基于组氨酸标记化学的 DNA-酶偶联物的适体基凝血酶微板检测分析。

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

机构信息

Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan.

出版信息

Anal Biochem. 2012 Feb 15;421(2):541-6. doi: 10.1016/j.ab.2011.11.028. Epub 2011 Dec 1.

DOI:10.1016/j.ab.2011.11.028

PMID:22178916

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.

摘要

我们报告了一种使用组氨酸标签（His-tag）化学制备 DNA-酶缀合物的方法。通过 Ni²⁺的络合作用，将 DNA 寡核苷酸修饰为氮川三乙酸（NTA），其对重组蛋白上的 His-tag 的 K(d)约为 10⁻⁶（M⁻¹）。碱性磷酸酶（His-AP）被用作模型酶。在微板上进行酶固定揭示了 His-AP 与通过 Ni²⁺络合的 NTA 修饰的 DNA 的缀合。SPR 测量也证明了 His-AP 与 NTA 修饰的 DNA 通过 Ni²⁺络合的缀合。然后，使用 DNA 适体通过 DNA-AP 缀合物检测凝血酶。DNA-AP 缀合物成功地放大了 DNA 适体与凝血酶之间的结合信号，并且信号通过来自 AP 催化反应的荧光强度来测量。检测限为 11 nM。最后，我们研究了从微板上释放固定的 His-AP 对 AP 活性的影响，因为本策略使用可切割的接头进行缀合和酶固定。DNase 催化的固定化 His-AP 的释放导致 AP 活性比表面固定化时观察到的活性高 1.7 倍。