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A versatile biosensing platform coupling CRISPR-Cas12a and aptamers for detection of diverse analytes

Science Bulletin 2021年第1期66卷 69-77,M0004页

作者： Xiangxiang Zhao Shanshan Li Guang Liu Zhong Wang Zhiheng Yang Quanwei Zhang Mindong Liang Jiakun Liu Zilong Li Yaojun Tong Guoliang Zhu Xinye Wang Lan Jiang Weishan Wang Gao-Yi Tan Lixin Zhang State Key Laboratory of Bioreactor Engineering and School of BiotechnologyEast China University of Science and Technology(ECUST)Shanghai 200237China State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijing 100193China State Key Laboratory of Microbial Resources Institute of MicrobiologyChinese Academy of SciencesBeijing 100101China Department of Urology Sixth People’s Hospital South Campus Affiliated to Shanghai Jiao Tong UniversityShanghai 201499China Shenzhen Institute of Synthetic Biology Shenzhen Institutes of Advanced TechnologyChinese Academy of SciencesShenzhen 518055China The Novo Nordisk Foundation Center for Biosustainability Technical University of DenmarkKgs Lyngby 2800Denmark University of Chinese Academy of Sciences Beijing 100049China

Rapid and sensitive detection of various analytes is in high *** from its application in genome editing,CRISPR-Cas also shows promises in nucleic acid detection *** further exploit the potential of CRISPR-Cas for detection of diverse analytes,we present a versatile biosensing platform that couples the excellent affinity of aptamers for broad-range analytes with the collateral single-strand DNA cleavage activity of CRISPR-Cas12 *** demonstrated that the biosensors developed by this platform can be used to detect protein and small molecule in human serum with a complicated background,i.e.,the tumor marker alpha fetoprotein and cocaine with the detection limits of 0.07 fmol/L and 0.34 lmol/L,respectively,highlighting the advantages of simplicity,sensitivity,short detection time,and low cost compared with the state-of-the-art biosensing ***,this biosensing platform with plug-and-play design show great potential in the detection of diverse analytes.

关键词： CRISPR–Cas12a aptamer Biosensing platform Diverse analyte Alpha fetoprotein detection Cocaine detection

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核酸适体修饰纳米金共振散射光谱探针快速检测痕量Pb^(2+)

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化学学报 2010年第4期68卷 339-344页

作者：凌绍明范燕燕蒋治良温桂清刘庆业梁爱惠广西师范大学环境与资源学院广西环境工程与保护评价重点实验室桂林541004 百色学院化学与生命科学系百色533000

以柠檬酸三钠做稳定剂,用硼氢化钠还原氯金酸制备了粒径为5nm的纳米金.用铅离子核酸适体aptamer保护纳米金获得了检测铅离子的适体纳米金(aptamer-NG)共振散射光谱探针.在pH7.0的Na2HPO4-NaH2PO4缓冲溶液中及30mmol·L-1NaCl存在下,apta... 详细信息

以柠檬酸三钠做稳定剂,用硼氢化钠还原氯金酸制备了粒径为5nm的纳米金.用铅离子核酸适体aptamer保护纳米金获得了检测铅离子的适体纳米金(aptamer-NG)共振散射光谱探针.在pH7.0的Na2HPO4-NaH2PO4缓冲溶液中及30mmol·L-1NaCl存在下,aptamer-NG稳定而不聚集.Pb2+可与该探针中的aptamer形成非常稳定的G-四分体结构,并释放出纳米金.在NaCl作用下纳米金聚集形成较大的微粒,导致552nm处共振散射峰强度增大.Pb2+浓度在0.07～42nmol·L-1范围内与552nm处共振散射强度增大值ΔI成线性关系,其回归方程为ΔI=12.0c+9.2,线性相关系数为0.9965,方法检出限为0.03nmol·L-1Pb2+.该方法用于水样中铅离子检测,结果与石墨炉原子吸收光谱法结果一致.

关键词： Pb2+ aptamer 纳米金共振散射光谱探针

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与细胞因子特异结合的寡聚核苷酸适配子及其在诊断和治疗中的应用

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生物工程学报 2004年第4期20卷 627-632页

作者：严馨蕊高绪文张智清中国疾病预防控制中心病毒病预防控制所病毒基因工程国家重点实验室北京100052

寡聚核苷酸适配子 (aptamer)是用指数富集式配基系统进化方法 (SELEX )筛选出的寡聚核苷酸 ,它能与靶分子特异性结合 ,具有识别和抑制靶物质生物学活性的作用。将体外筛选到的寡聚核苷酸适配子作为在动物或人体内应用的药剂 ,还需要进... 详细信息

寡聚核苷酸适配子 (aptamer)是用指数富集式配基系统进化方法 (SELEX )筛选出的寡聚核苷酸 ,它能与靶分子特异性结合 ,具有识别和抑制靶物质生物学活性的作用。将体外筛选到的寡聚核苷酸适配子作为在动物或人体内应用的药剂 ,还需要进行化学修饰来提高它的生物利用度和在血浆中的稳定性。 2 氟、2′ 烷氧基或 2′ 氨基修饰可以提高适配子的稳定性 ,使适配子的体外半衰期延长 ;5′端交联一个高分子量的PEG分子或脂质体分子 ,可以使它的血浆清除率由 1小时提高到几小时至 1天。修饰后仍保持生物学活性的适配子可用于治疗相应靶细胞因子引起的疾病。目前 ,国内外已经筛选到了十几种细胞因子的适配子 ,其中血管内皮生长因子已经用于临床疾病的治疗。除了用于临床治疗外 ,适配子还可以用于细胞因子的诊断 ,凡是涉及抗体的诊断领域 ,几乎都可以用寡聚核苷酸适配子代替。应用大规模机械化筛选技术 ,可以在短期内筛选到大量的高特异性、高亲和力适配子。

关键词： SELEX aptamer 细胞因子血管内皮生长因子寡聚核苷酸

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Molecular recognition of live methicillin-resistant staphylococcus aureus cells using DNA aptamers

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World Journal of Translational Medicine 2013年第3期2卷 67-74页

作者： Diane Turek Dimitri Van Simaeys Judith Johnson Ismail Ocsoy Weihong Tan Shands Cancer and Genetic Research Center Department of Physiology and Functional Genomics University of Florida Gainesville FL 32611 United States Emerging Pathogens Institute University of Florida Gainesville FL 32611 United States Department of Chemistry Center for Research at Bio/Nano Interface University of Florida Gainesville FL 32611 United States

AIM: To generate DNA-aptamers binding to Methicillinresistant Staphylococcus aureus(MRSA).METHODS: The Cell-Systematic Evolution of Ligands by Exponential Enrichment(SELEX) technology was used to run the selection against MRSA bacteria and develop target-specific aptamers. MRSA bacteria were targeted while Enterococcus faecalis bacteria were used for counter selection during that process. Binding assays to determine the right aptamer candidates as well as binding assays on clinical samples were performed through flow cytometry and analyzed using the FlowJ o software. The characterization of the aptamers was done by determination of their Kd values and determined by analysis of flow data at different aptamer concentration using Sigma Plot. Finally, the recognitionof the complex Gold-nanoparticle-aptamer to the bacteria cells was observed using transmission electron microscopy(TEM).RESULTS: During the cell-SELEX selection process, 17 rounds were necessary to generate enrichment of the pool. While the selection was run using fixed cells, it was shown that the binding of the pools with live cells was giving similar results. After sequencing and analysis of the two last pools, four sequences were identified to be aptamer candidates. The characterization of those aptamers showed that based on their Kd values, DTMRSA4 presented the best binding with a Kd value of 94.61 ± 18.82 nmol/L. A total of ten clinical samples of MRSA, S. aureus and Enterococcus faecalis were obtained to test those aptamers and determine their binding on a panel of samples. DTMRSA1 and DTMRSA3 showed the best results regarding their specificity to MRSA, DTMRSA1 being the most specific of all. Finally, those aptamers were coupled with gold-nanoparticle and their binding to MRSA cells was visualized through TEM showing that adduction of nanoparticles on the aptamers did not change their binding ***: A total of four aptamers that bind to MRSA were obtained with Kd values ranking from 94

关键词： aptamer Methicillin-resistant Staphylococcus aureus Gram-positve bacteria Cell recognition

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Nucleic Acid aptamers as Potential Therapeutic and Diagnostic Agents for Lymphoma

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Journal of Cancer Therapy 2013年第4期4卷 872-890页

作者： Ka-To Shum Jiehua Zhou John J. Rossi Department of Molecular and Cellular Biology Beckman Research Institute of City of Hope Duarte CA USA Irell and Manella Graduate School of Biological Sciences Beckman Research Institute of City of Hope Duarte CA USA

Lymphomas are cancers that arise from white blood cells and usually present as solid tumors. Treatment of lymphoma often involves chemotherapy, and can also include radiotherapy and/or bone marrow transplantation. There is an unquestioned need for more effective therapies and diagnostic tool for lymphoma. aptamers are single stranded DNA or RNA oligonucleotides whose three-dimensional structures are dictated by their sequences. The immense diversity in function and structure of nucleic acids enable numerous aptamers to be generated through an iterative in vitro selection technique known as Systematic Evolution of Ligands by EXponential enrichment (SELEX). aptamers have several biochemical properties that make them attractive tools for use as potential diagnostic and pharmacologic agents. Isolated aptamers may directly inhibit the function of target proteins, or they can also be formulated for use as delivery agents for other therapeutic or imaging cargoes. More complex aptamer identification methods, using whole cancer cells (Cell-SELEX), may identify novel targets and aptamers to affect them. This review focuses on recent advances in the use of nucleic acid aptamers as diagnostic and therapeutic agents and as targeted delivery carriers that are relevant to lymphoma. Some representative examples are also discussed.

关键词： Lymphoma Nucleic Acid aptamer SELEX Cell-Type Specific Drug Delivery Biosensor Nanotechnology

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Development of HBsAg-Binding aptamers that bind HepG2.2.15 cells via HBV surface antigen

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Virologica Sinica 2010年第1期25卷 27-35页

作者： Jia LIU Yan YANG Bin HU Zhi-yong MA Hong-ping HUANG Yuan YU Shen-pei LIU Meng-ji LU Dong-liang YANG Division of Clinical Immunology Tongji Hospital Tongji Medical College Huazhong University of Science and Technology Wuhan 430030 China Experimental Medicine Center Tongji Hospital Tongji Medical College Huazhong University of Science and Technology Wuhan 430030 China Department of Microbiology Tongji Medical College Huazhong University of Science and Technology Wuhan 430030 China Institute of Virology Medical School of Duisburg-Essen University Essen 45122 Germany

Hepatitis B virus surface antigen （HBsAg）, a specific antigen on the membrane of Hepatitis B virus （HBV）-infected cells, provides a perfect target for therapeutic drugs. The development of reagents with high affinity and specificity to the HBsAg is of great significance to the early-stage diagnosis and treatment of HBV infection. Herein, we report the selection of RNA aptamers that can specifically bind to HBsAg protein and HBsAg-positive hepatocytes. One high affinity aptamer, HBs-A22, was isolated from an initial 115 met library of -1.1 ×10^15 random-sequence RNA molecules using the SELEX procedure. The selected aptamer HBs-A22 bound specifically to hepatoma cell line HepG2.2.15 that expresses HBsAg but did not bind to HBsAg-devoid HepG2 cells. This is the first reported RNA aptamer which could bind to a HBV specific antigen. This newly isolated aptamer could be modified to deliver imaging, diagnostic, and therapeutic agents targeted at HBV-infected cells.

关键词： aptamer Systematic evolution of ligands by exponential enrichment (SELEX) Hepatitis B virus (HBV) HBsAg Hepatocytes

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Nucleic acids analysis

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Science China Chemistry 2021年第2期64卷 171-203页

作者： Yongxi Zhao Xiaolei Zuo Qian Li Feng Chen Yan-Ru Chen Jinqi Deng Da Han Changlong Hao Fujian Huang Yanyi Huang Guoliang Ke Hua Kuang Fan Li Jiang Li Min Li Na Li Zhenyu Lin Dingbin Liu Juewen Liu Libing Liu Xiaoguo Liu Chunhua Lu Fang Luo Xiuhai Mao Jiashu Sun Bo Tang Fei Wang Jianbin Wang Lihua Wang Shu Wang Lingling Wu Zai-Sheng Wu Fan Xia Chuanlai Xu Yang Yang Bi-Feng Yuan Quan Yuan Chao Zhang Zhi Zhu Chaoyong Yang Xiao-Bing Zhang Huanghao Yang Weihong Tan Chunhai Fan Institute of Analytical Chemistry and Instrument for Life Science The Key Laboratory of Biomedical Information Engineering of Ministry of EducationSchool of Life Science and TechnologyXi’an Jiaotong UniversityXi’an 710049China Institute of Molecular Medicine Shanghai Key Laboratory for Nucleic Acid Chemistry and NanomedicineRenji HospitalSchool of MedicineShanghai Jiao Tong UniversityShanghai 200127China School of Chemistry and Chemical Engineering Frontiers Science Center for Transformative MoleculesInstitute of Translational MedicineShanghai Jiao Tong UniversityShanghai 200240China Cancer Metastasis Alert and Prevention Center Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and ChemotherapyState Key Laboratory of Photocatalysis on Energy and EnvironmentCollege of ChemistryFuzhou UniversityFuzhou 350108China CAS Key Laboratory of Standardization and Measurement for Nanotechnology CAS Center for Excellence in NanoscienceNational Center for Nanoscience and TechnologyBeijing 100190China State Key Lab of Food Science and Technology International Joint Research Laboratory for Biointerface and BiodetectionSchool of Food Science and TechnologyJiangnan UniversityWuxi 214122China Faculty of Materials Science and Chemistry Engineering Research Center of Nano-Geomaterials of Ministry of EducationChina University of GeosciencesWuhan 430074China College of Chemistry and Molecular Engineering Biomedical Pioneering Innovation Center(BIOPIC)Beijing Advanced Innovation Center for Genomics(ICG)Peking-Tsinghua Center for Life SciencesPeking UniversityBeijing 100871China State Key Laboratory of Chemo/Biosensing and Chemometrics College of Chemistry and Chemical EngineeringHunan UniversityChangsha 410082China Division of Physical Biology CAS Key Laboratory of Interfacial Physics and TechnologyShanghai Institute of Applied PhysicsChinese Academy of SciencesShanghai 201800China Bioimaging Center Shanghai Synchrotron Radiation FacilityZhangjiang LaboratoryShanghai Advanced

Nucleic acids are natural biopolymers of nucleotides that store, encode, transmit and express genetic information, which play central roles in diverse cellular events and diseases in living things. The analysis of nucleic acids and nucleic acids-based analysis have been widely applied in biological studies, clinical diagnosis, environmental analysis, food safety and forensic *** the past decades, the field of nucleic acids analysis has been rapidly advancing with many technological *** this review, we focus on the methods developed for analyzing nucleic acids, nucleic acids-based analysis, device for nucleic acids analysis, and applications of nucleic acids analysis. The representative strategies for the development of new nucleic acids analysis in this field are summarized, and key advantages and possible limitations are discussed. Finally, a brief perspective on existing challenges and further research development is provided.

关键词： nucleic acids analysis signal amplification DNA nanotechnology aptamer biosensing

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Selection of aptamers to a non-purified target of native conformation by liquid-SELEX

Selection of aptamers to a non-purified target of native con...

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中国生物化学与分子生物学会第十届会员代表大会暨全国学术会议

作者： Shaohua Li Hui Li Hongmei Ding Fang Wang Ningsheng Shao Institute of Basic Medical Sciences Academy of Military Medical Science

Background:aptamers are short single stranded DNA or RNA molecules,which bind specifically to their targets based on their specific tertiary *** to the properties of precise discrimination,easy synthesis and modification, less immunogenicity and small size,aptames are emerging candidates for medical research and *** are evolved from an in vitro selection process known as SELEX(Systematic Evolution of Ligands by Exponential Enrichment) that involves the progressive selection of highly specific ligands by repeated rounds of selection and amplification from a large random synthetic nucleic acid *** traditional SELEX requires a purified

关键词： liquid-SELEX aptamer EMSA catalytic region of glucosyltransferase

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Identification of Phoxim and Omethoate Using α-hemolysin Nanopore and aptamers

Identification of Phoxim and Omethoate Using α-hemolysin Na...

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中国植物病理学会2024年学术年会

作者： LI Bin WANG Junxiao ZHANG Chengling LI Guangyue WANG Ying Shandong Provincial Key Laboratory of Detection Technology for Tumor Markers College of Life ScienceLinyi University Xuzhou Institute of Agricultural Sciences Shandong Dingyi Ecological Agriculture Co.LTD

Contamination of pesticides has inflicted substantial harm on human health,so it is great significance to develop rapid,ultra-sensitive,and no-labeling simultaneous detection methods for multiple ***,we show that α-hemolysin（α-HL） nanopore sensor can detect and discriminated organophosphorus pesticides of phoxim and omethoate in a single nanopore without the need for labels of the probes or purification of the pesticides in real *** specifically recognize and bind pesticides to obtain pesticide-aptamer complexes,which produce characteristic current signals during passing through the *** and omethoate are accurately distinguished by a portable instrument within minutes,and the detective sensitivity is up to femtomole level whether detected alone or *** detection limits of phoxim and omethoate are 8.13×10 M and 4.16×10 *** recoveries of phoxim and omethoate from pear,tomato,and cucumber samples are 82.0%～107.0 %and 80.0%-100.4 % respectively,with coefficient of variable below 8.0 %.The developed nanopore sensor has a powerful tool for food safety research and human health.

关键词： α-Hemolysin nanopore aptamer Phoxim Omethoate Sensor Detection

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PEGylation of therapeutic oligonucletides： From linear to highly branched PEG architectures

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Nano Research 2018年第10期11卷 5519-5534页

作者： Xueguang Lu Ke Zhang Department of Chemistry and Chemical Biology Northeastern University Boston Massachusetts 02115 USA

PEGylation, the attachment of poly（ethylene glycol）（PEG）, has been adopted to improve the pharmacokinetic properties of oligonucleotide therapeutics for nearly 30 years. Prior efforts mainly focused on the investigation of linear or slightly branched PEG having different molecular weights, terminal functional groups, and possible oligonucleotide sites for functionalization. Recent studies on highly branched PEG （including brush, star, and micellar structures） indicate superior properties in several areas including cellular uptake, gene regulation efficac~ reduction of side effects, and biodistribution. This review focuses on comparing the effects of PEG architecture on the physiochemical and biological properties of the PEGylated oligonucleotide.

关键词： PEGylation oligonucleotide brush polymer antisense small interfering RNA aptamer phosphorothioate

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