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Affinity interaction betwen phenylboronic acid-carrying self-assembled monolayers and FAD or HRP
(2005)
A method is provided for the recognition of glycated molecules based on their binding affinities to boronate- carrying monolayers. The affinity interaction of flavin adenine dinucleotide (FAD) and horseradish peroxidase (HRP) with phenylboronic acid monolayers on gold was investigated by using voltammetric and microgravimetric methods. Conjugates of 3-aminopherrylboronic acid and 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide ester) or 11-mercaptoundecanoic acid were prepared and self-assembled on gold surfaces to generate monolayers. FAD is bound to this modified sur-face and recognized by a pair of redox peaks with a formal potential of -0.433 V in a 0.1 m phosphate buffer solution, pH 6.5. Upon addition of a sugar to the buffer, the bound FAD could be replaced, indicating that the binding is reversible. Voltammetric, mass measurements, and photometric activity assays show that the HRP can also be bound to the interface. This binding is reversible, and HRP can be replaced by sorbitol or removed in acidic solution. The effects of pH, incubation time, and concentration of H2O2 were studied by comparing the catalytic reduction of H2O2 in the presence of the electron-donor thionine. The catalytic current of the HRP-loaded electrode was proportional to HRP concentrations in the incubation solution in the range between 5 mu g mL(-1) and 0.4 mg mL(-1) with a linear slope of 3.34 mu A mL mg(-1) and a correlation coefficient of 0.9945
Detection of subicomolar concentrations of human matrix metalloproteinase-2 by an optical biosensor
(2004)
We describe in this paper the development of a one-step sandwich assay for the highly sensitive and fast detection of human matrix metalloproteinase (MMP)-2 (EC 3.4.24.24), using surface plasmon resonance (SPR). For the assay, two ligands were selected: monoclonal anti-MMP-2 antibody Ab-2 and the tissue inhibitor of metalloproteinases (TIMP)-2. They were chosen on the basis of (1) their affinities to MMP-2, (2) the efficiency of immobilization to the sensor chip, (3) the efficiency of adsorption to colloidal gold, and (4) the stability of these protein-coated gold particles. The assay included mixing of MMP-2 with antibody Ab-2 adsorbed to colloidal gold with a diameter of about 20 rim and injection into the flowcell of the SPR instrument containing immobilized TIMP-2. By using colloidal gold particles an amplification factor of 114 and a detection limit of 0.5 pM for MMP-2 were obtained. The precision of the assay was high even at low analyte concentrations, the standard deviation being 8.3% for five determinations of 1 pM MMP- 2. No significant binding was observed with the structurally related MMP-9. The assay is far more sensitive and faster than commonly used methods for MMP-2 detection. As TIMP-bound MMP-2 is not detected by this method, the assay can be applied for measuring free MMP-2, reflecting the imbalance of free and inhibitor-bound enzyme in various pathological situations. (C) 2004 Elsevier Inc. All rights reserved
Characterization of a monoclonal antibody and its Fab fragment against diphenylurea hapten with BIA
(1998)
A novel electrochemical immunoassay based on the multiple affinity labeling of the indicator antibody with an electro-active tag is presented. The concept is illustrated for the determination of the glycated hemoglobin HbA1c in hemoglobin samples. Hemoglobin is adsorbed to the surfactant-modified surface of a piezoelectric quartz crystal. Whereas the quartz crystal nanobalance is used to validate the total Hb binding, the HbA1c on the sensor surface is recognized by an antibody and quantified electrochemically after the sugar moieties of the antibody have been labeled in-situ with ferroceneboronic acid. The sensitivity of this sensor is about threefold higher than the sensitivity of a hemoglobin sensor, where the ferroceneboronic acid is bound directly to HbA1c.