Method For Detection Of Residues Research Articles

Single-atom ruthenium nanozyme-induced signal amplification strategy in photoelectrochemical aptasensor for ultrasensitive detection of chloramphenicol

To develop ultrasensitive and rapid antibiotics residue detection method is crucial for ensuring food safety and protecting human health. Herein, a novel photoelectrochemical (PEC) aptasensor integrated with single-atom ruthenium (Ru) nanozyme-mediated catalytic precipitation as a valuable signal amplification strategy, have been established for ultrasensitive chloramphenicol (CAP) detection. Particularly, the exceptional peroxidase-mimicking activity of single-atom Ru nanozyme is responsible for accelerating the oxidation of 4-chloro-1-naphthol (4-CN) to produce insoluble precipitate on the electrode, which in turn causes a notable reduction in the photocurrent. Whereas, when CAP is present, the aptamer is liberated away the electrode because of its potent affinity with CAP, resulting in an elevation of the photocurrent signal, enhancing the detection sensitivity. Importantly, the signal amplification strategy combines the effective photoactive material of Au nanoparticles/CdS quantum dot/TiO2 composites, a PEC aptasensor for determination of CAP with an ultralow detection limit of 4.12 pM is achieved in a self-powered mode with great selectivity and accuracy. This work proposes a novel reasonable approach utilizing high-activity single-atom nanozyme to induce signal amplification strategy for the advancement of single-atom nanozyme in ultrasensitive PEC biosensor, and further creates new avenues for ultrasensitive detection beyond antibiotics residue.

Highly efficient Z-scheme CeO2/Bi2MoO6 heterojunction strengthened by redox mediator for photoelectrochemical detection of tetracycline with enhanced sensitivity

Accurate monitoring of antibiotics that pose risks to the environment and human health is essential. The photoelectrochemical system has emerged as a rapid and precise method for detecting environmental pollutants. However, current photoelectrochemical materials made of semiconductors face challenges due to poor photoelectric conversion efficiency. In this study, a Z-scheme CeO2/Bi2MoO6 heterojunction structure was successfully prepared by a cost-effective hydrothermal method. By leveraging built-in electric field and the Ce3+/Ce4+ redox mediator, this heterojunction achieved high photoelectrochemical conversion efficiency and functioned effectively as a sensor for tetracycline (TCH). The designed sensor demonstrated two linear ranges of 0.05–100 nM and 100–300 nM with an ultra-low detection limit of 15.4 pM for TCH. Additionally, the sensor exhibited excellent selectivity and high stability, demonstrating its potential for TCH detection in tap-water and milk samples. This study presents a simple and reliable method for the practical detection of antibiotic residues in environmental and food samples.

Multiresidue analysis of 200 pesticides in jasmine (Jasminum sambac (L.) Aiton) flowers

A multiresidue analysis method was established for 200 pesticides in jasmine (Jasminum sambac(L.) Aiton) flowers based on QuEChERS procedure and gas chromatography-tandem mass spectrometry (GC–MS/MS). For better recoveries and purification effects, extraction solvents, extractive salts and purification methods were optimized. The limits of quantification (LOQ) of test pesticides are in the range of 0.01 and 0.1 mg kg−1, most of which were below the maximum residue limits (MRLs) prescribed by EU and China. Recoveries at 0.01, 0.1, 1.0 mg kg−1 were predominantly in the range of 70–120 % (n = 3) with corresponding RSDs lower than 20 %, which suggests satisfactory accuracy. The multiresidue analysis method was applied in monitoring pesticide residues in 8 jasmine flower samples. 50 % of those samples present with 1–2 pesticides residues. This study provides a sensitive and high-throughput method for detection of multi-pesticide residues in jasmine flowers.

Research on detection and protection technology of DC residual current

Abstract To realize that the residual current device can accurately and quickly detect the fault when the fluctuating DC residual current occurs in the system and improve the system’s safety, a DC residual current detection and protection method based on VMD was proposed. Particle Swarm Optimization (PSO) was used to optimize the penalty factor and the number of decomposition layers of VMD. The minimum envelope entropy of the residual current signal is used as the fitness function to obtain the optimal combination. The criterion of fault occurrence is established based on the optimal modal component mutation characteristic of the residual current decomposed by VMD. Experiments show that this method can identify faults quickly and effectively when pulsating DC residual current is generated in the system.

β-Cyclodextrin based magnetic hyper-crosslinked polymer: A recyclable adsorbent for effective preconcentration of triazine herbicides in complex sample matrices

High efficiency enrichment and trace analysis of triazine herbicide residues are crucial for ensuring environmental and food safety. Herein, a series of magnetic hyper-crosslinked polymers (CD-gs-MHCPs) were synthesized with different crosslinkers, which might possess different pore structure and surface area, so they might dispay variable adsorption performance. CD-gs-MHCP2 with dichloroxylene as crosslinker delivered superior adsorption ability for triazine herbicides (THs). The synergistic effect of hydrogen bonds, hydrophobic interaction, π-π stacking interaction and pore adsorption were proved to be the main adsorption mechanism. Combined CD-gs-MHCP2 based magnetic solid-phase extraction (MSPE) with high-performance liquid chromatography, the quantitative analysis of THs in river water and vegetable samples (zucchini, pakchoi) was achieved. Under the optimal conditions, the enrichment factors for three different samples ranged from 94 to 244 and low detection limit (S/N = 3) of the four THs were obtained from 0.05 to 0.15 ng mL−1 for river water and 0.31–3.10 ng g−1 for vegetable samples. The method recoveries were in the range of 86.2 %–120 % with relative standard deviations lower than 7.4 %. This work not only offers a new strategy for fabrication β-CD-based HCPs, but also provided a practical and effective method for efficient isolation and sensitive detection of trace THs residues in complex samples.

A novel fluorescent and photothermal probe based on nanozyme-mediated cascade reaction for detecting organophosphorus pesticide residues

In this study, a nanozyme (ZIF-Co-Cys) with high oxidase-like catalytic activity was prepared, and a ratiometric fluorescent/photothermal dual-mode probe was constructed for organophosphorus pesticides (OPs) detection based on the competitive effect of ZIF-Co-Cys and the enzymatic reaction product of acid phosphatase (ACP) on o-phenylenediamine and the inhibition effect of OPs on ACP activity. Using dimethyl dichloroviny phosphate (DDVP) as the model, both the fluorescence intensity ratio and the temperature change of the probe solution exhibited an excellent correlation with OPs concentration. The detection limits were 1.64 ng/mL and 0.084 ng/mL, respectively. Additionally, the detection of DDVP residues in real samples verified the outstanding anti-interference and accuracy of the probe. This work not only provided a complementary dual-mode method for the accurate and rapid detection of OPs residues in complex samples, but also supplied a new insight into the design of a multi-mode sensing platform based on the cascade reaction of nanozyme.

Standardization of Gas chromatography method for detection of Deltamethrin residues in chicken meat

Standardization of Gas chromatography method for detection of Deltamethrin residues in chicken meat

Active ingredient identification and purification of organosilicon spray adjuvant and its uptake and translocation in rice (Oryza sativa L.).

The assessment of residue, absorption, conduction, and degradation of agricultural organosilicon surfactants in the environment is hindered by the lack of information on active ingredients and corresponding quantitative standards for organosilicon spray adjuvants. The spray adjuvant 'Jiexiaoli,' a primary organosilicon spray agent in China, was identified as hydroxy (polyethylene) propyl-heptamethyl trisiloxane (TSS-H) with 3-15 ethoxy (EO) groups. Purification of TSS-H was achieved through semi-preparative separation using high-performance liquid chromatography (HPLC), resulting in TSS-H purity exceeding 96%. An accurate residual detection method for nine oligomers (4-12 EO) of TSS-H in rice roots, stems, leaves, and culture solution samples was developed using HPLC tandem high-resolution mass spectrometry (HPLC-HRMS). Recoveries for nine oligomers of TSS-H in the four matrices ranged from 80.22% to 104.01%. Foliar application experiments demonstrated that TSS-H did not transfer from the upper to the lower parts of the rice plant. The half-lives of each oligomer (4-12 EO) in leaves were less than 3.21 days. Root application experiments revealed a root concentration factor (RCF) ranging from 0.20 to 0.56, a biological enrichment factor (BCF) ranging from 0.36 to 0.68, a transpiration factor (TSCF) ranging from 0.069 to 0.086, and a transport factor (TF) ranging from 0.08 to 0.43. These results indicated that TSS-H could be absorbed by rice roots and conducted to the above-ground parts of rice plants. This study fills the data gap in the environmental risk and food safety assessment of agricultural silicone spray adjuvants. © 2024 Society of Chemical Industry.

Stable detection of diazinon residues in vegetables by an electrochemiluminescent aptasensor based on the in-situ production of H2O2 from dual-catalytic glucose

Stable detection of diazinon (DZN) residues in vegetables is important for food safety. In this work, an electrochemiluminescence (ECL) aptasensor with dual-catalytic glucose in-situ production of H2O2 was constructed for the stable detection of DZN in vegetables. Firstly, MWCNTs@MB was prepared using π-π stacking interactions between methylene blue (MB) and multi-walled carbon nanotubes (MWCNTs) to enhance the loading of MB on an electrode and thus catalyze the generation of H2O2 from glucose. Secondly, Cu2O@AuNPs was formed by loading AuNPs on the surface of Cu2O through spontaneous reduction reaction, which improved the interfacial charge transfer, Cu2O nano-enzyme had glucose oxidase mimicking activity and could further catalyze the production of more H2O2 from glucose. MWCNTs@MB and Cu2O@AuNPs played a key role in the in-situ generation of co-reacting reagent H2O2, which solved the problem of unstable detection caused by the easy decomposition of the H2O2 solution added to the luminescence system. In addition, the aptamer was immobilized on the electrode surface by forming Au–S bonds with Cu2O@AuNPs. As a result, the ECL aptasensor performed good linearity in 1.00 pg mL−1-1.00 μg mL−1 and a low limit of detection (LOD) to 0.39 pg mL−1 (S/N = 3). This work provided an effective method for the accurate and stable detection of DZN residues in vegetables, which was of great significance in ensuring food safety and assessing the environmental risk of DZN.

Migration characteristics and risk assessment of chemical hazardous substances in infant teether toys

Teether is a special toy used for infants oral contact. In this paper, a residual and migration detection method was developed using gas chromatography–tandem mass spectrometry for 20 screened hazardous substances in teethers. Fifteen substances were detected in 59 samples, with residual amounts and detection rates ranging from 0.01 mg⋅kg−1 to 106.15 mg⋅kg−1 and from 3.39 % to 84.7 % respectively. Then, 12 substances were detected in simulated saliva at migration levels ranging from 0.0143 mg⋅kg−1 to 20.03 mg⋅kg−1, with detection rates ranging from 1.69 % to 76.3 %. Statistically, the average migration rate of each substance ranged from 8.18 % to 53.28 % depending on the properties of the substance and the sample. The exposure risk of infants to teethers was evaluated separately for two age groups. The hazard quotient (HQ) and hazard index (HI) values for the analytes were higher in the 3–12-month age group than in the 12–24-month age group. The HQ values of triphenylphosphine oxide, benzocaine, and N-methylformanilide were relatively high, with averages of 1.2 × 10−2, 2.5 × 10−3, and 1.6 × 10−3, respectively, and the max HI of the 12 substances was 0.04. The HI and HQ values of the analytes were all below 1, indicating that the non-carcinogenic risks of analytes in teethers are at an acceptable level.

Validation and application of a method for determination of multi-class pesticides in muscle chicken breast fillets using QuEChERS extraction and GC/MS.

The occurrence of pesticide residues in animal products deserves attention because of the contamination by environmental pollutants and pesticides that may be present in the food that animals are fed. The goal of this work was the validation of a method for detection of residues of multiple classes of pesticide and determination of their residues in chicken breast fillets. Gas chromatography with mass spectrometry was used for analysis. A modified quick, easy, cheap, effective, rugged and safe (QuEChERS) method was put into practice for its validation and applied to real samples. The study optimised mass detection and investigated the effect of a freezing step during the preparation of samples. Pesticides were determined in samples from conventional and organic production. The impact of the matrix effect decreased, with the largest number of pesticides and satisfactory recovery determined by the application of mixed solvent acetonitrile and ethyl acetate for extraction. Detection of pesticide residues was achieved in a linear range between 5 and 50 µg/kg with satisfactory excellent correlation coefficients greater than 0.99. The recovery of all the pesticide residues ranged between 71.2 and 118.80%. The relative standard deviation was from 2.9% to 18.1% for all validated pesticide residues. The limits of quantification were in the range of 3.0-4.9 µg/kg. Out of 56 pesticide residues analysed in real samples, 5 were detected: α endosulfan, cypermethrin, endosulfan sulphate, permethrin and p,p´-dichlorodiphenyltrichloroethane (DDT) and their concentrations ranged from 4.9 to 15.2 µg/kg. All tested samples were compliant with the evaluation criteria, and detected values of pesticide residues were lower than the maximum residual levels.

Mechanism and Method for Residual Flux Detection of Transformer Cores Based on Different Polarities Response Currents

This paper proposes a residual flux detection method based on the different polarities of response currents. When a positive–negative alternating DC voltage is loaded, the direction of the residual flux can be determined by the difference between the various polarities of the response current waveforms. As a result, residual flux value can be calculated using the empirical formula that relates residual flux to the responce current. In this formula, unknown parameters can be obtained using the field-circuit coupling method. Finally, this paper employs a closed iron core as an example to obtain the corresponding formula and then verifies its accuracy through experiments. The results show that the accuracy of the proposed method is less than 5%, which is higher than that of other existing methods. The method presented in this paper not only accurately detects the residual flux of a transformer but also requires no additional energy from the transformer.

Smartphone-based dual inverse signal MOFs fluorescence sensing for intelligent on-site visual detection of malachite green

The development of intelligent, sensitive, and visual methods for the rapid detection of veterinary drug residues is essential to ensure food quality and safety. Here, a smartphone-based dual inverse signal MOFs fluorescence sensing system was proposed for intelligent in-site visual detection of malachite green (MG). A UiO-66-NH2@RhB-dual-emission fluorescent probe was successfully synthesized in one step using a simple one-pot method. The inner filter effect (IFE) quenches the red fluorescence, while hydrogen bonding interaction enhances the blue fluorescence, enabling highly sensitive, accurate, and visual detection of MG dual inverse signals through fluorescence analysis. The probe showed great linearity over a wide range of 0.1–100 μmol/L, with a limit of detection (LOD) of 20 nmol/L. By integrating smartphone photography and RGB (red, green, and blue) analysis, accurate quantitative analysis of MG in water and actual fish samples can be achieved within 5 min. This developed platform holds great promise for the on-site detection of MG in practical applications, with the advantages of simplicity, cost-effectiveness, and rapidity. Consequently, it may open up a new pathway for on-site evaluation of food safety and environmental health.

Immunochromatographic method based on PCN-222 with dual-signal output for the rapid and sensitive detection of furazolidone metabolites in animal-derived food

The residues of furazolidone and its metabolites (3-amino-2-oxazolidinone [AOZ]) in food would have adverse effects on humans. Herein, a colorimetric and fluorescent dual-signal output immunochromatographic method with a zirconium-based porphyrin metal-organic framework (PCN-222) as the signal label was developed for the detection of 3-[(4-carboxyphenyl) monomethyl] amino-2-oxazolidinone (CPAOZ), a derivative of furazolidone metabolite. PCN-222 was synthesized using the solvothermal method, and the material was characterized via scanning electron microscopy, Fourier-transform-infrared spectroscopy, and powder X-ray diffraction, which proved the successful synthesis of PCN-222. Synthesized PCN-222, as a nanotag, was bound to antibodies to prepare PCN-222-mAbs with dual optical properties. Under the optimized conditions, PCN-222- lateral flow assay (LFA) exhibited a great linear relationship in the range of 0.25–100 ng/mL. The visual limit detection of CPAOZ using colorimetric and fluorescent signals were 1.0 ng/mL and 0.5 ng/mL, respectively, and the sensitivity was 5- and 10-fold higher than that of the colloidal gold-based LFA method. In addition, PCN-222-LFA was successfully applied to the detection of CPAOZ in shrimp, chicken and milk samples achieving satisfactory recoveries from 92.34% to 117.44%. This study provides an alternative, convenient and sensitive method for the rapid detection of veterinary drug residues.

Carboxymethyl chitosan–modified UiO-66 for the rapid detection of fenpropathrin in grains

Fenpropathrin residues in grain are potentially harmful to humans. Therefore, a fluorimetric lateral flow immunoassay using a zirconium-based organic skeleton (UiO-66) as a signal marker was developed for detecting fenpropathrin. Herein, carboxymethyl chitosan (CMCS) was used to modify UiO-66 and improve its water solubility to facilitate stable binding with sodium fluorescein (NaFL). This resulted in formation of a new fluorescent probe that is more suitable for lateral flow immunoassay (LFIA). The materials were characterized via electron microscopy, Fourier-transform infrared spectroscopy, and powder X-ray diffraction. CMCS and NaFL were successfully bound to UiO-66. Under optimized conditions, the constructed NaFL/UiO-66@CMCS-LFIA exhibited a good linear relationship within the range of 0.98–62.5 μg/L, with a detection limit of 3.91 μg/L. This probe was fourfold more sensitive than traditional colloidal gold nanoparticle-based LFIA. Finally, NaFL/UiO-66@CMCS-LFIA was successfully applied to detect fenpropathrin in wheat and maize samples. The detection limit was 1.56 μg/kg and recoveries ranged from 96.58 % to 118.56 %. This study provides a sensitive, stable, and convenient method for the rapid detection of pesticide residues.

Dissipation behaviours, residues, and health risk of six herbicides in sugar beets under field conditions

This study established a residue detection method based on the QuEChERS pre-treatment method and combined it with high-performance liquid chromatography–tandem mass spectrometry to test six herbicides (metamitron, clopyralid, desmedipham, phenmedipham, ethofumesate, and haloxyfop-p-methyl) in sugar beet plants, soil, and roots. The degradation dynamics and terminal residues of each herbicide in sugar beets were analysed. Finally, the dietary risks of various herbicides in sugar beets were evaluated based on the dietary structure of Chinese people, and the risk quotient values were below 100%. Using this detection method, all reagents exhibited good linearity (0.9724 ≤ R2 ≤ 0.9998), The limit of quantification (LOQ) ranged from 0.01 to 0.05 mg/L, the matrix effect ranged from −1.2% to −50%, the addition recovery rate ranged from 77.00% to 103.48%, and the relative standard deviation ranged from 1.61% to 16.17%; therefore, all indicators of this method met the residue detection standards. Under field conditions, the half-lives (t1/2) ranged about 0.65 ∼ 2.96 d and 0.38 ∼ 27.59 d in sugar beet plants and soil, respectively. All herbicides were easily degraded in sugar beet plants and soil (t1/2 < 30 d). The terminal residue amounts in the beet plants, soil, and roots ranged from < LOQ to 0.243 mg/kg. The dietary risk assessment of each pesticide was conducted based on the residual median of the terminal residues and the highest residual values on the edible part of the beetroot. The chronic exposure risk quotient (RQc) and acute exposure risk quotient (RQa) values were < 100%, indicating that the residue of each pesticide in beetroot posed low risks to consumers in China at the recommended dosage.

A Novel Approach for Single-Step Analyte Fractionation of Raw Milk Prior to Antibiotic Residue Trace Analysis as an Alternative to QuEchERS-Based Extraction.

Antibiotic residues in milk are a well-known hazard in the dairy food chain. Detection methods for these residues, such as nonspecific microbiological inhibitor tests or group-specific receptor tests, are relatively inexpensive, easy to use, and widely applied to ensure food safety. In contrast, specific detection by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-although a critical, complimentary method to confirm the results of nonspecific testing-is relatively costly, time-consuming, and laborious. Furthermore, sample processing before LC-MS/MS analysis requires unique preparation procedures for different groups of antibiotic compounds. To simplify and speed up specific antibiotic residue detection, a low-cost, passive, and single-step method to fractionate analytes in raw milk was developed. Untreated raw milk was fractionated into its water and fat/protein phases using a Fractionation of Milk for Trace Analysis of Contaminants and Residues for Antibiotics (FraMiTrACR® AB) fractionation unit. The water fraction was then analyzed by LC-MS/MS. The analyte fractionation method was evaluated against a Quick Easy Cheap Effective Rugged and Safe (QuEChERS)-based method for sample preparation. Our method allows qualitative and quantitative detection of substances from the penicillin, cephalosporin, macrolide, lincosamide, sulfonamide, tetracycline, and fluoroquinolone groups of antibiotics. Detection limits are below the legally prescribed maximum residue levels, allowing reliable, specific, and rapid validation of a positive result in nonspecific microbiological inhibitor tests. Analyte fractionation by FraMiTrACR AB is a faster alternative to QuEChERS-based sample preparation for the detection of antibiotic substances in milk. This method describes a low-cost, environmentally friendly, passive, and single-step milk analyte fractionation. As an alternative to QuEChERS-based preparation, this fractionation method simplifies and speeds up the process for specific antibiotic residue detection.

A Recycled-Material-Based Electrochemical Eco-sensor for Sensitive Detection of Antischistosomal Drug Residues in Bovine-Derived Food Samples

Veterinary drug residues in food have emerged as an urgent threat to consumer safety. Herein, we present the first square wave voltammetric method for the trace-level detection of cinnarizine residues, a recently used antischistosomal drug, in bovine food samples. The method depends on the electrochemical oxidation after modification of the carbon paste sensor with recycled Al2O3-NPs functionalized multi-walled carbon nanoparticles. The produced sensor (Al2O3-NPs/ MWCNTs/CPE) was characterized using the transmission electron microscope, scanning electron microscope, Fourier-transform infrared spectroscopy, energy-dispersive spectrometer, and X-ray diffractometer that confirm the successful incorporation of the Al2O3-NPs/MWCNTs composite into the modified electrode. As expected, the active surface area and electron transfer processes were accelerated by the modification, which was evidenced by cyclic voltammetry, chronoamperometric studies, scan rate studies, and electrochemical impedance spectroscopy. Compared to previous techniques, this facile sensor demonstrated enhancements across critical analytical criteria including the detection limit of 0.17 nM, linear response across 5–100 nM (r2 = 0.998), accuracy ranging from 96.5 to 103.2%, precision below 0.81% relative standard deviation, reproducibility within 0.36% range, 20 s response time and applicability in spiked food matrices. In addition, five different greenness and whiteness tools quantified exceptional environmental friendliness, economic feasibility and waste reduction of 63%–93%, reaffirming alignment with sustainability paradigms. These advantages support practical adoption in quality control especially laboratories lacking expensive instrumentation. Overall, the ingenious sensor reconciles nanotechnology innovation with the circular economy ethos to tackle an urgent food safety challenge, guided holistically by sustainability metrics.

Detection of doxycycline by using a tapered droplet structure fiber sensor.

The abuse of doxycycline (DC) can lead to residues in animals and water environments, which severely threaten human health; however, currently accepted detection methods are generally complicated and cannot be used for real-time detection. Therefore, developing a method for rapid real-time detection of DC microcontent residues is highly important. Herein, based on the Mach-Zehnder interference, we propose a simple tapered droplet structure fiber sensor with a high detection sensitivity. By modifying the sensing region with a molecularly imprinted polymer film of DC, this sensor realizes the specific detection of DC and has a detection sensitivity of 58.81 pm/ppm for DC in a large concentration range of 0-300 ppm. This sensor can be used to detect DC microcontent in aqueous solutions in real time.

Residual current detection method based on improved VMD-BPNN.

To further enhance the residual current detection capability of low-voltage distribution networks, an improved adaptive residual current detection method that combines variational modal decomposition (VMD) and BP neural network (BPNN) is proposed. Firstly, the method employs the envelope entropy as the adaptability function, optimizes the [k, ɑ] combination value of the VMD decomposition using the bacterial foraging-particle swarm algorithm (BFO-PSO), and utilizes the interrelation number R as the classification index with the Least Mean Square Algorithm (LMS) to classify, filter, and extract the effective signal from the decomposed signal. Then, the extracted signals are detected by BPNN, and the training data are utilized to predict the residual current signals. Simulation and experimental data demonstrate that the proposed algorithm exhibits strong robustness and high detection accuracy. With an ambient noise of 10dB, the signal-to-noise ratio is 16.3108dB, the RMSE is 0.4359, and the goodness-of-fit is 0.9627 after processing by the algorithm presented in this paper, which are superior to the Variational Modal Decomposition-Long Short-Term Memory (VMD-LSTM) and Normalized-Least Mean Square (N-LMS) detection methods. The results were also statistically analyzed in conjunction with the Kolmogorov-Smirnov test, which demonstrated significance at the experimental data level, indicating the high accuracy of the algorithms presented in this paper and providing a certain reference for new residual current protection devices for biological body electrocution.