Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, Shandong Key Laboratory of Biochemical Analysis, Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao, 266042, People's Republic of China.
Mikrochim Acta. 2019 Feb 2;186(3):149. doi: 10.1007/s00604-019-3269-0.
An electrochemical method is described for the sensitive detection of the activity of the enzyme T4 polynucleotide kinase (PNK) by using a DNA functionalized porphyrinic metal-organic framework (L/(Fe-P)n-MOF). In the presence of PNK, the hairpin oligonucleotide (HP1) becomes phosphorylated, and the trigger is released by lambda exonuclease (λ exo). The trigger DNA hybridizes with hairpin probe (immobilized on the gold electrode) to form a nicking endonuclease cleavage site. Thus, a single-strand capture probe is employed to hybridize with L/(Fe-P)n-MOF. The (Fe-P)n-MOF is a peroxidase mimicking material with high catalytic efficiency. By using this amplification strategy, an electrochemical signal is procured that allows for the determination of T4 PNK in the 1.0 mU·mL to 1.0 U·mL with a detection limit of 0.62 mU·mL. The method is selective and can be used to screen for enzyme inhibitors. Conceivably, the (Fe-P)n-MOF can also be used to detect other analytes via its peroxidase-mimicking activity. Graphical abstract Schematic presentation of T4 polynucleotide kinase (PNK) detection. Two hairpin DNAs (HP) and a porphyrinic metal-organic framework with peroxidase-mimicking activity are used. The detection limit is 0.62 mU mL with enzyme assisted signal amplification. This method is selective and can be used to screen for enzyme inhibitors.
电化学方法用于通过使用 DNA 功能化卟啉金属-有机骨架（L/（Fe-P）n-MOF）灵敏检测 T4 多核苷酸激酶（PNK）的活性。在 PNK 的存在下，发夹寡核苷酸（HP1）被磷酸化，并且触发物由λ核酸外切酶（λ exo）释放。触发 DNA 与发夹探针（固定在金电极上）杂交以形成切口内切酶切割位点。因此，使用单链捕获探针与 L/（Fe-P）n-MOF 杂交。（Fe-P）n-MOF 是一种具有高效催化效率的过氧化物酶模拟物。通过使用这种扩增策略，可以获得电化学信号，从而允许在 1.0 mU·mL 至 1.0 U·mL 的范围内测定 T4 PNK，检测限为 0.62 mU·mL。该方法具有选择性，可用于筛选酶抑制剂。可以想象，（Fe-P）n-MOF 也可以通过其过氧化物酶模拟活性来检测其他分析物。
