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Magnetic nanoparticles (MNPs) are widely used for immobilizing complex biomolecules such as proteins due to their high loading capacity, biocompatibility, and easy magnetic harvesting. However, there is not a universal solution to achieve the oriented immobilization of the protein of interest. One common strategy involves the selective binding of poly-histidine-tagged (His-tag) proteins to divalent transition metal centers (TM2+) chelated by organic molecules like nitrilotriacetic acid (NTA). This article describes an innovative procedure to functionalize MNPs by simply growing of a thin layer of TM2+ hydroxides on the MNP surface, which increases the number of coordination centers and enhances the surface interaction with the protein surface. The immobilization yields of two sets of MNPs coated with Co2+, Ni2+ and Cu2+ hydroxides were compared to the conventional coating using the same inorganic cores but coated and functionalized with NTA-TM2+ moieties. It is shown that the one-step Co coated MNPs showed the highest selectivity and specificity towards His-tags. Thus, this coating not only reduce the functionalization steps to drive an oriented protein binding but also achieve an increment of immobilization yields over 1.5-fold respect to NTA-Co2+ analogous for his-tagged proteins with different 3D complexity while preserving similar or even higher enzymatic activity.

It is well known that magnetic nanoparticles (MNPs) absorb magnetic energy when exposed to an alternating magnetic field (AMF) and therefore dissipate heat in their local environment. So far, the heat dissipated by the MNPs has been widely used in biomedicine for cancer treatment and drug delivery. The FET-OPEN project HOTZYMES proposes instead to exploit this property for the biotechnological production of pharmaceuticals and bio-commodities, by conjugating thermophilic enzymes on the MNPs surface in order to selectively tune their activity by triggering this highly localized magnetic heating while maintaining the reaction media at low temperature., To this aim superparamagnetic iron oxide nanoparticles coated with different organic shells (dimercaptusuccinic acid, DMSA; polyacrylic acid, PAA) have been selected due to their good colloidal stability, high density of functionalizable chemical groups (-COOH) and a good combination of different heating efficiencies. They have been further functionalized with NTA-Cu2+, a chelating agent commonly used in protein purification by metal affinity interaction. Herein, we demonstrate that we can selectively bind a His-tag variant of the superfolded GFP protein, thanks to the high affinity of the immobilized copper to the 6xhistidine tag genetically fused to the protein. Furthermore, using the same strategy, we were able to bind the tetrameric recombinant His-tag variant of the B. stearothermophilus alcohol dehydrogenase (ADH) with also high specificity. Additionally, we demonstrate that the application of an AMF to the selected protein@MNPs derivatives can lead to the generation of different temperature gradients on the MNPs surface by studying both the GFP fluorescence (that strongly depends on temperature), and the effect of the triggered local heating on ADH activity. These first results suggest the feasibility of tuning remotely protein properties by combining their coupling to MNPs with the application of alternating magnetic fields.

The histidine tag (His-tag) is one of the most used affinity-tag for protein purification due to its small size and versatility. Agarose and sepharose beads containing nitriloacetic acid (NTA) transition metal derivatives are widely used for the purification of His-tagged proteins, thanks to their high affinity to the His-tag genetically fused to the protein of interest [1]. The same chemistry can be used to conjugate enzymes to magnetic nanoparticles (MNPs) with the aim of tuning their activity by magnetic heating [2, 3]. Within the frame of the FET-OPEN project HOTZYMES (https://www.hotzymes.eu), different MNPs have been synthetized and coated with polyacrylic acid and dimercaptosuccinic acid, and then were further functionalized with NTA-Cu2+ as His-tag chelating agent. Different proteins were expressed as His-tag variants and immobilized on the MNPs, including monomeric (superfolded GFP), dimeric (C. violaceum transaminase, CvTA; C. uda cellobiose phosphorylase, CuCbP), and tetrameric (B. stearothermophilus alcohol dehydrogenase, ADH) variants. While for the monomeric protein selected as model no difficulties in the bioconjugation processes were observed, when using dimeric or tetrameric enzymes the aggregation of the MNPs occurs very easily due to crosslinking between the nanoparticles. This colloidal destabilization of the MNPs is favored due to its high surface area and the presence of several tags per enzyme molecule. To avoid this situation, different strategies have been developed: saturation of the binding site of the nanoparticles, presence of a small percentage of imidazole in reaction, changes in the incubation conditions (pH, ionic strength, …). First positive results confirm that by playing with different factors it is possible to conjugate different His-tagged enzymes to very different MNPs in terms of size, shape, surface area, and colloidal stability. Actually, it is possible to avoid protein and MNPs aggregation while obtaining good activity yiel

The contactless heating capacity of magnetic nanoparticles (MNPs) has been exploited in fields such as hyperthermia cancer therapy, catalysis, and enzymatic thermal regulation. Herein, we propose an advanced technology to generate multiple local temperatures in a single-pot reactor by exploiting the unique nanoheating features of iron oxide MNPs exposed to alternating magnetic fields (AMFs). The heating power of the MNPs depends on their magnetic features but also on the intensity and frequency conditions of the AMF. Using a mixture of diluted colloids of MNPs we were able to generate a multi-hot-spot reactor in which each population of MNPs can be selectively activated by adjusting the AMF conditions. The maximum temperature reached at the surface of each MNP was registered using independent fluorescent thermometers that mimic the molecular link between enzymes and MNPs. This technology paves the path for the implementation of a selective regulation of multienzymatic reactions.

There is a current need to fabricate new biobased functional materials. Bottom-up approaches to assemble simple molecular units have shown promise for biomaterial fabrication due to their tunability and versatility for the incorporation of functionalities. Herein, the fabrication of catalytic protein thin films by the entrapment of catalase into protein films composed of a scaffolding protein is demonstrated. Extensive structural and functional characterization of the films provide evidence of the structural integrity, order, stability, catalytic activity, and reusability of the biocatalytic materials. Finally, these functional biomaterials are coupled with piezoelectric disks to fabricate a second generation of bio-inorganic generators. These devices are capable of producing electricity from renewable fuels through catalase-driven gas production that mechanically stimulates the piezoelectric material.