Analytical Chemistry最新文献

{"title":"Hydrogen-Bonded Organic Framework Nanozyme with Multi-Enzyme Activity for Chemiluminescence Sensing of Acetylcholinesterase and Screening Its Inhibitors","authors":"Yunping Lai, Shuangshuang He, Yuanying Chen, Tianran Lin, Li Hou, Shulin Zhao","doi":"10.1021/acs.analchem.4c06917","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c06917","url":null,"abstract":"Strong and persistent chemiluminescence (CL) is essential for enhancing the detection accuracy and reproducibility of CL-based analytical methods. In this study, we explored the synergistic effects of amino groups present on the surface of hydrogen-bonded organic framework (HOF-PyTTA, where PyTTA is denoted as 4,4′,4″,4‴-(pyrene-1,3,6,8-tetrayl)tetraaniline) materials and <i>N</i>-(4-aminobutyl-<i>N</i>-ethylisoluminol) (ABEI) for reducing gold nanoparticles (AuNPs) on the surface of HOFs. Additionally, we utilized the substantial specific surface area and abundant amino groups of HOFs to sequester Co²⁺ ions, resulting in the synthesis of HAACo material. The resulting HAACo exhibited remarkable peroxidase, oxidase, and catalase mimetic activities, enabling the luminol-H₂O₂ chemiluminescence system to maintain a glow-type CL phenomenon for more than 1 h. Subsequently, we developed a CL point-of-care testing (POCT) sensor that integrated the CL characteristics of HAACo with smartphone technology and 3D printing for the determination of acetylcholinesterase (AChE) activity in serum samples, as well as the screening for AChE inhibitors. The sensor demonstrated a linear detection range for AChE activity from 0.001 to 40 mU mL^–1, with a detection limit of 0.00057 mU mL^–1. The calculated IC₅₀ for the AChE inhibitor tacrine was found to be 21.9 nmol L^–1, indicating good selectivity and stability of the sensor. This work not only expands the applications of glow-type CL in biosensing but also enriches the utilization of HOF materials in analytical chemistry, paving the way for the development of multifunctional HOF-based materials for future applications.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"34 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143813906","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Strategies for Acid and Amine Cross-linking and Labeling for Protein Structural Characterization Using Mass Spectrometry","authors":"Ashlyn N. Dollar, Ian K. Webb","doi":"10.1021/acs.analchem.4c03978","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c03978","url":null,"abstract":"Cross-linking and covalent labeling are common tools for protein structural elucidation by mass spectrometry. However, despite the importance of electrostatic interactions in proteins, there are not many tools for probing both acids and amines. Therefore, we introduce novel solution-phase amine-to-acid cross-linkers and single reagent amine and acid covalent labels utilizing carbodiimide chemistry. Cross-linking and labeling sites were determined using top-down and enzymatic digestion approaches. Ubiquitin was chosen as a model protein for isotopically labeled ¹³C–¹⁵N-glycine cross-linking/covalent labeling and glycyl-<span>l</span>-proline cross-linking/covalent labeling with top-down mass spectrometry as a proof of concept. Alpha synuclein, an intrinsically disordered protein that plays a role in multiple neurological disorders, was also cross-linked/covalent labeled with these methods/reagents. We expect that these structural characterization techniques will play a role in gaining insight into functionally and pathologically important structural tendencies of disordered proteins.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"39 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143813905","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Towards Generalizable In Silico Predictions of Differential Ion Mobility Using Machine Learning and Customized Fingerprint Engineering","authors":"Cailum M. K. Stienstra, Christopher R. M. Ryan, Daniel Demczuk, Justine R. Bissonnette, Anish Arjuna, J. Larry Campbell, W. Scott Hopkins","doi":"10.1021/acs.analchem.5c00737","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00737","url":null,"abstract":"Differential mobility spectrometry (DMS), a tool for separating chemically similar species (including isomers), is readily coupled to mass spectrometry to improve selectivity in analytical workflows. DMS dispersion curves, which describe the dynamic mobility experienced by an ion in a gaseous environment, show the maximum ion transmission for an analyte through the DMS instrument as a function of the separation voltage (SV) and compensation voltage (CV) conditions. To date, there exists no fast, general prediction tool for the dispersion behavior of ions. Here, we demonstrate a machine learning (ML) model that achieves generalized dispersion prediction using an <i>in silico</i> feature addition pipeline. We employ a data set containing 1141 dispersion curve measurements of anions and cations recorded in pure N₂ environments and in N₂ environments doped with 1.5% methanol (MeOH). Our feature addition pipeline can compute 1591 RDKit and Mordred descriptors using only SMILES codes, which are then normalized to sampled molecular distributions (<i>n</i> = 100 000) using cumulative density functions (CDFs). This tool can be thought of as a “learned” feature fingerprint generation pipeline, which could be applied to almost any molecular (bio)cheminformatics tasks. Our best performing model, which for the first time considers solvent-modified environments, has a mean absolute error (MAE) of 2.1 ± 0.2 V for dispersion curve prediction, a significant improvement over the previous state-of-the-art work. We use explainability techniques (<i>e.g.</i>, SHAP analysis) to show that this feature addition pipeline is a semideterministic process for feature sets, and we discuss “best practices” to understand feature sets and maximize model performance. We expect that this tool could be used for prescreening to accelerate or even automate the use of DMS in complex analytical workflows (<i>e.g.</i>, 2D LC×DMS separation) and perform automated identification of transmission windows and increase the “self-driving” potential of the instrument. We make our models available as a free and accessible tool at https://github.com/HopkinsLaboratory/DispersionCurveGUI.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"32 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143814036","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optical Photothermal Infrared Imaging Using Metabolic Probes in Biological Systems","authors":"Sydney O. Shuster, Anna E. Curtis, Caitlin M. Davis","doi":"10.1021/acs.analchem.4c03752","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c03752","url":null,"abstract":"Infrared spectroscopy is a powerful tool for identifying biomolecules. In biological systems, infrared spectra provide information on structure, reaction mechanisms, and conformational change of biomolecules. However, the promise of applying infrared imaging to biological systems has been hampered by low spatial resolution and the overwhelming water background arising from the aqueous nature of in-cell and <i>in vivo</i> work. Recently, optical photothermal infrared microscopy (OPTIR) has overcome these barriers and achieved both spatially and spectrally resolved images of live cells and organisms. Here, we determine the most effective modes of collection on a commercial OPTIR microscope for work in biological samples. We examine three cell lines (Huh-7, differentiated 3T3-L1, and U2OS) and three organisms (<i>Escherichia coli</i>, tardigrades, and zebrafish). Our results suggest that the information provided by multifrequency imaging is comparable to hyperspectral imaging while reducing imaging times 20-fold. We also explore the utility of IR active probes for OPTIR using global and site-specific noncanonical azide containing amino acid probes of proteins. We find that photoreactive IR probes are not compatible with OPTIR. We demonstrate live imaging of cells in buffers with water. ¹³C glucose metabolism monitored in live fat cells and <i>E. coli</i> highlights that the same probe may be used in different pathways. Further, we demonstrate that some drugs (e.g., neratinib) have IR active moieties that can be imaged by OPTIR. Our findings illustrate the versatility of OPTIR and, together, provide a direction for future dynamic imaging of living cells and organisms.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"28 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143813907","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Deep Learning-Assisted Multiplexed Electrochemical Fingerprinting for Chinese Tea Identification","authors":"Yuyu Tan, Mengli Luo, Chao Xu, Jiaoli Wang, Xinlin Wang, Lelun Jiang, Jian Yang","doi":"10.1021/acs.analchem.4c06651","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c06651","url":null,"abstract":"Selectively differential identification of natural components with similar chemical structures in complex matrices is still a challenging task by conventional analytical strategies. Herein, we developed a landmark (DaXing airport)-inspired laser engraving sensor array that combined multiplex electrochemical fingerprinting technology with a one-dimensional convolutional neural network (1D-CNN) for rapidly precise detection of three tea polyphenols and the differentiation of 24 distinct types of Chinese teas. This sensing strategy employs a diverse array of three different working electrode configurations as a multivariate sensor (bare electrode, nanoenzyme electrode, and bioenzyme electrode), generating distinct electrochemical fingerprints in complex samples. By utilizing a self-designed 1D-CNN algorithm for feature extraction, the identification of electrochemical fingerprints is significantly improved, thereby enhancing the predictive accuracy for tea polyphenols and Chinese teas. This platform successfully achieves detection of three tea polyphenols, distinguishing six Chinese tea series and 24 tea varieties with accuracy rates of 98.84 and 97.68%, respectively. Notably, the deep learning-assisted multiplexed electrochemical fingerprinting technique achieves better accuracy for tea identification compared with other representative machine learning methods. This advancement offers a rapid and reliable approach to enhancing the development of identification and authentication processes for agricultural products.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"108 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143814335","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dual-Mode Sensor Based on a Single-Atom Cobalt Catalyst for Simultaneous Electrochemical and Colorimetric Detection of Bioactive Small Molecules","authors":"Jin Jin, Jing-Jing Wei, Zhi-Yang Gu, Yong-Xin Zhang, Zhuang-Yi Lu, Qi-Wei Zhang, Jing-Jing Wan, Guo-Yue Shi, Xing-Hua Xia, Yi Shi","doi":"10.1021/acs.analchem.5c01216","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c01216","url":null,"abstract":"The design of single-atom catalysts with dual functions has emerged as a promising strategy for developing high-performance sensing platforms. Herein, we reported a facile host–guest strategy for synthesizing an atomically dispersed Co catalyst (Co–N–C), where Co atoms were uniformly anchored on the N-doped carbon matrix derived from zeolitic imidazolate framework-8. The as-prepared Co–N–C exhibits both excellent electrochemical sensing and peroxidase-like colorimetric activities toward the detection of three important bioactive small molecules, ascorbic acid (AA), dopamine (DA), and uric acid (UA). The electrochemical sensor demonstrated ultrahigh sensitivity with detection limits of 4.83, 1.36, and 0.371 μM for AA, DA, and UA, respectively, along with outstanding selectivity against common interferents and stable performance. Meanwhile, the colorimetric method also showed analytical performance with detection limits of 2.24 μM (AA), 3.09 μM (DA), and 2.97 μM (UA). The results indicate that the electronic modulation of Co through precise nitrogen coordination enhances the affinity of Co–N_<i>x</i> for target reactants, thereby promoting adsorption and electron transfer throughout the reaction. This improves catalytic efficiency and selectivity, establishing Co–N–C with dual-catalytic functionality as a promising material for biosensing applications.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"25 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143806366","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Low-Frequency Optical Signal Enhancement Device Combines CRISPR-Based Assay for Portable S. Pneumoniae Detection","authors":"Chen Yang, Menglu Gao, Yifan Wang, Tao Jiang, Yihan Ma, Xiaotong Si, Youping Deng, Yantong Liu, Minjing Mo, Xuan Xiao, Fubing Wang, Yi Yang","doi":"10.1021/acs.analchem.5c00634","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00634","url":null,"abstract":"Early detection and treatment of <i>Streptococcus pneumoniae</i> (SPN) is crucial for patients. However, since nucleic acid testing relies on large-scale equipment and specialized operators, challenges remain for accurate, fast, and low-cost SPN detection. Here, we present a point-of-care testing (POCT) device for rapid and accurate detection of SPN based on low-frequency optical signal enhancement and cluster of regularly interspaced short palindromic repeats (CRISPR). The spotlight tube enables the enhancement of the fluorescence signal, while the combination of an artificial intelligence-assisted autoexposure algorithm and a homomorphic filtering image processing method improves the signal-to-noise ratio of the fluorescence image, thus realizing highly sensitive detection. Nucleic acid identification is performed using CRISPR-based crRNAs, and fluorescent probes were constructed against the IytA gene of SPN. And they showed high specificity and sensitivity for the IytA gene. This device demonstrated excellent sensitivity in detecting the SPN using the developed CRISPR-based nucleic acid detection strategy. The detection threshold of SPN reached 0.1 fM, and the single detection time of the device was only 40 min. Specificity was validated using clinical samples, and the test showed 100% agreement with quantitative polymerase chain reaction results from clinical samples. This method provides a highly sensitive optical and signal processing device, which, in combination with a novel DNA probe for SPN, provides a novel indicator option for POCT of SPN.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"65 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143813978","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Direct Fluorescence Anisotropy Detection of miRNA Based on Duplex-Specific Nuclease Signal Amplification","authors":"Huilan Wu, Xiaoting Ling, Shoulong Huang, Qiang Zhao, Dapeng Zhang, Hailin Wang","doi":"10.1021/acs.analchem.5c00723","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00723","url":null,"abstract":"The dysregulation of microRNAs (miRNAs) is associated with various diseases, including cancer, so miRNAs are considered a potential biomarker candidate for disease diagnosis and therapy. However, the direct, rapid, sensitive, and specific detection of miRNAs remains quite challenging due to their short length, sequence homology, and low abundance. Herein, we propose a simple and homogeneous fluorescence anisotropy (FA) strategy for the direct and rapid (∼35 min) quantification of miRNA-21 based on duplex-specific nuclease (DSN)-assisted signal amplification. In the presence of target miRNA-21, the complementary single-stranded DNA (ssDNA) probes labeled with a single fluorophore, tetramethylrhodamine (TMR), are specifically hydrolyzed into small fragments by endonuclease DSN upon formation of the DNA/RNA hybrid, which leads to a reduction in FA due to the decrease in molecular size. However, the target miRNA remains intact during the enzymatic digestion process and is released in solution for the next round of binding, hydrolysis, and release for recycling. It is observed that the ssDNA probe labeled with TMR at the 5′-end, in which the fluorophore is nine nucleotides away from the nearest dG base to eliminate/reduce photoinduced electron transfer interaction between TMR and the dG base, exhibits the maximum FA change in response to the target miRNA-21. The change in FA enables the sensitive detection of miRNA-21 ranging from 0.050 to 2.0 nM, with a detection limit of 40 pM. In addition, this amplification strategy exhibits high selectivity and can even discriminate single-base mutations between miRNA family members. We further applied this method to detect miRNA-21 in the extract of various cancer cell lines. Therefore, this method holds great potential for miRNA analysis in tissues or cells, providing valuable information for biomedical research, clinical diagnostics, and therapeutic applications.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"65 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143814336","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fast Imaging of Mitochondria and Efficient Generation of Singlet Oxygen by Red Fluorescent BODIPY Photosensitizers","authors":"Wan-Ying Yao, Jiang-Lin Wang, Jin-Zhao Huang, Meng-Xin Li, Yan Huang, Feng-Lei Jiang","doi":"10.1021/acs.analchem.4c06673","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c06673","url":null,"abstract":"The biomedical applications of BODIPY fluorophores are limited by challenges such as short-wavelength emission, high hydrophobicity, and poor selectivity. To address these issues, two water-soluble red-emitting BODIPY derivatives, namely, PSPyBDP and I-PSPyBDP, were synthesized by conjugating pyridine units to the BODIPY core, followed by the ring-opening reactions with 1,3-propanesulfonate. Notably, PSPyBDP showed fast mitochondrial imaging capability (∼5 min), indicating its potential as an alternative to mitochondria tracker. I-PSPyBDP, with the heavy-atom effect, could effectively produce singlet oxygen (¹O₂) under irradiation at 660 nm in a short time (∼1 min) with a ¹O₂ quantum yield of 0.89. Cytotoxicity assays revealed that the BODIPY derivatives exhibited phototoxicity to HeLa cells while maintaining low dark toxicity. Interestingly, they had low toxicity against normal COS-7 cells. Confocal imaging and flow cytometry confirmed that the BODIPY derivatives could increase intracellular reactive oxygen species (ROS), reduce mitochondrial membrane potential, and induce apoptosis upon irradiation. These findings suggest their promising application in photodynamic therapy (PDT) for tumors.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"6 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143813910","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Self-Calibrated Stimulated Raman Scattering Spectroscopy for Rapid Cholangiocarcinoma Diagnosis","authors":"Jin Guo, Lingfu Zhang, Qiaozhi Yu, Yafeng Qi, Haojie Zhang, Lan Zhang, Chunhui Yuan, Muxing Li, Hanqing Xiong","doi":"10.1021/acs.analchem.5c00480","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00480","url":null,"abstract":"Cholangiocarcinoma (CCA) is an aggressive malignancy with poor clinical outcomes. The current “gold standard” diagnostic approach, endoscopic retrograde cholangiopancreatography (ERCP)-obtained biopsy, has a relatively low sensitivity (i.e., ∼50%). Here, we developed a bile-based diagnostic system using transient stimulated Raman scattering (T-SRS). Except for the tolerance to autofluorescence inherited from traditional SRS spectroscopy, T-SRS features quantum-limit spectral line shapes and is further improved with self-calibration ability in this research. These advantages make the acquired Raman spectra insensitive to the drifting of the excitation parameters, facilitating long-term reliability. Based on the T-SRS spectra in the C–H stretching region from 76 bile samples accumulated over more than 1 year, we demonstrated high accuracy (i.e., 85 ± 3%) and sensitivity (i.e., 87 ± 9%) for classification between CCA and benign diseases. The T-SRS acquisition only requires ∼9-μL bile samples and features a drastically improved time cost. This study suggests that the self-calibrated T-SRS analysis of the bile sample offers a promising approach for rapid CCA diagnosis.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"60 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143814039","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}