Microplate assay for aptamer-based thrombin detection using a DNA-enzyme conjugate based on histidine-tag chemistry

Josui Shimada, Tatsuo Maruyama, Momoko Kitaoka, Noriho Kamiya, Masahiro Goto

Research output: Contribution to journalArticle

10 Citations (Scopus)

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 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 Ni 2+ 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.

Original languageEnglish
Pages (from-to)541-546
Number of pages6
JournalAnalytical Biochemistry
Volume421
Issue number2
DOIs
Publication statusPublished - Feb 15 2012

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All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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