title: Microplate assay for aptamer-based thrombin detection using a DNA-enzyme conjugate based on histidine-tag chemistry.
abstract: We report a method to prepare a DNA-enzyme conjugate using histidine-tag (His-tag) chemistry. A DNA oligonucleotide was modified with nitrilotriacetate (NTA), whose K(d) was approximately 10⁻⁶ (M⁻¹) toward a His-tag present on a recombinant protein via the complexation of Ni²⁺. His-tagged alkaline phosphatase (His-AP) was used as the model enzyme. Enzyme immobilization on the microplate revealed the conjugation of His-AP and the NTA-modified DNA via an Ni²⁺ complex. SPR measurements also proved the conjugation of His-AP with the NTA-modified DNA via an Ni²⁺ complex. The DNA-enzyme conjugate was then used for the detection of thrombin using a DNA aptamer. The DNA-AP conjugate successfully amplified the binding signal between the DNA aptamer and the thrombin, and the signal was measured as the fluorescent intensity derived from the AP-catalyzed reaction. The detection limit was 11 nM. Finally, we studied the effect of the release of the immobilized His-AP from the microplate on the AP activity, because the present strategy used a cleavable linker for the conjugation and the enzyme immobilization. The DNase-catalyzed release of the immobilized His-AP resulted in a 1.7-fold higher AP activity than observed when the His-AP was surface-immobilized.
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan.
authors.forename: Josui
authors.initials: J
authors.lastname: Shimada
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Maruyama
authors.forename: Momoko
authors.initials: M
authors.lastname: Kitaoka
authors.forename: Noriho
authors.initials: N
authors.lastname: Kamiya
authors.forename: Masahiro
authors.initials: M
authors.lastname: Goto
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D004800:Enzymes, Immobilized / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D020349:Surface Plasmon Resonance; D013917:Thrombin / Q000032:analysis*
chemical_list: D052157:Aptamers, Nucleotide; D017931:DNA Primers; D004800:Enzymes, Immobilized; D006639:Histidine; D013917:Thrombin