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 K d was approximately 10 -6 (M -1) toward a His-tag present on a recombinant protein via the complexation of Ni 2+. 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 Ni 2+ 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

Fingerprint

Histidine
Thrombin
Alkaline Phosphatase
Assays
DNA
Enzymes
Nucleotide Aptamers
Enzyme immobilization
Immobilization
Deoxyribonucleases
Complexation
Recombinant Proteins
Oligonucleotides
Limit of Detection
thrombin aptamer

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Microplate assay for aptamer-based thrombin detection using a DNA-enzyme conjugate based on histidine-tag chemistry. / Shimada, Josui; Maruyama, Tatsuo; Kitaoka, Momoko; Kamiya, Noriho; Goto, Masahiro.

In: Analytical Biochemistry, Vol. 421, No. 2, 15.02.2012, p. 541-546.

Research output: Contribution to journalArticle

@article{8f73d5d055a045359dc9892c5ddca11d,
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 Ni 2+. 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 Ni 2+ 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.",
author = "Josui Shimada and Tatsuo Maruyama and Momoko Kitaoka and Noriho Kamiya and Masahiro Goto",
year = "2012",
month = "2",
day = "15",
doi = "10.1016/j.ab.2011.11.028",
language = "English",
volume = "421",
pages = "541--546",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Microplate assay for aptamer-based thrombin detection using a DNA-enzyme conjugate based on histidine-tag chemistry

AU - Shimada, Josui

AU - Maruyama, Tatsuo

AU - Kitaoka, Momoko

AU - Kamiya, Noriho

AU - Goto, Masahiro

PY - 2012/2/15

Y1 - 2012/2/15

N2 - 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 Ni 2+. 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 Ni 2+ 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.

AB - 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 Ni 2+. 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 Ni 2+ 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.

UR - http://www.scopus.com/inward/record.url?scp=84859711910&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84859711910&partnerID=8YFLogxK

U2 - 10.1016/j.ab.2011.11.028

DO - 10.1016/j.ab.2011.11.028

M3 - Article

C2 - 22178916

AN - SCOPUS:84859711910

VL - 421

SP - 541

EP - 546

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 2

ER -