A Polymeric Nanobeacon for Monitoring the Fluctuation of Hydrogen Polysulfides during Fertilization and Embryonic Development.

Fertilization and early embryonic development as the beginning of a new life are key biological events. Hydrogen polysulfide (H2 Sn ) plays important roles during physiological regulation, such as antioxidation-protection. However, no report has studied in situ H2 Sn fluctuation during early embryonic development because of the low abundance of H2 Sn and inadequate sensitivity of probes. We herein construct a polymeric nanobeacon from a H2 Sn -responsive polymer and fluorophores, which is capable of detecting H2 Sn selectively and of signal amplification. Taking the zebrafish as a model, the polymeric nanobeacon revealed that the H2 Sn level was significantly elevated after fertilization due to the activation of cell multiplication, suppressed partially during embryonic development, and finally kept steady up to zebrafish emergence. This strategy is generally accessible for biomarkers by altering the responsive unit and significant for facilitating biological analysis during life development.

Enhancing the Sensitivity of DNA and Aptamer Probes in the Dextran/PEG Aqueous Two-Phase System.

Increasing the local concentration of DNA-based probes is a convenient way to improve the sensitivity of biosensors. Instead of using organic solvents or ionic liquids that phase-separate with water based on hydrophobic interactions, we herein studied a classic aqueous two-phase system (ATPS) comprising polyethylene glycol (PEG) and dextran. Polymers of higher molecular weights and higher concentrations favored phase separation. DNA oligonucleotides are selectively enriched in the dextran-rich phase unless the pH was increased to 12. A higher volume ratio of PEG-to-dextran and a higher concentration of PEG also enrich more DNA probes in the dextran-rich phase. The partition efficiency of the T15 DNA was enriched around seven times in the dextran phase when the volume ratio of dextran and PEG reached 1:10. The detection of limit improved by 3.6-fold in a molecular beacon-based DNA detection system with the ATPS. The ATPS also increased the sensitivity for the detection of Hg2+ and adenosine triphosphate, although these target molecules alone distributed equally in the two phases. This work demonstrates a simple method using water soluble polymers to improve biosensors.

A Glucose-Powered Activatable Nanozyme Breaking pH and H2 O2 Limitations for Treating Diabetic Infections.

The peroxidase-like activity of nanozymes is promising for chemodynamic therapy by catalyzing H2 O2 into . OH. However, for most nanozymes, this activity is optimal just in acidic solutions, while the pH of most physiological systems is beyond 7.0 (even >8.0 in chronic wounds) with inadequate H2 O2 . We herein communicate an activatable nanozyme with targeting capability to simultaneously break the local pH and H2 O2 limitations under physiological conditions. As a proof of concept, aptamer-functionalized nanozymes, glucose oxidase, and hyaluronic acid constitute an activatable nanocapsule "APGH", which can be activated by bacteria-secreted hyaluronidase in infected wounds. Nanozymes bind onto bacteria through aptamer recognition, and glucose oxidation tunes the local pH down and supplements H2 O2 for the in-situ generation of . OH on bacteria surfaces. The activity switching and enhanced antibacterial effect of the nanocapsule were verified in vitro and in diabetic wounds. This strategy for directly regulating local microenvironment is generally accessible for nanozymes, and significant for facilitating biological applications of nanozymes.

Lipophilic G-Quadruplex Isomers as Biomimetic Ion Channels for Conformation-Dependent Selective Transmembrane Transport.

Transport selectivity is a challenge in the design of biomimetic transmembrane channels. In the present study, specific ion-dependent lipophilic G-quadruplexes displaying different conformations were designed for the construction of highly selective artificial transmembrane channels. The presence of Pb2+ and K+ ions prompted the folding of lipophilic PS2.M sequence into G-quadruplexes with antiparallel and parallel conformations. Membrane immobilization of the G-quadruplex channels restricted the reversible configurational changes between different topologies, which was confirmed by Förster resonance energy transfer (FRET) analysis. 8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS) transport assays revealed that Pb2+-stabilized antiparallel isomers, and K+-stabilized parallel isomers exhibited significant differences in the transmembrane transport. The former showed high Pb2+ transport activity (EC50 = 1.55 µM) and selectivity (Pb2+/K+ selectivity = 30.6), while the latter demonstrated high K+ transport activity (EC50 = 0.56 µM) and selectivity (K+/Pb2+ selectivity = 31.8). The cation selectivity of these channel mimics was also consistent with the outcomes of the conducted fluorescent probe assays. The results described herein provide a platform for effective development of conformation-dependent ion-selective biomimetic transmembrane channels. The G-quadruplex channels demonstrate high potential for application in the fields of molecular diagnostics, logic biocomputing, selective separation, and single-molecule biosensing.

Liposome-Boosted Peroxidase-Mimicking Nanozymes Breaking the pH Limit.

Peroxidase-mimicking nanozymes such as Fe3 O4 nanoparticles are promising substitutes for natural enzymes like horseradish peroxidase. However, most such nanozymes work efficiently only in acidic conditions. In this work, the influence of various liposomes on nanozyme activity was studied. By introducing negatively charged liposomes, peroxidase-mimicking nanozymes achieved oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in neutral and even alkaline conditions, although the activity towards anionic 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was inhibited. The Fe3 O4 nanoparticles adsorbed on the liposomes without disrupting membrane integrity as confirmed by fluorescence quenching, dye leakage assays, and cryo-electron microscopy. Stabilization of the blue-colored oxidized products of TMB by electrostatic interactions was believed to be the reason for the enhanced activity. This work has introduced lipids to nanozyme research, and it also has practically important applications for using nanozymes at neutral pH, such as the detection of hydrogen peroxide and glucose.

Flexible Assembly of an Enzyme Cascade on a DNA Triangle Prism Nanostructure for the Controlled Biomimetic Generation of Nitric Oxide.

Spatial organization of multiple enzymes at specific positions for a controlled reaction cascade has attracted wide attention in recent years. Here, we report the construction of a biomimetic enzyme cascade organized on DNA triangle prism (TP) nanostructures to enable the efficient catalytic production of nitric oxide (NO) on a single microbead. Two enzymes, glucose oxidase (GOx) and horseradish peroxidase (HRP), were assembled at adjacent locations on a DNA TP nanostructure by using DNA-binding protein adaptors with small interenzyme distances. In the cascade, the first enzyme, GOx, converts glucose into gluconic acid in the presence of oxygen. The produced H2 O2 intermediate is rapidly transported to the second enzyme, HRP, which oxides hydroxyurea into NO and other nitroxyl species. The pH near the surface of the negatively charged DNA nanostructures is believed to be lower than that in the bulk solution; this creates an optimal pH environment for the anchored enzymes, which results in higher yields of the NO product. Furthermore, the multienzyme system was immobilized on a microbead mediated by a DNA adaptor, and this enabled the efficient catalytic generation of gas molecules in the microreactor. Therefore, this work provides an alternative route for the biomimetic generation of NO through enzyme cascades. In particular, the dynamic binding capability of the DNA sequence enabled the positions of the protein enzyme and the DNA nanostructure to be reversed, which allowed the cascade catalysis to be modulated.

Design of a Modular DNA Triangular-Prism Sensor Enabling Ratiometric and Multiplexed Biomolecule Detection on a Single Microbead.

DNA nanostructures have emerged as powerful and versatile building blocks for the construction of programmable nanoscale structures and functional sensors for biomarker detection, disease diagnostics, and therapy. Here we integrated multiple sensing modules into a single DNA three-dimensional (3D) nanoarchitecture with a triangular-prism (TP) structure for ratiometric and multiplexed biomolecule detection on a single microbead. In our design, the complementary hybridization of three clip sequences formed TP nanoassemblies in which the six single-strand regions in the top and bottom faces act as binding sites for different sensing modules, including an anchor module, reference sequence module, and capture sequence module. The multifunctional modular TP nanostructures were thus exploited for ratiometric and multiplexed biomolecule detection on microbeads. Microbead imaging demonstrated that, after ratiometric self-calibration analysis, the imaging deviations resulting from uneven fluorescence intensity distribution and differing probe concentrations were greatly reduced. The rigid nanostructure also conferred the TP as a framework for geometric positioning of different capture sequences. The inclusion of multiple targets led to the formation of sandwich hybridization structures that gave a readily detectable optical response at different fluorescence channels and distinct fingerprint-like pattern arrays. This approach allowed us to discriminate multiplexed biomolecule targets in a simple and efficient fashion. In this module-designed strategy, the diversity of the controlled DNA assembly coupled with the geometrically well-defined rigid nanostructures of the TP assembly provides a flexible and reliable biosensing approach that shows great promise for biomedical applications.

Programmable Self-Assembly of DNA-Protein Hybrid Hydrogel for Enzyme Encapsulation with Enhanced Biological Stability.

A DNA-protein hybrid hydrogel was constructed based on a programmable assembly approach, which served as a biomimetic physiologic matrix for efficient enzyme encapsulation. A dsDNA building block tailored with precise biotin residues was fabricated based on supersandwich hybridization, and then the addition of streptavidin triggered the formation of the DNA-protein hybrid hydrogel. The biocompatible hydrogel, which formed a flower-like porous structure that was 6.7 ± 2.1 µm in size, served as a reservoir system for enzyme encapsulation. Alcohol oxidase (AOx), which served as a representative enzyme, was encapsulated in the hybrid hydrogel using a synchronous assembly approach. The enzyme-encapsulated hydrogel was utilized to extend the duration time for ethanol removal in serum plasma and the enzyme retained 78% activity after incubation with human serum for 24 h. The DNA-protein hybrid hydrogel can mediate the intact immobilization on a streptavidin-modified and positively charged substrate, which is very beneficial to solid-phase biosensing applications. The hydrogel-encapsulated enzyme exhibited improved stability in the presence of various denaturants. For example, the encapsulated enzyme retained 60% activity after incubation at 55 °C for 30 min. The encapsulated enzyme also retains its total activity after five freeze-thaw cycles and even suspended in solution containing organic solvents.

DNA nanotubes in coacervate microdroplets as biomimetic cytoskeletons modulate the liquid fluidic properties of protocells.

Coacervate microdroplets, formed via liquid-liquid phase separation, have been proposed as a compartment model for the construction of artificial cells or organelles. However, these microsystems are very fragile and demonstrate liquid-like fluidity. Here, an artificial cytoskeleton based on DNA nanotubes was constructed in coacervate microdroplets to modulate the liquid fluidic properties of the microdroplets. The coacervate microdroplets were obtained from the association of oppositely charged polyelectrolytes through liquid-liquid phase separation, and DNA nanotubes were constructed by molecular tile self-assembly from six clip sequences. The DNA nanotubes were efficiently sequestered in the liquid coacervate microdroplets, and the rigid structure of the DNA nanotubes was capable of modulating the liquid fluidic properties of the coacervate protocell models, as indicated by coalescence imaging and atomic force microscopy analysis. Therefore, artificial cytoskeletons made from DNA nanotubes worked in modulating the liquid fluidic properties of coacervate microdroplets, in a manner akin to the cytoskeleton in the cell. DNA cytoskeletons have the potential to become an ideal platform with which how the liquid fluidic properties of cells are modulated by their cytoskeletons can be investigated, and the cell-sized coacervate microdroplets containing artificial cytoskeletons might be critical in developing a stable liquid-phase protocell model.

DNA supersandwich assemblies as artificial receptors to mediate intracellular delivery of catalase for efficient ROS scavenging.

Aptamer-tailored DNA supersandwich assemblies are proposed as artificial receptors anchored onto a cell membrane, enabling catalase delivery with an 8-fold enhancement in efficiency. The catalase transfection led to inhibition of intracellular ROS, while the cell viability was also remarkably improved even when exposed to H2O2 and lipopolysaccharide environments.

Self-assembled DNA nanowires as quantitative dual-drug nanocarriers for antitumor chemophotodynamic combination therapy.

To combine cocktail chemotherapy and photodynamic therapy into one biocompatible and biodegradable nanocarrier, self-assembled DNA nanowires were fabricated and co-loaded with a photosensitizer chlorin e6 (Ce6) and a chemotherapeutic drug doxorubicin (DOX) for antitumor chemophotodynamic combination therapy. Two short DNA chains served as building blocks for the self-assembly of DNA nanowires in a supersandwich hybridization reaction that led to the successful formation of linear DNA nanowires of 500 bases, equal to a length of 167 nm. Ce6 and DOX were loaded onto the nanowires through covalent or noncovalent intercalation interactions, respectively. The DNA nanowires were taken up into cells, and the released Ce6 and DOX were ultimately distributed in different cellular compartments. The photosensitizer-loaded nanowires demonstrated increased generation of photodynamic reactive oxygen species (ROS) compared to that of free Ce6. In comparison with chemo- or photodynamic therapy alone, the combined treatment provided by DNA nanowires loaded with dual-drug significantly increased the incidence of HepG-2 cell death and produced a clear synergistic effect in the treatment of cancer cells. The DNA nanowire nanocarrier provided a flexible and quantitative drug-loading module that allowed for dose control of both drugs. More importantly, the DNA nanowires demonstrate a strong synergistic effect in antitumor chemophotodynamic combination therapy, likely because of increased photodynamic ROS generation and the distribution of Ce6 and DOX in different intracellular compartments. This work suggests that DNA nanowires may be useful as multifunctional and effective therapeutic nanocarriers for chemophotodynamic modalities in cancer therapy.