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A novel ultra-hydrophilic zwitterionic-HILIC (ZIC-HILIC) nanosphere (Fe3O4-CG) was synthesized via a one-step hydrothermal strategy, which significantly simplified the conventional multi-step procedures for the preparation of ZIC-HILIC materials. The dual-functional Fe3O4-CG nanosphere exhibited excellent selectivity (molar ratio BSAHRP = 50001), low detection limit (0.05 fmol/μL), satisfactory reusability (at least 5 times) and recovery rate (93.7 ± 2.1%). The binding constant of Fe3O4-CG for HRP is 2.45 ± 0.32 × 10-6 M and the theoretica