Affinity interactions between phenylboronic acid-carrying self-assembled monolayers and flavin adenine dinucleotide or horseradish peroxidase.
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-aminophenylboronic 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 surface 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 H(2)O(2) were studied by comparing the catalytic reduction of H(2)O(2) 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 microg mL(-1) and 0.1 mg mL(-1) with a linear slope of 3.34 microA mL mg(-1) and a correlation coefficient of 0.9945.
Liu
Songqin
S
University of Potsdam, Institute of Biochemistry and Biology, Department of Analytical Biochemistry, Golm, Germany.
Wollenberger
Ulla
U
Halámek
Jan
J
Leupold
Eik
E
Stöcklein
Walter
W
Warsinke
Axel
A
Scheller
Frieder W
FW
D001897:Boronic Acids / Q000737:chemistry*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry*; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure
D001897:Boronic Acids; D005182:Flavin-Adenine Dinucleotide; D006735:Horseradish Peroxidase; C010686:benzeneboronic acid