Biomedical subjects

Leonidas G Bachas

Publications and source records attributed to Leonidas G Bachas.

At least 19 recordsLinked to original sources

PubMed2006

Modified Fenton reaction for trichlorophenol dechlorination by enzymatically generated H2O2 and gluconic acid chelate.

Glucose oxidase is a well-known enzyme that catalyzes the oxidation of beta-D-glucose to produce gluconic acid and hydrogen peroxide. Fenton reaction is a powerful oxidation technology used for the oxidation of groundwater pollutants. For the application of Fenton reaction in groundwater remediation, successful operation of Fenton reaction near neutral pH, and on-site generation of both H2O2 and chelate will be beneficial. The focus of this experimental study was to couple the glucose oxidation reaction with chelate-based Fenton reaction. The idea was to use the hydrogen peroxide and chelate gluconic acid generated during glucose oxidation for the dechlorination of 2,4,6-trichlorophenol (TCP) by Fenton reaction. The oxidation of glucose was achieved using the enzyme in free and immobilized forms. The rate of production of hydrogen peroxide was determined for each system, and was used to estimate the time required for complete consumption of glucose during the process, thus avoiding any traces of glucose in the Fenton reaction. In the case of free enzyme reaction, separation of the enzyme was achieved using an ultrafiltration membrane before initiating the Fenton reaction. The oxidation of TCP by Fenton reaction was performed at varying ratios of gluconic acid/Fe, and its effect on the decomposition of TCP and H2O2 was studied. TCP degradation was studied both in terms of parent compound degradation and free chloride generation.

Chelating Agents↗

PubMed2006

Biosensor incorporating cell barrier architectures for detecting Staphylococcus aureus alpha toxin.

Alpha toxin is a common virulent factor of Staphylococcus aureus and is believed to play crucial roles in pathogenicity induced by S. aureus. Alpha toxin is also known to induce permeability to endothelial cell monolayers in vitro due to the formation of interendothelial gaps. The present study is directed towards measuring alpha toxin using a whole-cell-based biosensor. The biosensor, consisting of a confluent monolayer of human umbilical vein endothelial cells (HUVECs) on a potassium ion-selective electrode, takes advantage of cell permeability dysfunction to detect the presence of small quantities of alpha toxin. When a confluent monolayer of cells was formed on the membrane surface, the response of the electrode toward the marker ion, potassium, was inhibited. Upon exposing this sensor to varying concentrations of alpha toxin for 20 min, an increase in sensor response to potassium was observed. The response thus obtained was indirectly related to the concentration of alpha toxin. The detection limit of this sensor for alpha toxin was found to be 0.1 ng/ml. Cell monolayers were stained with silver nitrate to quantify the formation of intercellular gaps as well as to study the effect of this toxin on HUVECs morphology. A strong positive correlation was observed between the response obtained from the biosensor and the area of the intercellular gaps. Silver staining also revealed the tendency of cells to round up upon being exposed to alpha toxin.

Bacterial Toxins↗

PubMed2006

Microfabrication of screen-printed nanoliter vials with embedded surface-modified electrodes.

A self-contained ion-selective sensing system within a nanoliter-volume vial has been developed by integrating screen printing, laser ablation, and molecular imprinting techniques. Screen printing and laser ablation are used in tandem to fabricate nanoliter-volume vials with carbon and Ag/AgCl ring electrodes embedded in the sidewalls. Using multisweep cyclic voltammetry, the surface of the carbon electrode can be modified with a polypyrrole film. By polymerizing pyrrole in the presence of nitrate, pores complementary to the nitrate anion in size, shape, and charge distribution are formed in the resulting film. Electrochemical cells modified with this nitrate-imprinted polypyrrole film show a near-Nernstian response to nitrate, and excellent reproducibility. The integration of molecular recognition and electrochemical response in the nanoliter vials is demonstrated by the detection of as little as 0.36 ng nitrate in nanoliter-volume samples. The integration of tailored molecular recognition within nanoliter vials via established fabrication and imprinting protocols should result in a number of nanosensor devices with applications in BioMEMS and micro total analysis systems.

Electrochemistry↗

PubMed2006

ClcR-based biosensing system in the detection of cis-dihydroxylated (chloro-)biphenyls.

Polychlorinated biphenyls (PCBs) are a group of organic pollutants that are persistent when released into the environment. Among the metabolites of PCBs, dihydroxylated PCBs are also considered as toxic compounds. Various studies have shown that dihydroxylated PCBs affect the reproductive, immune, nervous, and endocrine systems. Detection of these chemicals in environmental and biological samples could provide first-hand information about their levels and lead to a better understanding of their role in toxicity. To that end, we developed a sensing system for the detection of dihydroxylated PCBs based on the clc operon. The Pseudomonas putida clc operon encodes a catabolic pathway for degradation of chlorocatechols, which are major metabolites of a large number of chlorinated compounds. In P. putida, the expression of these genes is regulated by a protein encoded by the gene clcR located upstream from the clcABD genes. We demonstrate here for the first time that dihydroxy PCBs can also induce the clc operon. Our sensing system employs P. putida bacteria harboring a plasmid in which the reporter gene, lacZ, is under the control of the regulatory protein ClcR. Consequently, when exposed to dihydroxy PCBs, the bacteria express beta-galactosidase in an amount related to the concentration of the corresponding dihydroxy PCB. Various dihydroxylated PCBs, differing in the number and position of chlorines and in the position of hydroxyls, were tested for their ability to induce expression of beta-galactosidase. Detection limits as low as 1 x 10(-6) mol L(-1) were obtained for various dihydroxylated PCBs.

Biosensing Techniques↗

PubMed2006

Integration of microcolumns and microfluidic fractionators on multitasking centrifugal microfluidic platforms for the analysis of biomolecules.

This work demonstrates the development of microfluidic compact discs (CDs) for protein purification and fractionation integrating a series of microfluidic features, such as microreservoirs, microchannels, and microfluidic fractionators. The CDs were fabricated with polydimethylsiloxane (PDMS), and each device contained multiple identical microfluidic patterns. Each pattern employed a microfluidic fractionation feature with operation that was based on the redirection of fluid into an isolation chamber as a result of an overflow. This feature offers the advantage of automated operation without the need for any external manipulation, which is independent of the size and the charge of the fractionated molecules. The performance of the microfluidic fractionator was evaluated by its integration into a protein purification microfluidic architecture. The microfluidic architecture employed a microchamber that accommodated a monolithic microcolumn, the fractionator, and an isolation chamber, which was also utilized for the optical detection of the purified protein. The monolithic microcolumn was polymerized "in situ" on the CD from a monolith precursor solution by microwave-initiated polymerization. This technique enabled the fast, efficient, and simultaneous polymerization of monoliths on disposable CD microfluidic platforms. The design of the CD employed allows the integration of various processes on a single microfluidic device, including protein purification, fractionation, isolation, and detection.

Microfluidic Analytical Techniques↗

PubMed2006

Decyl methacrylate-based microspot optodes.

Optode sensing membranes employing decyl methacrylate cross-linked with 1,6-hexanediol dimethacrylate as the polymer support were fabricated by a direct microspotting method on several surfaces. Photopolymerization was used to attach the microspots to the substrate. Using this method, diameters in the micrometer domain were obtained. Silanized glass, poly(methyl methacrylate) (PMMA), polycarbonate, and poly(dimethylsiloxane) were tested as possible substrates. Both polypropylene tips and the steel tips of drafting pens were used for spotting. It was determined that both silanized glass and PMMA gave working optodes, but the ones on PMMA did not fit the theoretical model. Diameters of 994 +/- 80 and 1279 +/- 85 microm were obtained on silanized glass and PMMA, respectively, using the polypropylene tips for spotting. Different size optodes were fabricated using 0.35- and 0.50-mm steel drafting pen tips. The 0.35-mm tips produced diameters of 895 +/- 26 and 688 +/- 54 microm on silanized glass and PMMA, respectively, and the 0.50-mm tips produced diameters of 1274 +/- 94 microm on silanized glass and 839 +/- 28 microm on PMMA. Thus, the microspot size can be controlled based on the hydrophobicity of the surface and the size of the tip used for spotting. Calibration plots of potassium optode microspots indicated that miniaturization does not alter response characteristics, such as selectivity, response time, and dynamic range, of the optodes.

Journal Article↗

PubMed2006

Intersubunit disulfide interactions play a critical role in maintaining the thermostability of glucose-6-phosphate dehydrogenase from the hyperthermophilic bacterium Aquifex aeolicus.

Proteins from thermophilic microorganisms are stabilized by various mechanisms to preserve their native folded states at higher temperatures. A thermostable glucose-6-phosphate dehydrogenase (tG6PDH) from the hyperthermophilic bacterium Aquifex aeolicus was expressed as a recombinant protein in Escherichia coli. The A. aeolicus G6PDH is a homodimer exhibiting remarkable thermostability (t1/2 = 24 hr at 90 degrees C). Based on homology modeling and upon comparison of its structure with human G6PDH, it was predicted that cysteine 184 of one subunit could form a disulfide bond with cysteine 352 of the other subunit resulting in reinforced intersubunit interactions that hold the dimer together. Site-directed mutagenesis was performed on tG6PDH to convert C184 and C352 to serines. The tG6PDH double mutant exhibited a dramatic decrease in the half-life from 24 hr to 3 hr at 90 degrees C. The same decrease in half-life was also found when either C184 or C352 was mutated to serine. The result indicates that C184 and C352 may play a crucial role in strengthening the dimer interface through disulfide bond formation, thereby contributing to the thermal stability of the enzyme.

Bacteria↗

PubMed2006

Poly(amino acid)-facilitated electrochemical growth of metal nanoparticles.

Poly(amino acids) are natural chelating agents for various metal ions. Zinc ions were encapsulated in situ in a conductive polypyrrole film using polyglutamic acid as a localized complexing agent within the film. The subsequent electrochemical reduction of the metal ions to zero-valent metal leads to the formation of the nanoparticles. The electrochemical approach demonstrated in this report provides facile regeneration of the particles and also prevents aggregation of nanoparticles in the conductive polymeric film. The correlation of the amount of zinc with the thickness of the film indicates that the zinc resides largely in the outer layer of the film. TEM and EDS data show that the nanoparticles formed are composed of zinc and are 18 +/- 7 nm in diameter. The nanoparticle/ polymer composite was used to reduce halogenated organics, indicating its potential usefulness in remediation applications.

Amino Acids↗

PubMed2005

Enhancing the blood compatibility of ion-selective electrodes.

In vivo monitoring of various analytes is important for many bioanalytical and biomedical applications. The crucial challenge in this type of applications is the interaction of the sensor with the host environment, which is qualitatively described by the term biocompatibility. This review discusses recent advances in methods and materials used for the improvement of the biocompatibility of ion-selective electrodes especially as it relates to their interaction with blood components.

Biocompatible Materials↗

PubMed2005

Vascular endothelial growth factor as a biomarker for the early detection of cancer using a whole cell-based biosensor.

Vascular endothelial growth factor (VEGF) is a cytokine and endothelial cell (EC) mitogen that has been studied for its role in angiogenesis of malignant tumors. Elevated quantities of VEGF in the serum and plasma of patients have been correlated with the presence of cancer and metastasis. Since VEGF induces hyperpermeability of EC monolayers, this protein can be detected in vitro with a whole cell-based biosensor. This biosensor consists of a confluent monolayer of human umbilical vein endothelial cells (HUVECs) attached to a cellulose triacetate (CTA) membrane of an ion-selective electrode (ISE). Previous studies regarding this biosensor have shown that when the biosensor was exposed to a model toxin, such as histamine, the response of the biosensor served as an indirect measurement of the presence of histamine. Similarly, the biosensor responds to the presence of VEGF, but is much more sensitive because VEGF is known to be 50,000-fold more potent than histamine when inducing EC hyperpermeability. The ISE response increased with increasing VEGF concentration. Since lower concentrations required more exposure time, the detection limit was established as a function of exposure time (2-10 h). The practical applicability of the biosensor was also established with cultured human melanoma cells WM793 (nonmetastatic) and 1205LU (metastatic). The resultant change in the potential values revealed significant production of VEGF from the 1205LU cells. A VEGF ELISA was performed to confirm the VEGF concentration in each sample. The biosensor closely predicted the concentrations determined through the ELISA. These results support the use of a cell-based ISE as a quick screening method for the presence of VEGF.

Biomarkers↗

PubMed2005

Magnetoelastic transducers for monitoring coagulation, clot inhibition, and fibrinolysis.

Magnetoelastic transduction has been used to detect and monitor the viscosity changes that occur during the biological reactions of coagulation and fibrinolysis. Magnetoelastic sensors can be used, because the characteristic resonance frequency of the magnetoelastic strip shifts in response to the changes in fluid viscosity. At a set frequency, the output signal can be obtained over time to develop a coagulation and/or dissolution profile, which display the change in viscosity of a plasma sample that has undergone either coagulation or fibrinolysis. For coagulation screening, an exogenous tissue factor is added to an anticoagulated plasma sample to initiate coagulation. Further studies were performed to investigate fibrinolysis through the addition of plasmin. Plasmin is used in two different ways-as a competitive inhibitor before the initiation of clotting and also as a protease to dissolve the previously formed clot. This method is a viable option for the monitoring of processes that are paramount to maintaining hemostasis.

Anticoagulants↗

PubMed2005

Genetically engineered protein in hydrogels tailors stimuli-responsive characteristics.

Certain proteins undergo a substantial conformational change in response to a given stimulus. This conformational change can manifest in different manners and result in an actuation, that is, catalytic or signalling event, movement, interaction with other proteins, and so on. In all cases, the sensing-actuation process of proteins is initiated by a recognition event that translates into a mechanical action. Thus, proteins are ideal components for designing new nanomaterials that are intelligent and can perform desired mechanical actions in response to target stimuli. A number of approaches have been undertaken to mimic nature's sensing-actuating process. We now report a new hybrid material that integrates genetically engineered proteins within hydrogels capable of producing a stimulus-responsive action mechanism. The mechanical effect is a result of an induced conformational change and binding affinities of the protein in response to a stimulus. The stimuli-responsive hydrogel exhibits three specific swelling stages in response to various ligands offering additional fine-tuned control over a conventional two-stage swelling hydrogel. The newly prepared material was used in the sensing, and subsequent gating and transport of biomolecules across a polymer network, demonstrating its potential application in microfluidics and miniaturized drug-delivery systems.

Calcium↗

PubMed2004

Investigation into the applicability of the centrifugal microfluidics platform for the development of protein-ligand binding assays incorporating enhanced green fluorescent protein as a fluorescent reporter.

The incorporation of a protein-ligand binding assay into a centrifugal microfluidics platform is described. The platform itself is a disc-shaped polymer substrate, upon which a series of microfluidic channels and reservoirs have been machined. Centrifugal microfluidics platforms require no internal moving parts, and fluid propulsion is achieved solely through rotation of the disc. Fluid flow is controlled by passive valves, the opening of which is dependent on the angular frequency of the rotating platform, the channel dimensions, and the physical properties of the fluid. To evaluate the effectiveness of incorporating a protein-based assay onto the centrifugal microfluidics analytical platform, a class-selective, homogeneous assay for the detection of phenothiazine antidepressants was employed. This class of drugs is known to bind to calmodulin, a calcium binding protein. Specifically, a fusion protein between calmodulin and enhanced green fluorescent protein was utilized. Calmodulin undergoes a conformational change upon binding to phenothiazines that alters the fluorescence properties of the attached fluorescent protein, which can be correlated to the concentration of the drug present. Another important aspect of this work was to study the efficacy of the platform to perform reconstitution assays. To do this, the biological reagent was dried on the platform and rehydrated to carry out the assay. The ability to prealiquot reagents on the platform should enhance its versatility and portability. The integration of protein-based assays in this platform should be useful in the design of analytical systems for high-throughput screening of pharmaceuticals and clinical diagnostics.

Antidepressive Agents↗

PubMed2004

Development of a whole-cell-based biosensor for detecting histamine as a model toxin.

A novel whole-cell potentiometric biosensor for screening of toxins has been developed. The constructed biosensor consists of a confluent monolayer of human umbilical vein endothelial cells (HUVECs) attached to an ion-selective cellulose triacetate (CTA) membrane modified with a covalently attached RGD (arginine-glycine-aspartic acid) peptide sequence. When the HUVECs form a confluent monolayer, ion transport is almost completely inhibited, thereby reducing the response of the ion-selective electrode (ISE). When the monolayer is exposed to agents that increase its permeability (e.g., toxins), ions can diffuse through the membrane, and a potential response from the ISE is achieved. Histamine, a model toxin that increases the permeability of HUVEC monolayers, was used in this study. When the cell-based membranes are exposed to varying concentrations of histamine, the overall response increases with increasing histamine concentration. Thus, the measured potential is an indirect measurement of the histamine concentration. Further experiments were performed for a similar molecule, l-histidine, to test for selectivity. The cell permeability was unaffected by l-histidine, and the sensor response remained unchanged. This type of sensor should find multiple applications in medical, food, and environmental fields and in homeland security.

Biosensing Techniques↗

PubMed2004

Carbon nanotube aqueous sol-gel composites: enzyme-friendly platforms for the development of stable biosensors.

A new type of composite material based on carbon nanotubes and an aqueous sol-gel process has been developed. The electrochemical characteristics of these composites were investigated and compared to composites made with an alkoxy silane sol-gel process. The use of carbon nanotubes, as the conductive part of the composite, facilitated fast electron transfer rates. The feasibility of this type of composite for the development of biosensors was demonstrated using l-amino acid oxidase. The stability of the enzyme was increased when it was encapsulated in the aqueous sol-gel, and the sensor retained more that 50% of its response after 1 month of testing.

Biosensing Techniques↗

PubMed2004

Emerging issues: nutritional awareness in environmental toxicology.

Based on recent evidence, we hypothesize that nutrition can modulate the toxicity of environmental pollutants and thus modulate health and disease outcome associated with chemical insults. For example, certain dietary fats may increase the risk to environmental insult induced by polychlorinated biphenyls (PCBs), and fruits and vegetables, rich in antioxidant and anti-inflammatory nutrients or bioactive compounds, may provide protection. Nutritional awareness in environmental toxicology is critical, because of opportunities to develop dietary guidelines which specifically target exposed populations. Nutrition may provide the most sensible means to develop primary prevention strategies of diseases associated with environmental toxicology.

Awareness↗

PubMed2004

Protein immobilization on carbon nanotubes through a molecular adapter.

A new approach to the modification of carbon nanotubes with biomolecules for the development of nanoscale biosensors is presented. Alkaline phosphatase was immobilized on the surface of multi-wall carbon nanotubes utilizing a layer-by-layer methodology. Carbon nanotubes were incubated with streptavidin, resulting in the formation of a protein layer on the surface of the nanotubes. Biotinylated alkaline phosphatase was then allowed to bind to streptavidin, anchoring the sensing protein onto the surface. Electrochemical biosensors were constructed by using carbon nanotubes compacted into pellets. 1-Naphthyl phosphate, which is hydrolyzed by alkaline phosphatase to the electroactive 1-naphthol, was used as a substrate. Electrodes constructed in this manner were observed to generate an electrochemical signal that was a function of substrate concentration.

Adsorption↗

PubMed2003

Covalent immobilization of beta-galactosidase onto a gold-coated magnetoelastic transducer via a self-assembled monolayer: toward a magnetoelastic biosensor.

The enzyme beta-galactosidase has been covalently immobilized onto a gold-coated magnetoelastic film via a self-assembled monolayer (SAM) of omega-carboxylic acid alkylthiol. Use of magnetoelastic transduction allows for the wireless monitoring of enzymatic activity through the associated change in the frequency and amplitude of magnetic fields. The formations of SAMs of 3-mercaptopropanoic acid and thioctic acid were monitored by magnetoelastic transduction. After coupling of beta-galactosidase to the SAMs, the enzyme activity was monitored by using a substrate that forms an insoluble product upon action of the enzyme. Specifically, an indolyl galactopyranoside substrate was employed in conjunction with an azo dye as the precipitating system. The immobilized enzyme was evaluated and found to have an apparent Michaelis-Menten constant (KM) of 1.2 mM for the indolyl galactopyranoside. Calibration plots for both substrates and inhibitors were generated to establish the versatility of this sensing system. Kinetic parameters for nonprecipitating substrates were determined in conjunction with a precipitating enzymatic substrate by way of a competitive inhibition study using beta-galactosidase attached to magnetoelastic strips. The methods developed within this work allow for the fabrication of wireless enzyme sensing systems, which can also be used as another means of screening for enzyme inhibitors.

Biosensing Techniques↗