Microplate assay for aptamer-based thrombin detection using a DNA-enzyme coniueate based on histidine-tag chemistry
Microplate assay for aptamer-based thrombin detection using a DNA-enzyme coniueate based on histidine-tag chemistry
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使用基于组氨酸标签化学的 DNA 酶结合物进行基于适体的凝血酶检测的微孔板测定
DOI：
10.1016/j.ab.2011.11.028
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发表时间：
2012
影响因子：
2.9
通讯作者：
et.al.
中科院分区：
文献类型：
--
作者：
J.Shimada;et.al.
我们报告了一种使用组氨酸标签（His-tag）化学制备DNA-酶结合物的方法。通过Ni2+的络合作用，用对重组蛋白质上存在的His标签的Kd约为10 − 6（M − 1）的次氮基三乙酸酯（NTA）修饰DNA寡核苷酸。His标记的碱性磷酸酶（His-AP）被用作模型酶。酶固定在微孔板上揭示了His-AP和NTA修饰的DNA通过Ni 2+络合物的缀合。SPR测量也证明了His-AP与NTA修饰的DNA通过Ni 2+络合物的缀合。DNA-酶缀合物然后用于使用DNA适体检测凝血酶。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，并且该信号被测量为AP催化反应产生的荧光强度。检测限为11 nM。最后，我们研究了从微孔板上释放固定化的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。DNA酶催化释放的固定化His-AP导致AP活性比His-AP表面固定化时观察到的高1.7倍。
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 Kdwas 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 11nM. 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.