Enhancing the reusability of endoglucanase-gold nanoparticle bioconjugates by tethering to polyurethane microspheres

The synthesis of polyurethane microsphere-gold nanoparticle "core-shell" structures and their use in the immobilization of the enzyme endoglucanase are described. Assembly of gold nanoparticles on the surface of polymer microspheres occurs through interaction of the nitrogens in the polymer with the nanoparticles, thereby precluding the need for modifying the polymer microspheres to enable such nanoparticle binding. Endoglucanse could thereafter be bound to the gold nanoparticles decorating the polyurethane microspheres, leading to a highly stable biocatalyst with excellent reuse characteristics. The immobilized enzyme retains its biocatalytic activity and exhibits improved thermal stability relative to free enzyme in solution. The high surface area of the host gold nanoparticles renders the immobilized enzyme "quasi free", while at the same time retaining advantages of immobilization such as ease of reuse, enhanced temporal and thermal stability, etc.     

Mechanism of gold nanoparticles-induced trypsin inhibition: a multi-technique approach

The binding interactions of gold nanoparticles with trypsin were investigated using multi-spectra methods and molecular modeling. The experiment data showed that trypsin modified the surface of gold nanoparticles. The fluorescence intensity of trypsin was quenched by gold nanoparticles that strongly associated with protein and induced the inhibition of enzyme activity. The electrostatic and hydrophobic interactions were the primary contributors to the binding forces between trypsin and gold nanoparticles. The covalent interactions might be also involved in the binding process. The modeling calculated results indicated that the binding site was near to the primary substrate-binding pocket and the active site of the enzyme substrate. This work elucidated the interaction mechanism of trypsin with gold nanoparticles from the theoretical and experimental angle.     

Immobilization and biocatalytic activity of fungal protease on gold nanoparticle-loaded zeolite microspheres

Gold nanoparticles are excellent biocompatible surfaces for the immobilization of enzymes. However, separation of the gold nanoparticle-enzyme bioconjugate material from the reaction medium is often difficult. In this study, we investigate the assembly of the gold nanoparticles on the surface of the amine-functionalized zeolite microspheres in the formation of zeolite-gold nanoparticle "core-shell" structures and, thereafter, the use of this structure in immobilization of fungal protease. The assembly of gold nanoparticles on the zeolite surface occurs through the amine groups present in 3-aminopropyltrimethoxysilane (3-APTS). The fungal proteases bound to the massive "core-shell" structures were easily separated from the reaction medium by mild centrifugation and exhibited excellent reuse characteristics. The biocatalytic activity of fungal protease in the bioconjugate was marginally enhanced relative to the free enzyme in solution. The bioconjugate material also showed significantly enhanced pH and temperature stability and a shift in the optimum temperature of operation.     

Modification of silicon nanoparticle surface with gold or silver attenuates its biocompatibility in vitro

The effects of crystalline silicon (Si) nanoparticles covered with gold and silver on the viability and state of cellular organelles of cultured human peripheral blood lymphocytes were investigated. After interaction with Si nanoparticles covered with gold (Si/Au) or with silver (Si/Ag), the reactive oxygen species in cells increased, but their viability was not decreased. The Si/Au nanoparticles decreased functional activity of lysosomes and mitochondria, while Si/Ag decreased the functional activity only of mitochondria. It is concluded that modification of a surface with gold or silver results in a reduction of biocompatibility of crystalline silicon nanoparticles.     

Direct electron transfer between cytochrome P450scc and gold nanoparticles on screen-printed rhodium-graphite electrodes

This paper is concerned with an investigation of electron transfer between cytochrome P450scc (CYP11A1) and gold nanoparticles immobilised on rhodium-graphite electrodes. Thin films of gold nanoparticles were deposited onto the rhodium-graphite electrodes by drop casting. Cytochrome P450scc was deposited onto both gold nanoparticle modified and bare rhodium-graphite electrodes. Cyclic voltammetry indicated enhanced activity of the enzyme at the gold nanoparticle modified surface. The role of the nanoparticles in mediating electron transfer to the cytochrome P450scc was verified using ac impedance spectroscopy. Equivalent circuit analysis of the impedance spectra was performed and the values of the individual components estimated. On addition of aliquots of cholesterol to the electrolyte bioelectrocatalytic reduction currents were obtained. The sensitivity of the nanoparticle modified biosensor to cholesterol was 0.13 microA microM-1 in a detection range between 10 and 70 microM of cholesterol. This confirms that gold nanoparticles enhance electron transfer to the P450scc when present on the rhodium-graphite electrodes.     

家蚕系统表达的重组人丁酰胆碱酯酶的生化性质

研究家蚕系统中高效表达的重组人丁酰胆碱酯酶(rhBChE)的生化性质并与天然人丁酰胆碱酯酶(nhBChE)进行比较。采用丁酰胆碱酯酶活性测定,抑制剂及重活化剂作用,免疫印迹等方法。实验结果表明,rhBChE及nhBChE在底物亲和力、抑制剂敏感性及可重活化性、稳定性、对抗体的反应性等方面均有相似的生化性质,重组人丁酰胆碱酯酶具有天然人丁酰胆碱酯酶的功能。但rhBChE的糖基化修饰程度较低,用于人体时应予考虑     

A real-time PCR-based method for determining the surface coverage of thiol-capped oligonucleotides bound onto gold nanoparticles

Here we report a real-time PCR-based method for determining the surface coverage of dithiol-capped oligonucleotides bound onto gold nanoparticles alone and in tandem with antibody. The detection of gold nanoparticle-bound DNA is accomplished by targeting the oligonucleotide with primer and probe binding sites, amplification of the oligonucleotide by PCR, and real-time measurement of the fluorescence emitted during the reaction. This method offers a wide dynamic range and is not dependant on the dissociation of the oligonucleotide strands from the gold nanoparticle surface; the fluorophore is not highly quenched by the gold nanoparticles in solution during fluorescence measurements. We show that this method and a fluorescence-based method give equivalent results for determining the surface coverage of oligonucleotides bound onto 13 or 30 nm gold nanoparticles alone and in tandem with antibody. Quantifying the surface coverage of immobilized oligonucleotides on metallic nanoparticle surfaces is important for optimizing the sensitivity of gold nanoparticle-based detection methods and for better understanding the interactions between thiol-functionalized oligonucleotides and gold nanoparticles.     

Electrochemical immunoanalysis of cardiac myoglobin

Methods of myoglobin determination based on electrochemical analysis by means of analysis of electrochemical parameters of modified electrodes have been proposed. The method of direct detection is based on interaction of myoglobin with anti-myoglobin with subsequent electrochemical registration of this hemoprotein. The electrode surface was modified by a membrane-like synthetic didodecyldimethylammonium bromide (DDAB), gold nanoparticles and antibodies to human cardiac myoglobin the electrochemical reduction of myoglobin heme was registered provided that the antigen (myoglobin) was present in the samples. The reaction of myoglobin binding to antibodies immobilized on the electrode surface was also registered using electrochemical impedance spectroscopy. The study of electro analytical characteristics revealed high specificity and sensitivity of the developed method. The biosensor was characterized by low detection limit and a high working range of the detected concentrations from 17.8 to 1780 ng/ml (from 1 to 100 nM). The method of myoglobin determination based on a signal of gold nanoparticles has also been proposed. The signal was detected with stripping voltammetry. There was a change in the cathodic peak area and the peak height of gold oxide reduction for the electrodes with antibodies and the electrodes with the antibody-myoglobin complex.     

Improved Method of Study on the Photothermal Effect of Plasmonic Nanoparticles by Dynamic IR Thermography

Efficient heat generation by plasmon-resonant gold nanoparticles, together with their biocompatibility and high specificity of biomolecular recognition, opens new possibilities for applications in biomedical applications. In this work, we present an improved method of monitoring surface temperature changes subjected to external stimulation by dynamic IR thermography. The method is based on the careful analysis of an IR image sequence recorded before, during, and after the stimulation that allows one to select areas with significant temperature variation and evaluate temporal behavior of the surface temperature. The method was applied for the experimental study on the photothermal effect in a gold hydrosol containing hollow gold nanoparticles heated with laser beam. Under these conditions, it was seen that the surface temperature of the gold hydrosol (measured with a FLIR SC655 InfraRed Camera, resolution 640 × 480 pixels) under the laser beam gradually increases and reaches a saturation level. It was shown that the developed method is capable of producing a quantitative analysis of the changes in the surface temperature distribution of the gold hydrosol, as well as characterizing the photothermal properties of the nanoparticles.

     

Monoclonal Antibodies Allow Precipitation of Esterasic but Not Peptidasic Activities Associated with Butyrylcholinesterase

Commercially available and affinity-purified butyrylcholinesterases isolated from human serum were examined for their esterasic activity and their ability to hydrolyze various neuropeptides, including neurotensin, substance P, and leucine-enkephalin. The three pools that displayed the lowest esterasic activities were shown to hydrolyze neurotensin with the same HPLC degradative pattern. By contrast, noticeable qualitative and quantitative discrepancies were observed when hydrolyses of substance P and leucine-enkephalin by these three butyrylcholinesterase pools were studied. The pool that exhibited the highest esterasic activity appeared to be homogeneously constituted by 90- and 180-kDa protein bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and was totally unable to hydrolyze these three neuropeptides. This suggested that the three other butyrylcholinesterase preparations could be contaminated by exogenous peptidases. This was confirmed by means of three distinct monoclonal antibodies directed toward human serum butyrylcholinesterase. The three IgG-purified fractions precipitated the esterasic activity, whereas they failed to precipitate the neuropeptide-hydrolyzing activities whatever the substrate examined. Altogether, these results demonstrate that peptidases associated with butyrylcholinesterase are contaminating enzymes that cannot be considered as intrinsic activities of this enzyme.     

Human serum albumin interaction,in silico and anticancer evaluation of Pine-Gold nanoparticles

Unique characteristics displayed by phytoconstituent conjugated nanoparticles and their crucial interactions with proteins serve to develop nanoparticle-bio-interface platform. Gold nanoparticles of 16 nm in size were generated using aqueous extracts of pine bark and further conjugated to human serum albumin. The gold nanoparticles-protein complex was characterized by surface plasmon resonance, UV–vis and emission spectroscopy techniques. Further, it was characterized for surface morphology and elemental composition, crystallographic quality, nanoparticles size, shape, stability, structural determination and the identification of capping agent. Moreover, the interaction of gold nanoparticles with human serum albumin was investigated using conventional spectroscopy techniques. Fluorescence quenching and absorption studies demonstrated an effective binding of human serum albumin with oleamide capped gold nanoparticles. The molecular docking study showed a binding affinity of -6.1 kcal/mol whereas the molecular dynamics simulation indicated that the binding of oleamide to human serum albumin. A biological evaluation of pine bark extract-gold nanoparticles showed cytotoxicity with increased cell mortality in lung cancer cells and minimal toxicity on non-cancerous human embryonic kidney cells, respectively.     

Screen-printed electrodes based on carbon nanotubes and cytochrome P450scc for highly sensitive cholesterol biosensors

This paper is concerned with an investigation of electron transfer between cytochrome P450scc (CYP11A1) immobilized on nanostructured rhodium-graphite electrodes. Multi-walled carbon nanotubes (MWCNT) were deposited onto the rhodium-graphite electrodes by drop casting. Cytochrome P450scc was deposited onto MWCNT-modified rhodium-graphite electrodes. Cytochrome P450scc was also deposited onto both gold nanoparticle-modified and bare rhodium-graphite electrodes, in order to have a comparison with our previous works in this field. Cyclic voltammetry indicated largest enhanced activity of the enzyme at the MWCNT-modified surface. The role of the nanotubes in mediating electron transfer to the cytochrome P450scc was verified as further improved with respect to the case of rhodium-graphite electrodes modified by the use of gold nanoparticles. The sensitivity of our system in cholesterol sensing is higher by orders of magnitude with respect to other similar systems very recently published that are based on cholesterol oxidase and esterase. The electron transfer improvement attained by the use of MWCNT in P450-based cholesterol biosensors was demonstrated to be larger than 2.4 times with respect to the use of gold nanoparticles and 17.8 times larger with respect to the case of simple bare electrodes. The sensitivity was equal to 1.12muA/(mMmm(2)) and the linearity of the biosensor response was improved with respect to the use of gold nanoparticles.     

Glial cells in coculture can increase the acetylcholinesterase activity in human brain endothelial cells.

The elements of the cholinergic system (acetylcholinesterase and choline acetyltransferase) and butyrylcholinesterase were studied in human cortical capillary samples, brain-derived endothelial cell cultures and glial cell cultures. It was shown that the elements of the cholinergic system are present in the microvessels, but the choline acetyltransferase activity may be due to contamination with cholinergic nerve terminals since no choline acetyltransferase could be demonstrated in endothelial cell cultures. The present results revealed that the activity of acetylcholinesterase is reduced in the cortical endothelial cell cultures after longer culture times, while butyrylcholinesterase activity is not altered. In a system where endothelial cells were cocultured with embryonic human brain astroglial cells for 12 days in vitro, the acetylcholinesterase activity was increased 2-fold. These results support a glial influence on the enzyme activity of the cerebral endothelium.     

Addressable self-immobilization of lactate dehydrogenase across multiple length scales

Successful nanobiotechnology implementation largely depends on control over the interfaces between inorganic materials and biological molecules. Controlling the orientations of biomolecules and their spatial arrangements on the surface may transform many technologies including sensors, to energy. Here, we demonstrate the self-organization of L -lactate dehydrogenase (LDH), which exhibits enhanced enzymatic activity and stability on a variety of gold surfaces ranging from nanoparticles to electrodes, by incorporating a gold-binding peptide tag (AuBP2) as the fusion partner for Bacillus stearothermophilus LDH (bsLDH). Binding kinetics and enzymatic assays verified orientation control of the enzyme on the gold surface through the genetically incorporated peptide tag. Finally, redox catalysis efficiency of the immobilized enzyme was detected using cyclic voltammetry analysis in enzyme-based biosensors for lactate detection as well as in biofuel cell energy systems as the anodic counterpart. Our results demonstrate that the LDH enzyme can be self-immobilized onto different gold substrates using the short peptide tag under a biologically friendly environment. Depending on the desired inorganic surface, the proposed peptide-mediated path could be extended to any surface to achieve single-step oriented enzyme immobilization for a wide range of applications.     

A novel approach to the ultrastructural localization of cell surface receptors: affinity-gold labeling of Na, K-ATPase

We describe here a novel method of affinity-gold labeling for the ultrastructural localization and biochemical characterization of functional cell surface receptors. This approach combines the widely used colloidal gold technique, a previously published method for coating the gold with a matrix of derivatized dextran, small receptor-specific ligands, and a photoactivatable cross-linker. The resulting gold-affinity probe directed to a selected receptor by the ligand, is subsequently attached to the receptor by light activation of the cross-linker. As a specific example, a gold affinity probe prepared with ouabain, a selective inhibitor of Na,K-ATPase, as the directing ligand was used to investigate the ultrastructural localization of this enzyme complex in cell membranes. The biological activity of ouabain covalently linked to derivatized dextran containing the photoactivatable cross-linker was examined by its action on ion transport across dog trachea epithelium and on the enzyme activity of Na,K-ATPase preparations obtained from the rectal gland of the elasmobranch, Mustelus californicus. By these tests the probe mimics the effects of free ouabain. Electron micrographs of labeled human erythrocytes and cultured human foreskin fibroblasts showed an apparent random distribution of Na,K-ATPase on the plasma membranes of these cells. Binding of the probe was blocked in the presence of excess ouabain, a result demonstrating that the affinity probe binds at the same sites as free ouabain. Covalent attachment of probe by light activation of the nitroarylazido groups greatly enhanced retention during washing and standard procedures of fixation and dehydration. The high density of the gold probe was utilized to isolate the covalently attached membrane components from labeled human foreskin fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)     

A chemiluminescent metalloimmunoassay based on copper‐enhanced gold nanoparticles for quantification of human growth hormone

A chemiluminescence (CL) immunoassay was developed to determine human growth hormone (hGH) based on copper‐enhanced gold nanoparticles. In this method, gold nanoparticles were deposited on polystyrene wells for adsorption of human growth antibodies as well as catalyst for reducing of copper ions from the copper enhancer solution. The reduction of copper ions was prevented where the gold nanoparticles were covered by the antibody–antigen immunocomplex. The deposited copper on Au nanoparticles was then dissolved in HNO₃ solution and quantified using the CL method. The CL intensity response was logarithmically dependent on the hGH concentrations over the range 0.2–50 ng/mL, with a detection limit (3σ) of 0.036 ng/mL. Copyright © 2012 John Wiley & Sons, Ltd.     

A subunit-sized butyrylcholinesterase present in high concentrations in pooled rabbit serum

A butyrylcholinesterase of mol.wt. approx. 83000 was observed in pooled rabbit serum. The enzyme was named monomeric butyrylcholinesterase to distinguish it from the larger oligomeric butyrylcholinesterase of horse and human serum whose subunits are the same size as the monomeric enzyme. The active-site concentration of monomeric butyrylcholinesterase in the pooled serum was 0.18mum, which is five times the concentration of butyrylcholinesterase in pooled horse serum. This was surprising, since the horse serum is regarded as a rich source of butyrylcholinesterase, whereas rabbit serum is not generally thought to contain significant amounts of any butyrylcholinesterase. The explanation, in large part, was the relatively low k(cat.) of the monomeric enzyme, which was approx. 57s(-1) with butyrylthiocholine as substrate and is one-thirtieth of the comparable k(cat.) of horse butyrylcholinesterase. The substrate specificity of monomeric butyrylcholinesterase also differed significantly from that of horse and human butyrylcholinesterase. For example, with the monomeric enzyme, the hydrolysis of 1mm-acetylthiocholine was only 4% the rate for 1mm-butyrylthiocholine, whereas human and horse butyrylcholinesterases hydrolysed 1mm-acetylthiocholine at 50% of the rate for 1mm-butyrylthiocholine. Moreover, monomeric butyrylcholinesterase generally hydrolysed aromatic esters more rapidly than choline esters, whereas the reverse is true of the butyrylcholinesterases. To facilitate the study of monomeric butyrylcholinesterase, it was separated from the larger butyrylcholinesterase and acetylcholinesterase, also present in rabbit serum, and purified 89-fold by fractionation with (NH(4))(2)SO(4) and ion-exchange chromatography.     

Catalytic properties of thioredoxin immobilized on superparamagnetic nanoparticles

Thioredoxin (Trx1), a very important protein for regulating intracellular redox reactions, was immobilized on iron oxide superparamagnetic nanoparticles previously coated with 3-aminopropyltriethoxysilane (APTS) via covalent coupling using the EDC (1-ethyl-3-{3-dimethylaminopropyl}carbodiimide) method. The system was extensively characterized by atomic force microscopy, vibrational and magnetic techniques. In addition, gold nanoparticles were also employed to probe the exposed groups in the immobilized enzyme based on the SERS (surface enhanced Raman scattering) effect, confirming the accessibility of the cysteines residues at the catalytic site. For the single coated superparamagnetic nanoparticle, by monitoring the enzyme activity with the Ellman reagent, DTNB = 5,5′-dithio-bis(2-15 nitrobenzoic acid), an inhibitory effect was observed after the first catalytic cycle. The inhibiting effect disappeared after the application of an additional silicate coating before the APTS treatment, reflecting a possible influence of unprotected iron-oxide sites in the redox kinetics. In contrast, the doubly coated system exhibited a normal in-vitro kinetic activity, allowing a good enzyme recovery and recyclability.     

Immobilization of glucose oxidase onto gold nanoparticles with enhanced thermostability

Immobilized proteins and enzymes were widely investigated in medical field as well as in food and environmental fields. In this paper, glucose oxidase (GOD) monolayer was covalently immobilized on the surface of gold nanoparticles (AuNPs) to fabricate bioconjugate complex. The citrate-stabilized AuNPs were first functionalized by a carboxyl-terminated alkanethiol and the terminal carboxyl groups were subsequently bonded with side-chain amino groups of protein surface through EDC/NHS coupling reaction. The enzyme activity assays of the obtained bioconjugates display an enhanced thermostability and similar pH-dependence behavior in contrast with that of free enzyme. Such GOD/AuNPs bioconjugates can be considered as a catalytic nanodevice to construct nanoreactor based on glucose oxidation reaction for biotechnological purpose.     

Immobilization of lipases on alkyl silane modified magnetic nanoparticles: effect of alkyl chain length on enzyme activity

Background

Biocatalytic processes often require a full recycling of biocatalysts to optimize economic benefits and minimize waste disposal. Immobilization of biocatalysts onto particulate carriers has been widely explored as an option to meet these requirements. However, surface properties often affect the amount of biocatalysts immobilized, their bioactivity and stability, hampering their wide applications. The aim of this work is to explore how immobilization of lipases onto magnetite nanoparticles affects their biocatalytic performance under carefully controlled surface modification.

Methodology/Principal Findings

Magnetite nanoparticles, prepared through a co-precipitation method, were coated with alkyl silanes of different alkyl chain lengths to modulate their surface hydrophobicity. Candida rugosa lipase was then directly immobilized onto the modified nanoparticles through hydrophobic interaction. Enzyme activity was assessed by catalytic hydrolysis of p-nitrophenyl acetate. The activity of immobilized lipases was found to increase with increasing chain length of the alkyl silane. Furthermore, the catalytic activities of lipases immobilized on trimethoxyl octadecyl silane (C18) modified Fe₃O₄ were a factor of 2 or more than the values reported from other surface immobilized systems. After 7 recycles, the activities of the lipases immobilized on C18 modified nanoparticles retained 65%, indicating significant enhancement of stability as well through hydrophobic interaction. Lipase immobilized magnetic nanoparticles facilitated easy separation and recycling with high activity retaining.

Conclusions/Significance

The activity of immobilized lipases increased with increasing alkyl chain length of the alkyl trimethoxy silanes used in the surface modification of magnetite nanoparticles. Lipase stability was also improved through hydrophobic interaction. Alkyl silane modified magnetite nanoparticles are thus highly attractive carriers for enzyme immobilization enabling efficient enzyme recovery and recycling.