Host-Guest Assembly-Crosslinked Nanoemulsions with Dual-Antimicrobial Mechanism for Treating Multidrug-Resistant Bacterial Biofilms.

Plant essential oils have good antimicrobial properties, but their poor stability and compatibility in aqueous solutions greatly limit their practical application. To address this issue, a dynamically crosslinked nanoemulsion based on host-guest assembly was developed in this study. First, a ß-cyclodextrin-functionalized quaternary ammonium surfactant (ß-CD-QA) and adamantane-terminated polyethylene glycol (APA) crosslinker were first synthesized. Then, the oil-in-water host-guest crosslinked nanoemulsions (HGCTNs) were formed by incorporating tea tree essential oils (TTO) as a natural antimicrobial agent. The results showed that HGCTNs significantly improved the stability of the essential oil nanoemulsions and extended their shelf life. Furthermore, HGCTNs demonstrated effective antimicrobial properties against both Gram-negative/positive bacterioplankton and bacterial biofilms. The results of antibacterial experiments showed that the dynamically crosslinked HGCTNs exhibit superior antibacterial efficacy, with a minimum inhibitory concentration (MIC) of 12.5 v/v % (0.13 µL/mL TTO) and could eradicate the biofilms. The electrical conductivity of the bacterial solution gradually increased within 5 h of treatment with the nanoemulsions, indicating that the HGCTNs have a slow-release effect of TTO and sustainable antibacterial ability. The antimicrobial mechanism can be attributed to the synergistic antibacterial action of the ß-CD-QA surfactant containing a quaternary ammonium moiety and TTO, which are stabilized by nanoemulsions.

Long-distance electron transfer in a filamentous Gram-positive bacterium.

Long-distance extracellular electron transfer has been observed in Gram-negative bacteria and plays roles in both natural and engineering processes. The electron transfer can be mediated by conductive protein appendages (in short unicellular bacteria such as Geobacter species) or by conductive cell envelopes (in filamentous multicellular cable bacteria). Here we show that Lysinibacillus varians GY32, a filamentous unicellular Gram-positive bacterium, is capable of bidirectional extracellular electron transfer. In microbial fuel cells, L. varians can form centimetre-range conductive cellular networks and, when grown on graphite electrodes, the cells can reach a remarkable length of 1.08 mm. Atomic force microscopy and microelectrode analyses suggest that the conductivity is linked to pili-like protein appendages. Our results show that long-distance electron transfer is not limited to Gram-negative bacteria.

Visualizing and Isolating Iron-Reducing Microorganisms at the Single-Cell Level.

Iron-reducing microorganisms (FeRM) play key roles in many natural and engineering processes. Visualizing and isolating FeRM from multispecies samples are essential to understand the in situ location and geochemical role of FeRM. Here, we visualized FeRM by a "turn-on" Fe2+-specific fluorescent chemodosimeter (FSFC) with high sensitivity, selectivity, and stability. This FSFC could selectively identify and locate active FeRM from either pure culture, coculture of different bacteria, or sediment-containing samples. Fluorescent intensity of the FSFC could be used as an indicator of Fe2+ concentration in bacterial cultures. By combining the use of the FSFC with that of a single-cell sorter, we obtained three FSFC-labeled cells from an enriched consortium, and all of them were subsequently shown to be capable of iron reduction; two unlabeled cells were shown to have no iron-reducing capability, further confirming the feasibility of the FSFC.IMPORTANCE Visualization and isolation of FeRM from samples containing multiple species are commonly needed by researchers from different disciplines, such as environmental microbiology, environmental sciences, and geochemistry. However, no available method has been reported. In this study, we provide a method to visualize FeRM and evaluate their activity even at the single-cell level. When this approach is combined with use of a single-cell sorter, FeRM can also be isolated from samples containing multiple species. This method can be used as a powerful tool to uncover the in situ or ex situ role of FeRM and their interactions with ambient microbes or chemicals.

Double-integrated mimic enzymes for the visual screening of Microcystin-LR: Copper hydroxide nanozyme and G-quadruplex/hemin DNAzyme.

Recently, mimic enzymes have obtained particular interest by their high activity, stability, and biocompatibility. In this work, by coupling copper hydroxide nanozyme and G-quadruplex/hemin DNAzyme to form a double-integrated mimic enzyme, a visual, sensitive and selective immunosensor was established to detect microcystin-LR (MC-LR). In this immunoassay, the microplates were modified with core-shell silica/nickel silicate as the substrate to capture MC-LR antigens. Then, Cu(OH)2 nanocages with fine regulation were used as the label to capture the secondary antibody for immunoreaction and the DNA primer for propagation, followed by using hybridization chain reaction to amplify the DNA primer, thus numerous DNAzymes (G-quadruplex/hemin) can be formed on the surface of Cu(OH)2 nanocages with the aid of hemin. Such double-integrated mimic enzyme including Cu(OH)2 nanozymes and DNAzymes showed excellent peroxidase activity for the chromogenic reaction of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), which realized the visual detection of MC-LR in the range from 0.007 to 75 µg/L with the detection limit of 6 ng/mL, and thus provided the probability for the portable assessment of MC-LR in real sample.

Multiple amplified enzyme-free electrochemical immunosensor based on G-quadruplex/hemin functionalized mesoporous silica with redox-active intercalators for microcystin-LR detection.

A novel multiple amplified enzyme-free immunosensor was developed for competitive immunoassay of microcystin-LR (MC-LR). Classical electrochemical immunosensors usually employ enzymes as biocatalysts to afford amplified signals, but the proteolytic degradation and poor stability are still crucial problem. Herein, monodisperse core-shell mesoporous silica (SiO2@MSN)-functionalized DNAzyme concatamers were synthesized to load hemin and methylene blue (MB) as the mimic enzyme. Firstly, the surface of SiO2@MSN was conjugated with secondary antibody as the recognition of MC-LR antibody and with a DNA strand as the initiator. Two auxiliary DNA strands were then selected for the in-situ propagation to form a double-helix DNA through hybridization chain reaction (HCR), forming numerous DNAzymes (G-quadruplex/hemin) after the addition of hemin. Secondly, MB was inserted into the formed double-helix DNA, and also loaded in the brush-like structure of mesoporous SiO2@MSN. The molecular docking study showed that electrons can transfer more effectively with π-π stack of hemin/G-quadruplex and intercalation of MB/DNA, thus the catalytic ability of DNAzymes can be greatly improved. With the aid of MB, DNAzymes can catalyze the reduction of H2O2 to produce the electrochemical signal. This enzyme-free electrochemical immunosensor can successfully detect MC-LR in a range of 0.5ng/L and 25µg/L with a detection limit of 0.3ng/L. This stable, sensitive and selective nonenzymatic electrochemical immunoassay shows promise for applications in food and environmental monitoring.

In-situ assembly of biocompatible core-shell hierarchical nanostructures sensitized immunosensor for microcystin-LR detection.

Microcystin-LR (MC-LR) is a kind of hepatotoxin which can cause functional and structural disturbances of the liver, accumulate in aquatic organisms and transfer to higher trophic levels, a biocompatible electrochemical immunosensor was constructed to detect MC-LR sensitively and selectively. The three-dimensional villiform-like carbon nanotube/cobalt silicate (CNT@Co silicate) core-shell nanocomposites were synthesized and firstly used as the substrate to immobilize the antigen of MC-LR (Ag), while Fe3O4 nanoclusters/polydopamine/gold nanoparticles (Fe3O4@PDA-Au) core-shell magnetic nanocomposites were prepared as the label carrier of the immunosensor to conjugate the second antibody (Ab2) and horse radish peroxidase (HRP). Since the toxicity of nanomaterials is important in the construction of biosensors including the immobilization of antigen or antibody, the biocompatibility of such nanocomposites were investigated by monitoring the cell viability after culturing with Hela cells. Due to the excellent biocompatibility, the immunosensor can immobilize more antigens by the large surface area of the three-dimensional villiform-like structure in CNT@Co silicate, and provide high electrochemical signals by Fe3O4@PDA-Au labeled Ab2 and HRP. After investigation of the binding capability of biomolecules on nanomaterials and optimization of the conditions in the competitive immunoassay, the proposed electrochemical immunosensor shows a linear response to MC-LR in the range from 0.005 µg/L to 50 µg/L with a detection limit of 0.004 µg/L. In addition, the specificity, reproducibility and stability of the immunosensor were also proved to be acceptable, indicating its potential application in environmental monitoring.

Sensitive electrochemical immunoassay for chlorpyrifos by using flake-like Fe3O4 modified carbon nanotubes as the enhanced multienzyme label.

A highly sensitive electrochemical immunoassay of chlorpyrifos (CPF) was developed by using a biocompatible quinone-rich polydopamine nanospheres modified glass carbon electrode as the sensor platform and multi-horseradish peroxidase-flake like Fe3O4 coated carbon nanotube nanocomposites as the signal label. Due to the quinone-rich polydopamine nanospheres, the platform exhibited excellent fixing capacity by simple coating of sticky polydopamine nanospheres and subsequent oxidization. By coprecipitation of Fe(3+) and Fe(2+) on polydopamine modified carbon nanotubes (CNTs) with the aid of ethylene glycol (EG), the flake-like Fe3O4 coated CNTs (CNTs@f-Fe3O4) were synthesized and chosen as the carrier of multi-enzyme label due to the high loading of secondary antibody (Ab2) and horseradish peroxidase (HRP) and also the peroxidase-mimic activity of Fe3O4. Under the optimum conditions, the immunosensor can detect CPF over a wide range with a detection limit of 6.3 pg/mL. Besides, the high specificity, reproducibility and stability of the proposed immunosensor were also proved. The preliminary application in real sample showed good recoveries, indicating it holds promise for fast analysis of CPF in aquatic environment.

Electrochemical immunosensor based on hydrophilic polydopamine-coated prussian blue-mesoporous carbon for the rapid screening of 3-bromobiphenyl.

A sensitive electrochemical immunosensor for 3-bromobiphenyl (3-BBP) detection was constructed by employing a new polydopamine coated prussian blue-mesoporous carbon (PDOP/PB/CMK-3) nanocomposite as the substrate platform and multi-horseradish peroxidase-double helix carbon nanotubes-secondary antibody (multi-HRP-DHCNTs-Ab2) as the signal label. PB/CMK-3 was firstly successfully in-situ synthesized with the aid of the CMK-3 reduction, which was characterized by transmission electron microscope (TEM), infrared spectroscopy (IR), X-ray diffraction (XRD) and N2 adsorption-desorption analysis. By using PDOP/PB/CMK-3 as the substrate, it can effectively enhance the specific surface for antigen loading due to the three-dimensional structure of the nanocomposites, while large amount of PB that fixed inside or outside the pore of CMK-3 successfully improved the electrochemical response and the PDOP film can provide a biocompatible environment to maintain the activity of antigen availability. Under the optimized conditions, the proposed immunosensor shows a good current response to 3-BBP in a linear range from 5 pM to 2 nM with a detection limit of 2.25 pM. In addition, the specificity, reproducibility and stability of the immunosensor were also proved to be acceptable, indicating its potential application in environmental monitoring.