ABSTRACT
In this short note we describe the conversion of the widely used fluorescence quenching azo-dyes DABCYL and HABA to fluorophores. The dyes were conjugated to the proteins RNase and human serum albumin (HSA) and subsequently reduced using sodium dithionite (Na2S2O4), thus forming amine-containing fluorophores. Since this chemical reaction can be applied to any azo-containing quencher compound, a great variety of substances can be readily obtained synthetically. This approach provides a promising tool in the use of fluorescence-based investigations of biomolecular interactions.
Subject(s)
Azo Compounds/chemistry , Fluorescent Dyes/chemistry , p-Dimethylaminoazobenzene/analogs & derivatives , Amino Acid Sequence , Binding Sites , Dithionite/chemistry , Fluorescence Resonance Energy Transfer , Humans , Molecular Structure , Oxidation-Reduction , Protein Binding , Ribonucleases/chemistry , Serum Albumin/chemistry , Structure-Activity Relationship , p-Dimethylaminoazobenzene/chemistryABSTRACT
The use of microfabricated cantilevers as bioaffinity sensors was investigated. Since many bioaffinity interactions involve proteins as receptors, we conducted studies of the magnitude, kinetics, and reversibility of surface stresses caused when common proteins interact with microcantilevers (MCs) with nanostructured (roughened) gold surfaces on one side. Exposure of nanostructured, unfunctionalized MCs to the proteins immunoglobulin G and bovine serum albumin (BSA) resulted in reversible large tensile stresses, whereas MCs with smooth gold surfaces on one side produced reversible responses that were considerably smaller and compressive. The response magnitude for nanostructured MCs exposed to BSA is shown to be concentration dependent, and linear calibration over the range of 1-200 mg/L is demonstrated. Stable, reusable protein bioaffinity phases based on unique enantioselective antibodies are created by covalently linking monoclonal antibodies to nanostructured MC surfaces. The direct (label-free) stereoselective detection of trace amounts of an important class of chiral analytes, the alpha-amino acids, was achieved based on immunomechanical responses involving nanoscale bending of the cantilever. The temporal response of the cantilever (delta deflection/delta time) is linearly proportional to the analyte concentration and allows the quantitative determination of enantiomeric purity up to an enantiomeric excess of 99.8%. To our knowledge, this is the first demonstration of chiral discrimination using highly scalable microelectromechanical systems.
Subject(s)
Antibodies, Monoclonal/chemistry , Biosensing Techniques/methods , Nanotechnology/methods , Amino Acids/analysis , Amino Acids/chemistry , Animals , Biosensing Techniques/instrumentation , Cattle , Humans , Immunoglobulin G/chemistry , Nanotechnology/instrumentation , Serum Albumin, Bovine/chemistry , Stereoisomerism , Stress, MechanicalABSTRACT
Stereoselective antibodies are excellent chiral selectors for the routine analysis of enantiopurity in, e.g., enzyme-linked immunosorbent assays and immunosensors. Here, we describe the application of stereoselective, group-specific antibodies to alpha-amino acids as tailor-made chiral selectors. The feasibility of chiral immunosensor techniques in space missions using these or other, newly designed antibodies is discussed.
ABSTRACT
We introduce a new nonradioactive, chromogenic label based on 4-hydroxyazobenzene-2-carboxylic acid (HABA), which is suitable for bioanalytical application, e.g., detection, localization, isolation, and purification. The HABA label is superior to other systems where it is difficult to separate labeled from unlabeled molecules or to determine the amount of label. HABA is readily detected spectroscopically by its absorption at 350 nm or by its interaction with avidin that results in a red shift to 500 nm. The HABA reagents described can be conjugated to a variety of functional groups on biomolecules and purified thereafter by affinity chromatography on an avidin column. The interaction of the HABAylated biomolecules with their corresponding targets is detected with high-affinity anti-HABA antibodies or with avidin. The nonradioactive, chromogenic HABA-based reagents form a homogeneous system that can complement or replace systems where facile quantification of the label is desired.
Subject(s)
Avidin/chemistry , Azo Compounds/chemistry , Biotin/chemistry , Indicators and Reagents/chemistry , Antibodies/isolation & purification , Blotting, Western , Chromatography, Affinity , Enzyme-Linked Immunosorbent Assay , Mass Spectrometry , Spectrophotometry, UltravioletABSTRACT
Based on the stereoselectivity of immunoglobulins, we have developed a new chiral sensor for the detection of low-molecular-weight analytes. Using surface plasmon resonance detection, enantiomers of free, underivatized alpha-amino acids can be monitored in a competitive assay by their interaction with antibodies specific for the chiral center of this class of substances. The sensitivity to the minor enantiomer in nonracemic mixtures exceeds currently available methods; therefore, such immunosensors can readily detect traces of enantiomeric impurities and are attractive for a range of applications in science and industry.
Subject(s)
Amino Acids/analysis , Antibodies/immunology , Stereoisomerism , Surface Plasmon Resonance/methods , Amino Acids/chemistry , Amino Acids/immunology , Animals , Antibodies/chemistry , Antigen-Antibody Reactions , Immunoglobulins/chemistry , Immunoglobulins/immunology , Rabbits , Sensitivity and SpecificityABSTRACT
Immobilization of low molecular weight haptens in ELISA usually involves their coupling to protein molecules or covalent binding to the solid phase. In this study we demonstrate that it is possible to directly bind the hapten p-aminophenylalanine to gamma-irradiated polystyrene microtiter plates for the detection of antibodies that stereospecifically recognize the chiral center of alpha-amino acids. Simple incubation of the hapten in aqueous buffer, without additional activation, results in a stable coating that is suited for use in ELISA.
