Dansyl Based "Turn-On" Fluorescent Sensor for Cu2+ Ion Detection and the Application to Living Cell Imaging.

A new "turn-on" fluorescent chemosensor based on dansyl derivative was prepared for Cu2+ ion sensing. Hydroxyl, imine and azomethine groups in Schiff base derived compound 1 were deliberately introduced for facilitating the binding of Cu2+ ion. Of screen metal ions, compound 1 showed a high degree of selectivity toward Cu2+ ion. Other interfering metal ions did not affect the fluorescence intensity of compound 1, except Hg2+ and Fe3+ ions exhibited a significant degree of fluorescence quenching. Upon binding of Cu2+ ion, compound 1 displayed a chelation enhanced fluorescence (CHEF) resulting in increasing of the fluorescence intensity. The molecular optimized geometry indicated the binding ratio between compound 1 and Cu2+ ion at 1:1 with the binding constant of 1.68 × 10- 7 M- 1. The optimized condition for sensing ability of compound 1 with a detection limit of 5 × 10- 7 M was found at the physiological pH 7.2 with the excitation wavelength of 366 nm. Due to no cytotoxicity and good photophysical properties, compound 1 was extended its application for the detection of Cu2+ ion in Vero cells. Compound 1 could be potentially used as an intracellular fluorescent chemosensor for tracking Cu2+ ion. Graphical Abstract.

Magnetic Nanoparticles PCR Enzyme-Linked Gene Assay for Quantitative Detection of BCR/ABL Fusion Gene in Chronic Myelogenous Leukemia.

BACKGROUND: Magnetic nanoparticles (MNPs) have been widely used in medical diagnostic research. In this work, two technologies, MNPs and polymerase chain reaction (PCR), were combined to increase detection sensitivity and specificity. A novel technique based on the MNPs-PCR enzyme-linked gene assay (MELGA) was developed for detection of the BCR/ABL abnormal gene in chronic myelogenous leukemia (CML) patients. METHODS: An MNPs-labeled BCR forward primer and a biotin-labeled ABL reverse primer were used to specifically amplify the target gene. After magnetic separation, the PCR product bound to MNPs labeled with streptavidin-conjugated horseradish peroxidase was incubated with the peroxidase substrate and hydrogen peroxide to generate the colorimetric signal. RESULTS: When compared with real-time quantitative-PCR (RQ-PCR), the MELGA technique exhibited an increased sensitivity of <1 fg with high specificity for the BCR/ABL fusion gene in CML patients. In addition, MELGA colorimetric results correlated well with the number of copies obtained from RQ-PCR. CONCLUSION: This simple and cost-effective technique is suitable for monitoring CML patients during targeted therapy (tyrosine kinase inhibitors) especially in rural hospitals.

Titania-functionalized graphene oxide for an efficient adsorptive removal of phosphate ions.

Titania-functionalized graphene oxide (T-F GO), synthesized by a sol-gel process, was used as a highly efficient material to remove phosphate ions from the simulated wastewater. X-ray diffraction spectra, Fourier transform infrared spectra and scanning electron micrographs of T-F GO confirmed that titania particles were successfully grown on graphene oxide (GO) surface. The phosphate ion adsorption capacities of GO, titania and T-F GO as a function of the contact time and the pH were investigated by a UV-visible spectrophotometer. Results showed that T-F GO could absorb phosphate ions better than titania and GO could. This indicated the synergistic effect between titania and GO in the phosphate ion adsorption. The pH increment lowered the absorption capacities due to increasing the repulsion between phosphate anions and the charges on the T-F GO surface, whereas the addition of sodium ions increased the adsorption capacities. Also, phosphate ions were absorbed by specific sites of T-F GO and formed a monolayer on its surface. Finally, the maximum adsorption capacity of T-F GO was 33.11 mg/g at pH 6, much higher than those of GO and titania. Therefore, T-F GO could be a promising material to remove phosphate ions from wastewater in the future.

Mesenchymal stem cell in vitro labeling by hybrid fluorescent magnetic polymeric particles for application in cell tracking.

Mesenchymal stem cells (MSCs) are a type of adult stem cell that contains multi-differentiation and proliferative properties and that shows high treatment implications for many clinical problems. The outcome of stem cell transplantation is still limited due to many factors, especially their survival and their interaction with the microenvironment after transplantation. Molecular imaging is a challenging technique that has been used to overcome this limitation and is based on the concept of labeling cells with tractable, visible, and non-toxic materials to track the cells after transplantation. In this study, magnetic polymeric nanoparticles (MPNPs) were used to directly label Wharton's jelly-derived MSCs (WJ-MSCs). After labeling, the growth rate and the viability of the MSCs as well as the time of exposure were determined. The 3D images of WJ-MSCs labeled with MPNPs for 24 h were created using confocal microscopy. The results showed that, after incubation with fluorescent MPNPs for over 8 h, the growth rate and cell viability of the WJ-MSCs was similar to those of the control. Three-dimensional imaging revealed that the fluorescent MPNPs could infiltrate into the cells and spread into the cytoplasm, which suggests that the synthesized fluorescent MPNPs could possibly label MSCs for cell tracking study and be further developed for in vivo applications.

Selective fluorescent detection of aspartic acid and glutamic acid employing dansyl hydrazine dextran conjugate.

Highly water soluble polymer (DD) was prepared and evaluated for its fluorescence response towards various amino acids. The polymer consists of dansyl hydrazine unit conjugated into dextran template. The conjugation enhances higher water solubility of dansyl hydrazine moiety. Of screened amino acids, DD exhibited selective fluorescence quenching in the presence of aspartic acid (Asp) and glutamic acid (Glu). A plot of fluorescence intensity change of DD against the concentration of corresponding amino acids gave a good linear relationship in the range of 1 × 10(-4) M to 25 × 10(-3) M. This establishes DD as a potential polymeric sensor for selective sensing of Asp and Glu.

Ag/Au-incorporated trimethyl chitosan-shell hybrid particles as reinforcing and antioxidant fillers for trimethyl chitosan hydrogel.

N,N,N-Trimethyl chitosan (TMC) is a quaternized chitosan with versatile biological features. However, low mechanical strength limits its uses, for example, as hydrogels for tissue engineering applications. This study illustrates a viable synthesis of metal/polymer hybrid, core-shell colloidal particles and their use as reinforcing and antioxidant fillers for TMC hydrogels. The core-shell particles were initially synthesized by surfactant-free emulsion polymerization, induced by a photo-redox initiating system of riboflavin assisted by a 3° amine and 2° alcohol co-initiators. The synthesized core-shell particles were based on two polymeric shells: TMC and chitosan, and two polymeric cores: poly (hydroxypropyl methacrylate) (PHPMA) and poly(2-hydroxy ethyl methacrylate) (PHEMA). The presence of both 3° amine on TMC and 2° alcohol on HPMA monomer enhanced the photopolymerization performance. The TMC-based particles had sizes of 122-154 nm and zeta potentials of 10-35 mV, bringing the colloidal stability in the 4-10 pH range. Furthermore, due to the presence of TMC on the shell layer, the core-shell particles could be used as templates to grow the Ag/Au bimetallic nanoparticles with alloy and core-shell types through a thermal reduction. The prepared hybrid particles were incorporated in TMC hydrogels as a multifunctional filler, improving their mechanical and antioxidant properties.

Detection of Vibrio cholerae using the intrinsic catalytic activity of a magnetic polymeric nanoparticle.

A novel and sensitive magnetic polymeric nanoparticle (MPNP)-polymerase chain reaction-colorimetry (magneto-PCR-colorimetry) technique was developed for detection of Vibrio cholerae ( V. cholerae ). The technique involved an amplification of V. cholerae DNA on the surface of an MPNP and then employed the intrinsic catalytic activity of the MPNP to detect the target gene by colorimetry. An amino-modified forward primer was covalently labeled onto the MPNP surface which would bind to PCR product during PCR cycling. By employing the catalytic activity of the MPNP, the analysis of PCR product bound MPNP yielded a sensitivity of 10(3) CFU/mL of V. cholerae in buffer system within 4 h. The specificity and efficiency of the technique were investigated by using various bacterial DNAs in drinking and tap water.

Selective FL quenching or enhancing of diimine ligands by guanine.

Diimine ligand (DL) 1 significantly exhibited the fluorescence quenching upon binding to guanine. Changing at the para-substituent of the phenyl ring from the hydroxyl to bromo groups reversely enhanced the fluorescence in the presence of guanine. The reverse in the fluorescence selectivity indicated the profound effect of the substituent at the para-position of the phenyl ring. The simple synthesis of DL 1 and DL 2 with good selectivity for guanine offers these DLs as promising compounds for chemosensors of other guanine derivatives.

Physical properties of polymer composite: Natural rubber glove waste/polystyrene foam waste/cellulose.

The polymer composite was prepared from the wastes of natural rubber glove (NRG) and polystyrene foam (PSF) blended with cellulose from sugar cane leaves via the laminate method. The NRG and PSF were firstly dispersed in toluene under continuous stirring. Then, maleic anhydride (MA) was added into the mixture. Effects of blend ratio and of MA content (0.5-15%, w/w) on physical properties of the polymer composite were investigated. The toluene resistance of the polymer blend was improved after adding MA and cellulose. The highest toluene resistance was achieved when using 12% cellulose. The chemical reactions of MA with polymer blend and with composite were confirmed by ATR-FTIR. The hardness of the polymer blend and composite increased as a function of PSF. In addition, their impact strength increased with increasing NRG and cellulose contents.

Solvent-sensitive nanoparticle-enhanced PCR assay for the detection of enterotoxigenic Escherichia coli.

Stimulus-responsive nanoparticles are among the most utilized nanoscale materials in biomedical applications. As these nanoparticles exhibit a manipulable response to a particular stimulus, such as pH, heat, and organic solvent, they are potential signalling units in diagnostic assays. This study aims to enhance the limit of detection and reduce the turnaround time of magnetic nanoparticle polymerase chain reaction (PCR) enzyme-linked gene assay (MELGA), an advanced PCR-based technique termed the solvent-sensitive nanoparticle (SSNP)-enhanced PCR assay. This technique was proposed to detect pathogenic enterotoxigenic Escherichia coli (ETEC) through applying stimulus-responsive nanoparticles. The SSNPs were elaborated with three main components, including mesoporous silica nanoparticles as a structural unit, organic dye (Nile red) as a payload, and the corresponding organic solvent-sensitive polymer shell as "gatekeeper" (poly(maleic anhydride-alt-methyl vinyl ether, PMAMVE). A suitable organic solvent capable of inducing polymer swelling and dye dissolution was investigated by considering a solubility parameter. Using ethanol, the encapsulated Nile red can diffuse out of the SSNPs faster than other solvents and reach a constant concentration within 15 min. For the PCR inhibition study, various SSNPs concentrations (10-30 µg/reaction) were mixed with the ETEC gene and PCR reagent. The results showed that the particles in this concentration range did not inhibit PCR. By comparing the efficacy of conventional PCR, MELGA, and SSNP-enhanced PCR assay, the proposed technique showed a better detection limit than that of PCR, whereas that of MELGA was the lowest. Moreover, compared to MELGA or conventional PCR, this technique provided remarkably faster results in the postamplification process.

DNA detection of chronic myelogenous leukemia by magnetic nanoparticles.

A novel tool for the detection of BCR/ABL fusion gene in chronic myelogenous leukemia (CML) was developed by a magneto-polymerase chain reaction (PCR)-enzyme linked gene technique. The forward primers covalently bound to the surface of magnetic nanoparticles allowed a convenient separation of PCR products with high sensitivity (0.5 pg ml(-1)) and high specificity using K562 cell line and CML patients. The results were obtained when the biotinylated-reverse primer bound to streptavidin-horseradish peroxidase (HRP) and hydrolysed the substrate. This novel readout system was approximately 1000-fold more sensitive than the conventional agarose gel electrophoresis. The present technique is practical and useful for following up CML patients and for providing appropriate treatment, particularly to patients in remote areas.

Selective fluorescence sensing of deoxycytidine 5'-monophosphate (dCMP) employing a bis(diphenylphosphate)diimine ligand.

A new bis(diphenylphosphate)diimine ligand (BP1) was prepared and evaluated for its ability for selective detection of deoxycytidine 5'-monophosphate (dCMP). BP1 exhibited off-type fluorescence in the presence of dCMP. The fluorescence of BP1 was significantly quenched upon the addition of 2.5 × 10(-4) M dCMP and the detection limit was 1.25 × 10(-5) M in MeCN-H(2)O (1:1, v/v). The binding ratio between BP1 and dCMP was determined to be 1:1 with the binding constant of 3.98 ± 0.60 × 10(-3) M(-1).

Facile Control of Structured ZnO Polymeric Nanoparticles through Miniemulsion Polymerization: Kinetic and UV Shielding Effects.

Zinc oxide polymeric nanoparticles (ZPPs) of poly (styrene-co-acrylic acid) P(St/AA), containing oleic acid modified zinc oxide nanoparticles (OA-ZnO NPs), were synthesized via miniemulsion polymerization. By simply adjusting the quantity of reactants, i.e., sodium dodecyl sulfate (SDS) surfactant, potassium persulfate (KPS) initiator, and divinyl benzene (DVB) crosslinking agent, the location of ZnO NPs were altered from the inner (core) to the outer (shell), leading to core-shell and Pickering-like morphologies, respectively. The Pickering-like ZPPs were obtained when using SDS at below or equal to the critical micelle concentration (CMC). At above the CMC, the complete encapsulation of OA-ZnO NPs within the ZPPs depicted a kinetically controlled morphology. The transition to Pickering-like ZPPs also occurred when reducing the KPS from 2 to 0.5-1%. Whereas the DVB accelerated the polymerization rate and viscosity in the growing monomer-swollen nanodroplets and, hence, contributed to kinetic parameters on particle morphology, i.e., an increase in the DVB content increased the rate of polymerization. A hollow structure was obtained by replacing styrene with the more hydrophilic monomer, i.e., methyl methacrylate. All ZPPs-incorporated poly (vinyl alcohol) (PVA) films greatly improved shielding performance over the UV region and were relatively transparent on a white paper background. Due to the large number of ZnO NPs in the central region and, hence, the ease of electron transfer, composite films containing core-shell ZPPs possessed the highest UV blocking ability. ZnO NPs in the outer part of the hollow and Pickering-like ZPPs, on the other hand, facilitated the multiple light scattering according to the difference of refractive indices between the inorganic shell and organic/air core. These results confirm the advantage of structured ZPPs and their potential use as transparent UV shielding fillers.

Heat-enhancing aggregation of gold nanoparticles combined with loop-mediated isothermal amplification (HAG-LAMP) for Plasmodium falciparum detection.

Malaria infection represents a major public health and economic issue that leads to morbidity and mortality globally. A highly effective and uncomplicated detection tool is required for malaria control in geographical hotspots of transmission. We developed a simple and more sensitive novel approach for the detection of the 18S rRNA gene of Plasmodium falciparum based on loop-mediated isothermal amplification (LAMP) and visualization using colorimetric, streptavidin-functionalized gold nanoparticles (SA-GNPs). Two loop primers of LAMP were biotinylated to produce biotin-containing products during amplification. After the addition of SA-GNPs, clusters of avidin-biotin complexes were established in the LAMP structure. While the positive reactions remained wine red, the negative reactions became colorless with partial aggregations induced by hydrochloric acid (HCl) under heat enhancement (60 °C). All steps of the assay were completed within 50 min, its detection limit was 1 parasite/µL, and it was highly specific for P. falciparum. This effortless detection system with high sensitivity and specificity could provide an alternative choice for malaria diagnostics in resource-limited regions.

Increased sensitivity of enterotoxigenic Escherichia coli detection in stool samples using oligonucleotide immobilized-magnetic nanoparticles.

PCR detection of enterotoxigenic Escherichia-coli (ETEC) can be used directly on stool sample. However, it still has limitations due to presence of PCR inhibitors and interferences. This study, oligonucleotide primer specific to ETEC was immobilized onto MNPs and applied for separation and enrichment of ETEC-DNA from contaminants in stool after boiling. DNA separation efficiency was evaluated using conventional PCR and magneto-PCR-enzyme linked-gene-assay (MELGA). Due to high specificity of primer and efficiency of nanoparticles to bring down PCR inhibitors, DNA separation using primer-immobilized-MNPs exhibited 100-fold increase of sensitivity compared to that using simple boiling. Moreover, the sensitivities in stool were increased from 108 to 106 CFU/mL and 104 to 102 CFU/mL when PCR products were detected by gel electrophoresis and MELGA, respectively. Results suggested that oligonucleotide-immobilized-MNPs combined with boiling DNA extraction method was successfully used to separate the DNA of ETEC in stool with high sensitivity using MELGA.

Self-assembly of amphiphilic poly(styrene-b-acrylic acid) on magnetic latex particles and their application as a reusable scale inhibitor.

The deposition of scale on membranes or container and pipe surfaces (clogging the system) is a costly issue in water treatment processes or water-cooling systems. To effectively cope with this issue, magnetic polymeric nanoparticles (MPNPs) have been developed and applied as promising scale inhibitors, due to their high surface-area-to-volume ratio, surface modifiability, and magnetic separation ability. Carboxylated MPNPs, having a monodisperse size distribution (236 ± 26 nm) with a high magnetic content of 70 wt% and superparamagnetic properties, were fabricated by using a 2-step process: (i) formation of clusters of hydrophobic magnetic nanoparticles stabilized by oleic acid (OA-MNPs), and (ii) self-assembly of the amphiphilic block copolymer of poly(styrene27-b-acrylic acid120) (PS27-b-PAA120) onto the cluster surfaces. With application of ultrasonication to 12.0 wt% OA-MNPs, a three-dimensional network was formed by particle-particle interactions, suppressing coalescence, and then creating stable magnetic clusters. The cluster surfaces were then adsorbed by amphiphilic PS27-b-PAA120 via the attractive force between hydrophobic PS blocks. This moves longer hydrophilic PAA blocks containing carboxylic acid groups into the water phase. The formulated MPNPs acted as a nanosorbent for calcium ion (Ca2+) removal with a removal efficiency of 92%. The MPNPs can be effectively reused for up to 4 cycles. Based on the electrostatic interactions between the negatively-charged polymer and the hydrated Ca2+, the resulting precipitation leads to the prevention of calcium carbonate scale formation. Insights into this mechanism open up a new perspective for magnetic-material applications as effective antiscalants.

Fabrication of functional hollow magnetic polymeric nanoparticles with controllable magnetic location.

Hollow magnetic polymeric particles (HoMPs) with controllable location of magnetic nanoparticles and functionality of polymeric double shell were fabricated by means of the facile soft templating method in one-pot. During the in situ miniemulsion polymerization, hexadecane, the Ostwald suppressing agent, acted as a soft template, which later formed a controllable void size. Adjusting ratio and polarity of monomers caused the difference in polymerization kinetics and phase separation, which resulted in HoMPs with two internal architectures, i.e., HoMPs-I with magnetic at the inner shell/void interface and HoMPs-II with magnetic-embedded shell. Both HoMPs-I and II contained high magnetic content (30-50%wt) with sufficient saturation magnetization (16-30 emu/g). With large void volume (>50%) and distinct shell polarity, either hydrophilic Rhodamine B or hydrophobic fluorescein isothiocyanate dye was selectively loaded. After functionalization with a cancer cell targeting ligand, folic acid (FA), the cellular uptake of HoMPs-FA in HeLa cell was improved with 100% cell viability and without cell destruction. This fabrication method provides a facile mean to tailor surface chemistry and architectures of hollow hybrid particles, which would be potentially used for efficient treatment of cancer tumors.

PMMA particles coated with chitosan-silver nanoparticles as a dual antibacterial modifier for natural rubber latex films.

The antibacterial activity in sulphur prevulcanized natural rubber (SPNR) latex film was effectively improved by deposition of poly(methyl methacrylate) (PMMA) particles encircled with chitosan-coated silver nanoparticles (AgNPs-CS). With the focus on a green process, CS was selected as a safe reducing and stabilizing agent for the one-step synthesis of AgNPs-CS (38 nm, +40.4 mV) in an autoclave. The adsorption of small-sized AgNPs-CS directly onto rubber film did not provide an inhibitory effect on S. aureus. It also had a low antibacterial effect on E. coli. This is because of the particles becoming completely/partially submerged into the soft rubber matrix upon drying. Hence, the AgNPs-CS were fabricated as a shell surrounding a rigid PMMA core (496 nm, -30.9 mV). This was done using a heterocoagulation technique prior to coating on SPNR film. The presence of PMMA/AgNPs-CS on the surface of SPNR film effectively increased the surface roughness from ca. 44 to 150 nm. This substantially promoted the antibacterial activity against E. coli and S. aureus by way of contact killing and repelling mechanisms. The cytotoxicity on L-929 fibroblasts was also suppressed. This study would be, therefore, applicable to the development of antibacterial SPNR film with high surface roughness, low cytotoxicity. It could also be applied for other soft substrates.

One-pot, large-scale green synthesis of silver nanoparticles-chitosan with enhanced antibacterial activity and low cytotoxicity.

Silver nanoparticles stabilized with chitosan (AgNPs-CS) were synthesized based on the one-pot green process in an autoclave, in which CS acts as reducing agent as well as stabilizer. Effects related to temperature and pressure input on particle formation were systematically investigated. Mechanism taking place during particle nucleation and growth was proposed. The data from UV-vis absorption, X-ray diffraction pattern and morphology confirmed the formation of AgNPs-CS with face-centered cubic (fcc) structure. The synthesized AgNPs-CS showed the effective antibacterial activity against both E. coli and S. aureus. The minimum bactericidal concentration values of 39.1 and 312.5 µg/ml for E. coli and S. aureus, respectively, did not show cytotoxicity to L-929 fibroblast. Moreover, the covering of CS on the surface of AgNPs-CS was proven to reduce the cytotoxicity when compared with commercial citrate-stabilized AgNPs. Considering simple and mild process, this synthesis approach would be helpful for development of benign AgNPs-based antibacterial agent.

Enhanced Sensitivity for Detection of Plasmodium falciparum gametocytes by magnetic nanoparticles combined with enzyme substrate system.

The highly sensitive and specific detection of Pfg377 gene of Plasmodium falciparum gametocyte using Magnetic Nanoparticles PCR Enzyme-Linked Gene Assay (MELGA) was successfully developed. The MELGA included amplification of the Pfg377 gene by polymerase chain reaction (PCR) using magnetic nanoparticles (MNPs)-conjugated forward primer and biotinylated reverse primer, followed by post-analytical process using horseradish peroxidase (HRP)-conjugated streptavidin (SA). The complexes of MELGA product were incubated with the peroxidase substrate and hydrogen peroxide to produce the signal for colorimetric measurement. Altogether, the MELGA technique provided a highly sensitive and specific detection at 1 P. falciparum gametocyte/µL, which was more efficient than that of microscopic examination and rapid diagnostic tests (RDTs). Additionally, the MELGA could detect target gene at femtogram level, which was greater sensitive than the conventional PCR, nested PCR and loop-mediated isothermal amplification (LAMP). The MELGA technique could become a novel and practical method that overcome limitation of traditional gametocyte detection.