Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10533710 | Analytical Biochemistry | 2012 | 6 Pages |
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 Kd was approximately 10â6 (Mâ1) 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 11Â 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.
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Josui Shimada, Tatsuo Maruyama, Momoko Kitaoka, Noriho Kamiya, Masahiro Goto,