Analyst最新文献

{"title":"Cationic and anionic phenothiazine derivatives: electrochemical behavior and application in DNA sensor development†","authors":"Anastasia N. Malanina, Yury I. Kuzin, Pavel L. Padnya, Alexey N. Ivanov, Ivan I. Stoikov and Gennady A. Evtugyn","doi":"10.1039/D5AN00164A","DOIUrl":"10.1039/D5AN00164A","url":null,"abstract":"<p >The global shift toward personalized medicine and point-of-care testing drives the need for improved analytical performance in sensor technologies. A critical aspect of developing voltammetric sensors lies in identifying novel materials for electrode modification. This study focuses on the electrochemical investigation of two phenothiazine derivatives with distinct terminal functional groups: a cationic compound, 3,7-bis(4-aminophenylamino)phenothiazin-5-ium chloride, and an anionic compound, 3,7-bis(4-carboxyphenylamino)phenothiazin-5-ium chloride. Cyclic voltammetry, electrochemical impedance spectroscopy, quartz crystal microbalance, and scanning electron microscopy were employed to characterize these novel materials. The mutual influence of phenothiazines on voltammetric signals in solutions was analyzed, revealing changes in the number of hydrogen ions transferred when transitioning from an individual cationic derivative solution to a mixed solution with the anionic derivative. Two approaches for modifying glassy carbon electrodes were studied: electropolymerization from a mixture of phenothiazines and consecutive electrodeposition of polymeric films from individual solutions. Morphological and quantitative differences in the resulting electrode films were observed, with the latter method yielding a more uniform and thicker layer of redox-active material. A DNA sensor based on the consecutive electrodeposited films for the detection of doxorubicin was developed. The redox peak currents of the electropolymerized phenothiazine products exhibited a linear response to the logarithm of doxorubicin concentration. The sensor displayed two distinct linear ranges from 0.1 fM to 1 nM and from 1 nM to 1 μM, with a limit of detection calculated from the first range at 0.6 fM. This DNA sensor offers promising applications for advancing point-of-care diagnostics.</p>","PeriodicalId":63,"journal":{"name":"Analyst","volume":" 10","pages":" 2087-2100"},"PeriodicalIF":3.6,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143713219","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Sensitive detection of small extracellular vesicles using a gold nanostar-based SERS assay†","authors":"Cao Hoang Long Ngo, Anastasiia Tukova, Wei Zhang, Simon Chang-Hao Tsao and Yuling Wang","doi":"10.1039/D5AN00110B","DOIUrl":"10.1039/D5AN00110B","url":null,"abstract":"<p >Small extracellular vesicles (sEVs) are lipid bilayer-bound vesicles that carry critical biomarkers for disease detection. However, the inherent heterogeneity and complexity of sEV molecular characteristics pose significant challenges for accurate and comprehensive molecular profiling. Traditional analytical methods, including immunoblotting, enzyme-linked immunosorbent assay (ELISA), and flow cytometry, exhibit several limitations, such as being time-consuming, requiring large sample volumes, and demonstrating relatively low sensitivity. Therefore, there is an urgent need to develop a highly sensitive and specific assay for the reliable detection of sEVs. Surface-enhanced Raman scattering (SERS) assays have emerged as a promising approach for sEV detection, offering advantages including high sensitivity and specificity. In the proposed SERS assay, SERS nanotags – comprising nanoparticles coated with Raman-active molecules and conjugated with antibodies – are employed to label surface-bound molecules on sEVs. This approach facilitates the generation of a high-intensity signal from molecules present in low abundance. Recently, anisotropic nanoparticles, such as star-shaped nanostructures, have garnered interest due to their ability to significantly amplify generated SERS signals for ultra-sensitive biomarker detection. In this study, we explore the application of gold nanostars (AuNSs) as SERS nanotags for the detection of sEVs. In principle, AuNS-based SERS nanotags were used to label the EpCAM protein, which can be found on the surface of cancer cell derived sEVs, and then sEV labelled SERS nanotags were captured by CD9-conjugated magnetic beads to form an immunocomplex, which exhibits a SERS signal. Our results demonstrate that the proposed SERS assay utilizing AuNSs provides high specificity and sensitivity, with a detection limit as low as 2.47 × 10<small>³</small> particles per μL. Furthermore, the assay was tested with spiked plasma samples (cancer cell-derived sEVs spiked into healthy plasma), showing that its specificity remains unaffected by the presence of plasma. These findings suggest that the SERS assay incorporating AuNSs holds significant promise as an effective and reliable detection method for potential clinical applications.</p>","PeriodicalId":63,"journal":{"name":"Analyst","volume":" 10","pages":" 2108-2117"},"PeriodicalIF":3.6,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143713220","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Tyramide signal amplification for a highly sensitive multiplex immunoassay based on encoded hydrogel microparticles†","authors":"Jun Hee Choi, Young Hee Kim, Jiwoo Kim, Yong Jun Lim, Min Jung Kim and Ki Wan Bong","doi":"10.1039/D5AN00078E","DOIUrl":"10.1039/D5AN00078E","url":null,"abstract":"<p >Proteins play a crucial role as mediators of immune regulation, homeostasis, and metabolism, making their quantification essential for understanding disease mechanisms in biomedical research and clinical diagnostics. However, conventional methods when used to detect proteins in clinical samples exhibit difficulty in terms of sensitivity, dynamic range, and multiplex capacity. In this study, we developed a highly sensitive multiplex immunoassay based on encoded hydrogel microparticles (MPs) utilizing tyramide signal amplification (TSA). The combination of the large multiplexing capacity of encoded hydrogel microparticles and the signal amplification of tyramide enables a highly sensitive multiplex immunoassay. By employing TSA, we are able to achieve larger detection signals with higher specificity. We effectively decreased the non-specific binding in the hydrogel network by blocking the unreacted acrylate double bonds remaining after the capture antibody-conjugation step and acquired a 3-fold increase in the signal-to-noise ratio. Also, we optimized three parameters mainly affecting the assay sensitivity: the detection antibody concentration, the biotinyl tyramide concentration, and the TSA reaction time. This approach leads to a significant improvement in assay sensitivity, achieving a limit of detection as low as 58 fg mL<small>⁻¹</small>. Compared to the previous method, the assay sensitivity is enhanced 10-fold. In addition, the multiplex capability of the assay is validated by detecting cytokines IL-4, IL-5, IL-6, IL-9, and IL-17, with no observed cross-reactivity. Finally, with enhanced sensitivity, we demonstrate the clinical applicability of our platform by successfully multiplexing these cytokines at concentrations down to several hundreds of fg mL<small>⁻¹</small> within human serum, which could not be detected using previous methods.</p>","PeriodicalId":63,"journal":{"name":"Analyst","volume":" 10","pages":" 2118-2127"},"PeriodicalIF":3.6,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143702960","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Paper and cloth-based microfluidic chips for rapid cysteine detection in deep-sea cold seeps†","authors":"Die Hu, Jiawen Xiang, Jingying Guo, Chao Wang, Ji Qi, Bowei Li, Xiaoyan Wang, Xin Zhang, Lingxin Chen and Xuming Zhuang","doi":"10.1039/D5AN00109A","DOIUrl":"10.1039/D5AN00109A","url":null,"abstract":"<p >In this study, we developed a simple and cost-effective paper and cloth-based microfluidic fluorescence sensing device for the selective and quantitative detection of <small>L</small>-cysteine (<small>L</small>-Cys) in deep-sea water, addressing the need for efficient monitoring of this critical metabolite of deep-sea creatures which acts as a biomarker for tracking these organisms. The device employs gold nanoparticles (Au NPs) immobilized on a cloth-based substrate and rhodamine B (Rh B) molecules. In the presence of <small>L</small>-Cys, strong interactions between <small>L</small>-Cys and the Au NPs release Rh B molecules, restoring fluorescence proportional to the cysteine concentration. The device achieves a low detection limit of 0.52 nM with a dynamic range of 1–100 nM. It demonstrates excellent selectivity over other amino acids, stability over 30 days, and reproducibility. Its practical applicability was confirmed using deep-sea cold seep water samples, yielding recoveries of 98.07%–102.62%. Compared to existing methods, this platform offers enhanced portability, lower cost, and faster response, making it suitable for <em>in situ</em> environmental monitoring.</p>","PeriodicalId":63,"journal":{"name":"Analyst","volume":" 10","pages":" 2066-2073"},"PeriodicalIF":3.6,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143702962","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A DNA concatemer-encoded CRISPR/Cas12a fluorescence sensor for sensitive detection of Pb2+ based on DNAzymes†","authors":"Shaoying He, Wei Lin, Xin Liu, Fei Li, Hong Liang, Huo Xu, Chunhua Lu and Chao Xing","doi":"10.1039/D5AN00189G","DOIUrl":"10.1039/D5AN00189G","url":null,"abstract":"<p >Lead pollution presents a significant threat to ecological systems and human health, underscoring the urgent need for highly sensitive detection methods. Herein, we introduce a novel DNA concatemer-encoded CRISPR/Cas12a fluorescence sensor (MDD-Cas12a) for sensitive detection of Pb<small>²⁺</small> based on DNAzymes. To accomplish this, we designed a substrate strand containing a long DNA concatemer encoding multiple protospacer adjacent motifs (PAMs) and protospacer sequences for activation of the CRISPR/Cas12a system. The DNA concatemer was subsequently anchored to the surface of magnetic beads (MBs) to fabricate a MBs-DNA concatemer nanoprobe. In the presence of Pb<small>²⁺</small>, the DNAzyme structure catalyzes the cleavage of the substrate strand, leading to the release of DNA concatemers. Following magnetic separation, the released DNA concatemers significantly activate the non-specific trans-cleavage activity of the Cas12a/crRNA complex. The fluorescence reporter DNA is then completely cleaved by the activated Cas12a/crRNA complex, and the Pb<small>²⁺</small> concentration in the sample can be quantified by measuring the fluorescence signal. By harnessing the specific recognition capability of DNAzymes for Pb<small>²⁺</small>, the programmability of DNA concatemers, and the self-amplification features of the CRISPR/Cas12a system, the MDD-Cas12a platform demonstrates high sensitivity and specificity for detecting Pb<small>²⁺</small> in milk and lake water samples.</p>","PeriodicalId":63,"journal":{"name":"Analyst","volume":" 9","pages":" 1778-1784"},"PeriodicalIF":3.6,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143702966","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a multiple reaction monitoring (MRM)-based LC-MS/MS method for the quantification of post-translational modifications on histone H3 variants in Arabidopsis thaliana†","authors":"Yajun Hu, Chenxi He, Lei Zhang and Hong Jin","doi":"10.1039/D4AN01563K","DOIUrl":"10.1039/D4AN01563K","url":null,"abstract":"<p > <em>Background</em>: although the canonical histone H3.1 and its variant H3.3 differ by only four amino acids, they exhibit distinct genome-wide binding patterns and regulate different biological pathways. Post-translational modifications (PTMs) on histone tails mediate diverse downstream regulatory processes, raising the question of whether H3.1 and H3.3 harbor variant-specific modifications. However, the minimal amino acid differences between H3.1 and H3.3 make it challenging to distinguish and quantify them using traditional methods. <em>Results</em>: in this study, we developed an integrated multiple reaction monitoring (MRM)-based LC-MS/MS method to accurately differentiate and quantify K27 and K36 modifications on H3.1 and H3.3 in <em>Arabidopsis thaliana</em>. Our findings show that H3.1 contains more K27 methylation marks, associated with gene silencing, whereas H3.3 is enriched in K36 methylation, a mark of active transcription. Additionally, we compared K36 methylation levels in wild-type and SDG8-depleted cells, revealing that the K36 methyltransferase SDG8 shows a strong preference for H3.3 in both <em>in vitro</em> and <em>in vivo</em> assays. By analyzing public datasets, we further identified a strong correlation between H3.3 and the regions where H3K36me3 levels were reduced in <em>sdg8</em> knockout cells. <em>Significance</em>: the MRM-based LC-MS/MS method established in this study provides a reliable and robust tool for the quantification of histone H3.1 and H3.3 PTMs in <em>Arabidopsis thaliana</em>. We demonstrate that the methyltransferase SDG8 shows a strong substrate preference for H3.3. This discovery highlights the importance of histone variant-specific modifications and suggests new avenues for research into their regulatory roles.</p>","PeriodicalId":63,"journal":{"name":"Analyst","volume":" 8","pages":" 1688-1697"},"PeriodicalIF":3.6,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143695769","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"LC-MS-based untargeted metabolomics reveals benzoic acid as a predictive biomarker for embryo implantation potential†","authors":"Xi Chen, Ying Liu, Sheng-Nan Duan, Ping Wang, Yu-Nan Chen, Min Fu, Rong Liang, Xin-Xiang Zhang, Huan Shen, Ying-Lin Zhou and Cheng Shi","doi":"10.1039/D4AN01552E","DOIUrl":"10.1039/D4AN01552E","url":null,"abstract":"<p >Evaluating the quality of embryos and implantation potential is a critical determinant of <em>in vitro</em> fertilization and embryo transfer, and it is also one of the main challenges of assisted reproductive technology. A reliable non-invasive method to choose the best candidate with real implantation potential for transfer from two day-3 embryos with equal morphological quality is still lacking clinically. In this article, a sensitive LC-MS method was developed and metabolomic profiling analysis in a 3-day embryo culture medium was performed. Differential metabolites were analysed in two kinds of commercial culture media, and a total of 66 common metabolites were obtained from 106 independent samples in 5 batches. The relationship between changes in key metabolite, benzoic acid, concentration and the embryo implantation result was discovered. This work improved coverage through conditional optimization, enhanced the reliability of omics data through multi-batch validation, and provided a potential biomarker for evaluating the implantation potential of day-3 embryos.</p>","PeriodicalId":63,"journal":{"name":"Analyst","volume":" 9","pages":" 1816-1822"},"PeriodicalIF":3.6,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143702969","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-sensitivity differential scanning calorimetry using a MEMS thermopile chip for analyzing polymer crystallization†","authors":"Zechun Li, Shaokui Tan, Ming Li, Yuhang Yang, Haozhi Zhang, Xinxin Li and Pengcheng Xu","doi":"10.1039/D5AN00246J","DOIUrl":"10.1039/D5AN00246J","url":null,"abstract":"<p >This paper introduces a high-sensitivity differential scanning calorimetry (DSC) technique based on a MEMS single-crystalline silicon thermopile chip and its application for analyzing the crystallization process of polyamide 6 (PA6) under various thermal processing conditions. The chip integrates 54 pairs of single-crystalline silicon thermocouples beneath a SiN<small>_<em>x</em></small>-suspended film, achieving a temperature responsivity of 31.5 mV per °C and a power responsivity of 147 V W<small>⁻¹</small>. Additionally, the chip's cooling time constant is only 2.4 ms. The non-isothermal experimental results of PA6 suggest that melt-crystallization is suppressed at cooling rates exceeding the critical rate of 50 °C s<small>⁻¹</small>, and cold-crystallization is suppressed at heating rates above the critical rate of 300 °C s<small>⁻¹</small>. Thanks to its high sensitivity, this chip can detect subtle exothermic signals associated with the γ–α phase transition in PA6. The critical heating rate for this phase transition is determined to be 25 °C s<small>⁻¹</small>. Isothermal experimental results show that PA6 undergoes crystallization within 70 °C to 170 °C, with the shortest half-crystallization time of ∼1.1 s at 120 °C. The high-sensitivity DSC technique proposed in this work holds great promise for studying the thermal behaviour of various materials at high heating and cooling rates.</p>","PeriodicalId":63,"journal":{"name":"Analyst","volume":" 11","pages":" 2231-2238"},"PeriodicalIF":3.6,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143695770","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"IS-SCP: enhanced single-cell proteomics using an in situ simplified strategy†","authors":"Zhuo Yang, Yi-Rong Jiang, Qin-Qin Xu, Jian-Bo Chen, Jian-Zhang Pan, Xin Di and Qun Fang","doi":"10.1039/D5AN00149H","DOIUrl":"10.1039/D5AN00149H","url":null,"abstract":"<p >Advances in single-cell proteomics have enabled the investigation of the distinctive proteomic makeup of individual cells, significantly impacting biomedical research. However, most existing approaches involve complex sample preparation workflows and are sensitive to potential sample loss, which limits their applicability. In this paper, we reported an advanced workflow for easy-to-use single-cell proteome analysis using an <em>in situ</em> simplified strategy, named “<em>in situ</em> simplified single-cell proteomics (IS-SCP)”. This workflow was developed following a comprehensive evaluation of reagent mix, volume, and reaction conditions, notably including the utilization of a cleavable surfactant, <em>n</em>-decyl-disulfide-β-<small>D</small>-maltoside (DSSM). In comparison to previous workflows that require multiple steps in sample preparation, the IS-SCP workflow simplifies the single-cell proteome pretreatment to a single step of adding single-cell samples into a mixed reagent, which increases the repeatability and depth of single-cell proteome analysis. The IS-SCP workflow was applied to the proteomic analysis of single mammalian tumor cells, specifically HeLa and A549 cells, resulting in the quantification of an average of 3021 and 3289 protein groups, respectively. These results showed the potential of this workflow for investigating cellular heterogeneity at a deep single-cell level.</p>","PeriodicalId":63,"journal":{"name":"Analyst","volume":" 8","pages":" 1679-1687"},"PeriodicalIF":3.6,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143677728","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Serum IgG galactosylation as a potential biomarker for the diagnosis of echinococcosis†","authors":"Zhuoer Lu, Xiaoxiao Feng, Bin Fu, Xiaojin Mo, Ting Zhang, Liming Wei, Zhonghua Li and Haojie Lu","doi":"10.1039/D4AN01578A","DOIUrl":"10.1039/D4AN01578A","url":null,"abstract":"<p >Echinococcosis is a serious and potentially fatal parasitic zoonosis, which can be divided into two subtypes in humans including cystic echinococcosis (CE) and alveolar echinococcosis (AE). It poses a great threat to patients’ lives, making timely diagnosis and subtype discrimination crucial. AE is easily confused with hepatocellular carcinoma (HCC) due to their highly similar features, so differential diagnosis is also imperative. In this work, the galactosylation level of serum IgG was analyzed by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) in a cohort comprising patients, including 100 diagnosed with CE, 27 with AE and 29 with HCC. The relative quantification of IgG digalactosylated (G2), monogalactosylated (G1), and agalactosylated (G0) <em>N</em>-glycans with the formula G0/(G1 + G2 × 2) (IgG Gal-ratio) was obtained and found to effectively distinguish between echinococcosis patients, CE and AE patients, and healthy controls, respectively. Meanwhile, the IgG Gal-ratio was evidently related to different types of CE (from CE1 to CE5) and the follow-up CE disease progress. Furthermore, the IgG Gal-ratio shows the potential differential diagnosis of AE and HCC. Thus, the results demonstrate that the IgG Gal-ratio has the potential to be a biomarker for diagnosis and discrimination of echinococcosis, which also needs to be verified in further studies.</p>","PeriodicalId":63,"journal":{"name":"Analyst","volume":" 10","pages":" 2058-2065"},"PeriodicalIF":3.6,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143677880","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}