层层自组装法制备普鲁士蓝修饰电极及对过氧化氢的测定

Fe3+可与电沉积在玻碳电极表面的对氨基苯磺酸发生静电吸附作用并与[Fe(CN)6]4-形成普鲁士蓝(PB),进一步交替重复吸附Fe3+和[Fe(CN)6]4-反应,形成PB晶体.该晶体对还原过氧化氢(H2O2)具有很高的电化学活性.通过循环伏安法、交流阻抗法和计时电流法对传感器进行了电化学表征.研究了该传感器对H2O2的电催化作用,探讨了工作电位,PH值及干扰物质对响应电流的影响.结果表明,在磷酸盐缓冲溶液中(pH =5.4,0.1 mol/L),响应电流与H2O2的浓度在0.97 ～ 32.33 mmol/L范围内具有良好的线性关系,相关系数为0.9993,传感器的响应时间小于5 s,检测限为0.48 mmol/L( S/N为3).     

Investigation of protein adsorption and electrochemical behavior at a gold electrode

The adsorption of two model proteins, human serum albumin and immunoglobulin G, on a gold electrode surface was investigated using 125I radiolabeling and cyclic voltammetry (CV). 125I radiolabeling was used to determine the extent of protein adsorption, while CV was used to ascertain the effect of the adsorbed protein layer on the electron transfer between the gold electrode and an electroactive moiety in solution, namely, K3Fe(CN)6. The adsorbed amounts of HSA and IgG agreed well with previous results and showed approximately monolayer coverage. The amount of adsorbed protein increased when a positive potential (700 mV) was applied to the electrode, while the application of a negative potential (-800 mV) resulted in a decrease. When the solution pH was varied to alter the charge on the protein, the adsorption trends appeared to follow electrostatic interaction, namely, greater adsorption when the electrode and the protein possessed opposite charge and vice versa. The adsorbed protein layer had the effect of blocking the electron transfer. It was possible to correlate the degree of electron blocking with the amount of adsorbed protein to show that the greater the adsorption, the larger the blocking effect. Of the two proteins used, HSA proved to be more efficient at blocking the electron transfer.     

Redox reactions of ferricyanide ions in layer-by-layer deposited polysaccharide films: a significant effect of the type of polycation in the films

Redox reactions of ferricyanide ions, [Fe(CN)6]3-, in polysaccharide thin films that were prepared by layer-by-layer (LbL) deposition on the surface of a gold electrode were studied electrochemically by cyclic voltammetry. LbL films composed of alginic acid (AGA) and carboxymethylcellulose (CMC) were successfully prepared using poly(ethyleneimine) (PEI) and poly(diallyldimethylammonium chloride) (PDDA) as the cationic counterparts in the electrostatic LbL deposition. The deposition behavior of the PEI-based films significantly depended on the pH of the solutions from which the LbL films were deposited, while the effects of pH were negligibly small for the PDDA-based films due to the pH-independent positive charges on the PDDA chains. The cyclic voltammograms (CVs) of [Fe(CN)6]3- ions on the LbL film-coated electrodes revealed that all the LbL films tested are permeable to [Fe(CN)6]3- ions and that the redox reactions of [Fe(CN)6]3- ions proceed smoothly in the LbL polysaccharide films. It was found that [Fe(CN)6]3- ions are concentrated in the films from the bulk solution, depending on the pH of the medium and on the type of polycations in the film. The PEI-based films concentrated [Fe(CN)6]3- ions more effectively in an acidic solution than in neutral and basic media, while the pH effect was not observed for the PDDA-based films. In addition, we found that the [Fe(CN)6]3- ions are confined in the LbL films due to a strong binding of the ions to the positively charged sites arising from the protonated amino groups in the films. The confined [Fe(CN)6]3- ions exhibited redox reactions in the films, with the redox potentials being shifted to the positive or negative direction in the PEI- or PDDA-based film, respectively, as compared to the redox potential of diffusing [Fe(CN)6]3- ions. Thus, significant effects of the type of polycation in the LbL films on the redox reactions of [Fe(CN)6]3- ions were observed.     

Reversible electrochemical switching of polyelectrolyte brush surface energy using electroactive counterions

Polyelectrolyte brushes with electroactive counterions provide an effective platform for surfaces with electrochemically switchable wetting properties. Polycationic poly(2-(methacryloyloxy)-ethyl-trimethyl-ammonium chloride) (PMETAC) brushes with ferricyanide ions ([Fe(CN)6] 3-) were used as the electrochemically addressable surface. After a negative potential of -0.5 V was applied to the [Fe(CN)6](3-)-coordinated PMETAC brushes, the [Fe(CN)6](3-) species were reduced to [Fe(CN)6](4-), and the surface became more hydrophilic. By application of alternating negative and positive potentials, PMETAC brushes were switched reversibly between the reduced state ([Fe(CN)6]4-) and oxidized state ([Fe(CN)6]3-), resulting in reversible changes in water contact angles. The time required for a complete contact angle change can be tuned from 1 to 20 s, by changing the brush thickness and the concentration of supporting electrolyte. We present an electrochemical brush transport model that includes the electrochemical reaction at the charged electrode and describes ion transport through the brush phase covering the electrode. The model quantitatively describes the response of the contact angle (hydrophilicity) to the applied voltage as a function of background ionic strength and brush thickness, supporting the proposed mechanism of ion transport through the brush and electrochemical reaction at the electrode. A typical diffusion constant for ferricyanide in a PMETAC brush of any thickness in 5 mM KCl supporting electrolyte was found to be 2 x 10(-15) m2 s(-1), 5 to 6 orders of magnitude smaller than its bulk solution value.     

Electrochemical characteristics of polyelectrolyte brushes with electroactive counterions

Cyclic voltammetry (CV) was employed to characterize the electrochemical behavior of polyelectrolyte brushes with immobilized electroactive counterions in response to external changes in concentration and composition of the supporting electrolyte and as a function of brush thickness. Poly(methacryloyloxy)ethyl-trimethyl-ammonium chloride (PMETAC) brushes were synthesized on Au substrates via atom transfer radical polymerization followed by ion-exchange with ferricyanide ions ([Fe(CN)6]3-) as redox probes. CV measurements of the modified PMETAC brushes showed the typical electrochemical response corresponding to a surface-confined electroactive species and the redox counterions, as [Fe(CN)6]3- species form stable ion pairs with the quaternary ammonium groups of the brush. The electron-transfer features of PMETAC brushes with different thicknesses, as characterized by CV and UV-vis spectroscopy, revealed that the charge density probed by CV was lower than the charge density measured by UV-vis spectroscopy. The electrode current decreased significantly with increasing concentration of supporting electrolyte due to the effect of the Donnan potential. Hydrophobic counterions, ClO4-, which induced brush collapse, lead to significantly reduced electrode currents.     

Determination of the hydrolysis rate constants and activation energy of aesculin with capillary electrophoresis end-column amperometric detection

Indirect chemiluminescence (ICL) detection for capillary electrophoresis (CE) of monoamines and catechol using luminol-K3 [Fe(CN)6] system was described. A strong and stable background chemiluminescence (CL) signal can be generated by luminol-K3 [Fe(CN)6] reaction. Based on the principle of that some phenolic compounds may be oxidized in the presence of K3 [Fe(CN)6], quenching effect of catecholamines for luminol-K3[Fe(CN)6] CL reaction results in a quantifiable decrease in the background signal. The conditions for CE separation and the CL detection for four standard catecholamines were systematically investigated using a homemade CE-ICL system. Under the optimum conditions, the detection limits of dopamine (DA), epinephrine (EP), norepinephrine (NE) and catechol (CA) were determined to be 0.18 mciroM 0.39 microM 0.48 microM and 0.09 microM, respectively. It also has been successfully applied to analyze seven pharmaceutical samples and seven human urine samples.     

Electroactive core-shell nanocluster films of heme proteins, polyelectrolytes, and silica nanoparticles

Novel protein core-shell nanocluster films were assembled layer by layer on solid surfaces. In the first step, positively charged heme protein hemoglobin (Hb) or myoglobin (Mb) and negatively charged poly(styrenesulfonate) (PSS) were alternately adsorbed on the surface of SiO2 nanoparticles, forming core-shell SiO2-(protein/PSS)m nanoclusters. In the second step, the SiO2-(protein/PSS)m nanoclusters and polycationic poly(ethylenimine) (PEI) were assembled layer by layer on various solid substrates, forming [[SiO2-(protein/PSS)m]/PEI]n films. Various techniques were used to characterize the nanoclusters and monitor the film growth. [[SiO2-(protein/PSS)m]/PEI]n films at pyrolytic graphite (PG) electrodes exhibited well-defined, chemically reversible cyclic voltammetric reduction-oxidation peaks characteristic of the heme Fe(III)/Fe(II) redox couples. The proteins in the films retained near native conformations in the medium pH range, and the films catalyzed electrochemical reduction of oxygen and hydrogen peroxide. Advantages of the nanocluster films over the simple [SiO2/protein]n layer-by-layer films include a larger fraction of electroactive protein and higher specific biocatalytic activity. Using this approach, biocatalytic activity can be tailored and controlled by varying the number of bilayers deposited on the nanoparticle cores and the number of nanocluster layers on electrodes.     

Comparative bioelectrochemical study of core-shell nanocluster films with ordinary layer-by-layer films containing heme proteins and CaCO3 nanoparticles

Negatively charged heme protein hemoglobin (Hb) or myoglobin (Mb) at pH 9.0 and positively charged poly(diallyldimethylammonium) (PDDA) were alternately adsorbed on the surface of CaCO(3) nanoparticles, forming core-shell CaCO(3)-[PDDA/(protein/PDDA)(m)] ([protein-m]) nanoclusters. Oppositely charged [protein-m] and poly(styrenesulfonate) (PSS) were then assembled layer by layer on various solid substrates, forming {[protein-m]/PSS}(n) films. In the meantime, ordinary layer-by-layer films of heme proteins with CaCO(3) nanoparticles ({protein/CaCO(3)}(n)) were also grown on solid surfaces. Transmission electron microscopy (TEM), ultraviolet-visible (UV-vis) spectroscopy, quartz crystal microbalance (QCM), and cyclic voltammetry (CV) were used to characterize the nanoclusters and monitor the growth of the two types of films. Both kinds of protein films assembled on pyrolytic graphite (PG) electrodes exhibited well-defined, nearly reversible CV reduction-oxidation peaks, characteristic of heme Fe(III)/Fe(II) redox couples, and were used to catalyze the electrochemical reduction of hydrogen peroxide. The {[protein-m]/PSS}(n) films demonstrate distinct advantages over the {protein/CaCO(3)}(n) films due to their larger fraction of electroactive proteins, higher catalytic efficiency, and better thermostability. The penetration experiments of the electroactive probe into these films indicate that the {[protein-m]/PSS}(n) nanocluster films possess more pores or channels than the simple {protein/CaCO(3)}(n) films, which may be beneficial to counterion transport in the charge-hopping mechanism and helpful for the diffusion of catalysis substrates into the films. In addition, the electrochemical and biocatalytic activity of protein nanocluster films can be tailored by controlling the number of bilayers assembled on the nanoparticle cores (m) as well as the film thickness or the number of nanocluster layers on the electrodes (n).     

Redox mediation at 11-mercaptoundecanoic acid self-assembled monolayers on gold

Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and digital simulation techniques were used to investigate quantitatively the mechanism of electron transfer (ET) through densely packed and well-ordered self-assembled monolayers (SAMs) of 11-mercaptoundecanoic acid on gold, either pristine or modified by physically adsorbed glucose oxidase (GOx). In the presence of ferrocenylmethanol (FcMeOH) as a redox mediator, ET kinetics involving either solution-phase hydrophilic redox probes such as [Fe(CN)6]3-/4- or surface-immobilized GOx is greatly accelerated: [Fe(CN)6]3-/4- undergoes diffusion-controlled ET, while the enzymatic electrochemical conversion of glucose to gluconolactone is efficiently sustained by FcMeOH. Analysis of the results, also including the digital simulation of CV and EIS data, showed the prevalence of an ET mechanism according to the so-called membrane model that comprises the permeation of the redox mediator within the SAM and the intermolecular ET to the redox probe located outside the monolayer. The analysis of the catalytic current generated at the GOx/SAM electrode in the presence of glucose and FcMeOH allowed the high surface protein coverage suggested by X-ray photoelectron spectroscopy (XPS) measurements to be confirmed.     

气相色谱法研究配位化合物的热稳定性XVIII: 配位CN离子的活化加氢

根据一系列金属(Cr、Mn、Fe、Co、Ni、Cu、Zn、Ag、Sn、La)的六氰合铁(II)、(III)酸盐在H2气分中热分解时所发生的CN^-加氢反应,从晶体结构和电子结构两方面探讨了双金属双端配位对CN^-的活化作用和CN^-的加氢反应机理.研究了CN^-的活化程度与配位金属离子的还原电位及d电子组态之间的关系.     

Redox properties of the ferricyanide ion on electrodes coated with layer-by-layer thin films composed of polysaccharide and poly(allylamine)

Polyelectrolyte multilayer thin films were prepared by an alternate deposition of poly(allylamine hydrochloride) (PAH) and anionic polysaccharides {carboxymethylcellulose (CMC) and alginic acid (AGA)} on the surface of a gold (Au) disk electrode, and the binding of ferricyanide [Fe(CN)(6)](3)(-) and hexaammine ruthenium ions [Ru(NH(3))(6)](3+) to the films was evaluated. Poly(acrylic acid) (PAA) was also employed as a reference polyanion bearing carboxylate side chains. A quartz-crystal microbalance study showed that PAH-CMC and PAH-AGA multilayer films grow exponentially as the number of depositions increases. The thicknesses of five bilayers of (PAH-CMC)(5) and (PAH-AGA)(5) films were estimated to be 150 +/- 20 and 90 +/- 15 nm, respectively, in the dry state. The PAH/polysaccharide multilayer film-coated Au electrodes exhibited a redox response to the [Fe(CN)(6)](3)(-) ion dissolved in solution, irrespective of the sign of the surface charge of the film, suggesting the high permeability of the films to the [Fe(CN)(6)](3)(-) ion. In contrast, the PAH-PAA film-coated Au electrodes exhibited a redox response only when the outermost surface of the film was covered with a positively charged PAH layer. However, the permeation of the [Ru(NH(3))(6)](3+) cation was severely suppressed for all of the multilayer films. It was possible to confine the [Fe(CN)(6)](3)(-) ion in the films by immersing the film-coated electrodes in a 1 mM [Fe(CN)(6)](3)(-) solution for 15 min. Thus, the [Fe(CN)(6)](3)(-)-confined electrodes exhibited a cyclic voltammetric response in the [Fe(CN)(6)](3)(-) ion-free buffer solution. The loading of the [Fe(CN)(6)](3)(-) ion in the films was higher when the surface charge of the film was positive and increased with increasing film thickness. It was also found that the [Fe(CN)(6)](3)(-) ion confined in the films serves as an electrocatalyst that oxidizes ascorbic acid in solution.     

Layer-by-layer films of hemoglobin or myoglobin assembled with zeolite particles: electrochemistry and electrocatalysis

Positively charged hemoglobin (Hb) or myoglobin (Mb) at pH 5.0 in solutions and negatively charged zeolite particles in dispersions were alternately adsorbed onto solid surfaces forming [zeolite/protein](n) layer-by-layer films, which was confirmed by quartz crystal microbalance (QCM) and cyclic voltammetry (CV). The protein films assembled on pyrolytic graphite (PG) electrodes exhibited a pair of well-defined, nearly reversible CV peaks at about -0.35 V vs. SCE at pH 7.0, characteristic of the heme Fe(III)/Fe(II) redox couples. Hydrogen peroxide (H(2)O(2)) and nitrite (NO(2)(-)) in solution were catalytically reduced at [zeolite/protein](7) film modified electrodes, and could be quantitatively determined by CV and amperometry. The shape and position of infrared amide I and II bands of Hb or Mb in [zeolite/protein](7) films suggest that the proteins retain their near-native structure in the films. The penetration experiments of Fe(CN)(6)(3-) as the electroactive probe into these films and scanning electron microscopy (SEM) results indicate that the films possess a great amount of pores or channels. The porous structure of ]zeolite/protein](n) films is beneficial to counterion transport, which is crucial for protein electrochemistry in films controlled by the charge-hopping mechanism, and is also helpful for the diffusion of catalysis substrates into the films. The proteins with negatively charged net surface charges at pH 9.0 were also successfully assembled with like-charged zeolite particles into layer-by-layer films, although the adsorption amount was less than that assembled at pH 5.0. The possible reasons for this were discussed, and the driving forces were explored.     

Electrochemical Behavior of K3[Fe(CN)6] on the Electrode Coated with Modified Multi‐Walled Carbon Nanotubes

The electrochemical behavior of K₃[Fe(CN)₆] was studied on an ITO electrode that was coated with β‐cyclodextrin (CD) modified multi‐walled carbon nanotubes (MWNTs) and with carboxyl modified multi‐walled carbon nanotubes (MWNT‐COOHs). MWNT‐COOHs showed an excellent electrocatalytic effect on the redox of K₃[Fe(CN)₆] while MWNT‐CDs had a subdued effect on the electrochemical response of K₃[Fe(CN)₆]. It is probably due to mismatching between K₃[Fe(CN)₆] and cyclodextrin, which hampers the contact of K₃[Fe(CN)₆] with carbon nanotubes. Moreover, the electrochemical behavior of K₃[Fe(CN)₆] on the MWNT‐COOHs coated ITO electrode at various scan rates also was measured. The results indicated that both potential difference between redox peaks and peak current of K₃[Fe(CN)₆] increased with increasing scan rate. A good linearity of peak current versus scan rate was observed.     

一种铂网栅薄层光谱电化学池的制作及表征

以铂网栅电极作工作电极,设计制作了一种应用于紫外-可见光谱电化学研究的新型光透薄层电化学池（OTTLE）,并以铁氰化钾-亚铁氰化钾体系对此光谱化学池进行了表征。铁氰化钾-亚铁氰化钾体系在该池中有良好的循环伏安特性,并可获得良好的紫外-可见光谱。测得的克式量电位E^0,电子转移数n与文献结果一致。     

Magnetism tuned by intercalation of various metal ions in coordination polymer

Insertion of various metal ions have changed the ferromagnetic coordination frameworks into paramagnetic, superparamagnetic, or ferromagnetic. The net charge and radius of the ions determined the magnetism of the host frameworks.     

On the Criteria of Instability for Electrochemical Systems

Both cyclic-voltammetry-based and impedance-based experimental criteria that have been developed recently for the oscillatory electrochemical systems are critically appraised with two typical categories of oscillators.Consistent conclusions can be drawn by the two criteria for the category of oscillators that involve the coupling of charge transfer mainly with surface steps(e.g.ad-and desorption)such as in the electrooxidation of C1 organic molecules.Whereas,impedance-based criterion is not applicable to the category of oscillators that involve the coupling of charge transfer mainly with mass transfer(e.g.diffusion and convection) such as in the Fe(CN)6^3- reduction accompanying periodic hydrogen evolution.The reason is that the negative impedance cannot include the feedback information of convection mass transfer induced by the hydrogen evolution.However,both positive and negative nonlinear feedbacks,i.e., the diffusion-limited depletion and convection-enhanced replenishment of the Fe(CN)6^3- surface concentration,that coexist between the bistability,i.e.,Fe(CN)6^3- reduction with and without hydrogen evolution at lower and higher potential sides respectively,are all reflected in the crossed cyclic voltammogram(CCV).It can be concluded that the voltammetry-based criterion(in time domain)is more intuitive,less time-consuming and has a wider range of applications than the impedancebased one (in frequency domain).     

Electrochemical characterization of polyelectrolyte/gold nanoparticle multilayers self-assembled on gold electrodes

Polyelectrolyte/gold nanoparticle multilayers composed of poly(l-lysine) (pLys) and mercaptosuccinic acid (MSA) stabilized gold nanoparticles (Au NPs) were built up using the electrostatic layer-by-layer self-assembly technique upon a gold electrode modified with a first layer of MSA. The assemblies were characterized using UV-vis absorption spectroscopy, cyclic and square-wave voltammetry, electrochemical impedance spectroscopy, and atomic force microscopy. Charge transport through the multilayer was studied experimentally as well as theoretically by using two different redox pairs [Fe(CN)(6)](3-/4-) and [Ru(NH(3))(6)](3+/2+). This paper reports a large sensitivity to the charge of the outermost layer for the permeability of these assemblies to the probe ions. With the former redox pair, dramatic changes in the impedance response were obtained for thin multilayers each time a new layer was deposited. In the latter case, the multilayer behaves as a conductor exhibiting a strikingly lower impedance response, the electric current being enhanced as more layers are added for Au NP terminated multilayers. These results are interpreted quite satisfactorily by means of a capillary membrane model that encompasses the wide variety of behaviors observed. It is concluded that nonlinear slow diffusion through defects (pinholes) in the multilayer is the governing mechanism for the [Fe(CN)(6)](3-/4-) species, whereas electron transfer through the Au NPs is the dominant mechanism in the case of the [Ru(NH(3))(6)](3+/2+) pair.     

Magnetic properties of cyano-bridged Ln3+-M3+ complexes. Part I: trinuclear complexes (Ln3+ = La, Ce, Pr, Nd, Sm; M3+ = FeLS, Co) with bpy as blocking ligand

The reaction of Ln(NO3)3(aq) with K3[Fe(CN)6] or K3[Co(CN)6] and 2,2'-bipyridine in water/ethanol led to eight trinuclear complexes: trans-[M(CN)4(mu-CN)2{Ln(H2O)4(bpy)2}2][M(CN)6].8H2O (M = Fe3+ or Co3+, Ln = La3+, Ce3+, Pr3+, Nd3+, and Sm3+). The structures for the eight complexes [La2Fe] (1), [Ce2Fe] (2), [Pr2Fe] (3), [Nd2Fe] (4), [Ce2Co] (5), [Pr2Co] (6), [Nd2Co] (7), and [Sm2Co] (8) have been solved; they crystallize in the triclinic space group P and are isomorphous. They exhibit a supramolecular 3D architecture through hydrogen bonding and pi-pi stacking interactions. A stereochemical study of the nine-vertex polyhedra of the lanthanide ions, based on continuous shape measures, is presented. No significant magnetic interaction was found between the lanthanide(III) and the iron(III) ions.     

Electrochemical Modification of Gold Electrodes with Azobenzene Derivatives by Diazonium Reduction

An electrochemical study of Au electrodes electrografted with azobenzene (AB), Fast Garnet GBC (GBC) and Fast Black K (FBK) diazonium compounds is presented. Electrochemical quartz crystal microbalance, ellipsometry and atomic force microscopy investigations reveal the formation of multilayer films. The elemental composition of the aryl layers is examined by X‐ray photoelectron spectroscopy. The electrochemical measurements reveal a quasi‐reversible voltammogram of the Fe(CN)₆^3?/4? redox couple on bare Au and a sigmoidal shape for the GBC‐ and FBK‐modified Au electrodes, thus demonstrating that electron transfer is blocked due to the surface modification. The electrografted AB layer results in strongest inhibition of the Fe(CN)₆^3?/4? response compared with other aryl layers. The same tendencies are observed for oxygen reduction; however, the blocking effect is not as strong as in the Fe(CN)₆^3?/4? redox system. The electrochemical impedance spectroscopy measurements allowed the calculation of low charge‐transfer rates to the Fe(CN)₆^3? probe for the GBC‐ and FBK‐modified Au electrodes in relation to bare Au. From these measurements it can be concluded that the FBK film is less compact or presents more pinholes than the electrografted GBC layer.     

Electron transport through a self-assembled monolayer of thiol-end-functionalized tetraphenylporphines and metal tetraphenylporphines

The monolayers of several thiol-end-functionalized tetraphenylphophines (SH-TPP) and metal tetraphenylporphines (SH-MTPP) were self-assembled on gold surfaces and identified by cyclic voltammetry (CV), electrochemical impedance spectroscopy, scanning electrochemical microscopy, and the contact angle. The CV peaks of the [Fe(CN)6]3-/ [Fe(CN)6]4- couple were used to identify the efficiency of electrons transferring through the self-assembled monolayer (SAM). The results suggested that SH-TPP and SH-MTPP could form high-quality SAMs on gold surfaces. The SAMs blocked electron transport from the gold electrode to solution. When the length of the thiol-end-link spacer (alkyl group) increased, the electron transport ability of the SAM decreased because of the increased insulator properties of the alkyl chain. With the insertion of metallic ions, the electron transport ability of the SAM of SH-MTPP increased compared to that of the SAM of SH-TPP, which was probably due to the fact that (i) the insertion of metallic ions changed the molecular structure and the molecular structure of SH-MTPP played an important role in electron transport through the SAM and (ii) the insertion of metallic ions increased the electron tunneling probability through the monolayer.