Fluorophore Label-Free Light-up Near Infrared Deoxyribonucleic Acid Nanosensor for Monitoring Extracellular Potassium Levels.

Fluorescent DNA nanosensors have been widely used due to their unique advantages, among which the near-infrared (NIR) imaging mode can provide deeper penetration depth and lower biological background for the nanosensors. However, efficient NIR quenchers require ingenious design, complex synthesis, and modification, which severely limit the development of NIR DNA nanosensors. Label-free strategies based on G-quadruplex (G4) and NIR G4 dyes were first introduced into in situ extracellular imaging, and a novel NIR sensing strategy for the specific detection of extracellular targets is proposed. The strategy avoids complex synthesis and site-specific modification by controlling the change of the NIR signal through the formation of a G4 nanostructure. A light-up NIR DNA nanosensor based on potassium ion (K+)-sensitive G4 chain PS2.M was constructed to verify the strategy. PS2.M forms a stable G4 nanostructure in the presence of K+ and activates the NIR G4 dye CSTS, thus outputting NIR signals. The nanosensor can rapidly respond to K+ with a linear range of 5-50 mM and has good resistance to interference. The nanosensor with cholesterol can provide feedback on the changes in extracellular K+ concentration in many kinds of cells, serving as a potential tool for the study of diseases such as epilepsy and cancer, as well as the development of related drugs. The strategy can be potentially applied to the NIR detection of a variety of extracellular targets with the help of functional DNAs such as aptamer and DNAzyme.

Hairpin Switches-Based Isothermal Transcription Amplification for Simple, Sensitivity Detection of MicroRNA.

The ability to simply, selectively, and sensitively detect low numbers of miRNAs in clinical samples is highly valuable but remains a challenge. In this work, we present a novel miRNA detection system by using the elaborately designed hairpin switch, where the T7 primer, template, target recognize sequence, and light-up RNA aptamer template are edited and embedded in one single-stranded DNA hairpin structure. In the beginning, the hairpin switch maintained the hairpin structure 1, in which the ds promoter of T7 polymerase was disrupted, thus the transcription reaction of T7 polymerase was inhibited. After binding to the target, the hairpin switch 1 was unfolded and turned to the hairpin structure 2. This switch initiates the in vitro T7 transcription reaction, producing plenty of RNA transcripts containing RNA aptamers. Consequently, transcribed tremendous RNA aptamers lighted up the fluorophore for quantitative analysis. Compared with the existing T7 polymerase-based amplification system, this strategy exhibits several advantages, including simplicity, convenience, and high selectivity and sensitivity. The experimental results demonstrated that we could achieve the quantification of miRNA in buffer and complex biological samples.

Chemical Composition and Potential Antimicrobial and Anti-Inflammatory Activities of Essential Oil from Fruits of Alpinia zerumbet (Pers.) B.L.Burtt & R.M.Sm.

Alpinia zerumbet (Pers.) B.L.Burtt & R.M.Sm. was extensively used in traditional medicine for its several properties, but continuous investigation is needed to discover the properties of its essential oils (EOs). This work evaluated the properties of an EO obtained by steam distillation (named ESD) as well as extracts obtained by petroleum ether (named EP) both from Alpinia zerumbet fruits. Gas chromatography-mass spectrometry (GC-MS) was chosen to identify the composition, and eleven compounds were identified as the main components of the EO and EP of Alpinia zerumbet fruits. The antimicrobial properties were investigated by minimum inhibitory concentration (MIC) and the inhibition area. The results identified the differences in antimicrobial activities attributed to different extraction methods. Enzyme-linked immunosorbent assay (ELISA) and Western Blot (WB) assay were conducted to assess the anti-inflammatory effects of ESD. In conclusion, our study suggested that EO from Alpinia zerumbet fruits might be a prospective candidate for antimicrobial and anti-inflammatory therapy.

Designing a CRISPR/Cas12a- and Au-Nanobeacon-Based Diagnostic Biosensor Enabling Direct, Rapid, and Sensitive miRNA Detection.

Direct, rapid, sensitive, and selective detection of nucleic acids in complex biological fluids is crucial for medical early diagnosis. We herein combine the trans-cleavage ability of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a with Au-nanobeacon to establish a CRISPR-based biosensor, providing rapid miRNA detection with high speed and attomolar sensitivity. In this strategy, we first report that the trans-cleavage activity of CRISPR/cas12a, which was previously reported to be triggered only by target ssDNA or dsDNA, can be activated by the target miRNA directly. Therefore, this method is direct, i.e., does not need the conversion of miRNA into its complementary DNA (cDNA). Meanwhile, as compared to the traditional ssDNA reporters and molecular beacon (MB) reporters, the Au-nanobeacon reporters exhibit improved reaction kinetics and sensitivity. In this assay, the miRNA-21 could be detected with very high sensitivity in only 5 min. Finally, the proposed strategy enables rapid, sensitive, and selective miRNA determination in complex biological samples, providing a potential tool for medical early diagnosis.

Nanoflare Couple: Multiplexed mRNA Imaging and Logic-Controlled Combinational Therapy.

Theranostics, which combines both diagnostic and therapeutic capabilities in one dose, has always been an intractable challenge in personalized cancer treatment. Herein, a versatile nanotheranostic platform "nanoflare couple (NC)" has been developed for in situ multiplex cancer-related mRNA imaging and subsequent logic-controlled aggregation of gold nanoparticles, leading to gene therapy and photothermal therapy upon irradiation with infrared light. As a proof of concept, TK1 and survivin mRNAs that are highly expressed in most tumor tissues are selected as endogenous cancer indicators and therapy triggers to design the NC. Mice bearing breast cancer cells MCF-7 are prepared as a model to test its efficacy. The in vitro and in vivo assays validate that the NC show the capability for multiplexed mRNA imaging and high efficiency for logic-controlled combinational therapy of breast cancer.

Bimetallic modified halloysite particle electrode enhanced electrocatalytic oxidation for the degradation of sulfanilamide.

The treatment of antibiotics wastewater by electrocatalytic oxidation has attracted much attention. In the paper, a novel halloysite bimetallic (HLS-Cu-Mn) particle electrode material was prepared and a bench-scale electrocatalytic reaction tank was designed. A three-dimensional electrocatalytic oxidation reactor composed of HLS-Cu-Mn and a bench-scale electrocatalytic reaction tank was used to degrade Sulfanilamide (SA) wastewater. Characterization of the synthesized material was conducted with Scanning electron microscopy (SEM), X-ray polycrystalline powder diffractometer (XRD), X-ray photoelectron spectroscopy (XPS), and Brunauer-Emmett-Teller (BET). The electron spin resonance spectroscopy test results confirmed that HLS-Cu-Mn produced a large number of •OH. The electrochemical workstation confirmed that HLS-Cu-Mn had strong electrocatalytic activity and repolarization ability. Under the optimum preparation conditions and degradation process parameters, the removal efficiency of SA and TOC was 99.84% and 88.95% respectively. The method also has good degradation efficiency for aniline, phenol, herbicides, antibiotics, and dyeing wastewater. It was found that 4 main intermediates appeared in the degradation process by Ultra-high performance liquid chromatography/triple tandem quadrupole mass spectrometry (LC-MS). In sum, it was believed that this work provides a new vision and idea for water treatment.

The Main Structural Unit Elucidation and Immunomodulatory Activity In Vitro of a Selenium-Enriched Polysaccharide Produced by Pleurotus ostreatus.

In recent years, the structure of selenium-enriched polysaccharides and their application in immunomodulation have attracted much attention. In previous studies, we extracted and purified a novel selenium-enriched Pleurotus ostreatus polysaccharide called Se-POP-21, but its structure and immunomodulatory activity were still unclear. In this study, the main structural unit formula of Se-POP-21 was characterized by methylation analysis and an NMR experiment. The results showed that the backbone of Se-POP-21 was →[2,6)-α-D-Galp-(1→6)-α-D-Galp-(1]4→2,4)-ß-L-Arap-(1→[2,6)-α-D-Galp-(1→6)-α-D-Galp-(1]4→, branched chain of ß-D-Manp-(1→ and ß-D-Manp-(1→4)-ß-L-Arap-(1→ connected with →2,6)-α-D-Galp-(1→ and →2,4)-ß-L-Arap-(1→,respectively, through the O-2 bond. In vitro cell experiments indicated that Se-POP-21 could significantly enhance the proliferation and phagocytosis of RAW264.7 cells, upregulate the expression of costimulatory molecules CD80/CD86, and promote RAW264.7 cells to secrete NO, ROS, TNF-α, IL-1ß, and IL-6 by activating the NF-κB protein. The results of this study indicate that Se-POP-21 can effectively activate RAW264.7 cells. Thus, it has the potential to be used in immunomodulatory drugs or functional foods.

Cell-Surface-Anchored DNA Sensors for Simultaneously Monitoring Extracellular Sodium and Potassium Levels.

The K+ and Na+ levels in cells have a synergistic effect on many biological processes (BPs); therefore, the simultaneous detection of them is important. Here, we propose a novel Y-shaped DNA sensor for simultaneous monitoring of Na+ and K+ in extracellular microenvironments. The designed sensor contributed to the selective response to the above two ions. In addition, it performed the imaging of the above two ions on the cell surface in a real-time, on-site manner, which would shed more light on the association of the Na+/K+ content with regulatory BPs. We believe that this new strategy will be a promising tool to investigate the synergy of Na+/K+ in regulating different BPs.

Dually acid- and GSH-triggered bis(ß-cyclodextrin) as drugs delivery nanoplatform for effective anticancer monotherapy.

The intrinsic poor solubility and limited load capacity of ß-cyclodextrins (ß-CDs) results in reduced bioavailability, rendering the material unsuitable in complex biological environments. In this work, a pair of ß-CDs was methylated and covalently linked with acid-sensitive acylhydrazone and GSH-sensitive disulfide bonds to ensure a precise drug release pattern. The hydrophobic anticancer drug doxorubicin (Dox) was encapsulated inside the hydrophobic core of bis(ß-CD) via hydrophobic association with loading capacity of 24% in weight and a hydrodynamic size of about 100 nm. When exposed to acidic and reductive environments, the acylhydrazone and disulfide bonds were found to be cleaved, resulting in Dox release. Using fluorescence imaging and flow cytometry analysis, the designed bis(ß-CD) were determined to activate the drug release behavior by specific intracellular stimuli (pH and GSH). In vivo studies demonstrated specific drug delivery characteristics and controlled drug release behaviors in the tumor sites, giving rise to high antitumor activity and low toxicity. Taken in concert, this dual stimuli-responsive bis(ß-CD) with superior amphiphilicity and biocompatibility features showed great potential for future clinical applications.

Intelligent Nanoprobe: Acid-Responsive Drug Release and In Situ Evaluation of Its Own Therapeutic Effect.

The design of an intelligent nanoprobe capable of intracellular controlled release of apoptosis inducers and subsequent high-fidelity imaging of the drug-induced apoptosis is highly desirable for precise cancer treatment. Herein, we report an intelligent nanoprobe that combined therapeutic and imaging functions in one agent. Briefly, a gold nanoparticle is designed to be conjugated with acid-responsive DNA duplexes (Dox intercalates in this region) and caspase-3-specific cleavable peptides (labeled with fluorophore). We demonstrated that the nanoprobe could efficiently deliver an anticancer drug (Dox) into cancer cells and achieve acid-responsive drug release. Furthermore, the apoptotic process was in situ-monitored by detection of fluorescence through the cleavage of the peptide linker by caspase-3, which is one of the executioner caspases involved in apoptosis. This newly developed nanoprobe could serve as a theranostic agent for targeted responsive chemotherapy and also provide feedback apoptosis imaging of the self-therapeutic effect.

Preparation and characterization of molecularly imprinted polymers based on ß-cyclodextrin-stabilized Pickering emulsion polymerization for selective recognition of erythromycin from river water and milk.

Molecularly imprinted polymers were prepared via ß-cyclodextrin-stabilized oil-in-water Pickering emulsion polymerization for selective recognition and adsorption of erythromycin. The synthesized molecularly imprinted polymers were spherical in shape, with diameters ranging from 20 to 40 µm. The molecularly imprinted polymers showed high adsorption capacity (87.08 mg/g) and adsorption isotherm data fitted well with Langmuir model. Adsorption kinetics study demonstrated that the molecularly imprinted polymers acted in a fast adsorption kinetic pattern and the adsorption features of molecularly imprinted polymers followed a pseudo-first-order model. Adsorption selectivity analysis revealed that molecularly imprinted polymers had a much better specificity for erythromycin than that for spiramycin or amoxicillin, and the relative selectivity coefficient values on the bases of spiramycin and amoxicillin were 3.97 and 3.86, respectively. The Molecularly imprinted polymers also showed a satisfactory reusability after four times of regeneration. In addition, molecularly imprinted polymers exhibited good adsorption capacities for erythromycin under complicated environment, that is, river water and milk. These results proved that the as-prepared molecularly imprinted polymers is a potent absorbent for selective recognition of erythromycin, and therefore it may be a promising candidate for practical applications, such as wastewater treatment and detection of erythromycin residues in food.

A nanoprobe for ratiometric imaging of glutathione in living cells based on the use of a nanocomposite prepared from dual-emission carbon dots and manganese dioxide nanosheets.

A ratiometric fluorescence assay for glutathione (GSH) was developed. The novel assay is based on a nanoprobe composed of manganese dioxide nanosheets (MnO2 NS) and dual-emission carbon dots (de-CDs) with intrinsic GSH-response property. After construction of the nanoprobe, two emission peaks of de-CDs were suppressed to varying degrees by MnO2 NS. The suppression was relieved and the two emission peaks recovered proportionally when MnO2 NS was decomposed by GSH, thus realizing the ratiometric assay for micromolar GSH. The intrinsic responsiveness of de-CDs to millimolar GSH broadens the analytical range of the nanoprobe. An appropriate precursor, calcon-carboxylic acid, was screened out to synthesize de-CDs via one-step hydrothermal treatment. The de-CD@MnO2 NS nanoprobe can measure GSH concentrations through the fluorescence intensity ratio between 435 and 516 nm excited at 365 nm. The range of response was from 1 µM to 10 mM and the detection limit reached 0.6 µM (3σ criterion). Benefiting from its good biocompatibility, the proposed nanoprobe has excellent applicability for intracellular GSH imaging.Graphical abstract Schematic representation of glutathione (GSH) ratiometric detection. The nanoprobe is prepared from dual-emission carbon dots (de-CDs) and manganese dioxide nanosheets (MnO2 NS). GSH removes quenching effect by decomposing MnO2 NS and induces intrinsic response of de-CDs, which realizes ratiometric detection.

Gold nanoparticle based fluorescent oligonucleotide probes for imaging and therapy in living systems.

Gold nanoparticles (AuNPs) with unique physical and chemical properties have become an integral part of research in nanoscience. In particular, the development of AuNP-based fluorescent oligonucleotide (AuFO) probes targeted at living systems through fluorescence are drawing increasing attention and have become a current hot topic in biomedical research. This review describes recent advances and developments in the use of AuFO probes from their preparation and features that allow the desired intracellular delivery to their applications in cellular imaging and drug delivery in living systems.

Molecularly imprinted polymers fabricated via Pickering emulsions stabilized solely by food-grade casein colloidal nanoparticles for selective protein recognition.

Novel molecularly imprinted polymers (MIPs) based on denatured casein nanoparticle (DCP)-stabilized Pickering emulsions were developed for the first time. Casein, a phosphoprotein, is the main protein in milk. In this work, DCPs were solely used as Pickering-type interfacial emulsifiers for fabrication of MIPs for the selective recognition of proteins for the first time. DCPs were prepared by acidification and heat denaturation (at 80 °C) of casein. Their dispersions have satisfactory colloidal stability over a wide pH range. The DCPs acted as natural, food-grade, and edible interfacial emulsifiers, and adsorbed at the oil-water interface to form Pickering emulsions. After the polymerization of monomers, the template protein was removed by elution. During the elution, the interfacial DCPs were also removed, allowing more imprinted cavities to become exposed. The interfacial imprinting technology causes nearly all the imprinted sites to locate on the surface of the polymeric material. Therefore, the MIPs obtained exhibit fast rebinding and excellent specific recognition ability toward the analytes. Overall, this work provides a promising method for designing and fabricating natural-protein-based structured emulsions to prepare MIPs and thus offers new insight into protein separation and purification. Graphical Abstract Pickering emulsions stabilized by denatured casein particles.

Synthesis of Size-Tunable Hollow Polypyrrole Nanostructures and Their Assembly into Folate-Targeting and pH-Responsive Anticancer Drug-Delivery Agents.

Chemotherapeutic drugs currently used in clinical settings have high toxicity, low specificity, and short half-lives. Herein, polypyrrole-based anticancer drug nanocapsules were prepared by tailoring the size of the nanoparticles with a template method, controlling drug release by means of an aromatic imine, increasing nanoparticle stability through PEGylation, and improving tumor-cell selectivity by folate mediation. The nanoparticles were characterized by TEM and dynamic light scattering. α-Folate receptor expression levelsof tumor cells and normal cells were investigated by western blot and quantitative polymerase chain reaction analyses. Flow cytometry and fluorescence imaging were used to verify the cell uptake of the different-sized nanoparticles. From the different-sized polypyrrole nanoparticles, the optimally functionalized nanoparticles of 180 nm hydrodynamic diameter were chosen and further usedfor in vitroandin vivotests. The nanoparticles showed excellent biocompatibility and the drug-loaded nanoparticles exhibited effective inhibition of tumor cell growth in vitro. Moreover, the drug-loaded nanoparticles showed substantially enhanced accumulation in tumor regions and effectively inhibitedin vivotumor growth. Furthermore, the nanoparticles showed reduced doxorubicin-induced toxicity andno significant side effects in normal organs of tumor-bearing mice, as measured by body-weight shifts and evaluationof drug distribution. Overall, the functionalized nanoparticles are promising nanocarriers for tumor-targeting drug delivery.

Multifunctional halloysite nanotubes for targeted delivery and controlled release of doxorubicin in-vitro and in-vivo studies.

The current state of cancer therapy encourages researchers to develop novel efficient nanocarriers. Halloysite nanotubes (HNTs) are good nanocarrier candidates due to their unique nanoscale (40-80 nm in diamter and 200-500 nm in length) and hollow lumen, as well as good biocompatibility and low cost. In our study, we prepared a type of folate-mediated targeting and redox-triggered anticancer drug delivery system, so that Doxorubicin (DOX) can be specifically transported to tumor sites due to the over-expressed folate-receptors on the surface of cancer cells. Furthermore, it can then be released by the reductive agent glutathione (GSH) in cancer cells where the content of GSH is nearly 103-fold higher than in the extracellular matrix. A series of methods have demonstrated that per-thiol-ß-cyclodextrin (ß-CD-(SH)7) was successfully combined with HNTs via a redox-responsive disulfide bond, and folic acid-polyethylene glycol-adamantane (FA-PEG-Ad) was immobilized on the HNTs through the strong complexation between ß-CD/Ad. In vitro studies indicated that the release rate of DOX raised sharply in dithiothreitol (DTT) reducing environment and the amount of released DOX reached 70% in 10 mM DTT within the first 10 h, while only 40% of DOX was released in phosphate buffer solution (PBS) even after 79 h. Furthermore, the targeted HNTs could be specifically endocytosed by over-expressed folate-receptor cancer cells and significantly accelerate the apoptosis of cancer cells compared to non-targeted HNTs. In vivo studies further verified that the targeted HNTs had the best therapeutic efficacy and no obvious side effects for tumor-bearing nude mice, while free DOX showed damaging effects on normal tissues. In summary, this novel nanocarrier system shows excellent potential for targeted delivery and controlled release of anticancer drugs and provides a potential platform for tumor therapy.

Halloysite-based dopamine-imprinted polymer for selective protein capture.

We describe a facile, general, and highly efficient approach to obtain polydopamine-coated molecularly imprinted polymer based on halloysite nanotubes for bovine serum albumin. The method combined surface molecular imprinting and one-step immobilized template technique. Hierarchically structured polymer was prepared in physiological conditions adopting dopamine as functional monomer. A thin layer of polydopamine can be coated on the surface of amino-modified halloysite nanotubes by self-polymerization, and the thickness of the imprinted shells can be controlled by the mass ratio of matrix and dopamine. The polymer was characterized by Fourier transform infrared spectrometry, transmission electron microscopy, and thermogravimetric analysis. The prepared material showed high binding capacity (45.4 mg/g) and specific recognition behavior toward the template protein. In addition, stability and regeneration analyses indicated that the imprinted polymer exhibited excellent reusability (relative standard deviation < 9% for batch-to-batch evaluation). Therefore, the developed polymer is effective for protein recognition and separation.

Water-compatible halloysite-imprinted polymer by Pickering emulsion polymerization for the selective recognition of herbicides.

A water-compatible molecularly imprinted polymer was prepared by Pickering emulsion polymerization using halloysite nanotubes as stabilized solid particles. During polymerization, we used 4-vinylpyridine as monomer, divinylbenzene as cross-linking agent, toluene as porogen, 2,2-azobisisobutyronitrile as initiator, 2,4-dichlorophenoxyacetic acid as template to form the oil phase, and Triton X-100 aqueous solution to form the water phase. The halloysite nanotubes molecularly imprinted polymer was characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. Kinetic and equilibrium bindings were also employed to evaluate the adsorption properties of the imprinted polymer. The imprinted polymer showed better selectivity, more rapid kinetic binding (60 min) for 2,4-dichlorophenoxyacetic acid in pure water compared with rebinding in toluene. The imprinted polymer was used as a sorbent to enrich and separate 2,4-dichlorophenoxyacetic acid from water, and was detected by high-performance liquid chromatography with UV detection.

Mobile phone-assisted imprinted nanozyme for bicolor colorimetric visual detection of erythromycin in river water and milk samples.

The residues of erythromycin (ERY) may have negative impacts on the ecological environment, health, and food safety. How to detect ERY effectively and visually is a challenging issue. Herein, we synthesized a molecularly imprinted polymer based nanozymes for selective detection of erythromycin (ERY-MIPNs) at neutral pH, and developed a mobile phone-assisted bicolor colorimetric detection system. This system produced a wide range of color changes from blue to pinkish purple as the ERY concentration increased, making it easy to capture the visualization result. Also, the system showed good sensitivity to ERY ranging from 15 to 135 µM, with a detection limit of 1.78 µM. In addition, the system worked well in the detection of ERY in river water and milk, with the recoveries of 95.57% âˆ¼ 103.20%. These data suggests that this strategy is of considerable potential for practical applications and it provides a new idea for visual detection with portable measurement.