM at 35 degrees C and pH 6.0. The effect of CNT addition into the immobilization matrix was also investigated. The linear relationships between sensor response (y; microA/cm(2)) and substrate concentration (x; mM) were defined by the equations of y=0.844x+0.029 (R(2)=0.999) and y=0.882x+0.0625 (R(2)=0.996) for AG/PyOx/CHIT and AG/PyOx/CHIT-CNT biosensors, respectively. All other data were also given as comparison of two systems one with CNT-modified and CNT-free. Finally, for the sample application, maltose was analyzed in beer samples. As a result, it has been found that; complex matrix of natural beer samples had no influence on the biosensing response. Also the results were in good agreement with those obtained by spectrophotometric measurements.

Development of an alcohol dehydrogenase biosensor for ethanol determination with toluidine blue O covalently attached to a cellulose acetate modified electrode.

In this work, a novel voltammetric ethanol biosensor was constructed using alcohol dehydrogenase (ADH). Firstly, alcohol dehydrogenase was immobilized on the surface of a glassy carbon electrode modified by cellulose acetate (CA) bonded to toluidine blue O (TBO). Secondly, the surface was covered by a glutaraldehyde/bovine serum albumin (BSA) cross-linking procedure to provide a new voltammetric sensor for the ethanol determination. In order to fabricate the biosensor, a new electrode matrix containing insoluble Toluidine Blue O (TBO) was obtained from the process, and enzyme/coenzyme was combined on the biosensor surface. The influence of various experimental conditions was examined for the characterization of the optimum analytical performance. The developed biosensor exhibited sensitive and selective determination of ethanol and showed a linear response between 1 × 10(-5) M and 4 × 10(-4) M ethanol. A detection limit calculated as three times the signal-to-noise ratio was 5.0 × 10(-6) M. At the end of the 20(th) day, the biosensor still retained 50% of its initial activity.

Noncovalently galactose imprinted polymer for the recognition of different saccharides.

Molecularly imprinted polymers (MIPs) represent a new class of materials possessing high selectivity and affinity for the target molecule. The main goal of this study was to prepare a galactose imprinted polymer and its potential application for the recognition of different saccharides. The selectivity of galactose imprinted polymer for several saccharides; glucose, mannose, fructose, maltose, lactose, sucrose and raffinose was investigated. Macroporous polymer was prepared utilizing ethyleneglycoldimethacrylate as a crosslinking agent, in the presence of galactose as a template molecule with acrylamide as a functional monomer. After the synthesis of polymer, galactose was removed by methanol:acetic acid washing. The selectivity of galactose imprinted polymer for other saccharides was utilized by batch rebinding assay. The arrangement of functional groups within cavities versus shape selectivity is discussed. The results showed that, the orientation of the functional groups was the dominating factor for the selectivity of galactose imprinted polymer. The dissociation constants of polymer were determined by Scatchard analysis.

A microbial biosensor based on bacterial cells immobilized on chitosan matrix.

A bio-electrochemical system consisting of Gluconobacter oxydans DSM 2343 cells as a biological material and carbon nanotube (CNT)-free and CNT-modified chitosan as immobilizing matrices has been developed. The measurement was based on the respiratory activity of the cells estimated by the oxygen consumption at -0.7 V (versus the Ag|AgCl reference electrode) due to the metabolic activity in the presence of substrates. The system was calibrated and dependence of signal amplitude on the measuring conditions and cell amount was studied as well as the substrate specificity, pH, temperature and working potential. The biosensors (CNT-modified and unmodified) were demonstrated for the quantification of glucose in the range of 0.05-1.0 mM, at 30 degrees C and pH 7.0 with the 40 s of response time. The linear relationships between sensor response (y; microA/cm(2)) and substrate concentration (x; mM) were defined by the equations of y=1.160x+0.151 (R(2)=0.990) and y=1.261x+0.197 (R(2)=0.982), respectively. All other data were also given as comparison of two systems one with CNT-modified and CNT-free.

Pseudomonas putida based amperometric biosensors for 2,4-D detection.

Amperometric biosensors using Pseudomonas putida cells as a bioelement were developed for 2,4-dichloro phenoxy acetic acid (2,4-D). After the adaptation process of Pseudomonas putida to 2,4-D, cells were immobilized onto the screen printed graphite electrodes (SPG) as well as Clark oxygen probe by gelatin and glutaraldehyde. Optimum pH, temperature, and stability of the biosensor were investigated. Substrate specificities for various phenolic compounds were also searched. In repeatability studies, variation coefficients and standard deviations for both type of systems were calculated; SPG and Clark electrodes were calculated and results are given as a comparison of two systems. Finally, the biosensors were applied to 2,4-D determination in a real herbicide sample.

Optimization of serotonin imprinted polymers and recognition study from platelet rich plasma.

Molecularly imprinted polymers using serotonin as the template molecule was prepared for selective recognition from platelet rich plasma by non-covalent imprinting approach. Four different monomers (methacrylic acid, acrylamide, 4-vinylpyridine and 2-acrylamido-2-methylpropane sulfonic acid) and acetonitrile and DMSO as porogen were investigated for the first time by bulk polymerization. The molecularly imprinted polymer which was prepared by acrylamide/methacrylic acid had the largest imprinting factor for serotonin. The affinity and specificity of these polymers were evaluated by equilibrium binding experiments. The effect of polarity of the solvents was examined by polymers binding capacity and imprinting factor. According to the Scatchard analysis the K(d) and Q(max) values were calculated as 1.95 micromoll(-1) and 19.129 micromolg(-1), respectively. The polymer was tested to evaluate serotonin from platelet rich plasma and 70% serotonin recovery was found.

A bio-imprinted urease biosensor: Improved thermal and operational stabilities.

Despite the increasing number of applications of biosensors in many fields, the construction of a steady biosensor remains still challenging. The high stability of molecularly bio-imprinted enzymes for its substrate can make them ideal alternatives as recognition elements for sensors. Urease (urea aminohydrolase, EC 3.5.1.5), which catalysis the hydrolysis of urea to ammonia and carbon dioxide, has been used in immobilized form in artificial kidney for blood detoxification. According to one report approximately half a million patients worldwide are being supported by haemodialysis. In this study, the enzyme of urease was first complexed by using a substrate analogue, thiourea, in aqueous medium and then this enzyme was immobilized on gelatin by crosslinking with glutaraldehyde on a glass electrode surface. Similarly, urease noncomplexed with thiourea was also immobilized on a glass electrode in the same conditions. The aim of the study was to compare the two biosensors in terms of their repeatability, pH stability and thermal stability, and also, linear ranges of two biosensors were compared with each other.

Biosensing approach for glutathione detection using glutathione reductase and sulfhydryl oxidase bienzymatic system.

Chitosan membrane with glutathione reductase and sulfhydryl oxidase (SOX) was subsequently integrated onto the surface of spectrographic graphite rods for obtaining a glutathione biosensor. The working principle was based on the monitoring of O(2) consumption that correlates the concentration of glutathione during the enzymatic reaction. A linear relationship between sensor response and concentration was obtained between 0.5 and 2.0 mM for oxidized glutathione (GSSG), and 0.2-1.0 mM for reduced glutathione (GSH) in the presence of 2 microM nicotinamide adenine dinucleotide phosphate (NADPH) under the optimum working conditions. Also, reduced/oxidized glutathione were separated by HPLC and utility of bienzymatic system was investigated as an electrochemical detector for the analysis of these compounds. All data were given as a comparison of two systems: biosensor and diode array detector (DAD).

Shell-core imprinted polyacrylamide crosslinked chitosan for albumin removal from plasma.

Molecularly imprinted polymeric beads were prepared using albumin as the imprinted molecule, acrylamide as the functional monomer, and epichlorhydrin crosslinked chitosan beads as the supporting matrix. The recognition and binding of the imprinted beads was also tested with human plasma for the targeted removal of HSA. Plasma is a rich source of biochemical products that can act as biomarkers of disease or physiological status of a patient. The application of current proteomic technologies in the search for potential diagnostic/prognostic indicators in the plasma of patients is limited by highly abundant albumin that constitute >60% of the total plasma proteins. Removal of abundant proteins will help in the discovery and detection of less abundant proteins that may prove to be informative. The adsorption capacities of the imprinted polymeric beads for pure human serum albumin and plasma albumin were estimated as 92% and 80%, respectively. The easy preparation protocol of derivatised beads and good protein recognition properties make them attractive for biotechnologic approaches.

Two biosensors for phenolic compounds based on mushroom (Agaricus bisporus) homogenate: comparison in terms of some important parameters of the biosensors.

Interest in molecular imprinted polymer techniques has increased because they allows for the improvement of some stability characteristics of enzymes. The high stability of molecularly imprinted enzymes for a substrate can make them ideal alternatives as recognition elements for sensors. A bioimprinted mushroom tissue homogenate biosensor was constructed in a very simple way. For this purpose, sulfite was used. The enzyme, polyphenol oxidase, was first complexed by using a competitive inhibitor, sulfite, in aqueous medium and then the enzyme was immobilized on gelatin by crosslinking with glutaraldehyde on a glass electrode surface. Similarly, polyphenol oxidase uncomplexed with sulfite was also immobilized on a glass electrode in the same conditions. The aim of the study was to compare the two biosensors in terms of their repeatability and thermal, pH, and operational stability; also, the linear ranges of the two biosensors were compared with each other.

Electrical wiring of Pseudomonas putida and Pseudomonas fluorescens with osmium redox polymers.

Two different flexible osmium redox polymers; poly(1-vinylimidazole)12-[Os-(4,4'-dimethyl-2,2'-di'pyridyl)2Cl2](2+/+) (osmium redox polymer I) and poly(vinylpyridine)-[Os-(N,N'-methylated-2,2'-biimidazole)3](2+/3+) (osmium redox polymer II) were investigated for their ability to efficiently "wire" Pseudomonas putida ATCC 126633 and Pseudomonas fluorescens (P. putida DSM 6521), which are well-known phenol degrading organisms, when entrapped onto cysteamine modified gold electrodes. The two Os-polymers differ in redox potential and the length of the side chains, where the Os(2+/3+)-functionalities are located. The bacterial cells were adapted to grow in the presence of phenol as the sole source of organic carbon. The performance of the redox polymers as mediators was investigated for making microbial sensors. The analytical characteristics of the microbial sensors were evaluated for determination of catechol, phenol and glucose as substrates in both batch analysis and flow analysis mode.

Determination of phenolic acids using Trametes versicolor laccase.

Two biosensors based on Trametes versicolor laccase (TvL) were developed for the determination of phenolic compounds. Commercial oxygen electrode and ferrocene-modified screen-printed graphite electrodes were used for preparation of laccase biosensors. The systems were calibrated for three phenolic acids. Linearity was obtained in the concentration range 0.1-1.0muM caffeic acid, 0.05-0.2muM ferulic acid, 2.0-14.0muM syringic acid for laccase immobilised on a commercial oxygen electrode and 2.0-30.0muM caffeic acid, 2.0-10.0muM ferulic acid, 4.0-30.0muM syringic acid for laccase immobilised on ferrocene-modified screen-printed electrodes. Furthermore, optimal pH, temperature and thermal stability studies were performed with the commercial oxygen electrode. Both electrodes were used for determination of a class of phenolic acids, achieving a cheap and fast tool and an easy to be used procedure for screening real samples of human plasma.

Effects of mediators on the laccase biosensor response in paracetamol detection.

An enzyme electrode suitable for paracetamol detection was developed by immobilizing laccase on a dissolved-oxygen probe surface. The immobilization procedure was achieved by means of gelatin, which was then cross-linked with glutaraldehyde. The measurement was based on the detection of oxygen consumption in relation to analyte oxidation. The optimum experimental conditions for the biosensor were investigated and the system was calibrated for paracetamol. Also the effects of three different mediators, namely HBT (1-hydroxybenzotriazole), VLA [violuric acid (5-isonitrosobarbituric acid)] and TEMPO (2,2',6,6'-tetramethylpiperidine-N-oxyl radical) were tested for the biosensor's response. As a result, it was observed that HBT has a remarkable effect on the signal by providing more oxygen consumption during the enzymatic reaction. A linear relationship between sensor responses and analyte concentrations was obtained over the concentration range 2.0-15.0 microM, whereas, in the presence of the mediator HBT, this range became 0.5-3.0 microM.