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<p>Immobilized enzyme reactors (IMERs) have gained increasing attention owing to their numerous advantages, including reusability, easy handling, cost saving, and prolonged lifetime. In this work, Fe<sub>3</sub>O<sub>4</sub> nanoparticles bearing different functional groups (-OH, -NH<sub>2</sub>, and -COOH) were modified with polyelectrolytes poly(ethyleneimine) and poly(acrylic acid), and systematically evaluated as magnetic supports for enzyme immobilization. Polyelectrolytes acting as spacer arms to minimize steric hindrance at the&#xa0;enzyme’s active sites and providing terminal carboxyl groups for covalent enzyme immobilization through amide coupling were selected for efficient surface chemistry. After optimization of the bonding chemistry, the synthesized IMERs were characterized using high-resolution surface imaging helium ion microscopy (HIM), Fourier transform infrared spectroscopy (FT-IR), zeta potential measurements, and energy dispersive X-ray spectroscopy (EDX). Trypsin, proteinase K, and ribonuclease (RNase) T1 as target enzymes were immobilized onto magnetic beads, respectively. The bioactivity of the immobilized enzymes was estimated by determination of the Michaelis-Menten parameters (<i>K</i><sub><i>m</i></sub>, <i>V</i><sub><i>max</i></sub>, <i>k</i><sub><i>cat</i></sub>, and <i>k</i><sub><i>cat</i></sub><i>/K</i><sub><i>m</i></sub>) of test substrates and compared to the&#xa0;corresponding enzyme in solution. The amount of bound enzyme was quantified by Micro BCA assay measuring the absorbance at 562&#xa0;nm. Furthermore, the developed IMERs were applied to protein and RNA digestion, and the resulting fragments were analyzed by HPLC–ESI-QTOF-MS/MS, demonstrating their applicability for biomolecular analysis. Finally, successful mRNA digestion isolated from a vaccine documented the practical applicability of the RNase T1 IMER and the suitability of the polyelectrolyte multilayer enzyme immobilization platform.</p> Graphical Abstract <p></p>

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Trypsin-, proteinase K-, and ribonuclease T1-functionalized magnetic nanoparticle-immobilized enzyme reactors based on polyelectrolyte multilayer platform for biopharmaceutical analysis

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月29日

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  • Yachao Hao,
  • Niklas Carstensen,
  • Ronny Loeffler,
  • Benjamin Junker-Reiss,
  • Dominique Lunter,
  • Michael Lämmerhofer

摘要

Immobilized enzyme reactors (IMERs) have gained increasing attention owing to their numerous advantages, including reusability, easy handling, cost saving, and prolonged lifetime. In this work, Fe3O4 nanoparticles bearing different functional groups (-OH, -NH2, and -COOH) were modified with polyelectrolytes poly(ethyleneimine) and poly(acrylic acid), and systematically evaluated as magnetic supports for enzyme immobilization. Polyelectrolytes acting as spacer arms to minimize steric hindrance at the enzyme’s active sites and providing terminal carboxyl groups for covalent enzyme immobilization through amide coupling were selected for efficient surface chemistry. After optimization of the bonding chemistry, the synthesized IMERs were characterized using high-resolution surface imaging helium ion microscopy (HIM), Fourier transform infrared spectroscopy (FT-IR), zeta potential measurements, and energy dispersive X-ray spectroscopy (EDX). Trypsin, proteinase K, and ribonuclease (RNase) T1 as target enzymes were immobilized onto magnetic beads, respectively. The bioactivity of the immobilized enzymes was estimated by determination of the Michaelis-Menten parameters (Km, Vmax, kcat, and kcat/Km) of test substrates and compared to the corresponding enzyme in solution. The amount of bound enzyme was quantified by Micro BCA assay measuring the absorbance at 562 nm. Furthermore, the developed IMERs were applied to protein and RNA digestion, and the resulting fragments were analyzed by HPLC–ESI-QTOF-MS/MS, demonstrating their applicability for biomolecular analysis. Finally, successful mRNA digestion isolated from a vaccine documented the practical applicability of the RNase T1 IMER and the suitability of the polyelectrolyte multilayer enzyme immobilization platform.

Graphical Abstract