Analysis & sensing最新文献

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The Lucigenin Assay: Measuring Anion Transport in Lipid Vesicles** 褐藻素测定法:测量脂质囊泡中的阴离子转运**
Analysis & sensing Pub Date : 2023-08-07 DOI: 10.1002/anse.202300044
Matúš Chvojka, Anurag Singh, Alessio Cataldo, Dr. Aaron Torres-Huerta, Dr. Marcin Konopka, Prof. Vladimír Šindelář, Dr. Hennie Valkenier
{"title":"The Lucigenin Assay: Measuring Anion Transport in Lipid Vesicles**","authors":"Matúš Chvojka,&nbsp;Anurag Singh,&nbsp;Alessio Cataldo,&nbsp;Dr. Aaron Torres-Huerta,&nbsp;Dr. Marcin Konopka,&nbsp;Prof. Vladimír Šindelář,&nbsp;Dr. Hennie Valkenier","doi":"10.1002/anse.202300044","DOIUrl":"10.1002/anse.202300044","url":null,"abstract":"<p>Synthetic anion transporters are developed to transport anions across lipid membranes with the long-term perspective of biological applications. The lucigenin assay is a popular tool to study their transport of chloride and other anions in liposomes. It relies on the quenching of the fluorescence of encapsulated lucigenin by anions, which can be monitored by fluorescence spectroscopy. This article provides a tutorial introduction to the practical use and understanding of the lucigenin assay. It describes in detail how to use this assay to monitor chloride/nitrate antiport in liposomes, process and interpret the data, and solve common issues. Variations of the assay enabling the investigation of the transport of other anions and transport mechanisms are discussed. Furthermore, a zwitterionic analogue of lucigenin is demonstrated to have advantages for use in experiments over longer time scales, as it does not leak out of the liposomes, or when studying chloride uniport, as it avoids interference from antiport with nitrate that is present in commercial lucigenin.</p>","PeriodicalId":72192,"journal":{"name":"Analysis & sensing","volume":"4 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"115655622","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
A Glutathione-Responsive Luminescence Sensor Based on Dual-Emissive Persistent Luminescent Nanoparticles for Ratiometric Tumor Imaging 基于双发光持久性发光纳米粒子的谷胱甘肽响应型发光传感器,用于肿瘤比率成像
Analysis & sensing Pub Date : 2023-07-23 DOI: 10.1002/anse.202300045
Xue-Mei Gao, Tian-Yue Gu, Ke-Lin Chen, Dr. Xu Zhao, Prof. Xiu-Ping Yan
{"title":"A Glutathione-Responsive Luminescence Sensor Based on Dual-Emissive Persistent Luminescent Nanoparticles for Ratiometric Tumor Imaging","authors":"Xue-Mei Gao,&nbsp;Tian-Yue Gu,&nbsp;Ke-Lin Chen,&nbsp;Dr. Xu Zhao,&nbsp;Prof. Xiu-Ping Yan","doi":"10.1002/anse.202300045","DOIUrl":"10.1002/anse.202300045","url":null,"abstract":"<p>Persistent luminescent nanoparticles (PLNP) have outstanding advantages in low-background imaging. However, avoiding exogenous interference and false positive signals to achieve precise imaging is still a problem. In addition, it is also a great challenge to intelligently control the size of PLNP. Herein, we report an intelligent luminescence ratiometric sensor based on ultra-small dual-emissive PLNP for precise tumor-targeted imaging. ZnGa<sub>2</sub>O<sub>4</sub>:Cr PLNP with a particle size of c.a. 5–10 nm and two emission peaks at 708 nm and 501 nm were firstly synthesized by thermal decomposition method along with systematical controlling the amount of chromium. [email protected] sensor was further constructed with a constant ratio (<i>I</i><sub>708</sub>/<i>I</i><sub>501</sub>) which is not interfered by exogenous factors such as detection time window and probe concentration. However, high concentrations of glutathione in the tumor microenvironment can specifically trigger the change of <i>I</i><sub>708</sub>/<i>I</i><sub>501</sub> and cause the sensor to self-assemble into clusters at tumor site, thus realizing long-term retention and specificity imaging.</p>","PeriodicalId":72192,"journal":{"name":"Analysis & sensing","volume":"4 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"133036572","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Morphological Transformation of Self-Assembled Peptide Nanostructures for Bioimaging Applications 用于生物成像应用的自组装多肽纳米结构的形态转变
Analysis & sensing Pub Date : 2023-07-12 DOI: 10.1002/anse.202300039
Ying Xu, Qiaochu Jiang, Dr. Xianbao Sun, Prof. Gaolin Liang
{"title":"Morphological Transformation of Self-Assembled Peptide Nanostructures for Bioimaging Applications","authors":"Ying Xu,&nbsp;Qiaochu Jiang,&nbsp;Dr. Xianbao Sun,&nbsp;Prof. Gaolin Liang","doi":"10.1002/anse.202300039","DOIUrl":"10.1002/anse.202300039","url":null,"abstract":"<p>Stimuli-triggered in-situ morphological transformation of peptide nanomaterials may enhance the on-site accumulation and retention of the imaging agent cargos in the stimuli-rich regions, thus enabling precise, sensitive, and prolonged imaging of diseases. Moreover, this strategy permits the co-delivery of contrast agents and drugs with the smart “turn-on” ability, allowing for efficient disease theranostics. In light of the significance of this strategy in designing smart biomedical peptide materials, which remains scarcely reviewed in recent years, we herein provide this review. We summarize the bioimaging applications (i. e., fluorescence imaging, magnetic resonance imaging, and photoacoustic imaging) of these smart morphological transformation-based peptide materials, and highlight the remarkable breakthroughs. Besides, challenges to be addressed in this field are discussed.</p>","PeriodicalId":72192,"journal":{"name":"Analysis & sensing","volume":"4 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"116802178","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Arylamino-substituted Rhodamine as a Fluorogenic Molecular Rotor for the Wash-free Imaging of Non-catalytic Proteins in Live Cells 芳基氨基取代的罗丹明作为荧光分子旋转剂,用于活细胞中非催化蛋白质的免洗成像
Analysis & sensing Pub Date : 2023-07-11 DOI: 10.1002/anse.202300037
Huiling Tang, Xia Yuan, Yefeng Chen, Dr. Yuyao Li, Prof. Dr. Xiaoyong Xu, Prof. Dr. Hexin Xie
{"title":"Arylamino-substituted Rhodamine as a Fluorogenic Molecular Rotor for the Wash-free Imaging of Non-catalytic Proteins in Live Cells","authors":"Huiling Tang,&nbsp;Xia Yuan,&nbsp;Yefeng Chen,&nbsp;Dr. Yuyao Li,&nbsp;Prof. Dr. Xiaoyong Xu,&nbsp;Prof. Dr. Hexin Xie","doi":"10.1002/anse.202300037","DOIUrl":"10.1002/anse.202300037","url":null,"abstract":"<p>Fluorescent probes are valuable tools to visualize non-catalytic proteins in live cells. Currently, the majority of imaging reagents for non-catalytic proteins are based on “always-on” fluorophores and the use of these reagents usually necessitate a wash step to remove unbounded fluorophores before microscope imaging. Herein, we report the use of arylamino-substituted rhodamine as an activatable fluorophore for the imaging of non-catalytic protein in live cells. We have shown the induction of an arylamino to structurally rigid rhodamine could significantly reduce the fluorescent emission in aqueous medium but the ligand-directed binding of this molecule to protein receptor could effective restrict its intramolecular motion and thus lead to enhancement in fluorescence intensity at 590 nm over 30-fold. With fluorescent probes based on this fluorophore, we could visualize integrin <i>α<sub>v</sub>β<sub>3</sub></i> and azido-functionalized glycans in living cells with high contrast in a wash-free manner.</p>","PeriodicalId":72192,"journal":{"name":"Analysis & sensing","volume":"4 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"124448192","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Translating Imaging Nanoprobes Design to the Clinic 将成像纳米探针设计应用于临床
Analysis & sensing Pub Date : 2023-07-10 DOI: 10.1002/anse.202300038
Wenbo Sun, Geng Tian, Prof. Dr. Lehui Lu
{"title":"Translating Imaging Nanoprobes Design to the Clinic","authors":"Wenbo Sun,&nbsp;Geng Tian,&nbsp;Prof. Dr. Lehui Lu","doi":"10.1002/anse.202300038","DOIUrl":"https://doi.org/10.1002/anse.202300038","url":null,"abstract":"<p>The use of nanoprobes has revolutionized the way we approach medical diagnosis and treatment. These tiny particles are designed to target specific cells or tissues within the body, allowing doctors to visualize and monitor disease progression in real-time. The paramount consideration in the utilization of nanoprobes is their safety. Unlike traditional contrast agents, which can cause adverse reactions in some patients, these probes are specifically engineered to minimize any potential harm. And it can promote the rapid development of new biological imaging techniques in various fields such as biological structure and functional imaging, disease diagnosis, in situ imaging, and real-time dynamic imaging at the in vivo level. Based on the principles and mechanisms of imaging, this study focuses on the recent applications of nanoprobes for several typical imaging techniques (magnetic resonance imaging, computed tomography imaging, fluorescence imaging, and acoustic imaging) with the aim of providing researchers with a fresh perspective on precise disease diagnosis and treatment through the development of nanoprobes.</p>","PeriodicalId":72192,"journal":{"name":"Analysis & sensing","volume":"3 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"109168887","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Construction of an Oxidative Cleavage-Activated DNAzyme Biosensor for Rapid Detection and Cellular Imaging of the Myeloperoxidase Activity 构建氧化裂解激活 DNA 酶生物传感器,用于快速检测和细胞成像髓过氧化物酶活性
Analysis & sensing Pub Date : 2023-07-06 DOI: 10.1002/anse.202300043
Dr. Ming-Hao Liu, Ning-Ning Zhao, Wan-Tong Yu, Jin-Zhi Zhang, Prof. Zi-Yue Wang, Prof. Chun-Yang Zhang
{"title":"Construction of an Oxidative Cleavage-Activated DNAzyme Biosensor for Rapid Detection and Cellular Imaging of the Myeloperoxidase Activity","authors":"Dr. Ming-Hao Liu,&nbsp;Ning-Ning Zhao,&nbsp;Wan-Tong Yu,&nbsp;Jin-Zhi Zhang,&nbsp;Prof. Zi-Yue Wang,&nbsp;Prof. Chun-Yang Zhang","doi":"10.1002/anse.202300043","DOIUrl":"10.1002/anse.202300043","url":null,"abstract":"<p>Myeloperoxidase (MPO) is a mammalian pro-oxidant protease and it is closely related to severe infections and diverse inflammatory diseases. Rapid and sensitive measurement of MPO activity is essential for anticancer drug discovery and inflammatory research. Herein, we demonstrate the construction of an oxidative cleavage-activated deoxyribozymes (DNAzyme) biosensor for rapid detection and cellular imaging of the MPO activity. When target MPO is present, the phosphorothioate (PS)-modified hairpin probe is site-specifically cleaved by MPO, releasing the intact Mg<sup>2+</sup>-dependent DNAzyme sequences. Subsequently, the activated DNAzyme initiates the cyclic cleavage of the signal probe with the assistance of cofactor Mg<sup>2+</sup>, liberating large numbers of Cy5 molecules. This assay possesses the characteristics of easy operation, low sample consumption, without the requirements of expensive radiolabeling, antibodies, and nanomaterials. Especially, this assay can be performed in one pot under isothermal conditions (37°C) within 60 min. Due to the high efficiency of DNAzyme-based cyclic cleavage reaction and the intrinsic advantages of single-molecule detection, this assay achieves high sensitivity with a limit of detection (LOD) of 2.74×10<sup>−3</sup> ng μL<sup>−1</sup>. It can be applied to screen MPO inhibitors, measure cellular MPO activity at the single-cell level, and image intracellular MPO in living cells, providing a powerful platform for early clinical diagnosis and drug discovery.</p>","PeriodicalId":72192,"journal":{"name":"Analysis & sensing","volume":"4 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"116422564","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
BODIPY-based Fluorescent Indicators for Lipid Droplets 基于 BODIPY 的脂滴荧光指示剂
Analysis & sensing Pub Date : 2023-07-04 DOI: 10.1002/anse.202300049
Dr. Jianping Zhu, Nian Kee Tan, Kai Kikuchi, Dr. Amandeep Kaur, Prof. Elizabeth J. New
{"title":"BODIPY-based Fluorescent Indicators for Lipid Droplets","authors":"Dr. Jianping Zhu,&nbsp;Nian Kee Tan,&nbsp;Kai Kikuchi,&nbsp;Dr. Amandeep Kaur,&nbsp;Prof. Elizabeth J. New","doi":"10.1002/anse.202300049","DOIUrl":"10.1002/anse.202300049","url":null,"abstract":"<p>Lipid droplets are essential for cellular lipid storage, playing critical roles in cellular lipid metabolism. Although lipid droplets have drawn intense research in recent years, much remains to be uncovered about the roles of this organelle in biology. Lipid droplet indicators exhibiting large Stokes shifts and improved brightness are therefore in demand. We report two asymmetric BODIPY derivatives, <b>BoL1</b> and <b>BoL2</b> bearing a benzothiazole group at the 6-position, which led to bathochromic shifts of 62 nm for <b>BoL1</b> and 37 nm for <b>BoL2</b> in fluorescence emission when compared to the reference molecules without the benzothiazole group. The incorporation of the benzothiazole moiety also resulted in a large Stokes shift of 40 nm. <b>BoL1</b> and <b>BoL2</b> have been demonstrated to operate as lipid droplet indicators in both confocal and STED imaging. <b>BoL2</b> showed particularly good cellular retention and was further applied to explore the impact of cellular starvation on the trafficking of lipid droplets.</p>","PeriodicalId":72192,"journal":{"name":"Analysis & sensing","volume":"4 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/anse.202300049","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"124467052","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Cover Feature: Catalyst-Free Three-Component Petasis Reactions Accelerated in Microdroplets: Reaction Optimization and Senstive Detection by Mass Spectrometry (Anal. Sens. 6/2023) 封面专题:微滴加速无催化剂三组分Petasis反应:反应优化和质谱灵敏度检测[j]。参议员6/2023)
Analysis & sensing Pub Date : 2023-06-29 DOI: 10.1002/anse.202300046
Xiaoxiao Jin, Yikang Wu, Jiannan Sun, Prof. Jinhua Liu, Prof. Heyong Cheng
{"title":"Cover Feature: Catalyst-Free Three-Component Petasis Reactions Accelerated in Microdroplets: Reaction Optimization and Senstive Detection by Mass Spectrometry (Anal. Sens. 6/2023)","authors":"Xiaoxiao Jin,&nbsp;Yikang Wu,&nbsp;Jiannan Sun,&nbsp;Prof. Jinhua Liu,&nbsp;Prof. Heyong Cheng","doi":"10.1002/anse.202300046","DOIUrl":"https://doi.org/10.1002/anse.202300046","url":null,"abstract":"<p><b>The cover feature image illustrates</b> two mail devices, drones (representing sprayed microdroplets) and in-person (representing bulk phase), to deliver a letter (representing the Petasis reaction involving aldehydes, arylboronic acids and amines) in sharply different efficiencies. This study supplies not only a mild, efficient and environmentally friendly methodology to constructing aryl amines in organic community but also a useful derivatization strategy for highly sensitive mass spectrometric detection of arylboronic acids and aryl aldehydes. More information can be found in the Research Article by Heyong Cheng, Jiannan Sun, and co-workers.\u0000 <figure>\u0000 <div><picture>\u0000 <source></source></picture><p></p>\u0000 </div>\u0000 </figure>\u0000 </p>","PeriodicalId":72192,"journal":{"name":"Analysis & sensing","volume":"3 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://chemistry-europe.onlinelibrary.wiley.com/doi/epdf/10.1002/anse.202300046","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"109232520","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
Front Cover: A Highly Accurate pH Detection Method for Sweat Analysis using a Printed 96-Microwell Colorimetric Sensor Array (Anal. Sens. 5/2023) 封面:使用印刷的96微孔比色传感器阵列进行汗液分析的高精度pH检测方法(Anal.Sens.5/2023)
Analysis & sensing Pub Date : 2023-06-07 DOI: 10.1002/anse.202300035
Dr. Yui Sasaki, Xiaojun Lyu, Prof. Tsuyoshi Minami
{"title":"Front Cover: A Highly Accurate pH Detection Method for Sweat Analysis using a Printed 96-Microwell Colorimetric Sensor Array (Anal. Sens. 5/2023)","authors":"Dr. Yui Sasaki,&nbsp;Xiaojun Lyu,&nbsp;Prof. Tsuyoshi Minami","doi":"10.1002/anse.202300035","DOIUrl":"https://doi.org/10.1002/anse.202300035","url":null,"abstract":"<p><b>The cover picture shows</b> a printed 96-microwell paper-based sensor array depicting various color patterns upon changing pH conditions, which enables the prediction of pH values below the decimal point in human sweat by imaging analysis and pattern recognition techniques. More information can be found in the Research Article by Yui Sasaki, Xiaojun Lyu, and Tsuyoshi Minami .\u0000 <figure>\u0000 <div><picture>\u0000 <source></source></picture><p></p>\u0000 </div>\u0000 </figure>\u0000 </p>","PeriodicalId":72192,"journal":{"name":"Analysis & sensing","volume":"3 5","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-06-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/anse.202300035","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50123924","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
A Highly Accurate pH Detection Method for Sweat Analysis using a Printed 96-Microwell Colorimetric Sensor Array 使用印刷96微孔比色传感器阵列进行汗液分析的高精度pH检测方法
Analysis & sensing Pub Date : 2023-06-07 DOI: 10.1002/anse.202300036
Dr. Yui Sasaki, Xiaojun Lyu, Prof. Tsuyoshi Minami
{"title":"A Highly Accurate pH Detection Method for Sweat Analysis using a Printed 96-Microwell Colorimetric Sensor Array","authors":"Dr. Yui Sasaki,&nbsp;Xiaojun Lyu,&nbsp;Prof. Tsuyoshi Minami","doi":"10.1002/anse.202300036","DOIUrl":"https://doi.org/10.1002/anse.202300036","url":null,"abstract":"<p>Invited for this month's cover are is the collaborating group of Prof. Minami at The University of Tokyo. The cover picture shows a printed 96-microwell paper-based colorimetric sensor array to detect pH changes below the decimal point in human sweat. More information can be found in the Research Article by Yui Sasaki, Xiaojun Lyu, and Tsuyoshi Minami.</p>","PeriodicalId":72192,"journal":{"name":"Analysis & sensing","volume":"3 5","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-06-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50123923","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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