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Biomedical subjects

L G Bachas

Publications and source records attributed to L G Bachas.

At least 19 recordsLinked to original sources
PubMed2001

Reducing the thrombogenicity of ion-selective electrode membranes through the use of a silicone-modified segmented polyurethane.

The susceptibility of segmented polyurethanes (SPUs) to in vivo oxidative cleavage and hydrolysis constitutes a drawback in the use of these materials in the fabrication of implantable devices. The introduction of poly(dimethylsiloxane) (PDMS) groups into the polymer main chain has been previously reported to enhance the stability of SPUs. Herein, we evaluated the use of BioSpan-S, a silicone-modified SPU, in the design of membranes for cation-selective electrodes. The resulting electrodes exhibited good potentiometric response with all of the tested ionophores (valinomycin, sodium ionophore X, and nonactin). The obtained selectivity coefficients meet the selectivity requirements for the determination of sodium and potassium in blood. Moreover, as reflected by SEM studies, membranes prepared with BioSpan-S showed less adhesion of platelets than membranes prepared with conventional poly(vinyl chloride) (PVC). These results lead to the conclusion that BioSpan-S would be an appropriate candidate for the fabrication of implantable ion-selective electrodes.

Coated Materials, Biocompatible↗
PubMed2001

Development of a fully integrated analysis system for ions based on ion-selective optodes and centrifugal microfluidics.

A fully integrated, miniaturized analysis system for ions based on a centrifugal microfluidics platform and ion-selective optode membranes is described. The microfluidic architecture is composed of channels, five solution reservoirs, a measuring chamber, and a waste reservoir manufactured onto a disk-shaped substrate of poly(methyl methacrylate). Ion-selective optode membranes, composed of plasticized poly(vinyl chloride) impregnated with an ionophore, a proton chromoionophore, and a lipophilic anionic additive, were cast, with a spin-on device, onto a support layer and then immobilized on the disk. Fluid propulsion is achieved by the centrifugal force that results from spinning the disk, while a system of valves is built onto the disk to control flow. These valves operate based on fluid properties and fluid/substrate interactions and are controlled by the angular frequency of rotation. With this system, we have been able to deliver calibrant solutions, washing buffers, or "test" solutions to the measuring chamber where the optode membrane is located. An analysis system based on a potassium-selective optode has been characterized. Results indicate that optodes immobilized on the platform demonstrate theoretical responses in an absorbance mode of measurement. Samples of unknown concentration can be quantified to within 3% error by fitting the response function for a given optode membrane using an acid (for measuring the signal for a fully protonated chromoionophore), a base (for fully deprotonated chromoionophore), and two standard solutions. Further, the ability to measure ion concentrations by employing one standard solution in conjunction with acid and base and with two standards alone were studied to delineate whether the current architecture could be simplified. Finally, the efficacy of incorporating washing steps into the calibration protocol was investigated.

Calibration↗
PubMed2001

Polycysteine and other polyamino acid functionalized microfiltration membranes for heavy metal capture.

Polycysteine and other polyamino acid functionalized microfiltration membrane sorbents work exceptionally well for the removal and recovery of toxic heavy metals from aqueous streams. These are high capacity sorbents (0.3-3.7 mg/cm2) with excellent accessibility and selectivity for heavy metals, such as Hg(II), Pb(II), and Cd(II) over nontoxic components such as calcium. Polycysteine functionalized membranes work particularly well for metals such as Hg(II) and Cd(II), even in high total dissolved solids containing streams. Parameters such as permeate flow rate,feed metal concentration, and counterion (for Hg(II)) have also been found to influence sorbent behavior. For multicomponent systems, polyglutamic acid functionalized membranes have been found to selectively sorb Pb(II) versus Cd(II). Selective sorption of Cr(III) has also been observed with actual waste streams containing several heavy metals, hardness, and high sodium (2,000 mg/L). The high capacity, site accessibility, and ease of regeneration of these membrane-based sorbents make them ideal for environmental separations when volume reduction or selective recovery is required.

Absorption↗
PubMed2001

Use of a biomimetic peptide in the design of a competitive binding assay for biotin and biotin analogues.

A competitive binding assay for biotin, biocytin, and desthiobiotin utilizing a genetically engineered enzyme-ligand conjugate is described herein. This assay is unique in that the enzyme-ligand conjugate consists of the streptavidin binding peptide Strep-tag II, which mimics the binding of biotin to streptavidin, rather than biotin itself. This allows for the construction of a well-defined, oligosubstituted enzyme-ligand conjugate for which the site of attachment of the ligand on the enzyme is known precisely. The assay has detection limits of 5 x 10(-8) M for biotin, 1 x 10(-7) M for biocytin, and 2 x 10(-6) M for desthiobiotin, and it serves as a model system in that it demonstrates the feasibility of using enzyme-ligand conjugates in which a peptide mimic of the analyte ligand is genetically fused to the enzyme. This avoids the problems associated with covalent attachment of the ligand to the enzyme, such as multiple substitution of the ligand and variability of the site of attachment. To our knowledge, this is the first example of using an enzyme-peptide mimic conjugate to detect a nonpeptide analyte.

Amino Acid Sequence↗
PubMed2001

Improving the activity of immobilized subtilisin by site-directed attachment through a genetically engineered affinity tag.

An octapeptide affinity tag, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (temied FLAG), was genetically fused to the C-terminus of subtilisin BPN' (SBT) from Bacillus amyloliquefaciens. The fusion protein SBT-FLAG was immobilized to nonporous polystyrene and silica beads both in a site-directed and a random fashion. Site-directed immobilization was achieved by employing the interaction between protein A and a monoclonal antibody specific for the FLAG peptide, while random immobilization was obtained by using glutaraldehyde as a cross-linking reagent. The activity of the immobilized enzymes was compared. It was found that the site-directed subtilisin had higher catalytic efficiency, kcat/KM, which was more than 7-fold of that of the randomly immobilized enzyme. It was also noted that the site-directly immobilized enzyme had superior storage stability over the homogeneous enzyme.

Amino Acid Sequence↗
PubMed2001

Monitoring of bacteria growth using a wireless, remote query resonant-circuit sensor: application to environmental sensing.

A new technique is presented for in-vivo remote query measurement of the complex permittivity spectra of a biological culture solution. A sensor comprised of a printed inductor-capacitor resonant-circuit is placed within the culture solution of interest, with the impedance spectrum of the sensor measured using a remotely located loop antenna; the complex permittivity spectra of the culture is calculated from the measured impedance spectrum. The remote query nature of the sensor platform enables, for example, the in-vivo real-time monitoring of bacteria or yeast growth from within sealed opaque containers. The wireless monitoring technique does not require a specific alignment between sensor and antenna. Results are presented for studies conducted on laboratory strains of Bacillus subtilis, Escherichia coli JM109, Pseudomonas putida and Saccharomyces cerevisiae.

Bacteria↗
PubMed2001

Orientation specific immobilization of organophosphorus hydrolase on magnetic particles through gene fusion.

Recombinant DNA technology has been utilized to fuse an octapeptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (FLAG), to the C-terminus of organophosphorus hydrolase (OPH, EC 3.1.8.1), an enzyme capable of hydrolyzing organophosphate compounds, such as insecticides and nerve gas agents. The recombinant OPH-FLAG was immobilized onto magnetic beads coated with protein A in the following ways: (a) site-directly through a monoclonal antibody (MAb) specific for the FLAG peptide; (b) through the MAb that was randomly tethered to the beads using glutaraldehyde; (c) randomly by cross-linking OPH-FLAG to protein-coated beads using glutaraldehyde. Kinetic studies demonstrated that the site-directly immobilized enzyme maintained the highest catalytic efficiency. The orientation specific immobilization strategy described in this article can be applied to other proteins, and therefore, it may find potential applications in the design of biosensors, biocatalysts, and bioreactors having immobilized proteins as their biorecognition elements.

Animals↗
PubMed2000

Tripodal ionophore with sulfate recognition properties for anion-selective electrodes.

Ionophore topology has a profound effect on the behavior of ion-selective electrodes. This is demonstrated with a new class of ionophores that incorporates aminochromenone moieties linked through urea spacers to different scaffolds that preorganize the ionophore binding cleft into tripodal topologies. Tris(2-aminoethylamine) and cis-1,3,5-tris(aminomethyl)cyclohexane were employed as the scaffolds. The two differ in their rigidity and in the size of ionophore cavity that they create. The electrodes based on the ionophore that incorporates the tris(2-aminoethylamine) scaffold show anti-Hofmeister behavior with an improved selectivity for sulfate. In contrast, the ionophore with the cis-1,3,5-tris(aminomethyl)cyclohexane scaffold exhibits a more Hofmeister-like response.

Anions↗
PubMed2000

A selective optical sensor based on [9]mercuracarborand-3, a new type of ionophore with a chloride complexing cavity.

A highly selective optical sensor for chloride, based on the multidentate Lewis acid ionophore [9]mercuracarborand-3, is described herein. This sensor is constructed by embedding the mercuracarborand ionophore, a suitable pH-sensitive lipophilic dye, and lipophilic cationic sites in a plasticized polymeric membrane. The multiple complementary interactions offered by the preorganized complexing cavity of [9]mercuracarborand-3 is shown to control the anion selectivity pattern of the optical film. The film exhibits a significantly enhanced selectivity for chloride over a variety of lipophilic anions such as perchlorate, nitrate, salicylate, and thiocyanate. Furthermore, the optical selectivity coefficients obtained for chloride over other biologically relevant anions are shown to meet the selectivity requirements for the determination of chloride in physiological fluids, unlike previously reported chloride optical sensors. In addition, the optical film responds to chloride reversibly over a wide dynamic range (16 microM-136 mM) with fast response and recovery times.

Algorithms↗
PubMed2000

Chlorocatechol detection based on a clc operon/reporter gene system.

A sensitive and selective sensing system for chlorocatechols (3-chlorocatechol and 4-chlorocatechol) was developed based on Pseudomonas putida bacteria harboring the plasmid pSMM50R-B'. In this plasmid, the regulatory protein of the clc operon, ClcR, controls the expression of the reporter enzyme beta-galactosidase. When bacteria containing components of the clc operon are grown in the presence of chlorocatechols, ClcR activates the clcA promoter, which is located upstream from the beta-galactosidase gene. Thus, the concentration of chlorocatechols can be related to the production of beta-galactosidase in the bacteria. The concentration of beta-galactosidase expressed in the bacteria was determined by measuring the chemiluminescence signal emitted with the use of a 1,2-dioxetane substrate. ClcR has a high specificity for chlorocatechols and provides the sensing system with high selectivity. This was demonstrated by evaluating several structurally related organic compounds as potential interfering agents. Both 3-chlorocatechol and 4-chlorocatechol can be detected with this sensing system at concentrations as low as 8 x 10(-10) and 2 x 10(-9) M, respectively, using a 2-h induction period. In the case of 3-chlorocatechol, a highly selective sensing system was developed that can detect this species at concentrations as low as 6 x 10(-8) M after a 5-min induction period; the presence of 4-chlorocatechol at concentrations as high as 2 x 10(-4) M did not interfere with this system.

Arabidopsis Proteins↗
PubMed1999

Controlled layer-by-layer immobilization of horseradish peroxidase.

Horseradish peroxidase (HRP) was biotinylated with biotinamidocaproate N-hydroxysuccinimide ester (BcapNHS) in a controlled manner to obtain biotinylated horseradish peroxidase (Bcap-HRP) with two biotin moieties per enzyme molecule. Avidin-mediated immobilization of HRP was achieved by first coupling avidin on carboxy-derivatized polystyrene beads using a carbodiimide, followed by the attachment of the disubstituted biotinylated horseradish peroxidase from one of the two biotin moieties through the avidin-biotin interaction (controlled immobilization). Another layer of avidin can be attached to the second biotin on Bcap-HRP, which can serve as a protein linker with additional Bcap-HRP, leading to a layer-by-layer protein assembly of the enzyme. Horseradish peroxidase was also immobilized directly on carboxy-derivatized polystyrene beads by carbodiimide chemistry (conventional method). The reaction kinetics of the native horseradish peroxidase, immobilized horseradish peroxidase (conventional method), controlled immobilized biotinylated horseradish peroxidase on avidin-coated beads, and biotinylated horseradish peroxidase crosslinked to avidin-coated polystyrene beads were all compared. It was observed that in solution the biotinylated horseradish peroxidase retained 81% of the unconjugated enzyme's activity. Also, in solution, horseradish peroxidase and Bcap-HRP were inhibited by high concentrations of the substrate hydrogen peroxide. The controlled immobilized horseradish peroxidase could tolerate much higher concentrations of hydrogen peroxide and, thus, it demonstrates reduced substrate inhibition. Because of this, the activity of controlled immobilized horseradish peroxidase was higher than the activity of Bcap-HRP in solution. It is shown that a layer-by-layer assembly of the immobilized enzyme yields HRP of higher activity per unit surface area of the immobilization support compared to conventionally immobilized enzyme.

Avidin↗
PubMed1999

Mercuracarborand "anti-crown ether"-based chloride-sensitive liquid/polymeric membrane electrodes.

Highly sensitive and selective chloride liquid/polymeric membrane electrodes are described that employ [9]-mercuracarborand-3 (MC3), a neutral preorganized macrocyclic Lewis acid, as the anion carrier. MC3-based chloride-sensitive membrane electrodes, doped with different mole percentages of cationic additives (5, 10, and 60 mol % tridodecylmethylammonium chloride) relative to the amount of the carrier, exhibit enhanced potentiometric selectivity for chloride over other anions, including more lipophilic anions such as perchlorate, nitrate, and thiocyanate. In addition, the selectivity coefficients obtained are shown to meet the requirement for clinical applications. The obtained selectivity pattern is shown to correlate very well with 199Hg NMR titrations of MC3 with various anions, performed in organic solvents. Optimized membrane electrodes show a near-Nernstian response toward chloride over a wide concentration range and have micromolar detection limits. MC3-based chloride sensors show a fast response time (in the order of few seconds), as well as short recovery time. The developed mercuracarborand-based sensors do not practically respond to pH changes over the pH range of 2.5-7.0. Response characteristics (e.g., detection limit, linear range, response slope, and selectivity) of the [9]mercuracarborand-3 based chloride sensors remain essentially the same over a period of approximately 2 months, reflecting remarkable stability and well-defined chemistry of the macrocyclic Lewis acid ionophore.

Chlorides↗
PubMed1999

Guanidinium-based potentiometric SO2 gas sensor.

An SO2 gas sensor was developed by using a hydrogen sulfite-selective electrode positioned behind a gas-permeable membrane (GPM). The hydrogen sulfite-selective electrode was prepared by incorporating a multicyclic guanidinium ionophore in a plasticized poly(vinyl chloride) membrane. This gas sensor presents important advantages over the conventional Severinghaus-type SO2 gas sensor that contains a pH electrode immersed in an internal solution behind the GPM. The Severinghaus gas sensor suffers interferences from weak acids that can cross the GPM as gases and change the pH of the internal solution. In contrast, in the proposed sensor, the excellent selectivity of the HSO3- electrode and the ability of the GPM to discriminate gaseous from nongaseous species combine to generate the most selective potentiometric SO2 gas sensor reported to date.

Air Pollutants↗
PubMed1998

Site-directed and random immobilization of subtilisin on functionalized membranes: activity determination in aqueous and organic media.

Kinetic comparisons have been made between a randomly immobilized and a site-specifically immobilized subtilisin BPN' on microfiltration membranes of varying hydrophilicities in both aqueous and organic media. Site-directed mutagenesis was employed to introduce a single cysteine into the amino acid sequence of subtilisin at a location away from the active site. Immobilization of this mutant enzyme was then carried out using the single cysteine residue to orient the active site of the enzyme away from the membrane surface. Kinetic comparison of the immobilized mutant enzyme with the randomly immobilized wild-type enzyme in aqueous media showed an activity enhancement on both hydrophilic silica-containing and hydrophobic poly(ether)sulfone membranes. Higher loading efficiencies were observed for the site-directed enzyme on immobilization. Optimal enzyme loading values were calculated for the randomly immobilized enzyme. An enhancement of activity was also observed for the site-directed immobilized systems using nearly anhydrous hexane as the solvent.

1-Butanol↗
PubMed1997

Improving the activity of immobilized subtilisin by site-specific attachment to surfaces.

Understanding the properties of immobilized proteins is critical to the optimal design of biosensors, bioseparations, and bioreactors. The protease subtilisin BPN' was used as a model protein to study how the orientation of immobilized enzyme molecules on surfaces affects their catalytic properties. To achieve this goal, a single cysteine residue was introduced into the cysteine-free enzyme by site-directed mutagenesis. This cysteine residue was designed to be away from the active site of the enzyme. The enzyme molecules were immobilized through the side-chain sulfhydryl group of the cysteine residue on several supports. This site-specific immobilization method leads to ordered two-dimensional arrays of enzyme molecules on the support surface with the active sites of the enzyme oriented toward the solution phase. Such oriented immobilized subtilisin demonstrated a higher catalytic efficiency compared to subtilisin that was immobilized by a conventional method that leads to random immobilization.

Bacillus↗
PubMed1996

Effect of surface-attached heparin on the response of potassium-selective electrodes.

Heparin (or hydrolyzed heparin) was covalently attached on the surface of derivatized cellulose triacetate membranes, which were subsequently impregnated with the potassium-selective ionophore valinomycin. The resulting ion-selective electrodes presented near-Nernstian response to potassium and had selectivity coefficients of the same order of magnitude as those of conventional poly-(vinyl chloride)-based electrodes. It was found that the heparin layer does not alter significantly the response characteristics of the electrodes. The biological activity of the immobilized heparin (or hydrolyzed heparin) was measured in terms of its inactivation of blood coagulation factor Xa. It was found that the covalently anchored hydrolyzed heparin was not biologically active, but the immobilized heparin was able to inactivate factor Xa. Therefore, by covalently attaching heparin on the surface of ion-selective electrodes, electrodes with improved blood compatibility characteristics may be prepared.

Cellulose↗
PubMed1994

Fiber optic sensor for Ca2+ based on an induced change in the conformation of the protein calmodulin.

A fiber optic sensor that exploits the natural selectivity of the Ca(2+)-binding protein calmodulin (CaM) is described. In this sensor, a dialysis membrane is used to entrap a fluorescein-labeled CaM (F-CaM) solution at the common end of a bifurcated fiber optic bundle. Ca2+ ions in a sample solution can diffuse through the membrane and bind to the F-CaM. Upon binding with Ca2+, CaM undergoes a conformational change that induces a change in the fluorescence of the attached fluorescein tag. This change in fluorescence can be related to the concentration of Ca2+ in the sample solution. The detection limit for the sensor is 5 x 10(-8) M Ca2+. The sensor has no interference by Mg2+ at concentrations as high as 10(-2) M.

Animals↗