Recent development in the design of artificial enzymes through molecular imprinting technology.

Enzymes, a class of proteins or RNA with high catalytic efficiency and specificity, have inspired generations of scientists to develop enzyme mimics with similar capabilities. Many enzyme mimics have been developed in the past few decades based on small molecules, DNA, and nanomaterials. These artificial enzymes are of great interest because of their low cost and high stability. However, most of these enzyme mimics do not have the desired substrate selectivity. The substrate selectivity of natural enzymes usually stems from a specific binding pocket. A powerful method to create substrate binding cavities is molecular imprinting technology (MIT). Molecularly imprinted polymers (MIPs) have three main characteristics: structural predictability, identification specificity, and application versatility compared with other identification systems. The MIP-based artificial enzymes have the advantages of simple preparation, low cost, and high stability and can realize excellent catalytic activity and selectivity. The development of MIP-based artificial enzymes has been further promoted by optimization methods such as imprinting transition state molecules, post-imprinting modification, opening cross-linked polymers' internal space, and some special preparation methods. Combining molecular imprinting technology with nanozymes, the synergistic effect of both solved the defect of lack of specificity of nanozymes and improved their catalytic activity. This paper summarizes the recent research progress in preparing high-performance artificial enzymes based on MIPs and molecularly imprinted nanozymes. We hope to provide a reference for the design of artificial enzymes, reduce the gap between artificial enzymes and natural enzymes, and thus broaden the application of artificial enzymes in human life and production.

"On/Off" Switchable Sequential Light-Harvesting Systems Based on Controllable Protein Nanosheets for Regulation of Photocatalysis.

A controllable protein nanostructures-based "On/Off" switchable artificial light-harvesting system (LHS) with sequential multistep energy transfer and photocatalysis was reported herein for mimicking the natural LHS in both structure and function. Single-layered protein nanosheets were first constructed via a reversible covalent self-assembly strategy using cricoid stable protein one (SP1) as building blocks to realize an ordered arrangement of pigments. Fluorescent chromophores like carbon dots (CDs) can be precisely distributed on the protein nanosheets superficially via electrostatic interactions and make the ratio between donors and acceptors adjustable. After being anchored with a photocatalysis center (eosin-5-isothiocyanate, EY), the constructed LHS could sequentially transfer energy between two kinds of chromophores (CD1 and CD2), and further transfer to EY center with a high efficiency of 84%. Interestingly, the Förster resonance energy transfer (FRET) process of our LHS could be reversibly "On/Off" switched by the redox regulated assembly and disassembly of SP1 building blocks. Moreover, the LHS has been further proved to promote the yield of a model cross-coupling hydrogen evolution reaction and regulate the process of the reaction with the FRET process "On/Off" state.

Single-Molecule Observation of Selenoenzyme Intermediates in a Semisynthetic Seleno-α-Hemolysin Nanoreactor.

The development of monitoring methods to capture short-lived intermediates is crucial for kinetic mechanism validation of enzymatic reaction steps. In this work, a semisynthetic selenoenzyme nanoreactor was constructed by introducing the unnatural amino acid (Sec) into the lumen of the α-hemolysin (αHL) nanopore. This nanoreactor not only created a highly confined space to trap the enzyme-substrate complex for a highly efficient antioxidant activity but also provided a single channel to characterize a series of selenoenzyme intermediates in the whole catalytic cycle through electrochemical analysis. In particular, the unstable intermediate of SeOH can be clearly detected by the characteristic blocking current. The duration time corresponding to the lifetime of each intermediate that stayed within the nanopore was also determined. This label-free approach showed a high detection sensitivity and temporal-spatial resolution to scrutinize a continuous enzymatic process, which would facilitate uncovering the mysteries of selenoenzyme catalysis at the single-molecule level.

Single-Cell VEGF Analysis by Fluorescence Imaging-Microfluidic Droplet Platform: An Immunosandwich Strategy on the Cell Surface.

Despite recent advances in single-cell analysis techniques, the ability of single-cell analysis platforms to track specific cells that secreted cytokines remains limited. Here, we report a microfluidic droplet-based fluorescence imaging platform that can analyze single cell-secreted vascular endothelial growth factor (VEGF), an important regulator of physiological and pathological angiogenesis, to explore cellular physiological clues at the single-cell level. Two kinds of silica nanoparticle (NP)-based immunoprobes were developed, and they were bioconjugated to the membrane proteins of the probed cell surface via the bridging of secreted VEGF. Thus, an immunosandwich assay was built above the probed cell via fluorescence imaging analysis of each cell in isolated droplets. This analytical platform was used to compare the single-cell VEGF secretion ability of three cell lines (MCF-7, HeLa, and H8), which experimentally demonstrates the cellular heterogeneity of cells in secreting cytokines. The uniqueness of this method is that the single-cell assay is carried out above the cell of interest, and no additional carriers (beads or reporter cells) for capturing analytes are needed, which dramatically improves the availability of microdroplets. This single-cell analytical platform can be applied for determining other secreted cytokines at the single-cell level by changing other immune pairs, which will be an available tool for exploring single-cell metabonomics.

Supramolecularly regulated artificial transmembrane signal transduction for 'ON/OFF'-switchable enzyme catalysis.

An artificial signal transduction model with a supramolecular recognition headgroup, a membrane anchoring group, and a pro-enzyme catalysis endgroup was constructed. The transmembrane translocation of the transducer can be reversibly regulated by competitive host-guest complexations as an input signal to control an enzyme reaction inside the lipid vesicles.

Hierarchical protein self-assembly into dynamically controlled 2D nanoarrays via host-guest chemistry.

A dynamically reversible two-dimensional (2D) protein assembly system was designed based on host-guest interactions and was triggered to disassemble via a competition mechanism. The artificially tunable and reversible protein assembly architectures hold great potential for on/off switches in bio-systems.

Difunctionalized pillar[5]arene-based polymer nanosheets for photodynamic therapy of Staphylococcus aureus infection.

Bacterial infections pose severe threats to global public health security. Developing antibacterial agents with both high efficiency and safety to handle this problem has become a top priority. Here, highly stable and effective polymer nanosheets have been constructed by the covalent co-assembly of a pillar[5]arene derivative and metalloporphyrin for photodynamic antibacterial therapy (PDAT). The monolayer nanosheets are strongly positively charged and thus capable of binding with Staphylococcus aureus (SA) through electrostatic interactions. Additionally, the nanosheets can be activated to generate reactive oxygen species (ROS) under white-light irradiation, and exhibit satisfactory antibacterial performance towards SA. More importantly, cell viability assays demonstrate that the nanosheets show little to no cytotoxicity impact on mammalian cells even when the concentrations are much higher than those employed in the antibacterial studies. The above results suggest that the polymer nanosheets could be an effective antibacterial agent to overcome bacterial infections and hold a broad range of potential applications in real life.

Construction of Ultralarge Two-Dimensional Fluorescent Protein Arrays via a Reengineered Rhodamine B-Based Molecular Tool.

The self-luminous property of enhanced green fluorescent protein (EGFP) makes it an extremely attractive building block for creating functional biomaterials. A practical challenge in the design of EGFP-based materials, however, stems from the structural and chemical heterogeneity of the EGFP surface. In this study, a maleimide-functionalized rhodamine B molecule (RhG2M) was designed as a versatile molecular tool to overcome this obstacle. Site-specific modification of an EGFP variant (EGFP-4C) with RhG2M allowed for the fabrication of a series of well-defined two-dimensional (2D) arrays that span nano- and micrometer scales. Furthermore, the resulting ultralarge 2D EGFP-4C arrays feature both structural uniformity and flexibility, together with the inherent optical properties, making them advanced materials with great potential for practical applications. In addition, this strategy can be further extended into three dimensions and applied to the modular generation of periodic functional materials with more complex structures.

Virus-Based Supramolecular Structure and Materials: Concept and Prospects.

Rodlike and spherelike viruses are various monodisperse nanoparticles that can display small molecules or polymers with unique distribution following chemical modifications. Because of the monodisperse property, aggregates in synthetic protein-polymer nanoparticles could be eliminated, thus improving the probability for application in protein-polymer drug. In addition, the monodisperse virus could direct the growth of metal materials or inorganic materials, finding applications in hydrogel, drug delivery, and optoelectronic and catalysis materials. Benefiting from the advantages, the virus or viruslike particles have been widely explored in the field of supramolecular chemistry. In this review, we describe the modification and application of virus and viruslike particles in surpramolecular structures and biomedical research.

Engineering Nonmechanical Protein-Based Hydrogels with Highly Mechanical Properties: Comparison with Natural Muscles.

The elegant elasticity and toughness of muscles that are controlled by myofilament sliding, highly elastic springlike properties of titin, and Ca2+-induced conformational change of the troponin complex have been a source of inspiration to develop advanced materials for simulating elastic muscle motion. Herein, a highly stretchable protein hydrogel is developed to mimic the structure and motion of muscles through the combination of protein folding-unfolding and molecular sliding. It has been shown that the protein bovine serum albumin is covalently cross-linked, together penetrated with alginate chains to construct polyprotein-based hydrogels, where polyproteins can act as the elastic spring titin via protein folding-unfolding and also achieve tunable sliding facilitated by alginate due to their reversible noncovalent interactions, thus providing desired mechanical properties such as stretchability, resilience, and strength. Notably, these biomaterials can achieve the breaking strain of up to 1200% and show massive energy dissipation. A pronounced expansion-contraction phenomenon is also observed on the macroscopic scale, and the Ca2+-induced contraction process may help to improve our understanding of muscle movement. Overall, these excellent properties are comparable to or even better than those of natural muscles, making the polyprotein-based hydrogels represent a new type of muscle-mimetic biomaterial. Significantly, the prominent biocompatibility of the designed biomaterials further enables them to hold potential applications in the biomedical field and tissue engineering.

Morphological Transformation between Orthogonal Dynamic Covalent Self-Assembly of Imine-Boroxine Hybrid Polymer Nanocapsules and Thin Films via Linker Exchange.

Orthogonal dynamic covalent self-assembly is used as a facile method for constructing polymer hollow nanocapsules (NCs) and thin films. The bifunctional precursor 4-formylphenylboronic acid is symmetrically installed with a boronic acid group for the boroxine linkage, and an aldehyde group for the Schiff base reaction which can react with twofold symmetry linkers ethylenediamine and para phenylenediamine to attain polymer NCs and nanosheets. Owing to the reversibility of the imine linkages, the mutual morphological transformation between polymer NCs and thin films via an amine-imine-exchange strategy is successfully achieved. Multiple reversible covalent bonds allow the control the release of the load in polymer NCs using different techniques. This may be useful for designing stimulus-responsive smart materials.

Constructing antibacterial polymer nanocapsules based on pyridine quaternary ammonium salt.

Excessive use of antibiotics accelerates the development and spread of drug-resistant strains, which is a huge challenge for the field of medical health worldwide. Quaternary ammonium salt polymers are considered to be membrane-active bactericidal groups with vast potential to control bacterial infections and inhibit drug resistance. Herein, we report on the creative synthesis and characterization of novel antimicrobial polymer nanocapsules based on pyridine quaternary ammonium salt. The antimicrobial polymer nanocapsules were formed by reaction of C3 symmetrical rigid monomer 2,4,6­tris(4­pyridyl)­1,3,5­triazine (TPT) and a flexible linker 1,2­dibromoethane. The polymer nanocapsule was constructed as a cationic hollow sphere composed of a two-dimensional sheet whose main chain was formed by the pyridine quaternary ammonium salt, and a part of the bromide ion was adsorbed on the sphere. This hollow nanocapsule was characterized in detail by DLS, SEM, TEM, AFM, EDS and EA. When the cationic polymer nanocapsules are close to the Gram-negative Escherichia coli, the negatively charged phospholipid molecules in the bacterial membrane are attracted to the cationic surface and lead to rupture of cells. SEM confirmed the breakage of Escherichia coli membranes. The minimum inhibitory concentration was found to be 0.04 mg/mL, and the minimum bactericidal concentration was 0.1 mg/mL. Our experiments demonstrated that the adsorption of negatively charged phospholipid molecules on the surface of the pyridine quaternary ammonium salt polymer can kill Gram-negative bacteria without inserting quaternary ammonium salt hydrophobic groups into the cell membrane.

Rational Design and Biological Application of Antioxidant Nanozymes.

Nanozyme is a type of nanostructured material with intrinsic enzyme mimicking activity, which has been increasingly studied in the biological field. Compared with natural enzymes, nanozymes have many advantages, such as higher stability, higher design flexibility, and more economical production costs. Nanozymes can be used to mimic natural antioxidant enzymes to treat diseases caused by oxidative stress through reasonable design and modification. Oxidative stress is caused by imbalances in the production and elimination of reactive oxygen species (ROS) and reactive nitrogen species (RNS). This continuous oxidative stress can cause damage to some biomolecules and significant destruction to cell structure and function, leading to many physiological diseases. In this paper, the methods to improve the antioxidant properties of nanozymes were reviewed, and the applications of nanozyme antioxidant in the fields of anti-aging, cell protection, anti-inflammation, wound repair, cancer, traumatic brain injury, and nervous system diseases were introduced. Finally, the future challenges and prospects of nanozyme as an ideal antioxidant were discussed.

Biomimetic Pulsating Vesicles with Both pH-Tunable Membrane Permeability and Light-Triggered Disassembly-Re-assembly Behaviors Prepared by Supra-Amphiphilic Helices.

The reversible unfolding-refolding transition is considerably important for natural elastomeric proteins (e.g., titin) to fulfill their biological functions. It is of great importance to develop synthetic versions by borrowing their unique stretchable design principles. Herein, we present a novel pulsating vesicle by means of the aqueous self-assembly of supra-amphiphilic helices. Interestingly, this vesicle simultaneously features dynamic swelling and shrinkage movements in response to external proton triggers. Titin-like unfolding-refolding transformation of artificial helices was proved to play a crucial role in this pulsatile motion. Moreover, the vesicular membrane of this vesicle has exhibited tunable permeability during reversible expansion and contraction circulation. Meanwhile, light can also be used as a driving force to further regulate the disassembly-reassembly transformation of the pulsating vesicle. In addition, the drug delivery system was also employed as an investigating model to estimate the permeability variation and disassembly-reassembly behaviors of the pulsating vesicles, which displayed unique dual quick- and sustained-release behaviors toward anti-cancer agents. It is anticipated that this work opens an avenue for fabricating novel stretchable biomimetics by using the exclusive unfolding-refolding nature of artificial foldamers.

Construction of a reconfigurable DNA nanocage for encapsulating a TMV disk.

A new reconfigurable DNA nanocage based on a DNA origami method has been constructed to capture a tobacco mosaic virus (TMV) disk. We used a hairpin to control the transformation of the nanocage and a strand of TMV RNA to attract the TMV disk. Our design could inspire new DNA-protein complex designs.

Template-Free Construction of Highly Ordered Monolayered Fluorescent Protein Nanosheets: A Bioinspired Artificial Light-Harvesting System.

Using biological materials for light-harvesting applications has attracted considerable attention in recent years. Such materials provide excellent environmental compatibility and often exhibit superior properties over synthetic materials. Herein, inspired by the outstanding energy transfer performance in coelenterates, we constructed a template-free, highly ordered two-dimensional light-harvesting system by covalent-induced coassembly of EBFP2 (donor) and EGFP (acceptor), in which the fluorescent chromophores were well distributed and adopted a fixed orientation. By introducing approximate square planar binding sites on the side surface of protein, assembly pattern was pin down and self-assembly extended in orthogonal directions to achieve monolayered and tessellated protein nanoarrays. The excellent antiself-quenching property of fluorescent proteins endowed the coassembled system with attractive light-harvesting capability. Even at high local concentrations, a low resonance energy transfer self-quenching was observed and, therefore, energy can be efficiently transferred. More importantly, the distance between adjacent chromophores is continuously adjustable. By making minor changes to the length of the inducing linker, we have achieved significant control over the size of the assembly. A micron-sized light-harvesting system with satisfactory energy transfer efficiency was finally obtained. This work developed a template-free light-harvesting system completely based on fluorescent proteins (FPs), which overcame the restriction of using templates. Not limited to this work, the special core-shell structure of FPs may be expected to direct the optimization of fluorescent dyes by cladding.

Self-constructing giant vesicles for mimicking biomembrane fusion and acting as enzymatic catalysis microreactors.

Self-constructing giant fused vesicles based on hydrazone-pillar[5]arene (HP5) were formed catalytically in weak acid via the formation of dynamic covalent bonds in water. The HP5 vesicles mimicked the process of biomembrane fusion and acted as biocatalysis microreactors induced by fusion.

Construction of Artificial Enzymes on a Virus Surface.

Combination of artificial enzyme design and self-assembly strategies leads to a novel way to construct supramolecular enzymes. To address this challenge, auxotrophic expression systems show great potential because they can introduce nonnatural catalytic groups into the subunits of protein assemblies. Among nonnatural amino acids, selenocysteine is the catalytic group of glutathione peroxidase (GPx). With the aid of computer simulation, we have incorporated selenocysteine into natural protein assemblies such as tobacco mosaic virus (TMV) and ferritin by cysteine auxotrophic technology, resulting in the conversion of TMV and ferritin into supramolecular enzymes.

Reductive-Responsive, Single-Molecular-Layer Polymer Nanocapsules Prepared by Lateral-Functionalized Pillar[5]arenes for Targeting Anticancer Drug Delivery.

Herein, a new reductive-responsive pillar[5]arene-based, single-molecule-layer polymer nanocapsule is constructed for drug delivery. The functionalized system shows good biocompatibility, efficient internalization into targeted cells and obvious triggered release of entrapped drugs in a reducing environment such as cytoplasm. Besides, this smart vehicle loaded with anticancer drug shows excellent inhibition for tumor cell proliferation and exhibits low side effect on normal cells. This work not only demonstrates the development of a new reductive-responsive single molecular layer polymer nanocapsule for anticancer drug targeting delivery but also extends the design of smart materials for biomedical applications.

Design of artificial enzymes by supramolecular strategies.

Enzymes are biomacromolecules with three-dimensional structures composed of peptide polymers via supramolecular interactions. Owing to the incredible catalytic efficiency and unique substrate selectivity, enzymes arouse considerable attention. To rival natural enzymes, various artificial enzymes have been developed over the last decades. Since supramolecular interactions play important roles in both substrate recognition and the process of enzymatic catalysis, designing artificial enzymes using supramolecular strategies is undoubtedly significant. Here we discuss the recent advances in constructing artificial enzymes using supramolecular platforms.