Key Laboratory of Synthetic and Biological Colloids, Ministry of Education, School of Chemical and Material Engineering, Jiangnan University, Wuxi, Jiangsu, 214122, People's Republic of China; College of Chemistry and Chemical Engineering, Anyang Normal University, Anyang, Henan, 455000, People's Republic of China.
College of Chemistry and Chemical Engineering, Anyang Normal University, Anyang, Henan, 455000, People's Republic of China; College of Chemistry and Chemical Engineering, Henan University of Technology, Zhengzhou, Henan, 450001, People's Republic of China.
Anal Chim Acta. 2023 Sep 8;1273:341540. doi: 10.1016/j.aca.2023.341540. Epub 2023 Jun 17.
Accurate and sensitive determination of recombinant glycoproteins is in great demand for the treatment of anemia-induced chronic kidney disease and the illegal use of doping agents in sports. In this study, an antibody and enzyme-free electrochemical method for the detection of recombinant glycoproteins was proposed via the sequential chemical recognition of hexahistidine (His) tag and glycan residue on the target protein under the cooperation interaction of nitrilotriacetic acid (NTA)-Nicomplex and boronic acid, respectively. Specifically, NTA-Ni complex-modified magnetic beads (MBs-NTA-Ni) are employed to selectively capture the recombinant glycoprotein through the coordination interaction between His tag and NTA-Ni complex. Then, boronic acid-modified Cu-based metal-organic frameworks (Cu-MOFs) were recruited by glycans on the glycoprotein via the formation of reversible boronate ester bonds. MOFs with abundant Cu ions acted as efficient electroactive labels to directly produce amplified electrochemical signals. By using recombinant human erythropoietin as a model analyte, this method showed a wide linear detection range from 0.01 to 50 ng/mL and a low detection limit of 5.3 pg/mL. With the benefits from the simple operation and low cost, the stepwise chemical recognition-based method shows great promise in the determination of recombinant glycoproteins in the fields of biopharmaceutical research, anti-doping analysis and clinical diagnosis.
准确、灵敏地测定重组糖蛋白对于治疗因贫血引起的慢性肾病和运动中非法使用兴奋剂具有重要意义。本研究提出了一种抗体和酶免的电化学方法，用于检测重组糖蛋白。该方法通过在氮川三乙酸（NTA）-镍配合物和硼酸的协同作用下，依次对目标蛋白上的组氨酸（His）标签和糖基残基进行化学识别。具体来说，采用 NTA-镍配合物修饰的磁珠（MBs-NTA-Ni）通过 His 标签与 NTA-镍配合物的配位相互作用选择性捕获重组糖蛋白。然后，通过糖基与硼酸形成可逆硼酸酯键，将硼酸修饰的铜基金属有机骨架（Cu-MOFs）募集到糖蛋白上。具有丰富 Cu 离子的 MOFs 可直接产生放大的电化学信号，用作有效的电活性标记物。以重组人促红细胞生成素（rhEPO）为模型分析物，该方法在 0.01 至 50ng/mL 的宽线性检测范围内表现出较低的检测限（5.3pg/mL）。基于逐步化学识别的方法具有操作简单、成本低等优点，有望在生物制药研究、反兴奋剂分析和临床诊断等领域用于测定重组糖蛋白。
