Ultrasensitive biotin assay of a noncompetitive format in a homogeneous solution based on resonance energy transfer induced by a protein-protein interaction.
Biotin is a water-soluble vitamin serving as a cofactor for several metabolic enzymes and plays crucial roles in every living cell. In the present study, we describe a noncompetitive assay for determination of biotin in a homogeneous solution. Our assay is based on a biotinylation reaction from archaeon Sulfolobus tokodaii. S. tokodaii biotinylation has a unique property that biotin protein ligase (BPL) forms a stable complex with its biotinylated substrate protein (BCCP). Determination of biotin was performed by monitoring the complexation reaction between BPL and BCCP through biotinylation, based on luminescence resonance energy transfer (LRET) from a Tb(3+) complex to fluorescein, where BPL and BCCP were labeled with a Tb(3+) complex and fluorescein, respectively. Our assay allows for ultrasensitive detection of biotin with a detection limit of approximately 1 pM (or 0.2 fmol in a 0.2 mL sample volume) by a simple procedure without use of radioactive materials or enzymatic signal amplification. In addition, owing to its noncompetitive format, our assay has a very wide measurement range of at least 3 orders of magnitude. Our assay is also beneficial as a model system for interaction analysis based on LRET.
Department of Bioscience and Bioinformatics, Kyushu Institute of Technology , 680-4 Kawazu, Iizuka 820-8502, Japan.
Tomohiro
T
Ikeda
Hiroki
H
Miyao
Shinji
S
Sueda
D001710:Biotin / Q000737:chemistry*; D004735:Energy Transfer; D057230:Limit of Detection; D011485:Protein Binding; D012996:Solutions
D012996:Solutions; D001710:Biotin