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H H Weetall

Publications and source records attributed to H H Weetall.

At least 19 recordsLinked to original sources
PubMed2001

Simultaneous multianalyte detection with a nanometer-scale pore.

It was recently shown that naturally occurring, genetically engineered or chemically modified channels can be used to detect analytes in solution. We demonstrate here that the overall range of analytes that can be detected by single nanometer-scale pores is expanded using a potentially simpler system. Instead of attaching recognition elements to a channel, they are covalently linked to polymers that otherwise thread through a nanometer-scale pore. Because the rate of unbound polymer entering the pore is proportional to its concentration in the bulk, the binding of analyte to the polymer alters the latter's ability to thread through the pore, and the signal that results from individual polymer translocation is unique to the polymer type; the method permits multianalyte detection and quantitation. We demonstrate here that two different proteins can be simultaneously detected with this technique.

Bacterial Toxins↗
PubMed2001

Photoinduced transformation of 14-F-bacteriorhodopsin gelatin films based on both wild type and D96N mutant.

Spectral and kinetic transformations were studied in gelatin films made with 14-F wild type (WT) bacteriorhodopsin (BR) and 14-F D96N mutant BR. Unlike the recent study of water suspensions of the same pigments, where a red shifted species at 660 nm was shown to form under the light in 14-F WT only, there are no drastic differences in photoinduced behavior between gelatin films based on 14-F WT and 14-F D96N. It is not observed any photoinduced formation of red shifted species at 660 nm for both types of films as it is observed for corresponding pigments in water suspension. The observed results are explained in a terms of relationship between the rates of two photoinduced processes that occur in suspensions and films of corresponding pigments. Kinetic characteristics of the photoinduced processes for the films with chemical additives suggest that there are no advantages in using 14-F D96N films when compared to films based on 14-F WT.

Bacteriorhodopsins↗
PubMed2000

Measurement of proton release and uptake by analogs of bacteriorhodopsin.

Proton release and subsequent uptake by several forms of bacteriorhodopsin (bR), including 4-keto analogs of wild-type (WT) and D96N and D85N mutants as well as the 9-demethylretinal analog of WT and D96N mutants, have been measured using a highly sensitive electrochemical technique. Release and uptake of protons by bR in membrane patches on a tin oxide electrode produce a current transient whose amplitude is proportional to the rate of pH change at the electrode surface. Profiles of proton release by the analogs vs. pH are substantially different from the profiles of the native proteins.

Bacteriorhodopsins↗
PubMed1998

Phototransformation and proton pumping activity of the 14-fluoro bacteriorhodopsin derivatives.

The photoinduced behavior and proton pumping characteristics of some bacteriorhodopsin (BR) analogs with fluorinated chromophores (all-trans 14-fluorinated [14-F] retinal and 13-cis 14-F retinal) derived from wild type (WT) and D96N mutant BR were investigated. These analogs were characterized using spectrophotometry and a highly sensitive electrochemical technique. Similar to the white membrane JW2N, the apomembranes WT ET 1000 and D96N form photoactive pigments with the 14-F chromophores. The resulting analogs have a major absorption band at 588 nm. Red-shifted pigment (lambdamax</=680 nm) has been previously observed as a minor component of the major 587-nm pigment in 14-F BR made with white membrane JW2N. A similar red-shifted pigment is formed under yellow light (lambda>500 nm) only in the 14-F analogs derived from WT ET 1000. The measurements of the photoinduced transformation in 14-F WT analogs show that the photocycle of the major pigment occurs simultaneously with the process in the red region and is partially masked by the formation of the red-shifted species. The 14-F D96N samples have a significantly slower and more complicated photoinduced behavior. Electrochemical measurements show that the photoinduced transformation of the red species is not accompanied by proton transport.

Bacteriorhodopsins↗
PubMed1996

Retention of bacteriorhodopsin activity in dried sol-gel glass.

The D96N mutant form of bacteriorhodopsin (bR) purple membrane fragments isolated from Halobacterium salinarium has been entrapped in sol-gel glass and dried. The photo-activity of the bR was monitored during drying over a period of 60 days. The entrapped bR was initially dried at 4 degrees C followed by room temperature storage. Samples showing constant activity after storage at 4 degrees C lost further activity at room temperature, eventually reaching a level where the activity remained constant. Upon further drying at 100 degrees in a vacuum oven the glass lost an additional 11.4 +/- 1.3% weight and all remaining activity.

Bacteriorhodopsins↗
PubMed1995

Peroxidase- and tetracyanoquinodimethane-modified graphite paste electrode for the measurement of glucose/lactate/glutamate using enzyme-packed bed reactor.

A flow injection analysis sensor for the measurement of glucose/lactate/glutamate is reported. The glucose oxidase/glutamate oxidase/lactate oxidase was immobilized on silanized controlled pore glass particles and packed into a Teflon column (i.d., 1.2 mm; length, 40 mm) to give a bed for glucose/lactate/glutamate. The hydrogen peroxide formed by the enzymatic reaction in the packed bed was monitored by a horseradish peroxidase- and tetracyanoquinodimethane (TCNQ)- modified graphite paste electrode at 50 mV vs Ag/AgCl. The glucose oxidase/lactate oxidase/glutamate oxidase were regenerated in the packed bed, whereas peroxidase was regenerated in the TCNQ-mediated graphite paste electrode by the oxidation of TCNQ. The oxidized TCNQ was electrochemically reduced at 50 mV vs Ag/AgCl. The cathodic current obtained by the reduction of TCNQ determined the concentration of the injected analytes in the packed bed. The system showed very rapid response. Response curves for the analysis of peroxide, glucose, lactate, and glutamate are reported.

Biosensing Techniques↗
PubMed1995

Evanescent fluorobiosensor for the detection of polyaromatic hydrocarbon based on DNA intercalation.

A flow-injection analysis (FIA) system coupled with an evanescent wave (EW) biosensor employing total internal reflection of fluorescence radiation (TIRF) for the detection of polyaromatic hydrocarbon that intercalates into DNA is reported. A highly fluorescent intercalator, "ethidium bromide," has been used as the reference compound for detection. The EW biosensor was developed according to the procedure described earlier (1,2). Data on the analysis of Naphthalene, 3-methylcholanthrene, 7,12-dimethylbenz(a)anthracene, 1,2-benzanthracene, and some standard reference materials supplied by the National Institute of Standards and Technology are reported. The relative ability of the polyaromatic hydrocarbon to displace ethidium bromide, based on the relative binding ratio, is found to be on the order of 7,12-dimethylbenz[a]anthracene > 3-methylcholanthrene > 1,2-benzanthracene > napthalene.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1995

Tetracyanoquinodimethane mediated glucose sensor based on a self-assembling alkanethiol/phospholipid bilayer.

An amperometric tetracyanoquinodimethane (TCNQ) mediated biosensor for glucose is described, based on a self-assembling alkanethiol/phospholipid bilayer laid down onto a gold surface. Gold was sputter deposited onto chromium coated silicon wafers to a thickness of 200 nm. A monolayer of alkanethiol was allowed to self-assemble from an ethanolic solution of dodecanethiol onto a freshly cleaned gold electrode in an overnight incubation. The monolayer was characterized by ellipsometric, impedance and cyclic voltammetry measurements (capacitance = 1.60 +/- 0.06 microF/cm2, and thickness 1.34 +/- 0.15 nm). A mixture of phospholipid liposomes containing free amino groups was placed on the monolayer and allowed to incubate overnight. The self-assembly of a phospholipid monolayer and allowed to incubate overnight. The onto the alkanethiol monolayer, resulted in the formation of a bilayer. The formation of bilayer was again characterized by impedance and cyclic voltammetry measurements (capacitance = 0.98 +/- 0.09 microF/cm2, and thickness = 1.85 +/- 0.22 nm). TCNQ has been incorporated into the liposomes before the formation of the bilayer. Glucose oxidase was cross-linked with the amino-groups of the phospholipids using bis [2-(sulphosuccinimiidooxicarbonyloxy) ethyl] sulphone. TCNQ which was incorporated in the bilayer acted as an efficient mediator to regenerate glucose oxidase. Cyclic voltammetry of the modified electrode and a response curve for the glucose sensor are reported.

Biosensing Techniques↗
PubMed1994

Application of photochemical reaction in electrochemical detection of DNA intercalation.

A flow injection analysis (FIA) system for the detection of the compounds that intercalate within DNA is reported. A derivative of 9,10-anthraquinone has been used as the reference compound for photoelectrochemical detection. The sodium salts of 9,10-anthraquinone-2,6-disulfonic acid and 9,10-anthraquinone-2-sulfonic acid are photochemically activated and then reduced in the presence of an electron donor (glucose). The electrochemical signal is based on the measurement of the anodic current resulting from the oxidation of the reduced form of 9,10-anthraquinone. The reduced form of the 9,10-anthraquinone is oxidized through a mediated mechanism at the surface of a tetracyanoquinodimethane-(TCNQ)-modified graphite paste electrode covered by a Nucleopore membrane. TCNQ acts as an efficient mediator for the oxidation of reduced 9,10-anthraquinone. Cyclic voltammetry, photocyclic voltammetry, and the photoelectrochemical FIA response of 9,10-anthraquinone are reported. Experimental results show that these anthraquinones can be intercalated within the helix of double-stranded calf thymus DNA. The anthraquinone molecules that are intercalated within DNA cannot be oxidized due to their limited transport to the modified electrode surface. This results in a decrease in the anodic current at a constant concentration of anthraquinone after intercalation. There is a linear relation between the decrease in electrochemical response and the DNA concentration at a constant concentration of anthraquinone. The intercalated anthraquinone molecules can be completely replaced by another intercalating agent (i.e., ethidium bromide) that is more strongly intercalated within DNA, thereby regenerating the electrochemical response.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Aggregation kinetics of colloidal particles destabilized by enzymes.

Dynamic light scattering was used to measure enzymatic activity by monitoring the rate of destabilization of colloidal particles coated with proteins or carbohydrates. The photon autocorrelation spectrum was analyzed using a cumulant expansion or direct inversion. In all cases, the measured average diffusion coefficient and the standard deviation of the diffusion coefficient decreased with time. A kinetic model with a bimodal particle distribution function was used to represent the time dependence of the cumulants. The cumulants are very sensitive to the details of the particle distribution.

Amylases↗
PubMed1993

An amperometric flow-injection analysis biosensor for glucose based on graphite paste modified with tetracyanoquinodimethane.

A biosensor system using flow injection analysis (FIA) has been developed for the analysis of glucose in human serum. The system consists of the enzyme glucose oxidase incorporated into graphite paste modified with the electroactive material tetracyanoquinodimethane (TCNQ). TCNQ acts as an efficient mediator for oxidation of the reduced enzyme at 200 mV vs Ag/AgCl. The flow injection assay described has detection limits of 2 mM glucose using a 100-microliters sample injection through a 250-microliters sample loop. Data are presented to show the effect of sample injection volume and flow rate on the response of the FIA sensor. The biosensor exhibited excellent reproducibility for 800 injections. The loss of response after 800 injections was due to leaching of TCNQ from the graphite paste. Each assay takes 3 min giving a sample throughput of 20 per hour at a flow rate of 30 ml/h. The sensor was applied to the determination of glucose in human serum. The glucose measurements are in good agreement with those of a commercially available spectrophotometric method. Data showing the effect of interfering substances, ascorbic acid and acetaminophen, on the response of the sensor are also reported.

Acetaminophen↗
PubMed1993

Preparation of immobilized proteins covalently coupled through silane coupling agents to inorganic supports.

Enzymes were first immobilized on inorganic supports through silane coupling agents over 25 yr ago. Since that initial report, literally hundreds of laboratories have utilized this methodology for the immobilization of enzymes, antigens, antibodies, receptors, and other high and low mol wt compounds. Today silane coupling is one of the commonly used techniques in the arsenal of the biochemist for the binding of material of all sorts to inorganic surfaces. Inorganic materials come in a variety of shapes, sizes, and characteristics. Today silane coupling is one of the most used coupling methods for the preparation of biosensing devices. Sol-gel entrapped enzymes are also produced by the application of silane technology by the polymerization of the silane to form glass-like materials with entrapped protein. This review will discuss the general preparation and characterization of silane coupled proteins with special emphasis on enzymes and describe in detail the actual methods for the silanization and specific chemical coupling of proteins to the silanized carrier.

Antibodies↗
PubMed1991

Antibodies in water immiscible solvents. Immobilized antibodies in hexane.

Anti-progesterone antibodies covalently coupled to paramagnetic particles (PMPs) retain their immunologic activity to progesterone in hexane as solvent. Although reaction rates appear slowed, activity is not lost. The antibodies still bind similar quantities of antigen to those bound in aqueous solution. It is most likely that the increased reaction time is the result of a phase transfer. The immobilized antibodies are present in an aqueous phase, while the antigen is present in the more soluble organic solvent phase.

Antibodies↗
PubMed1989

Antibodies immobilized on inorganic supports.

Antibodies and antigens can be covalently coupled to a variety of carriers, both organic and inorganic. The methods for coupling these proteins may be found scattered throughout the technical literature. This report, although it concentrates on inorganic supports, describes several of the more successful methods used in laboratories today. Each of these methods is described in enough detail for the reader to carry out the coupling method of interest in his or her own laboratory. The coupling methods have been divided into two groups, direct and silane. Under each of these general headings are described the specific methodologies.

Antibodies↗
PubMed1982

Enzyme immunoassay for free thyroxin.

We describe a mathematical model for a single-tube enzyme immunoassay for free thyroxin (FT4), involving use of a thyroxin/horseradish peroxidase (EC 1.11.1.7) conjugate that does not interact with thyroxin-binding globulin. In the presence of serum two populations of unassociated, or free, immunologically active constituents are present: FT4 and the conjugate. The concentrations of the former are determined by the serum constituents and of the latter by the albumin concentration. When a small quantity of antibody is added, it reacts with the variable amount of FT4 and with the constant amount of the conjugate, thus giving a measure of the FT4. We constructed a mathematical model based on thermodynamic binding constants and adsorption data. The model gives satisfactory agreement with the experimental data under a variety of experimental conditions. Results for 19 patients' serum samples demonstrate the validity of the concept.

Horseradish Peroxidase↗
PubMed1981

Detection of Neisseria gonorrhoeae by ultraviolet illumination of NADH generated by an immobilized antibody-gonococcal enzyme complex.

Frozen, cervical swabs were placed in a lysis buffer containing an immobilized antibody to the gonococcal enzyme, 1,2-propanediol oxidoreductase. The immobilized antibody--enzyme complex that formed was active after the addition of substrate (1,2-propanediol and NAD) and this activity could be detected by visual inspection of NADH fluorescence under ultraviolet illumination.

Alcohol Oxidoreductases↗
PubMed1980

The isolation and characterization of a 1,2-propanediol oxidoreductase from Neisseria gonorrhoeae.

An enzyme which oxidizes 1,2-propanediol in the presence of NAD+ has been purified from lysates of Neisseria gonorrhoeae. The enzyme was activated by monovalent cations, had a pH optimum between 9 and 10, and showed a substrate specificity unlike any known alcohol or glycerol dehydrogenase. The enzyme had an apparent Km of 17 mM for 1,2-propanediol and 0 . 37 mM for NAD+. When chromatographed on a Sephadex G-150 column, the enzyme eluted as a single peak in the molecular weight region of a bovine serum albumin marker. An antibody to the purified enzyme was prepared in goats. When antiserum was reacted with the enzyme in immunodiffusion experiments, a single precipitin band was detected. When the enzyme was mixed with an excess of antibody and then reacted with substrate, enzyme activity was completely inhibited.

Alcohol Oxidoreductases↗