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 (His6) tag and glycan residue on the target protein under the cooperation interaction of nitrilotriacetic acid (NTA)-Ni2+complex and boronic acid, respectively. Specifically, NTA-Ni2+ complex-modified magnetic beads (MBs-NTA-Ni2+) are employed to selectively capture the recombinant glycoprotein through the coordination interaction between His6 tag and NTA-Ni2+ 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 Cu2+ 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.
Keywords: Boronic acid; Electrochemical biosensor; Hexahistidine tags; Metal-organic framework; Recombinant glycoproteins.
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