Cobalt and zinc nanoparticles from pyrolysis of their MOF precursors exhibiting potent organophosphorus hydrolase-mimicking activities.

Cobalt and zinc nanoparticles from pyrolysis of cobalt-containing ZIF-67 and zinc-containing ZIF-90 exhibited potent organophosphorus hydrolase-mimicking activities for the hydrolysis of organophosphorus compounds within minutes at pH 9.0 and 25-40 °C. The resulting nanozymes could find potential applications in many areas such as chemical decontamination, environmental protection and defense of chemical weapons.

Au nanoparticle-embellished UiO-66 on reduced graphene oxide as a non-enzymatic electrocatalyst at a remarkably low oxidation potential for glucose oxidation and sensing.

Au nanoparticle-embellished metal-organic framework UiO-66 on reduced graphene oxide (Au/UiO-66/rGO) was synthesized. Au/UiO-66/rGO displayed strong electrocatalytic activity for oxidation of glucose in alkaline solution at a remarkably low oxidation potential of +0.20 V vs. Ag/AgCl. Au nanoparticles played a paramount role in the catalytic oxidation of glucose at the electrode, while both rGO and UiO-66 can significantly enhance the current responses to glucose. The resulting non-enzymatic glucose sensor exhibited a wide range of linear response, high sensitivity and selectivity for the determination of glucose. The sensor was successfully applied for the determination of glucose in honey products.

A Critical Review on Organic Small Fluorescent Probes for Monitoring Carbon Monoxide in Biology.

Endogenous carbon monoxide (CO) is an important intracellular gas messenger that is intimately involved in many physiological and pathological processes. The abnormal concentration of CO in living organisms can cause many diseases. Therefore, it is of great significance to monitor CO in biological samples. Fluorescent probe technology provides an effective and convenient method for CO monitoring, with the advantages of high selectivity and sensitivity, fast response time and in situ fluorescence imaging in biological tissues, which is favored by the majority of researchers. In this paper, the research progress of CO fluorescent probes since 2018 is reviewed, and the design, detection mechanism and biological application of the related fluorescent probes are summarized. And the relationship between the structure and performance of the probes is discussed. Furthermore, the development trend and application prospect of CO fluorescent probes are prospected.

A near infrared fluorescent probe for detection and bioimaging of zinc ions and hypochlorous acid.

It is of great significance to monitor zinc ions (Zn2+) and hypochlorous acid (HClO) conveniently in disease diagnosis and environmental protection. Herein, we successfully prepared a near-infrared fluorescent probe based on dicyanoisophorone derivative and methyl hydrazate by Schiff base reaction for effective detection of Zn2+ and HClO. After binding with Zn2+ at a molar ratio of 1:1, the fluorescence intensity (λex = 482 nm, λem = 653 nm) of the probe was significantly enhanced, thus achieving quantitative detection and optical cell imaging of Zn2+. The complexation constant (K) of the probe with Zn2+ was 2.34 × 104 M-1. The response time of the probe for Zn2+ was less than 20 s and the detection limit was 15.3 nM. Moreover, this probe also showed a specific response to HClO. After interacting with HClO, the fluorescence intensity of the probe was increased significantly with a red shift of the emission wavelength from 670 nm to 705 nm (λex = 550 nm). The response time and the detection limit of probe for HClO were 4 min and 1.39 µM, respectively. The probe was successfully applied for visual bioimaging of Zn2+ and HClO inside living cells. The recoveries of Zn2+ and HClO using the probe in actual water samples were satisfactory.

Polyethyleneimine-Functionalized Carbon Nanotubes Enabling Potent Antimycotic Activity of Lyticase.

In this work, the positively-charged polymer polyethyleneimine was used to functionalize carbon nanotubes and activated carbon to load antimycotic enzyme lyticase. Interestingly, polyethyleneimine played a dual role functionalizing carbon materials to synergistically enhance antimycotic activity of loaded lyticase as well as exhibiting its own apparent antimycotic activity, where the enhanced enzymatic activity of loaded lyticase on functionalized carbon nanotubes was more than 2.8 times as high as the activity of free enzyme in solution. The actual activity of loaded lyticase on functionalized carbon nanotubes was applied with Penicillium janthinellum, exhibiting much faster digesting lysis of the bacteria in comparison with free lyticase. The synergistic and potent antimycotic activities from combined action of antimycotic lyticase and polyethyleneimine on carbon nanotubes provides a new antimycotic protection for medicine, food industry, and other biochemical processes.

Reduced activity of intestinal surface Na+/H+ exchanger NHE3 is a key factor for induction of diarrhea after PEDV infection in neonatal piglets.

Porcine epidemic diarrhea virus (PEDV; family Coronaviridae, genus Alphacoronavirus) causes acute diarrhea and vomiting, dehydration, and high mortality in neonatal piglets. Despite extensive research focusing on the pathogenesis of PEDV infection, the molecular pathogenesis of PEDV-induced diarrhea in piglets remains unclear. Na+/H+ exchanger 3 (NHE3), the main exchanger of electroneutral sodium in intestinal epithelial cells, is closely associated with the occurrence of diarrhea. To date, there is no study on whether diarrhea caused by PEDV infection is related to the activity of NHE3. In the present study, it was found that the expression level of cell membrane protein NHE3 significantly decreased after PEDV infection, whereas the total level of protein expression was not significantly changed. The Na+/H+ transport rate and the mRNA abundance of NHE3 decreased; the NHE3 activity decreased gradually with increasing infection time. In vivo, after PEDV infection of newborn piglets, rupture of intestinal villi and interstitial degeneration of intestinal epithelial cells in different intestinal segments were observed by hematoxylin-eosin staining. Immunohistochemical and immunofluorescence methods were used to observe the decreased expression of NHE3 protein on the membrane of intestinal epithelial cells in the jejunum and ileum. Taken together, these data indicate that PEDV infection reduces NHE3 activity in intestinal epithelial cells, hindering Na+ transport and thus causing diarrhea.

High Throughput Mapping of Single Molecules' Redox Potentials on Electrode.

By synchronizing electrochemical potential scanning with a single-molecule localization super-resolution fluorescence microscope, kinetic fluorescence changes of hundreds of single molecular redox events were tracked simultaneously with high throughput, and subsequent cross-correlation function analysis mapped single molecules' redox potentials (times) out on the imaging area from site to site in unprecedented detail by extracting electrochemically induced fluorescence change from apparently random fluorescence on/off blinking. This work paves the way toward mapping redox states at single-molecule levels in high throughput in chemical and biological systems.

Non-destructively shattered mesoporous silica for protein drug delivery.

Mesoporous silicas have been extensively used for entrapping small chemical molecules and biomacromolecules for drug delivery. We hypothesize that the loading density of biomacromlecules such as proteins in mesoporous silicas could be limited due to disordering in the pore structure and long diffusion time in the pore channels. We shattered mesoporous silicas non-destructively resulting in improved intramesoporous structures and reduced particle sizes in aqueous solutions by a powerful sonication, where the mesoporous structures were still well maintained. The sonication-shattered mesoporous silica can increase the protein loading density to nearly 2.7 times as high as that of the non-shattered one, demonstrating that significantly more mesopore space of the silica could be accessible by the protein molecules, which may result in more sustained protein drug delivery.

Heated proteins are still active in a functionalized nanoporous support.

Even under heated conditions, the nearly native conformation and activity of a protein can be hoarded in a functionalized nanoporous support via non-covalent interaction. Surprisingly, the protein released from the heated protein-nanoporous composite can maintain its nearly native conformation and activity, while free proteins are permanently denatured under the same treatment.

Intramesoporous silica structure differentiating protein loading density.

We report that hydrothermal aging temperature had a critical effect on intramesoporous structure of mesoporous silica and thus the intramesoporous structure affected protein loading in the mesoporous silica significantly. For a neutral protein Immunoglobulin G with a Y-like molecular shape, the larger desorption pore size allowed the larger protein loading. For a charged protein glucose oxidase with an elliptical molecular shape, the larger surface area resulted in the larger protein loading. Fluorescence emission spectra from tyrosinyl and tryptophanyl residues of the proteins in mesoporous silicas indicated that the charged protein was electrostatically attached inside the mesopores in a way of monolayer, while the neutral protein IgG could continue to aggregate after the monolayer occupancy.

In situ regeneration of NADH via lipoamide dehydrogenase-catalyzed electron transfer reaction evidenced by spectroelectrochemistry.

NAD/NADH is a coenzyme found in all living cells, carrying electrons from one reaction to another. We report on characterizations of in situ regeneration of NADH via lipoamide dehydrogenase (LD)-catalyzed electron transfer reaction to regenerate NADH using UV-vis spectroelectrochemistry. The Michaelis-Menten constant (K(m)) and maximum velocity (V(max)) of NADH regeneration were measured as 0.80±0.15 mM and 1.91±0.09 µM s(-1) in a 1-mm thin-layer spectroelectrochemical cell using gold gauze as the working electrode at the applied potential -0.75 V (vs. Ag/AgCl). The electrocatalytic reduction of the NAD system was further coupled with the enzymatic conversion of pyruvate to lactate by lactate dehydrogenase to examine the coenzymatic activity of the regenerated NADH. Although the reproducible electrocatalytic reduction of NAD into NADH is known to be difficult compared to the electrocatalytic oxidation of NADH, our spectroelectrochemical results indicate that the in situ regeneration of NADH via LD-catalyzed electron transfer reaction is fast and sustainable and can be potentially applied to many NAD/NADH-dependent enzyme systems.

Enzymatic conversion of CO(2) to bicarbonate in functionalized mesoporous silica.

We report here a concept converting carbon dioxide to biocarbonate in a biomimetic nanoconfiguration. Carbonic anhydrase (CA), the fastest enzyme that can covert carbon dioxide to bicarbonate, can be spontaneously entrapped in carboxylic acid group-functionalized mesoporous silica (HOOC-FMS) with super-high loading density (up to 0.5 mg of protein/mg of FMS) in sharp contrast to normal porous silica. The binding of CA to HOOC-FMS resulted in a partial conformational change comparing to the enzyme free in solution, but it can be overcome with increased protein loading density. The higher the protein loading density, the less conformational change, hence the higher enzymatic activity and the higher enzyme immobilization efficiency (up to >60%). The released enzyme still displayed the native conformational structure and the same high enzymatic activity as that prior to the enzyme entrapment, indicating that the conformational change resulted from the electrostatic interaction of CA with HOOC-FMS was not permanent. This work may provide a new approach converting carbon dioxide to biocarbonate that can be integrated with the other part of biosynthesis process for the assimilation of carbon dioxide.

Delivery of MicroRNA-10b with Polylysine Nanoparticles for Inhibition of Breast Cancer Cell Wound Healing.

Recent studies revealed that micro RNA-10b (mir-10b) is highly expressed in metastatic breast cancer cells and positively regulates breast cancer cell migration and invasion through inhibition of HOXD10 target synthesis. In this study we designed anti-mir-10b molecules and combined them with poly L-lysine (PLL) to test the delivery effectiveness. An RNA molecule sequence exactly matching the mature mir-10b minor antisense showed strong inhibition when mixed with PLL in a wound-healing assay with human breast cell line MDA-MB-231. The resulting PLL-RNA nanoparticles delivered the anti-microRNA molecules into cytoplasm of breast cancer cells in a concentration-dependent manner that displayed sustainable effectiveness.

In vitro release of organophosphorus acid anhydrolase from functionalized mesoporous silica against nerve agents.

We report here that under different physiological conditions, biomolecular drugs can be stockpiled in a nanoporous support and afterward can be instantly released when needed for acute responses, and the biomolecular drug molecules can also be gradually released from the nanoporous support over a long time for a complete recovery. Organophosphorus acid anhydrolase (OPAA) was spontaneously and largely entrapped in functionalized mesoporous silica (FMS) due to the dominant electrostatic interaction. The OPAA-FMS composite exhibited a burst release in a pH 9.0 NaHCO3-Na2CO3 buffer system and a gradual release in pH 7.4 simulated body fluid. The binding of OPAA to NH2-FMS can result in less tyrosinyl and tryptophanyl exposure OPAA molecules to aqueous environment. The bound OPAA in FMS displayed lower activity than the free OPAA in solution prior to the enzyme entrapment. However, the released enzyme maintained the native conformational structure and the same high enzymatic activity as that prior to the enzyme entrapment. The in vitro results in the rabbit serum demonstrate that both OPAA-FMS and the released OPAA may be used as a medical countermeasure against the organophosphorus nerve agents.

Conformational variability of organophosphorus hydrolase upon soman and paraoxon binding.

The bacterial enzyme organophosphorus hydrolase (OPH) exhibits both catalytic and substrate promiscuity. It hydrolyzes bonds in a variety of phosphotriester (P-O), phosphonothioate (P-S), phosphofluoridate (P-F), and phosphonocyanate (F-CN) compounds. However, its catalytic efficiency varies markedly for different substrates, limiting the broad-range application of OPH as catalyst in the bioremediation of pesticides and chemical war agents. In the present study, pK(a) calculations and multiple explicit-solvent molecular dynamics (MD) simulations were performed to characterize and contrast the structural dynamics of OPH bound to two substrates hydrolyzed with very distinct catalytic efficiencies: the nerve agent soman (O-pinacolylmethylphosphonofluoridate) and the pesticide paraoxon (diethyl p-nitrophenyl phosphate). pK(a) calculations for the substrate-bound and unbound enzyme showed a significant pK(a) shift from standard values (ΔpK(a) = ±3 units) for residues His254 and Arg275. MD simulations of protonated His254 revealed a dynamic hydrogen bond network connecting the catalytic residue Asp301 via His254 to Asp232, Asp233, Arg275, and Asp235, and is consistent with a previously postulated proton relay mechanism to ferry protons away from the active site with substrates that do not require activation of the leaving group. Hydrogen bonds between Asp301 and His254 were persistent in the OPH-paraoxon complex but not in the OPH-soman one, suggesting a potential role for such interaction in the more efficient hydrolysis of paraoxon over soman by OPH. These results are in line with previous mutational studies of residue His254, which led to an increase of the catalytic efficiency of OPH over soman yet decreased its efficiency for paraoxon. In addition, comparative analysis of the molecular trajectories for OPH bound to soman and paraoxon suggests that binding of the latter facilitates the conformational transition of OPH from the open to the closed substate promoting a tighter binding of paraoxon.

Local release of highly loaded antibodies from functionalized nanoporous support for cancer immunotherapy.

We report that antibodies can be spontaneously loaded in functionalized mesoporous silica (FMS) with superhigh density (0.4-0.8 mg of antibody/mg of FMS) due to their comprehensive noncovalent interaction. The superhigh loading density and noncovalent interaction between FMS and antibodies allow long-lasting local release of the immunoregulatory molecules from FMS under physiological conditions. Preliminary data indicate that FMS-anti-CTLA4 antibody injected directly into a mouse melanoma induces much greater and extended inhibition of tumor growth than the antibody given systemically. Our findings open up a novel approach for local delivery of therapeutically active proteins to tumors and, potentially, other diseases.

The role of nonbonded interactions in the conformational dynamics of organophosphorous hydrolase adsorbed onto functionalized mesoporous silica surfaces.

The enzyme organophosphorous hydrolase (OPH) catalyzes the hydrolysis of a wide variety of organophosphorous compounds with high catalytic efficiency and broad substrate specificity. The immobilization of OPH in functionalized mesoporous silica (FMS) surfaces increases significantly its catalytic specific activity, as compared to the enzyme in solution, with important applications for the detection and decontamination of insecticides and chemical warfare agents. Experimental measurements of immobilization efficiency as a function of the charge and coverage percentage of different functional groups have been interpreted as electrostatic forces being the predominant interactions underlying the adsorption of OPH onto FMS surfaces. Explicit solvent molecular dynamics simulations have been performed for OPH in bulk solution and adsorbed onto two distinct interaction potential models of the FMS functional groups to investigate the relative contributions of nonbonded interactions to the conformational dynamics and adsorption of the protein. Our results support the conclusion that electrostatic interactions are responsible for the binding of OPH to the FMS surface. However, these results also show that van der Waals forces are detrimental for interfacial adhesion. In addition, it is found that OPH adsorption onto the FMS models favors a protein conformation whose active site is fully accessible to the substrate, in contrast to the unconfined protein.

Probing mechanisms for enzymatic activity enhancement of organophosphorus hydrolase in functionalized mesoporous silica.

We have previously reported that organophosphorus hydrolase (OPH) can be spontaneously entrapped in functionalized mesoporous silica (FMS) with HOOC- as the functional groups and the entrapped OPH in HOOC-FMS showed enhanced enzyme specific activity. This work is to study the mechanisms that why OPH entrapped in FMS displayed the enhanced activity in views of OPH-FMS interactions using spectroscopic methods. The circular dichroism (CD) spectra show that, comparing to the secondary structure of OPH free in solution, OPH in HOOC-FMS displayed increased alpha-helix/beta-strand transition of OPH with increased OPH loading density. The fluorescence emission spectra of Trp residues were used to assess the tertiary structural changes of the enzyme. There was a 42% increase in fluorescence. This is in agreement with the fact that the fluorescence intensity of OPH was increased accompanying with the increased OPH activity when decreasing urea concentrations in solution. The steady-state anisotropy was increased after OPH entrapping in HOOC-FMS comparing to the free OPH in solution, indicating that protein mobility was reduced upon entrapment. The solvent accessibility of Trp residues of OPH was probed by using acrylamide as a collisional quencher. Trp residues of OPH-FMS had less solvent exposure comparing with free OPH in solution due to its electrostatical binding to HOOC-FMS thereby displaying the increased fluorescence intensity. These results suggest the interactions of OPH with HOOC-FMS resulted in the protein immobilization and a favorable conformational change for OPH in the crowded confinement space and accordingly the enhanced activity.

Clay nanoparticle-supported single-molecule fluorescence spectroelectrochemistry.

Here we report that clay nanoparticles allow formation of a modified transparent electrode, spontaneous adsorption of fluorescent redox molecules on the clay layer, and thus the subsequent observation of single-molecule fluorescence spectroelectrochemistry. We can trace single-molecule fluorescence spectroelectrochemistry by probing the fluorescence intensity change of individually immobilized single redox molecules modulated via cyclic voltammetric potential scanning. This work opens a new approach to explore interfacial electron transfer mechanisms of redox reactions.

Single-molecule fluorescence spectroelectrochemistry of cresyl violet.

Here we report a new path to study single molecule electron transfer dynamics by coupling scanning fluorescence microscopy with a potentiostat via a conventional electrochemical cell to enable single-molecule fluorescence spectroelectrochemistry of cresyl violet in aqueous solution, demonstrating that the single-molecule fluorescence intensity of cresyl violet is modulated synchronously with the cyclic voltammetric potential scanning.