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-methy cholanthrene, 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.     

Random peptide synthesis using immobilized enzymes in high concentrations of organic solvents

Trypsin, leucine aminopeptidase, and carboxypeptidase B were separately immobilized on controlled pore glass and reacted with a dipeptide substrate in high concentrations of either acetone or 1-propanol. Hydrolytic activity was demonstrated and evidence for the possible synthesis of peptide polymer is presented. Directed synthesis using amino acids and blocked amino acids as substrates was not successful.     

Preparation and characterization of molecularly imprinted electropolymerized carbon electrodes

Molecularly imprinted polymers (MIPs) selective for fluorescein, rhodamine or 2,4-dichlorophenoxyacetic acid (2,4-D) were electropolymerized onto graphite electrodes using an aqueous solution equimolar in resorsinol/ortho-phenylenediamine and in the presence of the template molecule. For the dyes, the MIP-coated electrodes showed higher affinity for their template molecule than for a non-template dye. The 2,4-D-MIP-coated electrode showed a concentration dependent response for 2,4-D as compared to the polymer-coated electrode prepared in the absence of template molecule.     

An Azulenic Bacteriorhodopsin Analog Has Photoinduced Activity

Abstract— The photoinduced changes of absorbance as well as proton release and uptake have been observed for an azulenic analog of wild-type bacteriorhodopsin. The release and uptake of protons have been measured using a highly sensitive electrochemical technique. Bacteriorhodopsin membrane patches on a tin-oxide electrode produce a transient photocurrent that is negative for proton release and positive for proton uptake. For azulenic bacteriorhodopsin the photocurrent is approximately 20% of the transient observed with native bacteriorhodopsin. The existence of the photoinduced absorbance changes and the transient photocurrent are important results for gaining further insight into the photoinduced function of bacteriorhodopsin.     

A method for the assay of hydrolytic enzymes using dynamic light scattering

Dynamic light-scattering techniques have been successfully used for the assay of several hydrolytic enzymes. The enzymes were assayed using substrate-coated colloidal particles. Hydrolysis of the substrate coat causes destabilization of the particles followed by particle aggregation. The rate of particle aggregation can be related to the initial concentration of added enzyme.     

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.     

An optical tweezers-based immunosensor for detection of femtomoles-per-liter concentrations of antigens

We used optical tweezers—optical trapping with focused laser beams—to pull microspheres coated with antigens off of an antibody-coated surface. Using this technique, we could quantify the force required to separate antigen to antibody bonds. At very low surface density of antigen, we were able to detect the single antigen to antibody binding. The force required to break the antigen-antibody bonds and pull the microsphere off the surface was shown to increase monotonically with increasing surface density of antigens. Using the force determination as a transducer, we were able to detect concentrations of free antigens in solution as small as 10⁻¹⁵ mol/L in a competitive binding assay.     

Effect of dehydration on photoinduced transformation in gelatin films made with 14-fluoro bacteriorhodopsin derivatives

Photoinduced transformation in gelatin films made with 14-fluoro bacteriorhodopsin derivatives, both wild-type (WT) and D96N mutant, were studied. Spectral and kinetic peculiarities for these two types of samples were compared over a wide range of relative humidity (9–92%). Analysis of the results considered two existing photoinduced processes that occur in suspensions and films of corresponding pigments. It was demonstrated that there is a range of humidity in which the performance of fluorine WT bacteriorhodopsin gelatin films may offer a technological advantage compared with fluorine D96N bacteriorhodopsin.     

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.