Electrochemical behavior of dopamine and ascorbic acid at osmium(II) complex cationic monolayer modified gold electrodes.

The electrooxidation of dopamine (DA) and ascorbic acid (AA) was studied using gold electrodes modified by a cationic self-assembled monolayer of [Os(bpy)2(bpy-(CH2),3SH)]2+ by cyclic voltammetry. At an [Os(bpy)2(bpy-(CH2)13SH)]2+/Au electrode, the oxidation peak of DA shifted to a much more positive potential as compared with that of a bare gold electrode, while the oxidation peak potential of AA showed a slightly negative shift due to their different electrostatic interactions with the cationic monolayer. Thus, a sufficient potential difference was achieved for distinguishing the electrochemical responses of DA and AA. However, when CH3(CH2)11SH was mixed into the cationic monolayer, the enhanced packing of the mixed monolayer blocked the access of DA or AA to the electrode, resulting in further positive shifts for both oxidation-peak potentials.     

Facilitated electrochemical oxidation of NADH and its model compound at gold electrode modified with terminally substituted electroinactive self-assembled monolayers

The electrochemical oxidation of NADH and its model compound, N-benzyl-1,4-dihydronicotinamide (DHN), has been studied at gold electrode modified with self-assembled monolayer of terminally substituted thiols/disulfide, i.e., cystamine (CYST), mercaptopropionic acid (MPA) and mercaptoethanol (ME). A substantial decrease in the overpotential (approximately 250 mV) when compared to the bare electrode has been observed for the oxidation of NADH at the monolayer-modified electrodes, containing no so-called redox mediator. The bare electrode shows an ill-defined voltammetric peak for the oxidation of DHN, whereas the monolayer-modified electrodes showed a well-defined voltammetric peak. The monolayer assembly on the gold electrode prevents the fouling of electrode surface by the oxidation products, which favors the oxidation at the less positive potential. The square-wave voltammograms showed a sharp voltammetric signal for the oxidation of NADH at all the monolayer-modified electrodes. All the monolayer-modified electrodes showed a linear current response to change in the NADH concentration in its range of 25-300 microM and their sensitivities were found to be 0.005+/-0.0003, 0.0063+/-0.0002 and 0.0052+/-0.0003 microA/microM for CYST-Au, ME-Au and MPA-Au electrodes, respectively. The hydrodynamic voltammograms obtained at the rotating CYST-Au electrode for the oxidation of NADH and DHN were used to estimate the diffusion coefficient of DHN, and the number of electrons involved in the oxidation process of NADH.     

Docosyltrimethylammonium chloride modified glassy carbon electrode for simultaneous determination of dopamine and ascorbic acid

A glassy carbon electrode (GCE) modified with docosyltrimethylammonium chloride (DCTMACl) is used for simultaneous determination of dopamine (DA) and ascorbic acid (AA) using differential pulse voltammetry (DPV) technique in 0.10 mol·L^?1 phosphate buffer solution of pH 5.0. The cationic surfactant DCTMACl modified film has a positive charge. DA exists as the positively charged species, whereas AA is the negatively charged one in the solution. Thus, at DCTMACl film-modified GCE, the oxidation peak potential of AA shifts toward less negative potential and the peak current of AA increases a little, while the oxidation peak potential of DA shifts toward more positive potential and peak current decreases greatly in comparison with that on bare electrode. The two anodic peaks are separated around 200 mV. Under optimal conditions, the catalytic peak currents obtained from DPV increase linearly with concentrations of DA and AA in the ranges of 1.0?×?10^?5 to 1.0?×?10^?3?mol·L^?1. This electrode has good reproducibility, high stability in its voltammetric response, and low detection limit (micromolar) for both AA and DA. The modified electrode has been applied to the determination of DA and AA in injection.     

Simultaneous electroanalysis of dopamine and ascorbic acid using poly (N,N-dimethylaniline)-modified electrodes

Glassy carbon (GC) electrode is modified with an electropolymerized film of N,N-dimethylaniline (DMA). This polymer (PDMA) film-coated GC electrode is used to electrochemically detect dopamine (DA) in the presence of ascorbic acid (AA). Polymer film has the positive charge in its backbone, and in neutral solution DA exists as the positively charged species whereas AA exists as the negatively charged one. In cyclic voltammetric measurements, favorable ionic interaction (i.e., electrostatic attraction) between AA and PDMA film causes a large negative shift of the oxidation potential for AA compared to that at the bare electrode. Oxidation potential for DA is positively shifted due to the electrostatic repulsion. The PDMA film shows hydrophobicity by incorporating uncharged hydroquinone molecule within the film. DA is also incorporated into the film due to hydrophobic attraction even though DA has a positive charge. The responses of DA and AA at polymer-modified electrodes largely change with the concentration of the monomer (i.e., 0.2, 0.1 and 0.05 M DMA) used in electropolymerization and thus with the film thickness. Hydrophobicity of the polymer film shows great influence on the voltammetric responses of both DA and AA. In square wave voltammetric measurements, the PDMA film-coated electrode can separate the DA and AA oxidation potentials by about 300 mV and can detect DA at its low concentration (e.g., 0.2 microM) in the presence of 1000 times higher concentration of AA, which is close to the physiological level. AA oxidizes at more negative potential than DA. The electrode response is not affected by the oxidized product of AA. So unlike the bare electrode, the fouling effect as well as the catalytic oxidation of AA by the oxidized form of DA are eliminated at the PDMA film-coated GC electrode. The electrode exhibits the stable and sensitive response to DA.     

Exploiting micellar environment for simultaneous electrochemical determination of ascorbic acid and dopamine

A simple and reliable method for simultaneous electrochemical determination of ascorbic acid (AA) and dopamine (DA) is presented in this work. It was based on the use of the cationic surfactant cetylpyridinium chloride (CPC) that enables the separation of the oxidation peaks potential of AA and DA. Cyclic voltammetry (CV) as well as pulse differential voltammetry (PDV) were used in order to verify the voltammetric behaviour in micellar media. In the cationic surfactant CPC, a remarkable electrostatic interaction is established with negatively charged AA, as a consequence, the oxidation peak potential shifted toward less positive potential and the peak current increased. On the other hand, the positively charged DA is repelled from the electrode surface and the oxidation peak potential shifts toward more positive potential in comparison to the bare electrode. Therefore, the common overlapped oxidation peaks of AA and DA can be circumventing by using CPC. Parameter that affects the E_pa and I_pa such as CPC concentration and pH were studied. Under optimised conditions, the method presented a linear response to AA and DA in the concentration range from 5 to 75 μmol L⁻¹ and 10 to 100 μmol L⁻¹, respectively. The proposed method was successfully applied to the simultaneous determination of AA and DA in dopamine hydrochloride injection (DHI) samples spiked with AA.     

Polystyrene sulphonate wrapped multiwalled carbon nanotubes modified graphite electrode for simultaneous determination of ascorbic acid, dopamine and uric acid

Graphite electrode is modified by casting multi-walled carbon nanotubes (MWCNTs) wrapped with polystyrene sulphonate (PSS) onto the surface of the bare graphite electrode. The modified electrode was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The behavior of the modified electrode towards the oxidation of ascorbic acid (AA), dopamine (DA) and uric acid (UA) has been determined by cyclic voltammetry (CV), differential pulse voltammetry (DPV) and chronoamperometry (CA). The modified electrode showed better electrocatalytic activity towards AA, DA and UA compared to bare graphite electrode. The electrochemical oxidation signals of AA, DA and UA are well separated into three distinct peaks with peak potential difference of 222, 128 and 350 mV between AA-DA, DA-UA and AA-UA respectively in CV studies and corresponding peak potential separation in DPV are 228, 120 and 348 mV. This modified electrode was successfully used for simultaneous determination of AA, DA and UA in ternary mixture.     

Electrochemical determination of dopamine in the presence of ascorbic acid and uric acid using the synergistic effect of gold nanoflowers and L-cysteamine monolayer at the surface of a gold electrode

This paper describes a facile and effective method to synthesize gold nanoflowers (AuNFs) by a controllable electrodeposition method induced by a L-cysteamine (L-Cys) monolayer self-assembled on the surface of a gold electrode. The AuNFs/L-Cys/Au electrodes were characterized by field emission scanning electron microscopy (FE-SEM), cyclic voltammetry, and AC impedance spectroscopy methods. This obtained AuNFs/L-Cys/Au electrode exhibits excellent electrocatalytic activity towards the oxidation of dopamine (DA) due to the synergistic effect of AuNFs and a L-Cys monolayer. Differential pulse voltammetry (DPV) experiment results show that the oxidation peak of DA is separated from the oxidation peaks of ascorbic acid (AA) and uric acid (UA), which can be used to detect DA in the presence of AA and UA, and the results are satisfactory.     

Simultaneous determination of dopamine and ascorbic acid on poly (3,4-ethylenedioxythiophene) modified glassy carbon electrode

Detection of dopamine (DA) in the presence of excess of ascorbic acid (AA) has been demonstrated using a conducting polymer matrix, poly (3,4-ethylenedioxythiophene) (PEDOT) film in neutral buffer (PBS 7.4) solution. The PEDOT film was deposited on a glassy carbon electrode by electropolymerization of EDOT from acetonitrile solution. Atomic force microscopy studies revealed that the electrodeposited film was found to be approximately 100 nm thick with a roughness factor of 2.6 nm. Voltammetric studies have shown catalytic oxidation of DA and AA on PEDOT modified electrode and can afford a peak potential separation of ∼0.2 V. It is speculated that the cationic PEDOT film interacts with the negatively charged ascorbate anion through favorable electrostatic interaction, which results in pre-concentration at a less anodic value. The positively charged DA tends to interact with the hydrophobic regions of PEDOT film through hydrophobic–hydrophobic interaction thus resulting in favorable adsorption on the polymer matrix. Further enhancement in sensitivity to micro molar level oxidation current for DA/AA oxidation was achieved by square wave voltammetry (SWV) which can detect DA at its low concentration of 1 μM in the presence of 1000 times higher concentration of AA (1 mM). Thus the PEDOT modified electrode exhibited a stable and sensitive response to DA in the presence of AA interference.     

Selective Detection of Dopamine and Its Metabolite,DOPAC, in the Presence of Ascorbic Acid Using Diamond Electrode Modified by the Polymer Film

The surface of boron‐doped diamond (BDD) electrode is modified by the polymer film for the first time. The cationic polymer film of N,N‐dimethylaniline (DMA) is electrochemically deposited on BDD electrode surface. This polymer (PDMA) film‐coated BDD electrode is used as a sensor which selectively detect dopamine (DA) in the presence of ascorbic acid (AA). This electrode also can detect both DA and its metabolite, 3,4‐dihydroxy phenyl acetic acid (DOPAC) in the presence of AA in the range of the physiological concentrations of these species. Favorable ionic interaction (i.e., electrostatic attraction) between the PDMA film and AA or DOPAC lowers their oxidation potentials and enhances the current response for AA and DOPAC compared to that at the bare electrode. The PDMA film also shows a hydrophobic interaction with DA and DOPAC. In cyclic voltammetric measurements, the PDMA film‐coated electrode can successfully separate the oxidation potentials for AA and DA coexisting in the same solution and the separation is about 200 mV. AA oxidizes at more negative potential than DA. In square‐wave voltammetry, the sensitivity of the PDMA film‐coated BDD electrode for DA in the presence of higher concentration of AA is higher than that of the PDMA film‐coated glassy carbon electrode. The hydrodynamic amperometric experiments confirm that the oxidation of AA is not affected by the oxidized product of DA and vice versa. So, unlike the bare electrode the catalytic oxidation of AA by the oxidized DA is eliminated at the PDMA film‐coated BDD electrode. The sensitivities of the modified electrode for AA, DA and DOPAC, which are present in the same solution with their physiological concentration ratios, are calculated to be 0.070, 0.363 and 0.084 μA μM^?1, respectively. The modified electrode exhibits a stable and sensitive response to DA.     

Detection of Uric Acid in the Presence of Dopamine and High Concentration of Ascorbic Acid Using PDDA Modified Graphite Electrode

The properties of graphite electrode (Gr) modified with poly(diallyl dimethyl ammonium chloride) (PDDA) for the detection of uric acid (UA) in the presence of dopamine (DA) and high concentration of ascorbic acid (AA) have been investigated by cyclic voltammetry, differential pulse voltammetry and chronoamperometry. The polymer modified graphite electrode was prepared by a very simple method just by immersing the graphite electrode in PDDA solution for 20 minutes. The PDDA/Gr modified electrode displayed excellent electrocatalytic activity towards the oxidation of UA, DA and AA compared to that at the bare graphite electrode. The electrochemical oxidation signals of UA, DA and AA are well resolved into three distinct peaks with peak potential separations of 220 mV, 168 mV and 387 mV between AA‐DA, DA‐UA and AA‐UA respectively in cyclic voltammetry studies and the corresponding peak potential separations are 230 mV, 130 mV and 354 mV respectively in differential pulse voltammetry. The lowest detection limits obtained for UA, DA and AA were 1×10^?7 M, 2×10^?7 M and 800×10^?9 M respectively. The PDDA/Gr electrode efficiently eliminated the interference of DA and a high concentration of AA in the determination of UA with good selectivity, sensitivity and reproducibility. The modified electrode was also successfully applied for simultaneous determination of UA, DA and AA in their ternary mixture.     

γ-Oryzanol-loaded calcium pectinate microparticles reinforced with chitosan: Optimization and release characteristics

The cationic surfactant, cetyltrimethyl ammonium bromide (CTAB) effect on accurate determination of dopamine in the presence of ascorbic acid (as natural obtrusive in determination of dopamine) using chemically modified electrode based on tin hexacyanoferrate (SnHCF) as a modifier at carbon paste electrode (CPE) is described. The electrochemical response of bare CPE and SnHCF modified electrode (ME) examined in different pHs, in absence and presence of different concentration of dopamine (DA) and ascorbic acid (AA). In addition a simple and reliable method for simultaneous electrochemical determination of AA and DA was presented. It was based on the use of the cationic surfactant CTAB that enables the separation of the oxidation peaks potential of AA and DA. The experimental results showed that DA and AA have two separate peaks. In pH 3 and at presence of 3 mM of CTAB the separation of DA and AA peaks and the eminence of them increased evidently. Good linear response to AA and DA was observed in the range of 0.4–50 and 0.2–25 mM with the correlation of 0.9912 and 0.9955, respectively.     

The Influence of Gold Nanoparticles on Simultaneous Determination of Uric Acid and Ascorbic Acid

A three-dimensional L-cysteine (L-cys) monolayer assembled on gold nanoparticles (GNP) providing simultaneous detection of uric acid (UA) and ascorbic acid (AA) was studied in this work. The cyclic voltammetry demonstrated that, at a bare glassy carbon electrode (GCE) or planar gold electrode, the mixture of UA and AA showed one overlapped oxidation peak; whereas when the electrode was modified with GNP, the oxidation peaks for UA and AA were separated. While a GNP modified electrode was further modified with L-cys monolayer (L-cys/GNP/GCE), namely, three-dimensional L-cys monolayer, a better separation for UA and AA response was obtained. Interestingly, the L-cys monolayer-modified planar gold electrode presented a block effect on the oxidation of AA, which was facilitated by the three-dimensional L-cys monolayer attributed to its distinct structure. The pH of solution presented a noticeable effect on the separation of UA and AA at GNP modified electrodes with or without L-cys monolayer. Wide concentration ranges from 2 × 10^?6?1 × 10^?3 M to UA and 2 × 10^?6?8 × 10^?4 M to AA could be obtained at L-cys/GNP/GCE.     

Electrocatalytic response of dopamine at a dl-homocysteine self-assembled gold electrode

The gold electrode self-assembled with the homocysteine monolayer (Hcy/Au) is demonstrated to catalyze the electrochemical response of dopamine (DA) by cyclic voltammetry. A pair of well-defined redox waves was obtained and the calculated standard rate constant (k_s) is 2.1×10⁻² cm/s at the self-assembled electrode. The reduction peak of DA can be used to determine the concentration of DA in presence of ascorbic acid (AA) owing to the Hcy/Au also catalyzing the electrochemical oxidation of AA.     

Electrochemical detection of ascorbic acid and serotonin at a boron-doped diamond electrode modified with poly(N,N-dimethylaniline)

The electrochemical oxidation of ascorbic acid (AA) and serotonin (5-HT) at a boron-doped diamond (BDD) electrode modified with poly(N,N-dimethylaniline) (PDMA) has been studied. The oxidation potentials of 5-HT and AA overlapped after mixing of the two chemicals, due to interference of AA at the bare BDD electrode. However, after modifying the BDD electrode with a cationic polymer (PDMA), the oxidation peaks of 5-HT and AA were separated. PDMA-coated BDD electrodes can be used for simultaneous detection of these species.     

Functionalized-graphene modified graphite electrode for the selective determination of dopamine in presence of uric acid and ascorbic acid

Graphene is chemically synthesized by solvothermal reduction of colloidal dispersions of graphite oxide. Graphite electrode is modified with functionalized-graphene for electrochemical applications. Electrochemical characterization of functionalized-graphene modified graphite electrode (FGGE) is carried out by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The behavior of FGGE towards ascorbic acid (AA), dopamine (DA) and uric acid (UA) has been investigated by CV, differential pulse voltammetry (DPV) and chronoamperommetry (CA). The FGGE showed excellent catalytic activity towards electrochemical oxidation of AA, DA and UA compared to that of the bare graphite electrode. The electrochemical oxidation signals of AA, DA and UA are well separated into three distinct peaks with peak potential separation of 193mv, 172mv and 264mV between AA-DA, DA-UA and AA-UA respectively in CV studies and the corresponding peak potential separations in DPV mode are 204mv, 141mv and 345mv. The FGGE is successfully used for the simultaneous detection of AA, DA and UA in their ternary mixture and DA in serum and pharmaceutical samples. The excellent electrocatalytic behavior of FGGE may lead to new applications in electrochemical analysis.     

Quasi-reversible two-electron reduction of oxygen at gold electrodes modified with a self-assembled submonolayer of cysteine

The electrochemical reduction of molecular oxygen (O₂) has been performed at gold electrodes modified with a submonolayer of a self-assembly (sub-SAM/Au) of a thiol compound (typically cysteine (CYST)) in O₂-saturated 0.5 M KOH. At bare gold electrode O₂ reduction reaction proceeds irreversibly, while this reaction is totally hindered at gold electrodes with a compact structure of CYST over its surface. The partial reductive desorption of the compact CYST monolayer was achieved by controlling the potential of the CYST/Au electrode, leading to the formation of a submonolayer coverage of the thiol compound over the Au electrode surface (sub-SAM/Au), at which the CYST molecules selectively block the Au(1 0 0) and Au(1 1 0) fractions (the so-called rough domains) of the polycrystalline Au while the Au(1 1 1) component (the so-called smooth domains) remains bare (i.e., uncovered with CYST). This sub-SAM/Au electrode extraordinarily exhibits a quasi-reversible two-electron reduction of molecular oxygen (O₂) in alkaline medium with a peak separation (ΔE_p) between the cathodic and anodic peak potentials (E_p^c,E_p^a) of about 60 mV. The ratio of the anodic current to the cathodic one is close to unity. The formal potential (E^o′) of this reaction is found to equal −150 mV vs. Ag/AgCl/KCl(sat.).     

DNA/Poly(p-aminobenzensulfonic acid) composite bi-layer modified glassy carbon electrode for determination of dopamine and uric acid under coexistence of ascorbic acid

A nano-composite of DNA/poly(p-aminobenzensulfonic acid) bi-layer modified glassy carbon electrode as a biosensor was fabricated by electro-deposition method. The DNA layer was electrochemically deposited on the top of electropolymerized layer of poly(p-aminobenzensulfonic acid) (Pp-ABSA). Scanning electron microscopy, X-ray photoelectron spectroscopy and electrochemical impedance spectrum were used for characterization. It demonstrated that the deposited Pp-ABSA formed a 2-D fractal patterned nano-structure on the electrode surface, and which was further covered by a uniform thin DNA layer. Cyclic voltammetry and electrochemical impedance spectrum were used to characterize the deposition, and demonstrated the conductivity of the Pp-ABSA layer. The biosensor was applied to the detection of dopamine (DA) and uric acid (UA) in the presence of ascorbic acid (AA). In comparison with DNA and Pp-ABSA single layer modified electrodes, the composite bi-layer modification provided superior electrocatalytic actively towards the oxidation of DA, UA and AA, and separated the originally overlapped differential pulse voltammetric signals of UA, DA and AA oxidation at the bare electrode into three well-defined peaks at pH 7 solution. The peak separation between AA and DA, AA and UA was 176 mV and 312 mV, respectively. In the presence of 1.0 mM AA, the anodic peak current was a linear function of the concentration of DA in the range 0.19-13 microM. The detection limit was 88 nM DA (s/n=3). The anodic peak current of UA was also a linear function of concentration in the range 0.4-23 microM with a detection limit of 0.19 microM in the presence of 0.5 mM AA. The superior sensing ability was attributed to the composite nano-structure. An interaction mechanism was proposed.     

Electropolymerized film of functionalized thiadiazole on glassy carbon electrode for the simultaneous determination of ascorbic acid, dopamine and uric acid

The present study reports the simultaneous determination of ascorbic acid (AA), dopamine (DA) and uric acid (UA) in 0.20 M phosphate buffer solution (pH 5.0) using electropolymerized ultrathin film of 5-amino-2-mercapto-1,3,4-thiadiazole (AMT) on glassy carbon (GC) electrode. The bare GC electrode does not separate the voltammetric signals of AA, DA and UA. However, electropolymerized AMT (p-AMT) modified GC electrode not only resolved the voltammetric signals of AA, DA and UA but also dramatically enhanced their oxidation peak currents when compared to bare GC electrode. The enhanced oxidation currents for AA, DA and UA at p-AMT modified electrode are due to the electrostatic interactions between them and the polymer film. Using amperometric method, we achieved the lowest detection of 75 nM AA, 40 nM DA and 60 nM UA at p-AMT modified electrode. The amperometric current was linearly increased from 200 nM to 0.80 mM for each AA, DA and UA and the lowest detection limit was found to be 0.92, 0.07 and 0.57 nM, respectively (S/N = 3). The practical application of the modified electrode was demonstrated by the determination of DA in dopamine hydrochloride injection.     

Poly(4-amino-1-1'-azobenzene-3, 4'-disulfonic acid) coated electrode for selective detection of dopamine from its interferences

Here, we described a new method for electrochemically selective detection of dopamine (DA). In this report, for the first time, electrochemical polymerization of 4-amino-1-1'-azobenzene-3,4'-disulfonic acid (acid yellow 9 dye (AY)) was carried out onto the surface of glassy carbon (GC) electrode and indium tin oxide coated electrode (ITO) from acidic solution containing AY monomers. A polymerized film of acid yellow on the surface of a glassy carbon electrode was characterized by cyclic voltammetry (CV). The redox response of the poly(AY) film on the GC electrode showed a couple of redox peak in 0.1M sulfuric acid solution and the pH dependent peak potential was -58mV/pH which was close to the Nernst behavior. The poly(AY) film-coated GC electrode (GC/PAY) exhibited excellent electrocatalytic activity towards the oxidations of dopamine (DA) in 0.1M phosphate buffer solution (PBS, pH 7.0) and increased the anodic peak current three time higher than bare GC electrode. GC/PAY did not reduce the considerable overpotential for oxidation of DA when compare to bare GC electrode. However, in contrast to other polymer modified electrode, due to the strong negatively charged back bone of poly(AY) highly repelled the important interference of DA, such as ascorbic acid (AA), uric acid (UA) and reduced form of nicotinamide adenine dinucleotide (NADH) in 0.1M PBS (pH 7.0) and did not showed any response for oxidation of these interferences. This behavior makes the GC/PAY for selective detection of DA in the presence of higher concentrations AA, UA and NADH. Using differential pulse voltammetry the calibration curves for DA were obtained over the range of 1-100muM with good selectivity and sensitivity. The proposed method provides a simple method for selective detection of DA from its interferences.     

多壁碳纳米管修饰碳黑微电极同时测定多巴胺和抗坏血酸

制备了多壁碳纳米管修饰碳黑微电极,研究了多巴胺(DA)和抗坏血酸(AA)在该修饰电极上的电化学行为.实验表明,在pH 7.0的PBS缓冲溶液中,该修饰电极对DA和从均具有显著的催化氧化作用,AA与DA的氧化电位分别为30 mV和280 mV(vs.SCE).利用二次导数线性扫描伏安法测定,DA与AA的线性范围分别为6.0×10-9～2.0×10-4 mol/L和2.0×10-7～1.0×10-3mol/L,检出限为2.0×10-9mol/L 和1.0×10-7mol/L.方法已用于人工合成样品的分析.