Biotinylated Glucose Oxidase Research Articles

Designing electrochemical interfaces based on nanohybrids of avidin functionalized-carbon nanotubes and ruthenium nanoparticles as peroxidase-like nanozyme with supramolecular recognition properties for site-specific anchoring of biotinylated residues

We are reporting an original supramolecular architecture based on a rationally designed new nanohybrid with enhanced peroxidase-like activity and site-specific biorecognition properties using avidin-functionalized multi-walled carbon nanotubes (MWCNTs-Av) and Ru nanoparticles (RuNPs). The nanohybrid-electrochemical interface was obtained by drop-coating of MWCNTs-Av dispersion at glassy carbon electrodes (GCE) followed by solvent evaporation and further electrodeposition of RuNPs (50 ppm RuCl2 for 15 s at -0.600 V). The simultaneous presence of MWCNTs and RuNPs produces a synergic effect on the non-enzymatic catatalytic reduction of H2O2 and allows the quantification of H2O2 in a wide linear range (from 5.0 × 10−7 M to 1.75 × 10−3 M) with a low limit of detection (65 nM). The avidin residues present in MWCNTs-Av/RuNPs hybrid nanomaterial allowed the anchoring by bioaffinity of biotinylated glucose oxidase (biot-GOx) as proof-of-concept of the analytical application of MWCNTs-Av platform for biosensors development. The resulting nanoarchitecture behaves as a bienzymatic-like glucose biosensor with a competitive analytical performance: linear range between 2.0 × 10−5 M and 1.23 × 10−3 M, sensitivity of (0.343 ± 0.002) μA mM−1 or (2.60 ± 0.02) μA mM−1 cm−2, detection limit of 3.3 μM, and reproducibility of 5.2% obtained with five different GCE/MWCNTs-Av/RuNPs/biot-GOx bioplatforms prepared the same day using the same MWCNTs-Av dispersion, and 9.1% obtained with nine biosensors prepared in different days with nine different MWCNTs-Av dispersions. The average concentrations of glucose in Gatorade®, Red bull® and Pepsi® with the biosensor demonstrated excellent agreement with those reported in the commercial beverages.

Reactive bienzyme systems fabricated through immobilization of biotinylated glucose oxidase and peroxidase molecules onto neutralized avidin-conjugated liposomes

Immobilization of multiple enzymes onto solid surface is of significance for efficient consecutive reactions. In this work, the neutralized avidin (NA) molecules were covalently conjugated at pH 7.5 to the liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 20 mol% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and 15 mol% N-(succinimidyloxy-glutaryl)-l-α-phosphatidylethanolamine, 1-palmitoyl-2-oleoyl (POPE-NHS). The apparent number of NA molecules conjugated per liposome, nNA was estimated on the basis of fluorescence quenching of biotin-4-fluorescein (B4F) in the presence of neutralized avidin-conjugated liposomes (NALs). The NALs possessed the nNA value of (4.2±1.8)×102 and the mean hydrodynamic diameter of about 150nm. The biotinylated glucose oxidase and horse radish peroxidase molecules (b-GO and b-HRP) were mixed with NALs at pH 7.5 under the condition that the total concentration of b-GO and b-HRP ([b-GO]=[b-HRP]) is equal to the apparent concentration of NA (500nM) in the NAL suspension. When the immobilization was performed at [Lipid]=0.35mM, a NAL possessed the enzyme activities corresponding to 1.4 free GO and 50 free HRP molecules. The b-GO and b-HRP-immobilized NALs could continuously catalyze the oxidation of glucose with molecular oxygen producing H2O2 which was consumed for the catalytic oxidation of 2,2′-Azino-bis(3-ethylbenzthiazoline 6-sulfonic acid) (ABTS2–). The results obtained demonstrate that the reactive bienzyme system can be fabricated under mild conditions by using the NALs as colloidal scaffolds.

Nanotubes and nanoparticles based 3D scaffolds for the construction of high performance Biosensors

ABSTRACT3D scaffolds with different pore sizes, using single-walled carbon nanotubes (SWCNTs) and nanoparticles of different size were constructed. Biotinylated glucose oxidase (GOX-B) and anti-cholera toxin (anti-CT) were immobilized onto the one and two level nanoscaffolds, functionalized with pyrene-β-cyclodextrin for the construction of glucose based enzyme sensors and immunosensors, respectively. For enzyme sensors, highest current density and sensitivity (41.72 μA cm-2, 3 mA M-1 cm-2) were obtained with two level scaffolds made with 100 nm nanoparticles. In contrast to this, for immunosensors, highest current density and sensitivity (11.71 μA cm-2, 116.2 μA M-1 cm-2) were obtained with two level scaffolds made with 500 nm nanoparticles, indicating that the pore sizes can be adjusted using different size of nanoparticles for the respective applications.

Apoferritin nanoparticle: a novel and biocompatible carrier for enzyme immobilization with enhanced activity and stability

Apoferritin is a uniform spherical nano-size biomaterial with excellent bio-compatibility. In this work, we report the use of apoferritin as a novel biocompatible carrier for stabilizing enzymes and enhancing their activities. We used glucose oxidase (GOx) as a model enzyme in this study. GOx was immobilized on the surface of the apoferritin through a green synthetic approach, taking advantage of bioaffinity binding between streptavidin and biotin. As a result, a glucose oxidase–biotin/streptavidin/biotin–apoferritin conjugate (Apo–GOx) was prepared using streptavidin as the bridge. The synthesized Apo–GOx was characterized by transmission electron microscopy, ultraviolet and fluorescence spectroscopy. The activity and stability of GOx on the surface of the apoferritin were investigated and challenged by different environmental factors, such as the temperature, chemicals and pH, in comparison with the biotinylated GOx (B-GOx). The results demonstrate that the activity of Apo-GOx is significantly enhanced while the thermal and chemical stabilities of Apo-GOx are also greatly improved compared to free B-GOx. For instance, the activity of the Apo-GOx only lost 30% after 2 h incubation at 50 oC in comparison to a 70% loss of free B-GOx. The activity of Apo-GOx remains intact after 30 min incubation in 5 M urea solution while B-GOx lost 80% activity after the same treatment. Furthermore, glucose detection was used as a model application for the enzyme immobilization method developed in this work. The GOx immobilized apoferritin nanoparticles exhibited high sensitivity for glucose detection with a detection limit of 3 nM glucose. This work offers a novel approach for immobilizing enzymes with enhanced stability and activity, thus holds the promising advantage for a number of applications, such as in enzyme catalysis, DNA assays and immunoassays.

Immobilization of biotinylated biomolecules onto electropolymerized poly(pyrrole-nitrilotriacetic acid)–Cu 2+ film

A novel and simple immobilization strategy for biotinylated biological macromolecules onto electropolymerized poly(pyrrole-nitrilotriacetic acid)(NTA)–Cu 2+ films without avidin as connecting bridge is reported. After complexation of Cu 2+ by the polymerized NTA chelator, biotinylated biomolecules were immobilized by coordination of the biotin groups on the NTA–Cu 2+ complex. The anchoring of biotinylated glucose oxidase was demonstrated by fluorescent characterization via FITC-labeled avidin and amperometric measurement of glucose. The resulting calibration curve led to a sensitivity and maximum current density values of 0.6 mA mol − 1 L cm − 2 and 13.2 μA cm − 2 , respectively. Thus, biotinylated polyphenol oxidase was fixed leading to a catechol sensor with a sensitivity of 656 mA mol − 1 L cm − 2 and maximum current density of 25.4 μA cm − 2 . This system was also applied to the efficient immobilization of biotinylated DNA, illustrated by impedimetric detection of the formation of the DNA duplex.

Pyrene functionalized single-walled carbon nanotubes as precursors for high performance biosensors

The attachment of biotin to nanotube modified electrodes by formation of π-stacking interactions using a biotinylated pyrene derivative is reported. The specific anchoring of biological macromolecules via avidin bridges was investigated using biotinylated glucose oxidase (GOX-B) as model enzyme. Further studies focus the possibility of the electropolymerization of pyrene derivatives. Four types of biotin-SWCNT modified electrodes were investigated: (1) deposits of biotin-pyrene functionalized nanotubes (B-SWCNTs); (2) deposits of regular nanotubes, functionalized by incubation in a biotin-pyrene solution; (3) the latter configuration and subsequent electropolymerization of the adsorbed biotin-pyrene; and (4) deposits of regular nanotubes subsequently modified by electropolymerization of a pyrene-biotin monomer (2 mM). The nanotubes deposits were characterized by SEM imaging and the electropolymerization of the pyrene derivatives were investigated using cyclic voltammetry. These types of biotin-SWCNT modified electrodes were tested due to their capacity to immobilize biotinylated biomolecules (GOX-B) via avidin bridges and their performances in glucose detection were examined using amperometry. The best configuration (3) was obtained with SWCNT coatings modified by specific adsorption and electropolymerization of biotin-pyrene. A maximum current density of 584 μA cm −2 could be realized with a sensitivity of 37 mA M −1 cm −2 for a linear range between 5 μM and 13 mM.

Amperometric Biosensors Based on Biotinylated Single-Walled Carbon Nanotubes

One challenging goal for the development of biosensors is the conception of three dimensional biostructures on electrode surfaces. In this context, single-walled carbon nanotube coatings (SWCNTs), functionalized by biotin groups, were investigated to develop 3D conductive nanostructures allowing a post-functionalization by biological macromolecules. This specific anchoring of biomolecules was carried via the affinity interactions using the avidin-biotin system. For this purpose, a biotinylated pyrene was specially synthesized to develop a non-covalent functionalization based on pi-interactions between pyrene and the nanotube sidewall. SWCNT coatings were also biotinylated via electropolymerization of biotin-pyrrole derivatives at 0.95 V in CH3CN electrolyte. The resulting biotinylated SWCNTs were modified by an avidin protein via affinity interactions and characterized with scanning electron microscopy. The biofunctionalization by a biotinylated glucose oxidase (GOX) was performed by successive incubation in avidin and GOX aqueous solutions via avidin bridges. The efficiency of the enzyme anchoring was examined through the electro-enzymatic activity of the modified electrodes towards the detection of glucose at 0.7 V versus SCE. The glucose sensitivity and maximum current density were 1.6 mAM(-1) cm(-2) and 131 microAcm(-2) respectively for pyrene biotin-SWCNT electrode and 2.5 mAM(-1) cm(-2) and 178 microAcm(-2) respectively for the poly(pyrrole biotin)-SWCNT.

Electrochemical Characterization of Biotin Functionalized and Regular Single-Walled Carbon Nanotube Coatings. Application to Amperometric Glucose Biosensors

Single-walled carbon nanotubes (SWCNTs) are considered nowadays as promising alternatives for sensor and biosensor development. In this study, the analytical capabilities of this material as sensor material are reported. Biotin functionalized and pristine SWCNTs were deposited onto electrodes out of dispersions with different concentrations. The amount of deposited SWCNTs can be controlled by the determination of capacitive charges of the layers. The electrochemical behaviour of SWCNTs coatings was investigated by cyclic voltammetry with potassium hexacyanoferrate as redox probe. In addition, their electro-catalytic effect towards the electrochemical H2O2 oxidation was evaluated. Moreover, the strong interactions between pyrene and SWCNTs have been exploited for the non-covalent attachment of a pyrene-biotin derivative on the pristine nanotube surface. The latter allowed the immobilization of biotinylated glucose oxidase via an avidin bridge by affinity interactions. The amperometric response of the biosensor to glucose was recorded at 0.7 V versus a saturated calomel electrode. The glucose sensitivity and maximum current density were 560 AM −1 cm −2 and 14 A/cm 2 , respectively.

New approach of constructing biosensing matrices by physical and chemical crosslinking of biotin-alginate with alginate-pyrrole

A new matrix was designed and constructed; enabling the physical and chemical co-crosslinking of alginate-biotin (Alg-B) with alginate-pyrrole (Alg-py), namely biotinylated-glucose oxidase (B-GOx) was conjugated to Alg-B through avidin (Av) bridges, thereafter mixed with Alg-Py. The alginate was physically crosslinked via calcium ions followed by pyrrole covalent crosslinking through electro-generation of polypyrrole networks. The pyrrole electropolymerization on the electrode surface created spatial wires which tightened the polymeric coating to the electrode surface, packed the 3D structure and reinforced its structure by the covalent bonds. These matrices exhibited better stability than those without Alg-Py even under vigorous stirring applied during the amperometric measurements. When the concentration of the glucose analyte was determined by hydrogen peroxide oxidation, the current was conducted faster to the electrode surface, therefore providing a low response time and higher sensitivity value; using a relatively low enzyme load.

Amperometric biosensor based on the electro-copolymerization of a conductive biotinylated-pyrrole and alginate-pyrrole

A new matrix was designed and produced, which entailed the co-electropolymerization of pyrrole-biotin (Py-B) with pyrrole-alginate (Alg-Py). The aim was to demonstrate that improved performance could be obtained in a classical model system such as monitoring for the presence of glucose. The sensor construction consisted of the conjugation of biotinylated-glucose oxidase (B-GOx) to B-Py through avidin (Av) bridges, followed by copolymerization with Alg-Py. When the aforementioned set-up did not include the pyrrole-modified alginate but unmodified alginate, its performance values were significantly less. During measurement of the concentration of the model glucose analyte via hydrogen peroxide oxidation, the current was observed to be conducted faster to the electrode surface, therefore providing a lower response time, and a higher sensitivity value, using a relatively low enzyme load.

Non-covalent biofunctionalization of single-walled carbon nanotubes via biotin attachment by π-stacking interactions and pyrrole polymerization

Single-walled carbon nanotubes were functionalized with biotin using either electropolymerization or formation of pi-stacking interactions for the construction of biosensors. Thanks to the high affinity of the avidin-biotin interactions, a biotinylated glucose oxidase (B-GOX) as a biomolecule model was immobilized on the biotinylated nanotubes. The influence of the biosensor configuration on their amperometric performances was investigated by changing the amount of nanotubes and the numbers of avidin/B-GOX layers. By increasing the amount of nanotube and avidin/B-GOX layers, both sensor setups show a perfect linear increase of immobilized enzymes reflecting a high reproducibility of our systems. The highest sensitivities (up to 5.2 mA M(-1) cm(-2)) and maximum current densities (up to 55 microA cm(-2)) were obtained using nanotube deposits modified by electrochemical coatings. In contrast, non-covalently functionalized biotin-nanotubes show a better permeability for the enzymatically generated hydrogen peroxide.

Glucose Oxidase-immobilized Glass Disks for Imaging of d-Glucose in Acute Brain Slices

A biotinylated glucose oxidase (bGOD)-immobilized glass disk was prepared for visualizing D-glucose fluxes in acute brain slices. A mouse hippocampal slice was placed on the bGOD disk and stimulated with a stimulant solution containing horseradish peroxidase (HRP) and a substrate DA-64, followed by capturing digital images of Bindschedler's Green (BG), an oxidized form of DA-64, with a CCD camera. The bGOD membranes responded proportionally to D-glucose, ranging from 2.0 to 5.0 mM. Sucrose, GABA, L-glutamic acid, L-aspartic acid, glycine, acetylcholine and L-ascorbic acid at 10 mM did not cause any responses. The D-glucose fluxes in mouse hippocampal slices stimulated by a hypoxia solution were neuronal region-dependent, i.e., dentate gyrus (DG), cornu ammonis 1 (CA1) and cornu ammonis 3 (CA3), while those stimulated by KCl was independent of the neuronal regions. The response of bGOD disks is discussed in terms of the principle, concentration dependence and selectivity.

A polypyrrole cDNA electrode for the amperometric detection of the West Nile Virus

The determination of a short cDNA sequence from West Nile Virus (WNV) was carried out with an amperometric DNA sensor. The latter was constituted by an amino-21-mer oligonucleotide probe covalently anchored to a poly(pyrrole-NHS), previously electro-polymerized on a platinum electrode. After incubation with a target model of the WNV cDNA, the hybridization events on the sensor surface were detected by an additional hybridization process with a complementary biotinylated 15-mer WNV cDNA followed by the specific attachment of a biotinylated glucose oxidase via an avidin bridge. The hybridization event was then monitored at 0.6 V vs Ag/AgCl by amperometric detection of H 2O 2, generated by the enzyme marker in the presence of glucose. A relatively short (2 h) hybridization period allows the convenient quantification of the WNV DNA target in the range 10 −10–10 −15 g ml −1.

A simple strategy based on photobiotin irradiation for the photoelectrochemical immobilization of proteins on electrode surfaces

A photoactivable organic polymer was prepared first by electrogeneration of a conductive biotinylated polypyrrole film in acetonitrile electrolyte. The successive anchoring of avidin and photobiotin led to a multilayer configuration. The latter was illuminated with light (wavelength 370–400 nm) in the presence of proteins adsorbed onto its surface. The irradiation allowed the covalent linking of the proteins to the modified electrode. As a result of the photochemical reaction, a monolayer of enzyme (glucose oxidase, GOX or alkaline phosphatase, AP) was covalently bound to the photobiotin-modified surface with retention of their catalytic activities. The surfacic activities were 34 and 1.69 mU cm − 2 for GOX and AP photobiotin electrodes, respectively. These enzyme electrodes were compared to similar configurations obtained through the immobilization of biotinylated glucose oxidase or avidin-conjugated alkaline phosphatase on biotinylated polypyrrole film. Our results suggest that both procedures led to the immobilization of the same enzyme amount, namely a protein monolayer. This novel photo-immobilization methodology was also successfully applied to the anchoring of an anti-cholera toxin antibody which was then detected by a secondary antibody labelled with a peroxidase.

Redox labelled avidin for enzyme sensor architectures

Conjugates of avidin with ferrocene and with microperoxidase 8 have been used as electrochemically active molecular building blocks. Assemblies of the conjugates with biotinylated glucose oxidase or lactate oxidase on gold electrodes were tested as enzyme sensors for glucose and lactate. The electrochemical detection is based either on ferrocene-mediated oxidation of the substrate in oxygen-free solution, or on microperoxidase-catalysed reduction of H 2O 2 which is enzymatically produced from the substrate and molecular oxygen. Glucose and lactate were detectable with both detection principles in concentrations down to 1 or 0.1 mM, respectively. The molecular architecture concept allows quick adaptation of the sensors to other analytes, and it provides a platform for arrays of sensors with different selectivity.

Comparison by QCM and photometric enzymatic test of the biotin-avidin recognition on a biotinylated polypyrrole

By gravimetric measurements using a quartz cristal microbalance (QCM), we have studied the immobilization of biotinylated glucose oxidase enzymes (B-GOx) bound through on an intermediate avidin layer to a biotinylated polypyrrole film. The aim is to assess the amount of B-GOx specifically anchored on the biotinylated polypyrrole/avidin assembly thank to the biotin/avidin interaction between avidin and B-GOx. Indeed the estimated amount from the QCM measurement corresponds to the specific recognition of avidin/B-GOx added to a non-specific recognition (adsorption) of B-GOx. In order to discriminate these two phenomena, we have carried out a study by QCM of the anchoring of B-GOx on an avidin layer linked by adsorption to a polypyrrole free from biotin units. From QCM measurements we have deduced for the biotinylated polypyrrole/avidin assembly that the amount of B-GOx bound via the biotin/avidin interaction and those due to the avidin adsorption process correspond to 3.9 pmol cm −2 (1.3 equivalent of B-Gox monolayer) and 1.4 pmol cm −2 (0.46 equivalent of B-GOx monolayer) respectively. These values have been corroborated by measurements of the enzymatic activity of GOx.

Reversible Association/Dissociation Reaction of Avidin on the Dendrimer Monolayer Functionalized with a Biotin Analogue for a Regenerable Affinity-Sensing Surface

A new approach to a repeatedly regenerable affinity-sensing surface was developed based on the reversible association/dissociation reactions between avidin and biotin analogues. For the affinity surface, a fourth generation poly(amidoamine) dendrimer monolayer was first constructed on the 11-mercaptoundecanoic acid self-assembled monolayer on gold. The dendritic surface amine groups then were functionalized with biotin analogues, desthiobiotin (1), or newly synthesized desthiobiotin amidocaproate (2), an extended form of 1, which shows lower affinity toward avidin. To test the association/dissociation reaction cycles at the affinity surface, avidin adlayer was formed onto the biotin analogue functionalized surface and displaced with free biotin. To trace the stepwise reactions, biotinylated glucose oxidase (b-GOx) as a model enzyme was loaded onto the affinity surface, and cyclic voltammetric measurements were performed by registering the activity of the associated b-GOx. The efficient association/dissociation reaction cycles were registered, especially for the 2-modified electrodes, implying steric hindrance from the ligand length for biospecific interaction. With the optimized affinity-surface construction steps and reaction conditions, continuous association/dissociation reaction cycles were achieved, resulting in a regenerable affinity surface.

Specific cell targeting for delivery of toxins into small-cell lung cancer using a streptavidin fusion protein complex.

New modalities of treatment for small-cell lung cancer (SCLC) are needed, because the majority of patients continue to die of disseminated disease despite an initial response to conventional chemotherapy. Abnormal surface expression of the neural-cell adhesion molecule (NCAM) has been noted to be highly associated with SCLC. We examined the ability and efficiency of a streptavidin-Protein A (ST-PA) fusion protein complexed with an anti-NCAM monoclonal antibody (Mab) to transfer biotinylated beta-galactosidase into human SCLC cell lines NCI-H69, NCI-H526, and NCI-H446. When the surface molecule NCAM was targeted with this system, more than 99% of the targeted cells internalized and exhibited beta-galactosidase activity. In addition, we evaluated cytotoxic activity against SCLC lines NCI-H69 and NCI-H526 by efficient delivery of biotinylated glucose oxidase using the same ST-PA/anti-NCAM Mab complex. Cytotoxicity of the transduced cells (SCLC) was 10-fold and 100-fold greater, respectively, than the glucose oxidase control. This system could be widely applied for specific therapy of cancer cells by targeting unique surface molecules (antigens) using the corresponding Mab/ST-PA complex to transfer a variety of effector molecules; e.g., immunotoxic compounds, into target cells with a high degree of efficiency and specificity.

Affinity Biosensor for Avidin Using a Double Functionalized Dendrimer Monolayer on a Gold Electrode

We have developed an affinity biosensor system based on avidin–biotin interaction on a gold electrode. As the building block of an affinity-sensing monolayer, a fourth-generation (G4) poly(amidoamine) dendrimer having partial ferrocenyl-tethered surface groups was prepared and used. The unmodified surface amine groups from dendrimers were functionalized with biotinamidocaproate, and the biotinylated and electroactive dendritic monolayer was constructed on a gold electrode for the affinity-sensing surface interacting with avidin. An electrochemical signal from the affinity biosensor was generated by free glucose oxidase in electrolyte, depending on the degree of coverage of the sensing surface with avidin. The sensor signal decreased correlatively with increasing avidin concentration and approached a minimum level when the sensing surface was fully covered with avidin. The detection limit of avidin was about 4.5 pM, and the sensor signal was linear ranging from 1.5 pM to 10 nM under optimized conditions. From the kinetic analysis using the biotinylated glucose oxidase, an active enzyme coverage of 2.5 × 10−12 mol/cm2 on the avidin-pretreated surface was registered, which demonstrates the formation of a spatially ordered and compact protein layer on the derivatized electrode surface.

Layer-by-layer electrostatic deposition of biomolecules on surfaces for molecular recognition, redox mediation and signal generation.

Layer-by-layer supramolecular structures composed of alternate layers of negatively charged enzymes and cationic redox polyelectrolyte have been assembled. Glucose oxidase (GOx), lactate oxidase (LOx) and soybean peroxidase (SBP) have been electrically wired to the underlying electrode by means of poly(allylamine) with [Os(bpy)2ClPyCOH]+ covalently attached (PAA-Os) in organized structures with high spatial resolution. Biotinylated glucose oxidase has also been used to assemble step-by-step on antibiotin goat immunoglobulin (IgG) layers and the enzyme was electrically wired by PAA-Os. These spatially organized multilayers with mono- and bienzymatic schemes can work efficiently in molecular recognition, redox mediation and generation of an electrical signal. The concentration of redox mediator integrated into the multilayers, obtained from the voltammetric charge and an estimation of the layer thickness, exceeds by 100-fold the amount of deposited enzyme assessed by quartz crystal microbalance. Differences in GOx electrical wiring efficiency have been detected with the different assembling strategies. The surface concentration of electrically wired enzyme represents a small proportion of all the enzyme molecules present in the multilayers which can be oxidized by the soluble mediator [Os(bpy)2Cl PyCOOH]Cl. This proportion, as well as the rate of FADH2 oxidation by PAA-Os, increases with the number of electrically wired enzyme layers and with the spatial accessibility of the Os moiety to the enzyme active center.