Noncovalent, extrinsic fluorescent dyes are applied in various fields of protein analysis, e.g. to characterize folding intermediates, measure surface hydrophobicity, and detect aggregation or fibrillation. The main underlying mechanisms, which explain the fluorescence properties of many extrinsic dyes, are solvent relaxation processes and (twisted) intramolecular charge transfer reactions, which are affected by the environment and by interactions of the dyes with proteins. In recent time, the use of extrinsic fluorescent dyes such as ANS, Bis-ANS, Nile Red, Thioflavin T and others has increased, because of their versatility, sensitivity and suitability for high-throughput screening. The intention of this review is to give an overview of available extrinsic dyes, explain their spectral properties, and show illustrative examples of their various applications in protein characterization.

/pdf/extrinsic-fluorescent-dyes-as-tools-for-protein-nmhkb08wui.pdf

Extrinsic fluorescent dyes as tools for protein characterization.

http://dbkgroup.org/Papers/davey_kell_micrev96.pdf

Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses.

The immobilization of antibodies on solid-phase materials has been used in many areas such as purification, diagnostic immunoassays and immunosensors. Problems associated with the loss of biological activity of the antibodies upon immobilization have been noticed in many cases. One of the main reasons for such loss is attributed to the random orientation of the asymmetric macromolecules on support surfaces. In this paper, the approaches for achieving oriented coupling of antibodies to increase the antigen binding capacity are reviewed. Some issues such as steric hindrance caused by neighbouring antibody molecules, the distance between an antibody and the support surface and the use of antibody fragments are dealt with. Some applications of the oriented immobilized antibodies in immunoassays and immunosensors are examined.

Tutorial review. Oriented immobilization of antibodies and its applications in immunoassays and immunosensors

Immunosensors: electrochemical sensing and other engineering approaches.

Affinity chromatography is a type of liquid chromatography that makes use of biological-like interactions for the separation and specific analysis of sample components. This review describes the basic principles of affinity chromatography and examines its use in the testing of clinical samples, with an emphasis on HPLC-based methods. Some traditional applications of this approach include the use of boronate, lectin, protein A or protein G, and immunoaffinity supports for the direct quantification of solutes. Newer techniques that use antibody-based columns for on- or off-line sample extraction are examined in detail, as are methods that use affinity chromatography in combination with other analytical methods, such as reversed-phase liquid chromatography, gas chromatography, and capillary electrophoresis. Indirect analyte detection methods are also described in which immunoaffinity chromatography is used to perform flow-based immunoassays. Other applications that are reviewed include affinity-based chiral separations and the use of affinity chromatography for the study of drug or hormone interactions with binding proteins. Some areas of possible future developments are then considered, such as tandem affinity methods and the use of synthetic dyes, immobilized metal ions, molecular imprints, or aptamers as affinity ligands for clinical analytes.

/pdf/affinity-chromatography-a-review-of-clinical-applications-kbbk9lt6ls.pdf

Affinity Chromatography: A Review of Clinical Applications

A competitive electrochemical enzyme immunoassay has been developed for the antiasthmatic drug theophylline, utilizing a controlled-pore glass–protein A immunoreactor and flow injection techniques. p-Aminophenyl phosphate, a substrate for alkaline phosphatase, has been used in this assay, and its hydrolysis product p-aminophenol was determined at +0.2 V versus the saturated calomel electrode. For each sample the antibody–protein A reaction takes place at near-neutral pH, and the complexes are eluted at acid pH. Serum theophylline has been determined by this method, and good relative standard deviations and percentage recoveries have been achieved.

Flow injection electrochemical enzyme immunoassay for theophylline using a protein A immunoreactor and p-aminophenyl phosphate–p-aminophenol as the detection system

The involatile residues remaining after closed-vessel microwave digestion of various food samples, using nitric acid, with and without post-digestion treatment with hydrogen peroxide, have been studied. Decomposition products were found to include aliphatic and aromatic acids, nitro-compounds, oxalates and inorganic nitrates and phosphates. Measures of digestion completeness were provided by appearance, carbon content, infrared spectra and thin layer chromatograms of the residues, enabling a comparison of different digestion methods and sample types. Residual carbon levels varied linearly with the relative amounts of carboxylic acid and inorganic nitrate, as measured by infrared spectrometry of the residues. The formation of calcium oxalate was also a function of the degree of decomposition: the carboxylic acid: oxalate ratio increased in a logarithmic fashion with increasing residual carbon content.Particularly high levels of residual carbon from nitric acid digestion of milk powder, due largely to carboxylic acid residues, were substantially decreased by post-digestion treatment with hydrogen peroxide. However, nitrobenzoic acids, which proved major interferents in electrochemical analysis, were only removed by treatment with perchloric acid.

Investigation of decomposition products of microwave digestion of food samples

A heterogeneous on line electrochemical enzyme immunoassay has been developed for the antiasthmatic drug theophylline using a flow injection analysis system containing a protein A immunoreactor to separate antibody bound and unbound theophylline-alkaline phosphatase. In this assay the antibody, label and sample are sequentially immobilised onto the protein A immunoreactor. The antibody-protein A and antibody-protein A label/sample reaction takes place at near neutral pH, and the complexes are eluted at acid pH. p-Aminophenyl phosphate (PAPP) and p-aminophenol (PAP) have been used as the detection system. Experimental variations such as flow rate and reactant concentration have been studied and serum theophylline has been determined by this method and good r.s.d values and % recoveries have been achieved.

Flow Injection Immunosensor for Theophylline

The fluorimeter includes means (4, 5) for receiving a sample of a fluorescent material, a source of electromagnetic radiation (11) arranged to illuminate a sample in the receiving means (4, 5), and a detector (16) for detecting electromagnetic radiation emitted from the sample. At least one filter device (19, 20) is provided, having a plurality of filter portions each for interposition in the path of the incident or the emitted radiation to select a respective wavelength of electromagnetic radiation which is allowed to pass through the device. A motor drive means repeatedly positions selected ones of the filter portions in turn in the path of the electromagnetic radiation.

A fluorimeter and a method for carrying out a fluorescent assay of a plurality of analytes

A high wavelength fluorescent probe, Nile Red, was added to four proteins, viz., bovine albumin, α1-acid glycoprotein, β-lactoglobulin and ovomucoid. Nile Red showed an enhancement in fluorescence and a shift in emission wavelength, suggesting it was bonding hydrophobically to these proteins. Drug displacement of Nile Red from α1-acid glycoprotein was achieved with both D, L-propranolol and flufenamic acid, showing that the binding site is less electrostatic and more hydrophobic in nature. In order to monitor these interactions, a simple spectrofluorimeter was constructed from solid-state components; the sensitivity of this instrument compared well with that of standard laboratory spectrofluorimeters.

Novel instrumentation and biomedical applications of very near infrared fluorescence.

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