Functionalization Strategies for Protease Immobilization on Magnetic Nanoparticles

Abstract

A comprehensive study on the general functionalization strategies for magnetic nanoparticles (MNPs) is presented in this work. Using well‐established techniques as well as modified protocols, the wide range of functional moieties grafted on γ‐Fe2O3 (maghemite) nanosurfaces include those of amine, aldehyde, carboxylic, epoxy, mercapto, and maleimide ends. Among the modified protocols are the one‐step water‐catalyzed silanization with mercaptopropyltrimethoxysilane, resulting in dense distal thiols, and the direct functionalization with a heterogeneous bifunctional linker N‐[p‐maleimidophenyl]isocynanate (PMPI). The former results in a protective Stöber type coating while simultaneously reducing the iron oxide core to magnetite (Fe3O4). The conjugation of trypsin, hereby chosen as model biomolecule, onto the differently functionalized MNPs is further demonstrated and assessed based on its activity, kinetics, and thermo‐/long‐term stability as well as reusability. Besides aqueous stability and ease in recovery by magnetic separation, the immobilized trypsin on MNPs offers superior protease durability and reusability, without compromising the substrate specificity and sequence coverage of free trypsin. The MNP‐based proteases can be used as valuable carriers in proteomics and miniaturized total analysis devices. The applicability of the functional surfaces devised in the current study is also relevant for the conjugation of other biomolecules beyond trypsin.

Covalent immobilization of porcine pancreatic lipase on carboxyl-activated magnetic nanoparticles: Characterization and application for enzymatic inhibition assays

“…Magnetic nanoparticles (MNPs) have gained popularity as immobilizing supports for bio-macromolecules because of their unique magnetic responsibility, low toxicity, and chemically modifiable surface, large enzyme loading capacity, and good reusability. Various enzymes have been immobilized on magnetic nanoparticle surfaces through a variety of functional groups including amine, aldehyde, carboxylic, epoxy, mercapto, and maleimide ends, and both enzymatic activities and stabilities were significantly improved [11]. In previous reports, amine and epoxy group functionalized silica-coated magnetic particles were used to covalently immobilize lipase [12,13].…”

Light Harvesting Proteins for Solar Fuel Generation in Bioengineered Photoelectrochemical Cells

“…of active site residues [65]. Enzyme-based crosslinking strategies represent a milder method with the potential to obtain higher amounts of active protein per surface area [58].…”

Effect of surface charge on the colloidal stability and in vitro uptake of carboxymethyl dextran-coated iron oxide nanoparticles

“…Factors such as magnetic structure, particle size, surface chemistry, and surface charge typically determine the magnetic response and physical or chemical interactions of the nanoparticles with biological entities during application. Emerging novel applications are focused on the development of magnetic nanoparticles as targeted magnetic carriers for drug delivery (Klostergaard and Seeney 2012; Gautier et al 2012; Manju and Sreenivasan 2011; Chen et al 2011; Veiseh et al 2010), the usage of magnetic nanoparticles as specific contrast enhancement agents for in vivo magnetic resonance imaging (MRI) (Jung et al 2011; Bhattacharya et al 2011), protein immobilization (Huang et al 2010; Li et al 2010), magnetic cell sorting (Xu et al 2011; Chen et al 2011), and biocompatible magnetic nanoparticles for cancer treatment mediated by hyperthermia (Creixell et al 2011; Mikhaylov and Vasiljeva 2011; Laurent et al 2011; Arias et al 2011). …”