Fluorescent dye for detecting human serum albumin in urine as well as preparation method and application of fluorescent dye
By developing a fluorescent probe that forms a hydrogen bond between the fluorescent dye DCTB and human serum albumin HSA, the problems of slowness and complexity of existing detection methods were solved, and rapid and sensitive urine albumin detection was achieved.
Patent Information
- Application Number
- CN202510680261.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-26
- Publication Date
- 2025-09-23
AI Technical Summary
Existing urine albumin detection methods are slow, complex to operate, and have poor anti-interference performance.
A fluorescent dye DCTB was developed to form a fluorescent probe by hydrogen bonding with human serum albumin HSA, enabling rapid quantitative or qualitative detection.
It achieved an ultrafast response speed (5 seconds) and high sensitivity (detection limit of 0.1304 μM) for human serum albumin, and can perform on-site rapid testing in real samples.
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Figure CN120682182A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of urine albumin detection, and in particular to a fluorescent dye for detecting human serum albumin in urine, and a preparation method and application thereof. Background Art
[0002] Human serum albumin (HSA) is the most abundant protein in the human circulatory system, accounting for approximately 50% to 60% of total plasma protein. Synthesized by the liver, it performs numerous physiological functions, including maintaining plasma osmotic pressure, transporting and storing fatty acids, hormones, and metal ions, and providing buffering. In healthy individuals, the kidneys filter and reabsorb HSA, resulting in urinary HSA concentrations below 30 mg / L. However, various diseases, such as renal tubular acidosis, diabetes, cardiovascular disease, acute renal failure, cancer, and polycystic kidney disease, can impair renal HSA absorption. Because the glomeruli filter metabolic products from the blood and absorb protein for reuse, renal abnormalities can allow protein to leak into the urine. Therefore, urinary HSA levels are considered an early biomarker for disease diagnosis. In clinical settings, urinary albumin levels of 30 to 300 mg / L are considered grade A2 proteinuria, which typically occurs in diabetic patients with decreased renal function. Meanwhile, urine albumin concentrations exceeding 300 mg / L are classified as A3 proteinuria, indicating kidney damage or the development of kidney disease, cardiovascular disease, and diabetes. Therefore, developing an effective detection method for urine albumin is crucial for the accurate diagnosis of proteinuria and its related diseases.
[0003] In recent decades, a variety of viable analytical methods have been developed, including immunoassays, liquid chromatography, colorimetry, and fluorometry. Fluorescence assays, with their rapid response, high sensitivity, high selectivity, and direct in situ detection of targets, have attracted the interest of numerous researchers. Based on the changes in fluorescence signal following interaction between the probe and the target, fluorescent probe systems can be generally categorized as: fluorescence quenching, enhancement, and ratiometric. While traditional quenching and enhancement fluorescent probes are widely used, they all detect targets through changes in the intensity of a single-wavelength fluorescence signal, are susceptible to the effects of the excitation light source and the efficiency of the detection instrument, and exhibit poor anti-interference performance.
[0004] Therefore, it is urgent to develop a method that can quickly detect urine albumin levels. Summary of the Invention
[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a fluorescent dye for detecting human serum albumin in urine, and its preparation method and application, aiming to solve the technical problems of slow detection speed and complicated operation when detecting urine albumin by the existing detection method.
[0006] Specifically, the technical solutions of the present invention are as follows:
[0007] In a first aspect of the present invention, a fluorescent dye for detecting human serum albumin in urine is provided, wherein the chemical structural formula of the fluorescent dye is as follows:
[0008]
[0009] A second aspect of the present invention provides a method for preparing the fluorescent dye for detecting human serum albumin in urine according to the present invention, comprising the steps of:
[0010] 2-Hydroxyacetophenone and 4-(dimethylamino)cinnamaldehyde react to obtain the intermediate product HSF;
[0011] The intermediate product HSF is subjected to an esterification reaction with 5-bromo-N,N,N-trimethylpentane-1-ammonium bromide to obtain the fluorescent dye, which is denoted as DCTB;
[0012] The synthetic route of the fluorescent dye is as follows:
[0013]
[0014] Optionally, the step of reacting 2-hydroxyacetophenone and 4-(dimethylamino)cinnamaldehyde to obtain the intermediate product HSF comprises:
[0015] 2-Hydroxyacetophenone and 4-(dimethylamino)cinnamaldehyde are dissolved in ethanol to obtain a reaction solution. Potassium hydroxide solution is added to the reaction solution, stirred at room temperature, and then placed in an ice bath. A hydrogen peroxide solution is added, and the solution is stirred at room temperature again. Ice water and hydrochloric acid are added to obtain a precipitate. The precipitate is filtered and washed to obtain the intermediate product HSF.
[0016] Optionally, the step of subjecting the intermediate product HSF to an esterification reaction with 5-bromo-N,N,N-trimethylpentane-1-ammonium bromide to obtain the fluorescent dye comprises:
[0017] The intermediate product HSF and 5-bromo-N,N,N-trimethylpentane-1-ammonium bromide are dissolved in N,N-dimethylformamide to obtain a mixed solution, cesium carbonate is added to the mixed solution, and the mixture is stirred at room temperature to carry out an esterification reaction, and then extracted with ethyl acetate, dried, distilled under reduced pressure, and purified by a chromatographic column to obtain the fluorescent dye.
[0018] A third aspect of the present invention provides a use of the fluorescent dye according to the present invention in preparing a fluorescent probe for detecting human serum albumin in urine.
[0019] Optionally, the fluorescent dye is combined with human serum albumin through hydrogen bonds to form the fluorescent probe.
[0020] Optionally, the molar ratio of the fluorescent dye to human serum albumin is 1:1.
[0021] Beneficial Effects: The present invention provides a fluorescent dye for detecting human serum albumin (HSA), its preparation method, and application. The complex formed by the fluorescent dye DCTB and human serum albumin (HSA) through hydrogen bonding can be used for rapid quantitative or qualitative detection of human serum albumin (HSA). The human serum albumin detection method of the present invention has an ultra-fast response speed (5 seconds), high sensitivity (detection limit of 0.1304 μM), and high selectivity. When detecting human serum albumin, the fluorescent probe formed by the fluorescent dye DCTB and human serum albumin (HSA) will change its fluorescence signal from red to yellow. This response signal can be directly recognized by a smartphone and has been successfully applied to on-site rapid testing of real samples such as urine. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 This is the H NMR spectrum of the chemical structure of the fluorescent dye DCTB in Example 1 of the present invention.
[0023] Figure 2 This is the C NMR spectrum of the chemical structure of the fluorescent dye DCTB in Example 1 of the present invention.
[0024] Figure 3 This is a high-resolution mass spectrum of the chemical structure of the fluorescent dye DCTB in Example 1 of the present invention.
[0025] Figure 4 (a) is the fluorescence spectrum (λex = 440 nm) of human serum albumin HSA (0-10 μM) at different concentrations added to a PBS buffer solution containing the fluorescent dye DCTB in Example 2 of the present invention; (b) is the calculated detection limit of the fluorescent dye DCTB for human serum albumin in a PBS buffer solution of 0.1304 μM; (c) is the fluorescence decay curve before and after the binding of the fluorescent dye DCTB to human serum albumin HSA (IRF: instrument response function, the laser source is a NanoLED with a wavelength of 453 nm); (d) is the fluorescence spectrum (λex = 280 nm) of different concentrations of the fluorescent dye DCTB (0-10 μM) added to a PBS buffer solution containing 10 μM human serum albumin; (e) is the calculated binding stoichiometry n and binding constant K of the fluorescent dye DCTB to human serum albumin HSA based on the fluorescence spectrum in (d). b .
[0026] Figure 5Figures 2A and 2B show the binding mode of the fluorescent probe DCTB@HSA and the sensing performance test of the fluorescent dye DCTB for human serum albumin HSA in Example 2 of the present invention, including: (a) AutoDock 4.0 molecular docking experiment results; (b) a two-dimensional graph of the fluorescent probe DCTB@HSA after Ligplot processing; (c) a fluorescence response time graph; (d) a detection specificity graph; (e) a detection anti-interference graph; and (f) a detection anti-drug interference graph.
[0027] Figure 6 The sensing performance test diagram of the fluorescent dye DCTB to HSA in diluted urine samples in Example 3 of the present invention includes: (a) excitation-emission matrix fluorescence spectra (EEMF) of urine samples, urine samples with 10 μM DCTB added, and urine samples with 10 μM DCTB and 25 μM HSA added simultaneously; (b) fluorescence spectrum response of the fluorescent dye to HSA in urine samples; (c) fluorescence intensity ratio (I 580 / I 620 ) and the linear relationship between HSA concentration; (d) Qualitative detection results of HSA in urine samples, the upper part of the figure shows the control group (only HSA at different concentrations was added to the urine sample), and the lower part shows the test group (DCTB and HSA were added to the urine sample at the same time). DETAILED DESCRIPTION
[0028] The present invention provides a fluorescent dye for detecting human serum albumin in urine, as well as its preparation method and application. To clarify the objectives, technical solutions, and effects of the present invention, the present invention is described in further detail below. It should be understood that the specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the scope of protection of the present invention.
[0029] An embodiment of the present invention provides a fluorescent dye for detecting human serum albumin. The chemical structural formula of the fluorescent dye is as follows:
[0030]
[0031] Fluorescent dye DCTB molecules can bind to the drug site (DS1) of human serum albumin (HSA) through non-covalent interactions such as hydrogen bonding, forming a complex DCTB@HSA. Binding of DCTB to HSA restricts DCTB's intramolecular rotation, extending its fluorescence lifetime. HSA, a protein abundant in serum with excellent water solubility and stability, contains a variety of semi-flexible asymmetric binding cavities within the HSA molecule. These cavities can bind to a variety of ligands, such as amino acids, lipids, drugs, and small fluorescent dyes, through size matching and multiple secondary bonds. Therefore, HSA can bind to small fluorescent dye DCTB molecules through hydrogen bonding to construct a host-guest supramolecular system.
[0032] Experimental calculations showed that the stoichiometric binding coefficient of the fluorescent dye DCTB to human serum albumin HSA was close to 1, indicating that the fluorescent dye DCTB and human serum albumin HSA formed a complex with a molar ratio of 1:1.
[0033] The present invention provides a method for preparing a fluorescent dye, comprising the steps of:
[0034] 2-Hydroxyacetophenone and 4-(dimethylamino)cinnamaldehyde react to obtain the intermediate product HSF;
[0035] The intermediate product HSF is subjected to an esterification reaction with 5-bromo-N,N,N-trimethylpentane-1-ammonium bromide to obtain the fluorescent dye DCTB.
[0036] In the above preparation method, a new fluorescent dye DCTB is synthesized through a series of chemical reactions including Claisen-Schmidt condensation reaction, Algar-Flynn-Oyamada reaction and esterification reaction. Its full name is: (E)-5-((2-(4-(dimethylamino)styryl)-4-oxo-4H-chromen-3-yl)oxy)-N,N,N-trimethylpentan-1-aminium bromide.
[0037] In some embodiments, the step of reacting the 2-hydroxyacetophenone and 4-(dimethylamino)cinnamaldehyde to obtain the intermediate product HSF comprises:
[0038] 2-Hydroxyacetophenone and 4-(dimethylamino)cinnamaldehyde are dissolved in ethanol to obtain a reaction solution. Potassium hydroxide solution is added to the reaction solution, stirred at room temperature, and then placed in an ice bath. A hydrogen peroxide solution is added, and the solution is stirred at room temperature again. Ice water and hydrochloric acid are added to obtain a precipitate. The precipitate is filtered and washed to obtain the intermediate product HSF.
[0039] In some embodiments, the step of esterifying the intermediate product HSF with 5-bromo-N,N,N-trimethylpentane-1-ammonium bromide to obtain the fluorescent dye comprises:
[0040] The intermediate product HSF and 5-bromo-N,N,N-trimethylpentane-1-ammonium bromide are dissolved in ultra-dry N,N-dimethylformamide to obtain a mixed solution, cesium carbonate is added to the mixed solution, and the mixture is stirred at 75° C. for 24 hours under nitrogen protection to carry out an esterification reaction, and then extracted with ethyl acetate, dried, distilled under reduced pressure, and purified by a chromatographic column to obtain the fluorescent dye.
[0041] An embodiment of the present invention provides a use of the fluorescent dye described above in preparing a fluorescent probe for detecting human serum albumin in urine.
[0042] The fluorescent dye DCTB binds to the human serum albumin through hydrogen bonding to form the fluorescent probe. In some embodiments, the molar ratio of the fluorescent dye DCTB to the human serum albumin is 1:1.
[0043] The present invention provides a method for quantitatively detecting the content of human serum albumin in urine, comprising:
[0044] PBS buffer was added to the urine sample of healthy subjects, and the urine sample diluted 10 times was used as the test solution;
[0045] Adding the fluorescent dye DCTB to the test solution, then adding human serum albumin of different concentration gradients, measuring the fluorescence spectrum, establishing a standard curve of the fluorescence intensity ratio of the emission peak and the human serum albumin concentration, and determining the detection limit of human serum albumin;
[0046] The standard curve is used to quantitatively detect the content of human serum albumin in an unknown urine sample.
[0047] In some embodiments, the volume ratio of the PBS buffer to the healthy human urine sample is 9:1.
[0048] In some embodiments, the fluorescence intensity ratio of the emission peaks is the ratio of the fluorescence intensities of the emission peaks at 580 nm and 620 nm in the fluorescence spectrum.
[0049] The present invention also provides a method for qualitatively detecting human serum albumin in urine, comprising:
[0050] Different concentrations of human serum albumin were added to 10-fold diluted urine as a blank group, and fluorescent dye and different concentrations of human serum albumin were added to 10-fold diluted urine as a test group. UV light was irradiated and the fluorescent colors of the blank group and the test group were photographed.
[0051] In some embodiments, the blank group is a urine sample diluted 10 times with human serum albumin of different concentrations added, and the test group is a urine sample diluted 10 times with 10 μM fluorescent dye DCTB and human serum albumin of different concentrations added simultaneously, and ultraviolet light is irradiated, and the fluorescent colors of the blank group and the test group are photographed.
[0052] In some embodiments, the ultraviolet light irradiation uses an excitation wavelength of 365 nm.
[0053] The aforementioned method for detecting human serum albumin in urine utilizes DCTB@HSA as a supramolecular recognition system based on host-guest interactions. The fluorescent dye, due to its positive ions, exhibits hydrophilicity and forms stable micelles in water. Upon binding to human serum albumin, the DCTB micelles dissociate into monomers, enabling specific ratiometric detection of human serum albumin in real urine samples.
[0054] The present invention will be further described below with reference to specific examples.
[0055] Example 1
[0056] The synthetic route of fluorescent dye DCTB is:
[0057]
[0058] According to the above synthetic route, the specific preparation steps are as follows:
[0059] 2-Hydroxyacetophenone (30 mmol) and 4-(dimethylamino)cinnamaldehyde (30 mmol) were dissolved in 100 mL of ethanol to obtain a reaction solution. Subsequently, 10 mL (75 mmol / L) of a potassium hydroxide aqueous solution was added to the reaction solution. After stirring at room temperature for 12 hours, the solution was placed in an ice bath. 5 mL of a 30% hydrogen peroxide aqueous solution was slowly added, and the solution was stirred at room temperature for 12 hours. The solution was then poured into ice water and neutralized with dilute hydrochloric acid until neutral. The resulting precipitate was filtered, collected, and washed with cold ethanol to obtain the intermediate product HSF in a yield of 56.5%.
[0060] 1.5 mmol of the intermediate product HSF and 3 mmol of 5-bromo-N,N,N-trimethylpentane-1-ammonium bromide were dissolved in 15 mL of ultra-dry N,N-dimethylformamide (DMF) to obtain a mixed solution. 3 mmol of cesium carbonate was added to the mixed solution, and the mixture was stirred at 75°C under nitrogen for 24 hours. After the reaction, the mixture was filtered and the filtrate was poured into ice water. The mixture was extracted three times with 20 mL of ethyl acetate. The crude product was purified by column chromatography to obtain the final red product DCTB in a yield of 41.5%.
[0061] Structural characterization results: The nuclear magnetic resonance hydrogen spectrum of fluorescent dye DCTB is as follows Figure 1 As shown, the nuclear magnetic resonance carbon spectrum of fluorescent dye DCTB is as follows Figure 2 As shown, the high-resolution mass spectrum of the fluorescent dye DCTB is as follows Figure 3 The above synthesis steps show that the fluorescent dye DCTB is synthesized through a series of chemical reactions such as Claisen-Schmidt condensation reaction, Algar-Flynn-Oyamada reaction and esterification reaction.
[0062] Example 2
[0063] The fluorescence spectrum of the fluorescent dye DCTB (prepared in Example 1) changes before and after binding with human serum albumin HSA (purchased from Sigma-Aldrich, product number A9731). The binding mechanism and fluorescence spectrum changes are as follows: Figure 4 As shown in (a), the emission peak of the fluorescent dye DCTB in water is at 620 nm, and the fluorescence color is red. However, after adding human serum albumin HSA (the molar ratio of fluorescent dye DCTB to human serum albumin HSA is 1:1), its fluorescence gradually increases and blue-shifts. When HSA is added to 10 μM, the fluorescence spectrum emission peak is at 580 nm, and the fluorescence color is yellow. Figure 4 (b) shows the fluorescence intensity ratio of the fluorescent probe DCTB@HSA at 580nm and 620nm (I 580 / I 620 ) showed a good linear relationship with the concentration of HSA, and the detection limit of the fluorescent dye DCTB for detecting human serum albumin in PBS buffer was calculated to be 0.1304 μM. In addition, after interacting with HSA, the fluorescence lifetime of the fluorescent dye DCTB was extended from 0.069 ns to 1.79 ns, indicating that the intramolecular rotation (RIR) of the fluorescent dye DCTB was restricted after binding to HSA, as shown in Figure 2. Figure 4 As shown in (c). Figure 4 As shown in (d) and (e), the back titration experiment calculated that the binding stoichiometric coefficient of the fluorescent dye DCTB and human serum albumin HSA is close to 1, indicating that the fluorescent dye DCTB and human serum albumin HSA form a complex with a molar ratio of 1:1.
[0064] The fluorescent probe DCTB@HSA solution was stored in a dark place and at a low temperature (below 10°C).
[0065] Study on the mechanism of rapid and highly selective detection of human serum albumin (HSA) using fluorescent dye DCTB
[0066] The possible binding modes of the fluorescent probe DCTB@HSA were calculated by molecular docking technology, such as Figure 5 As shown in (a) and (b). The results show that the fluorescent dye DCTB binds to the drug site (DS1) of albumin through hydrogen bonding, with a binding energy of -6.45kcal / mol. Figure 5 As shown in (c), the fluorescent dye DCTB itself has good photostability to ultraviolet light. After adding human serum albumin HSA, the fluorescent probe DCTB@HSA quickly responded and reached the peak within 5 seconds, and the signal output time was stable for more than 10 minutes. Figure 5 As shown in (d), the fluorescent dye DCTB does not change the fluorescence intensity of common biological substances present in the human body, such as ions (sodium ions, chloride ions, calcium ions, potassium ions, etc.), amino acids (cysteine, arginine, tryptophan, etc.) and enzymes (carbonic anhydrase, lysozyme), proving that the fluorescent probe DCTB@HSA has good detection specificity. Figure 5 As shown in (e), substances commonly found in urine such as uric acid, urea, creatinine, cortisol, glucose, lactose, trypsin, protease, vitamin B1, vitamin B6 and vitamin C will not change the fluorescence intensity of DCTB@HSA, proving that the fluorescent probe DCTB@HSA has good anti-interference properties. In order to prove that the fluorescent probe DCTB@HSA also has good anti-drug interference properties, by adding different concentrations of common drugs such as cardiovascular drugs (amlodipine, digitoxin), anti-inflammatory drugs (budesonide), sedatives (chloral hydrate, phenytoin) and anti-diabetic drugs (chlorpropamide) to DCTB@HSA, the fluorescence intensity was observed to see if it changed. The results showed that the addition of different common drugs had little effect on the fluorescence intensity of DCTB@HSA, proving that the fluorescent probe DCTB@HSA has good anti-drug interference properties. Figure 5 As shown in (f).
[0067] Example 3
[0068] Rapid quantitative detection of human serum albumin in urine samples
[0069] Add PBS buffer to the urine of healthy people and take the urine diluted 10 times as the test solution. First add fluorescent dye DCTB to the test solution, then add different concentrations of human serum albumin, such as Figure 6As shown in (b), under 440nm excitation light, as the concentration of human serum albumin HSA increases, the intensity of the fluorescence spectrum continues to increase and blue shifts. Figure 6 As shown in (c), the fluorescence intensity ratio at 580nm and 620nm (I 580 / I 620 ) showed a good linear relationship with the concentration of human serum albumin, and the detection limit (LOD) for HSA in urine was calculated to be 0.0855 μM.
[0070] Example 4
[0071] Rapid qualitative detection of human serum albumin in urine samples
[0072] Urine diluted 10 times was used as the test solution.
[0073] Blank group: Add 2 ml of test solution to each of 6 cuvettes of the same specifications. Then add human serum albumin of different concentrations to the cuvettes as a blank control. Use a handheld UV lamp (365 nm excitation wavelength) to irradiate and photograph the color of the blank solution in the cuvette.
[0074] Control group: Add 2 ml of test solution to each of 6 cuvettes of the same specifications, then add fluorescent dye DCTB and different concentrations of human serum albumin to the cuvettes as the test group. Use a handheld UV lamp (365 nm excitation wavelength) to irradiate and photograph the color of the test group solution in the cuvettes. Figure 6 As shown in (d), the blank group, spiked with varying concentrations of human serum albumin, exhibited a uniform blue fluorescence, while the test group exhibited a shift in fluorescence from blue to pink. This difference in fluorescence color between the blank and test groups was used to establish a standard (RGB) for the qualitative analysis of human serum albumin. This color comparison allows for qualitative detection of human serum albumin in urine samples.
[0075] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.
Claims
1. A fluorescent dye for detecting human serum albumin in urine, characterized in that: The chemical structural formula of the fluorescent dye is shown below:
2. A method for preparing a fluorescent dye for detecting human serum albumin in urine according to claim 1, characterized in that: Including steps: 2-Hydroxyacetophenone and 4-(dimethylamino)cinnamaldehyde react to obtain the intermediate product HSF; The intermediate product HSF is subjected to an esterification reaction with 5-bromo-N,N,N-trimethylpentane-1-ammonium bromide to obtain the fluorescent dye, which is denoted as DCTB; The synthetic route of the fluorescent dye is as follows:
3. The method for preparing a fluorescent dye for detecting human serum albumin in urine according to claim 2, wherein: The step of reacting 2-hydroxyacetophenone and 4-(dimethylamino)cinnamaldehyde to obtain the intermediate product HSF comprises: 2-Hydroxyacetophenone and 4-(dimethylamino)cinnamaldehyde are dissolved in ethanol to obtain a reaction solution. Potassium hydroxide solution is added to the reaction solution, stirred at room temperature, and then placed in an ice bath. A hydrogen peroxide solution is added, and the solution is stirred at room temperature again. Ice water and hydrochloric acid are added to obtain a precipitate. The precipitate is filtered and washed to obtain the intermediate product HSF.
4. The method for preparing a fluorescent dye for detecting human serum albumin in urine according to claim 2, wherein: The step of subjecting the intermediate product HSF to an esterification reaction with 5-bromo-N,N,N-trimethylpentane-1-ammonium bromide to obtain the fluorescent dye comprises: The intermediate product HSF and 5-bromo-N,N,N-trimethylpentane-1-ammonium bromide are dissolved in N,N-dimethylformamide to obtain a mixed solution, cesium carbonate is added to the mixed solution, and the mixture is stirred at room temperature to carry out an esterification reaction, and then extracted with ethyl acetate, dried, distilled under reduced pressure, and purified by a chromatographic column to obtain the fluorescent dye.
5. Use of the fluorescent dye according to claim 1 in preparing a fluorescent probe for detecting human serum albumin in urine.
6. The use according to claim 5, characterized in that The fluorescent dye is combined with human serum albumin through hydrogen bonds to form the fluorescent probe.
7. The use according to claim 6, characterized in that The molar ratio of the fluorescent dye to human serum albumin is 1:1.