Trypsin-, proteinase K-, and ribonuclease T1-functionalized magnetic nanoparticle-immobilized enzyme reactors based...

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 (K_m, V_max, k_cat, and k_cat/K_m) 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.