Effects of long and short carboxylated or aminated multiwalled carbon nanotubes on blood coagulation

In this work the effects of four different multiwalled carbon nanotubes (MWCNTs), including long carboxylated (L-COOH), short carboxylated (S-COOH), long aminated (L-NH(2)) and short aminated (S-NH(2)) ones, on the integrity of red blood cells, coagulation kinetics and activation of platelets were investigated with human whole blood. We found that the four MWCNTs induced different degrees of red blood cell damage as well as a mild level of platelet activation (10-25%). L-COOH and L-NH(2) induced a higher level of platelet activation than S-COOH and S-NH(2) respectively; meanwhile L-NH(2) caused marked reductions in platelet viability. The presence of the four MWCNTs led to earlier fibrin formation, L-NH(2) increased the clots hardness significantly, while L-COOH and S-NH(2) made the clots become softer. It was concluded that the four MWCNTs affected blood coagulation process and the clots mechanical properties; they also altered the integrity of the red blood cells and the viability of the platelets, as well as induced platelets activation. The effects of MWCNTs depended on the size and chemistry of the nanotubes and the type of cells they contacted.     

Preparation of thiolated polymeric nanocomposite for sensitive electroanalysis of dopamine

We report on the thiol-ene chemistry guided preparation of novel thiolated polymeric nanocomposite films of abundant anionic carboxylic groups for electrostatic enrichment and sensitive electroanalysis of cationic dopamine (DA) in neutral solution. Briefly, the thiol-ene nucleophilic reaction of a carboxylated thiol with oxidized polypyrrole (PPy), which was electrosynthesized on an Au electrode in the presence of solution-dispersed acidified multiwalled carbon nanotubes (MWCNTs), produced an a PPy-thiol-MWCNTs/Au electrode, and the PPy can be electrochemically overoxidized (OPPy) to form an OPPy-thiol-MWCNTs/Au electrode. The carboxylic groups of the polymeric nanocomposite film originate from the acidified MWCNTs, PPy-tethered carboxylated thiol, and OPPy. The carboxylated thiols examined are mercaptosuccinic acid (MSA) and thioglycolic acid, with β-mercaptoethanol as a control. Electrochemical quartz crystal microbalance, scanning electron microscopy, Fourier transform infrared spectroscopy and ultraviolet-visible spectroscopy were used for film characterization and process monitoring. Under the optimized condition, the differential pulse voltammetry peak current of DA oxidation at OPPy-MSA-MWCNTs/Au electrode is linear with DA concentration from 1.00×10(-9) to 2.87×10(-6) mol L(-1), with a limit of detection of 0.4 nmol L(-1), good anti-interferent ability and stability.     

A surface acoustic wave sensor functionalized with a polypyrrole molecularly imprinted polymer for selective dopamine detection

A surface acoustic wave sensor operating at 104 MHz and functionalized with a polypyrrole molecularly imprinted polymer has been designed for selective detection of dopamine (DA). Optimization of pyrrole/DA ratio, polymerization and immersion times permitted to obtain a highly selective sensor, which has a sensitivity of 0.55°/mM (≈550 Hz/mM) and a detection limit of ≈ 10 nM. Morphology and related roughness parameters of molecularly imprinted polymer surfaces, before and after extraction of DA, as well as that of the non imprinted polymer were characterized by atomic force microscopy. The developed chemosensor selectively recognized dopamine over the structurally similar compound 4‐hydroxyphenethylamine (referred as tyramine), or ascorbic acid,which co‐exists with DA in body fluids at a much higher concentration. Selectivity tests were also carried out with dihydroxybenzene, for which an unexpected phase variation of order of 75% of the DA one was observed. Quantum chemical calculations, based on the density functional theory, were carried out to determine the nature of interactions between each analyte and the PPy matrix and the DA imprinted PPy polypyrrole sensing layer in order to account for the important phase variation observed during dihydroxybenzene injection. Copyright © 2015 John Wiley & Sons, Ltd.     

Enhanced conjugation of Candida rugosa lipase onto multiwalled carbon nanotubes using reverse micelles as attachment medium and application in nonaqueous biocatalysis

Three liquid phases (viz. aqueous, nonaqueous, and reverse micelles) were scrutinized as medium for attachment of the enzyme Candida rugosa lipase (CRL) onto multiwalled carbon nanotubes (CNTs). The nanotubes were functionalized to attain carboxyl and amino groups on their surfaces before enzyme conjugation. Transmission electron microscopy and Fourier transformation infrared spectroscopic studies were used for characterization of the nanotubes during the course of functionalization. High enzyme loadings associated with the functionalized CNTs were observed when reverse micelles were used as the attachment medium. In addition, high activity in terms of ester synthesis in organic solvents was also observed while using those preparations. The nanobioconjugates prepared using reverse micelles were found to be highly sturdy and exhibited appreciable operational stability of around 95 ± 3% at 20th cycle (in case of carboxylated nanotubes) and 90 ± 5% at 10th cycle (in case of aminated nanotubes) for esterification. This shows the potential application of reverse micelles as the attachment medium for surface active enzymes such as CRL onto CNTs. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:828–836, 2014     

An in situ electrochemical surface plasmon resonance immunosensor with polypyrrole propylic acid film: comparison between SPR and electrochemical responses from polymer formation to protein immunosensing

A novel in situ electrochemical surface plasmon resonance (EC-SPR) immunosensor is presented in this paper. The EC-SPR measurement can be used to in situ monitor the polymer formation, probe immobilization, antigen-antibody interaction and protein immunosensing process. A sandwich immunosensor based on permeable polypyrrole propylic acid (PPA) film is constructed using mouse IgG as a model analyte. The results show that the introduction of capture antibody conjugated enzyme not only enhances the current responses but also increases the SPR angle shift. The calibration curves of electrochemical (EC) and surface plasmon resonance (SPR) measurement exhibit a similar dependence on the bulk concentration of antigen. An approximate linear relationship can be obtained by plotting the data in semi-logarithmic reference frame. Compared with SPR, EC shows higher sensitivity with prolonged time. The in situ EC-SPR immunosensor described herein could have important potentials for diagnostics and medicine applications.     

Overoxidized polypyrrole film directed single-walled carbon nanotubes immobilization on glassy carbon electrode and its sensing applications

In this paper, the films of overoxidized polypyrrole (PPyox) directed single-walled carbon nanotubes (SWNTs) have been electrochemically coated onto glassy carbon electrode (GCE). Electroactive monomer pyrrole was added into the solution containing sodium dodecyl sulfate (SDS) and SWNTs. Then, electropolymerization was proceeded at the surface of GCE, and a novel kind of conducting polymer/carbon nanotubes (CNTs) composite film with the orientation of CNTs were obtained correspondingly. Finally, this obtained polypyrrole (PPy)/SWNTs film modified GCE was oxidized at a potential of +1.8 V. It can be found that this proposed PPyox/SWNTs composite film modified GCE exhibited excellent electrocatalytic properties for some species such as nitrite, ascorbic acid (AA), dopamine (DA) and uric acid (UA), and could be used as a new sensor for practical applications. Compared with previous CNTs modified electrodes, SWNTs were oriented towards the outside of modified layer by PPyox and SDS, which made the film easily conductive. Moreover, this proposed film modified electrode was more stable, selective and applicable.     

Effects of acetylation in vapor phase and mercerization on the properties of sugarcane fibers

Chemical modification of sugarcane bagasse fiber was achieved by mercerization reaction and esterification reaction with anhydride acetic vapor. This is a new acetylation procedure. The results show that the fiber length and diameter are reduced after the reactions. Fourier transform infrared spectroscopy (FT-IR) studies produced clear evidence of the partial acetylation reaction. Optical microscopy revealed fibrillation in the acetylated fiber attributed to hemicellulose dissolution. The thermal stability measured by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) increased after acetylation and decreased after mercerization. The higher thermal stability of the acetylated fiber as compared with modified fibers in liquid medium was attributed to the small quantity of water and acetic acid present for the reaction in vapor phase. The lesser tensile strength of the acetylated fiber was due to fibrillation. The porous structure obtained favors migration of the polymer chains into the fiber acetylated, and thus it should enhance the polymer–fiber adhesion in polymer composites.     

Specific Uptake and Genotoxicity Induced by Polystyrene Nanobeads with Distinct Surface Chemistry on Human Lung Epithelial Cells and Macrophages

Nanoparticle surface chemistry is known to play a crucial role in interactions with cells and their related cytotoxic effects. As inhalation is a major route of exposure to nanoparticles, we studied specific uptake and damages of well-characterized fluorescent 50 nm polystyrene (PS) nanobeads harboring different functionalized surfaces (non-functionalized, carboxylated and aminated) on pulmonary epithelial cells and macrophages (Calu-3 and THP-1 cell lines respectively). Cytotoxicity of in mass dye-labeled functionalized PS nanobeads was assessed by xCELLigence system and alamarBlue viability assay. Nanobeads-cells interactions were studied by video-microscopy, flow cytometry and also confocal microscopy. Finally ROS generation was assessed by glutathione depletion dosages and genotoxicity was assessed by γ-H2Ax foci detection, which is considered as the most sensitive technique for studying DNA double strand breaks. The uptake kinetic was different for each cell line. All nanobeads were partly adsorbed and internalized, then released by Calu-3 cells, while THP-1 macrophages quickly incorporated all nanobeads which were located in the cytoplasm rather than in the nuclei. In parallel, the genotoxicity study reported that only aminated nanobeads significantly increased DNA damages in association with a strong depletion of reduced glutathione in both cell lines. We showed that for similar nanoparticle concentrations and sizes, aminated polystyrene nanobeads were more cytotoxic and genotoxic than unmodified and carboxylated ones on both cell lines. Interestingly, aminated polystyrene nanobeads induced similar cytotoxic and genotoxic effects on Calu-3 epithelial cells and THP-1 macrophages, for all levels of intracellular nanoparticles tested. Our results strongly support the primordial role of nanoparticles surface chemistry on cellular uptake and related biological effects. Moreover our data clearly show that nanoparticle internalization and observed adverse effects are not necessarily associated.     

Facile surface functionalization of multiwalled carbon nanotubes by soft dielectric barrier discharge plasma: Generate compatible interface for lipase immobilization

To create compatible interface for enzyme immobilization, the surface of multi-walled carbon nanotubes (MWCNTs) was functionalized using soft technique dielectric barrier discharge plasma (DBDP) for carboxylation and amination; followed by further amidation of carboxyl group with alkylamine. Successful functionalization and enzyme immobilization were structurally confirmed using spectroscopic analysis Fourier-Transform Infrared Spectroscopy (FTIR) and X-ray Photoelectron Spectroscopy (XPS). The immobilization of Candida rugosa lipase (CRL) on functionalized MWCNTs was evidenced by clearly viewing with Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) imaging. CRL showed more Freundlich equilibrium behavior upon immobilization on annealed and octadecylamidated MWCNTs, which suggested a multilayer adsorption; while upon physical adsorption on aminated and carboxylated MWCNTs, CRL, to more extent, demonstrated a Langmuir equilibrium property, producing an enzyme monolayer. It was proven that DBDP-mediated surface-functionalization could create compatible microenvironments for enzyme immobilization, resulted in improved specific activity and thermostability. The immobilized CRL on octadecylamidated MWCNTs displayed excellent reusability and operation stability, indicating its potential for industrial application.     

Amperometric and impedimetric characterization of a glutamate biosensor based on Nafion and a methyl viologen modified glassy carbon electrode

An electrochemical biosensor based on a glassy carbon (GC) electrode chemically modified with the perfluorinated cation-exchange polymer Nafion and methyl viologen (MV) is described. The enzyme was immobilized by cross-linking with glutaraldehyde in the presence of bovine serum albumin (BSA), methyl viologen and Nafion. Operating variables such as the enzyme/BSA ratio, cross-linking time in glutaraldehyde vapor, methyl viologen and Nafion percentages were investigated with regard to their influence on the biosensor sensitivity by using glucose oxidase as the enzyme model due to its high stability and low cost. The glutamate biosensor was elaborated by using optimized parameters and its electrochemical properties were investigated by cyclic voltammetry, amperometry and by electrochemical impedance spectroscopy. The glutamate biosensor shows a detection limit of 20 microM and a linear range extended to 0.75 mM. Its selectivity was tested with 15 different amino acids, each with a concentration of 20 microM, 25 microM acetaminophen, 20 microM uric acid and 200 microM ascorbic acid. No amperometric response was observed for the interfering species. This good selectivity allows glutamate detection in biological media without previous separation of the analyte.     

An amperometric oxalate biosensor based on sorghum oxalate oxidase bound carboxylated multiwalled carbon nanotubes-polyaniline composite film

A highly sensitive, specific and rapid electrochemical oxalate biosensor was constructed by covalently immobilizing sorghum leaf oxalate oxidase on carboxylated multiwalled carbon nanotubes and conducting polymer, polyaniline nanocomposite film electrodeposited over the surface of platinum (Pt) wire using N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry. The modified electrode was characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectrophotometry. The optimized oxalate biosensor showed linear response range of 8.4-272 μM with correlation coefficient of 0.93 and rapid response within 5 s at a potential of 0.4 V vs Ag/AgCl. The sensitivity was approximately 0.0113 μA/μM with a detection limit of 3.0 μM. Proposed oxalate biosensor was successfully applied to human urine sample.     

Development of a sensor prepared by entrapment of MIP particles in electrosynthesised polymer films for electrochemical detection of ephedrine

A voltammetric sensor for (-)-ephedrine has been prepared by a novel approach based on immobilisation of an imprinted polymer for ephedrine (MIPE) in an electrosynthesised polypyrrole (PPY) film. Composite films were grown potentiostatically at 1.0 V vs. Pt (QRE) on a glassy carbon electrode using an unconventional "upside-down" (UD) geometry for the three-electrode cell. As a consequence, a high MIP loading was obtained, as revealed by SEM. The sensor response was evaluated, after overoxidation of PPY matrix, by cyclic voltammetry after pre-concentration in a buffered solution of analyte in 0.5-3 mM concentration range. An ephedrine peak at approximately 0.9 V increasing with concentration and saturating at high concentrations was evident. PPY-modified electrode showed a response, which was distinctly lower than the MIP response for the same concentration of the template. The effect of potential interferences including compounds usually found in human fluids (ascorbic acid, uric acid, urea, glucose, sorbitol, glycine, dopamine) was examined.     

Regeneration of an electropolymerised polypyrrole layer for the amperometric detection of ammonia

During recent years, attempts have been undertaken to utilise electrochemically polymerised pyrrole for amperometric detection of ammonia. However, these attempts suffer from gradual decrease of sensitivity of the polypyrrole layer with increasing ammonia exposure, and trials to regenerate the polypyrrole failed until now. In this communication, we present recovery of polypyrrole sensitivity by treatment with perchloric acid and subsequent oxidation. Consequently, a higher durability of polypyrrole-based ammonia sensors could be achieved.     

Application of Polypyrrole Multi-Walled Carbon Nanotube Composite Layer for Detection of Mercury,Lead and Iron Ions Using Surface Plasmon Resonance Technique

Polypyrrole multi-walled carbon nanotube composite layers were used to modify the gold layer to measure heavy metal ions using the surface plasmon resonance technique. The new sensor was fabricated to detect trace amounts of mercury (Hg), lead (Pb), and iron (Fe) ions. In the present research, the sensitivity of a polypyrrole multi-walled carbon nanotube composite layer and a polypyrrole layer were compared. The application of polypyrrole multi-walled carbon nanotubes enhanced the sensitivity and accuracy of the sensor for detecting ions in an aqueous solution due to the binding of mercury, lead, and iron ions to the sensing layer. The Hg ion bonded to the sensing layer more strongly than did the Pb and Fe ions. The limitation of the sensor was calculated to be about 0.1 ppm, which produced an angle shift in the region of 0.3° to 0.6°.     

Sensitive detection of p53 tumor suppressor gene using an enzyme-based solid-state electrochemiluminescence sensing platform

An enzyme-based solid-state electrochemiluminescence (ECL) sensing platform for sensitive detection of a single point mutation is developed successfully using p53 tumor suppressor gene as a model analyte. A composite of multiwalled carbon nanotubes and Ruthenium (II) tris-(bipyridine) (MWNTs-Ru(bpy)(3)(2+)) was prepared and coated on an electrode surface, which was covered by polypyrrole (PPy) to immobilize ssDNA. Then, the ssDNA recognized the gold nanoparticle (AuNP)-labeled p53 tumor suppressor gene, and produced AuNP-dsDNA electrode with AuNP layer. The surface adsorbed the glucose-dehydrogenase (GDH) molecules for producing ECL signal. This system combined enzyme reaction with ECL detection, and it can recognize sequence-specific wild type p53 sequence (wtp53) and muted type p53 sequence (mtp53) with discrimination of up to 56.3%. The analytic results were sensitive and specific. It holds promise for the diagnosis and management of cancer.     

A novel impedimetric aptasensor,based on functionalized carbon nanotubes and prussian blue as labels

A simple and feasible electrochemical sensing protocol was developed for the detection of bisphenol A (BPA) by employing the gold nanoparticles (AuNPs), prussian blue (PB) and functionalized carbon nanotubes (AuNPs/PB/CNTs-COOH). An aminated complementary DNA as a capture probe and specific aptamer against BPA as a detection probe was immobilized on the surface of a modified glassy carbon (GC) electrode via the formation of covalent amide bond and hybridization, respectively. The proposed nanoaptasensor combined the advantages of the in situ formation of PB as a label, the deposition of neatly arranged AuNPs, and the covalent attachment of the capture probe to the surface of the modified electrode. Upon addition of target BPA, the analyte reacted with the aptamer and caused the steric/conformational restrictions on the sensing interface. The formation of BPA–aptamer complex at the electrode surface retarded the interfacial electron transfer reaction of the PB as a probe. Sensitive quantitative detection of BPA was carried out based on the variation of electron transfer resistance which relevant to the formation of BPA– aptamer complex at the modified electrode surface. Under the optimized conditions, the proposed aptasensor exhibited a high sensitivity, wide linearity to BPA and low detection limit. This aptasensor also displayed a satisfying electrochemical performance with good stability, selectivity and reproducibility.     

ProteoChip: a highly sensitive protein microarray prepared by a novel method of protein immobilization for application of protein-protein interaction studies

We have developed a highly sensitive microarray protein chip, ProteoChip, coated with ProLinker, novel calixcrown derivatives with a bifunctional coupling property that permits efficient immobilization of capture proteins on solid matrixes and makes high-throughput analysis of protein-protein interactions possible. The analysis of quartz crystal microbalance showed that both monoclonal antibody (mAb) and antigen (Ag) bound to the gold film of the sensor surface coated with ProLinker B and that it is useful for studies of Ab-Ag interactions. ProteoChip, aminated glass slide coated with ProLinker A, was also demonstrated to be useful for preparation of high-density array spots by using a microarrayer and for analysis of analyte Ags either by direct or sandwich methods of fluorescence immunoassay. The detection sensitivity of ProteoChip was as low as 1-10 femtogram/mL of analyte protein, useful for detection of tumor markers. ProteoChip was also useful for studies of direct protein-protein interactions as demonstrated by analysis of integrin-extracellular matrix protein interaction. These experimental results suggest that ProteoChip is a powerful tool for development of chip-based lead screening microarrays to monitor protein-protein interactions (i.e. drug target) as well as for biomarker assays which require high detection sensitivity.     

Enzymatic synthesis of phenol polymer and its functionalization

In phosphate buffer (pH = 7.0) containing sodium dodecyl sulfate (SDS), an environmentally friendly system, enzymatic polymerization of phenol catalyzed by horseradish peroxidase (HRP) was efficiently performed. The obtained phenol polymer is partly soluble in common solvents, such as acetone, THF and DMF. IR analysis shows that the polymer is composed of phenylene and oxyphenylene units. The functionalization of the phenol polymer was performed by reacting with epoxy chloropropane and triethylene-tetramine following, and then insoluble aminated phenol polymer was obtained. The aminated phenol polymer was adopted as carrier to prepare a novel supported palladium catalyst (PP-N-Pd) for Heck reaction. PP-N-Pd shows high catalytic performance for Heck reactions of aryl iodides with acrylic acid or styrene and the yields of trans-products were higher than 90%. Under the optimized conditions, aryl bromides and activated aryl chloride also reacted with alkenes to give the yields of above 80%. XPS analysis indicates that the main coordination atom in PP-N-Pd is N and the chemical valence of palladium in PP-N-Pd is Pd²⁺. The novel supported catalyst also shows good recyclability for Heck reaction.     

Directed covalent immobilization of aminated DNA probes on aminated plates

A new protocol that enables the immobilization of DNA probes on aminated micro-titer plates activated with aldehyde-dextran via an amino group artificially introduced in the 3' end of the oligonucleotide probe is reported in this work. The method is based on the use of hetero-functional-dextran as a long and multifunctional spacer arm covalently attached to an aminated surface capable of immobilizing DNA oligonucleotides. The immobilization occurred only via the amino introduced in the 3' end of the probe, with no implication of the DNA bases in the immobilization, ensuring that the full length of the probe is available for hybridization. These plates having immobilized oligonucleotide probes are able to hybridize complementary DNA target molecules. The tailor-made hetero-functional aldehyde-aspartic-dextran together with the chemical blocking of the remaining primary amino groups on the support using acetic anhydride avoid the nonspecific adsorption of DNA on the surface of the plates. Using these activated plates, (studying the effect of the probe concentration, temperature, and time of the plate activation on the achieved signal), thus, the covalent immobilization of the aminated DNA probe was optimized, and the sensitivity obtained was similar to that achieved using commercial biotin-streptavidin systems. The new DNA plates are stable under very drastic experimental conditions (90% formamide, at 100 degrees C for 30 min or in 100 mM NaOH).