Enzymatic micro/nanomotors in biomedicine: from single motors to swarms.

Micro/nanomotors (MNMs) have evolved from single self-propelled entities to versatile systems capable of performing one or multiple biomedical tasks. When single MNMs self-assemble into coordinated swarms, either under external control or triggered by chemical reactions, they offer advantages that individual MNMs cannot achieve. These benefits include intelligent multitasking and adaptability to changes in the surrounding environment. Here, we provide our perspective on the evolution of MNMs, beginning with the development of enzymatic MNMs since the first theoretical model was proposed in 2005. These enzymatic MNMs hold immense promise in biomedicine due to their advantages in biocompatibility and fuel availability. Subsequently, we introduce the design and application of single motors in biomedicine, followed by the control of MNM swarms and their biomedical applications. In the end, we propose viable solutions for advancing the development of MNM swarms and anticipate valuable insights into the creation of more intelligent and controllable MNM swarms for biomedical applications.

Metal-Organic Enzyme Nanogels as Nanointegrated Self-Reporting Chemobiosensors.

A fluorometric glucose biosensor based on fine-tuned chemoenzymatic nanohybrids is herein proposed. The successful integration of an engineered glucose oxidase enzyme and an optically responsive polymeric nanogel in a single entity has led to the fabrication of a highly efficient glucose chemobiosensor. The optical responsiveness has been achieved by the loading of preactivated polymeric hydrogel with fluorescent lanthanide, i.e., cerium (III), cations. A comprehensive investigation of the responsiveness of the biomaterial revealed the interplay between the oxidation state of the cerium lanthanide and the fluorescence emission of the polymer. Finally, a full structural, chemical, and biochemical characterization of the reported system supports the chemobiosensors as robust, specific, and sensitive materials that could be utilized to faithfully quantify the amount of glucose in tear fluids.

A Versatile Chemoenzymatic Nanoreactor that Mimics NAD(P)H Oxidase for the In Situ Regeneration of Cofactors.

Herein, we report a multifunctional chemoenzymatic nanoreactor (NanoNOx) for the glucose-controlled regeneration of natural and artificial nicotinamide cofactors. NanoNOx are built of glucose oxidase-polymer hybrids that assemble in the presence of an organometallic catalyst: hemin. The design of the hybrid is optimized to increase the effectiveness and the directional channeling at low substrate concentration. Importantly, NanoNOx can be reutilized without affecting the catalytic properties, can show high stability in the presence of organic solvents, and can effectively oxidize assorted natural and artificial enzyme cofactors. Finally, the hybrid was successfully coupled with NADH-dependent dehydrogenases in one-pot reactions, using a strategy based on the sequential injection of a fuel, namely, glucose. Hence, this study describes the first example of a hybrid chemoenzymatic nanomaterial able to efficiently mimic NOx enzymes in cooperative one-pot cascade reactions.

Tunable Polymeric Scaffolds for Enzyme Immobilization.

The number of methodologies for the immobilization of enzymes using polymeric supports is continuously growing due to the developments in the fields of biotechnology, polymer chemistry, and nanotechnology in the last years. Despite being excellent catalysts, enzymes are very sensitive molecules and can undergo denaturation beyond their natural environment. For overcoming this issue, polymer chemistry offers a wealth of opportunities for the successful combination of enzymes with versatile natural or synthetic polymers. The fabrication of functional, stable, and robust biocatalytic hybrid materials (nanoparticles, capsules, hydrogels, or films) has been proven advantageous for several applications such as biomedicine, organic synthesis, biosensing, and bioremediation. In this review, supported with recent examples of enzyme-protein hybrids, we provide an overview of the methods used to combine both macromolecules, as well as the future directions and the main challenges that are currently being tackled in this field.

Long-living and highly efficient bio-hybrid light-emitting diodes with zero-thermal-quenching biophosphors.

Bio-hybrid light-emitting diodes (Bio-HLEDs) based on color down-converting filters with fluorescent proteins (FPs) have achieved moderate efficiencies (50 lm/W) and stabilities (300 h) due to both thermal- and photo-degradation. Here, we present a significant enhancement in efficiency (~130 lm/W) and stability (>150 days) using a zero-thermal-quenching bio-phosphor design. This is achieved shielding the FP surface with a hydrophilic polymer allowing their homogenous integration into the network of a light-guiding and hydrophobic host polymer. We rationalize how the control of the mechanical and optical features of this bio-phosphor is paramount towards highly stable and efficient Bio-HLEDs, regardless of the operation conditions. This is validated by the relationships between the stiffness of the FP-polymer phosphor and the maximum temperature reached under device operation as well as the transmittance of the filters and device efficiency.

Immobilization of Enzymes in Protein Films.

Heterogeneous biocatalysis usually involves the use of immobilized enzymes on solid supports. Enzymes have suitable properties in terms of efficiency and selectivity for use as immobilized catalysts. Different approaches have been developed for effective immobilization, including adsorption, covalent binding, entrapment, encapsulation, and cross-linking. Those systems offer some advantages with regard to homogeneous catalysts in solution, such as low costs, easy separation and recovery of the catalyst, reusability, and enzymatic stability. Here, we describe a new approach for the immobilization of active enzymes into homogenous films composed solely of scaffolding proteins that differs from the standard methods of enzyme immobilization on solid supports.

Biocatalytic Protein-Based Materials for Integration into Energy Devices.

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.

Engineered protein-based functional nanopatterned materials for bio-optical devices.

The development of new active biocompatible materials and devices is a current need for their implementation in multiple fields, including the fabrication of implantable devices for biomedical applications and sustainable devices for bio-optics and bio-optoelectronics. This paper describes a simple strategy to use designed proteins to develop protein-based functional materials. Using simple proteins as self-assembling building blocks as a platform for the fabrication of new optically active materials takes previous work one step further towards the design of materials with defined structures and functions using naturally occurring protein materials, such as silk. The proposed fabrication strategy generates thin and flexible nanopatterned protein films by letting the engineered protein elements self-assemble over the surface of an elastomeric stamp with nanoscale features. These nanopatterned protein films are easily transferred onto 3D objects (flat and curved) by moisture-induced adhesion. Additionally, flexible nanopatterned protein films are prepared by incorporating a thin polymeric layer as a back support. Finally, taking advantage of the tunability of the selected protein scaffold, the flexible protein-based surfaces are endowed with optical functions, achieving efficient lasing features. As such, this work enables the simple and cost-effective production of flexible and nanostructured, protein-based, optically active biomaterials and devices over large areas toward emerging applications.

Self-assembly of repeat proteins: Concepts and design of new interfaces.

In nature, assembled protein structures offer the most complex functional structures. The understanding of the mechanisms ruling protein-protein interactions opens the door to manipulate protein assemblies in a rational way. Proteins are versatile scaffolds with great potential as tools in nanotechnology and biomedicine because of their chemical, structural, and functional versatility. Currently, bottom-up self-assembly based on biomolecular interactions of small and well-defined components, is an attractive approach to biomolecular engineering and biomaterial design. Specifically, repeat proteins are simplified systems for this purpose. In this work, we provide an overview of fundamental concepts of the design of new protein interfaces. We describe an experimental approach to form higher order architectures by a bottom-up assembly of repeated building blocks. For this purpose, we use designed consensus tetratricopeptide repeat proteins (CTPRs). CTPR arrays contain multiple identical repeats that interact through a single inter-repeat interface to form elongated superhelices. Introducing a novel interface along the CTPR superhelix allows two CTPR molecules to assemble into protein nanotubes. We apply three approaches to form protein nanotubes: electrostatic interactions, hydrophobic interactions, and π-π interactions. We isolate and characterize the stability and shape of the formed dimers and analyze the nanotube formation considering the energy of the interaction and the structure in the three different models. These studies provide insights into the design of novel protein interfaces for the control of the assembly into more complex structures, which will open the door to the rational design of nanostructures and ordered materials for many potential applications in nanotechnology.