[Objective] The aim was to construct a plasmid reference molecule (PRM) for detection of transgenic soybean MON89788. [Method] the lectin gene sequence,3'-junction and 5'-junction sequence between host plant D...[Objective] The aim was to construct a plasmid reference molecule (PRM) for detection of transgenic soybean MON89788. [Method] the lectin gene sequence,3'-junction and 5'-junction sequence between host plant DNA integrated DNA of MON89788 soybean were amplified independently,and the three fragments were cloned into the cloning vector pMD18-T in order through molecular manipulation method to construct pMD-LM3M5,the applicability of the constructed novel PRM was tested. [Result] Sequencing confirmation result showed that the PRM was 3 700 bp in length,containing 1 029 bp of recombined DNA fragment. The limits of qualitative detection of the PRM were 10 copies. [Conclusion] The PRM constructed in this study was suitable for the identification of MON89788 event.展开更多
It is important and challenging to analyze nanocluster structure with atomic precision.Herein,α-hemolysin nanopore was used to identify nanoclusters at the single molecule level by providing two-dimensional(2D)dwell ...It is important and challenging to analyze nanocluster structure with atomic precision.Herein,α-hemolysin nanopore was used to identify nanoclusters at the single molecule level by providing two-dimensional(2D)dwell time–current blockage spectra and translocation event frequency which sensitively depended on their structures.Nanoclusters such as Anderson,Keggin,Dawson,and a few lacunary Dawson polyoxometalates with very similar structures,even with only a two-atom difference,could be discriminated.This nanopore device could simultaneously measure multiple nanoclusters in a mixture qualitatively and quantitatively.Furthermore,molecular dynamics(MD)simulations provided microscopic understandings of the nanocluster translocation dynamics and yielded 2D dwell time–current blockage spectra in close agreement with experiments.The nanopore platform provides a novel powerful tool for nanocluster characterization.展开更多
DNA sequencers have become increasingly important research and diagnostic tools over the past 20 years.In this study,we developed a single-molecule desktop sequencer,GenoCare 1600(GenoCare),which utilizes amplificatio...DNA sequencers have become increasingly important research and diagnostic tools over the past 20 years.In this study,we developed a single-molecule desktop sequencer,GenoCare 1600(GenoCare),which utilizes amplification-free library preparation and two-color sequencing-by-synthesis chemistry,making it more user-friendly compared with previous single-molecule sequencing platforms for clinical use.Using the GenoCare platform,we sequenced an Escherichia coli standard sample and achieved a consensus accuracy exceeding 99.99%.We also evaluated the sequencing performance of this platform in microbial mixtures and coronavirus disease 2019(COVID-19)samples from throat swabs.Our findings indicate that the GenoCare platform allows for microbial quantitation,sensitive identification of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)virus,and accurate detection of virus mutations,as confirmed by Sanger sequencing,demonstrating its remarkable potential in clinical application.展开更多
Owing to their structural dispersion,the catalytic properties of nanoparticles are challenging to characterize in ensemble-averaged measurements.The single-molecule approach enables studying the catalysis of nanoparti...Owing to their structural dispersion,the catalytic properties of nanoparticles are challenging to characterize in ensemble-averaged measurements.The single-molecule approach enables studying the catalysis of nanoparticles at the single-particle level with real-time single-turnover resolution.This article reviews our single-molecule fluorescence studies of single Au-nanoparticle catalysis,focusing on the theoretical formulations for extracting quantitative reaction kinetics from the single-turnover resolution catalysis trajectories.We discuss the single-molecule kinetic formulism of the Langmuir-Hinshelwood mechanism for heterogeneous catalysis,as well as of the two-pathway model for product dissociation reactions.This formulism enables the quantitative evaluation of the heterogeneous reactivity and the differential selectivity of individual nanoparticles that are usually hidden in ensemble measurements.Extension of this formulism to single-molecule catalytic kinetics of oligomeric enzymes is also discussed.展开更多
目的：建立乌头霜霉病病原菌快速分子检测方法,为乌头种苗检疫及栽培环境土壤安全性评价提供依据。方法：使用真菌DNA提取试剂盒提取病原菌DNA,采用真菌核糖体内转录间隔区（ITS）通用引物ITS1/ITS4,对病原菌的r DNA-ITS区间序列进行扩...目的：建立乌头霜霉病病原菌快速分子检测方法,为乌头种苗检疫及栽培环境土壤安全性评价提供依据。方法：使用真菌DNA提取试剂盒提取病原菌DNA,采用真菌核糖体内转录间隔区（ITS）通用引物ITS1/ITS4,对病原菌的r DNA-ITS区间序列进行扩增,将聚合酶链式反应（PCR）产物回收纯化并进行序列测定,并将测得的ITS序列同Gene Bank中搜索到的相关ITS序列进行比较,运用DNAMAN比对出特异序列片段,利用特异序列片段通过Primer Premier 5.0设计并筛选特异性引物,建立乌头霜霉病病原菌快速PCR检测方法。结果：从Primer Premier 5.0设计的8对引物中筛选出了灵敏度高、特异性强的引物对L1A/L1B,该引物能够从乌头霜霉病病原菌DNA扩增出670 bp的具有检测价值的明亮条带,利用该引物也能够检测出乌头种苗、成株以及栽培土壤是否存在乌头霜霉病原菌。结论：筛选出的引物对L1A/L1B能够快速、简便、有效地检测出乌头霜霉病病原菌,建立的方法能用于对乌头种苗霜霉病的提前检测或检疫。展开更多
Formaldehyde(FA,a typical reactive carbonyl species)is a well-known environmental pollutant and a disease-related biomarker,making its sensitive and selective detection significant.Fluorescent probes have been explore...Formaldehyde(FA,a typical reactive carbonyl species)is a well-known environmental pollutant and a disease-related biomarker,making its sensitive and selective detection significant.Fluorescent probes have been explored for FA perception in environment,intracellular media and in vivo.In this review,we majorly conclude the recently represented fluorescence FA analysis based on small molecule probes.The general FA sensing mechanisms are first introduced.Regarding the FA detection in various environments,sensing tactics and performances are discussed in order of natural environment,living cells and in vivo.In the end,this review discusses the challenges and future trends of FA detection based on fluorescent probes.展开更多
Virus is a kind of microorganism and possesses simple structure and contains one nucleic acid,which must be replicated using the host cell system.It causes large-scale infectious diseases and poses serious threats to ...Virus is a kind of microorganism and possesses simple structure and contains one nucleic acid,which must be replicated using the host cell system.It causes large-scale infectious diseases and poses serious threats to the health,social well-being,and economic conditions of millions of people worldwide.Therefore,there is an urgent need to develop novel strategies for accurate diagnosis of virus infection to prevent disease transmission.Quantum dots(QDs)are typical fluorescence nanomaterials with high quantum yield,broad absorbance range,narrow and size-dependent emission,and good stability.QDs-based nanotechnology has been found to be effective method with rapid response,easy operation,high sensitivity,and good specificity,and has been widely applied for the detection of different viruses.However,until now,no systematic and critical review has been published on this important research area.Hence,in this review,we aim to provide a comprehensive coverage of various QDs-based virus detection methods.The fundamental investigations have been reviewed,including information related to the synthesis and biofunctionalization of QDs,QDs-based viral nucleic acid detection strategies,and QDs-based immunoassays.The challenges and perspectives regarding the potential application of QDs for virus detection is also discussed.展开更多
A wavelength-dependent three-dimensional(3D)superlocalization imaging method on gold nanoislands(GNIs)chip was developed as a supersensitive single-molecule thyroid-stimulating hormone(TSH)nanobiosensor.Scattered and ...A wavelength-dependent three-dimensional(3D)superlocalization imaging method on gold nanoislands(GNIs)chip was developed as a supersensitive single-molecule thyroid-stimulating hormone(TSH)nanobiosensor.Scattered and fluorescent signals from gold nanoislands on the substrate and quantum dots(QDs)nanoprobes were simultaneously isolated and acquired within an evanescent field layer generated by total internal reflection(TIR)of incident light using a dual-view device.The 3D TIR fluorescence images of TSH-bound QDs on the GNIs were obtained using z-axis optical sectioning at 10nm intervals before/after immunoreaction to identify the optimal conditions for detection.The localized centroid position of QD nanoprobes and GNI were distinguished at a subdiffraction limit resolution using 3D Gaussian fitting to the point spread function.The QD TSH nanobiosensor using wavelength-dependent 3D TIR fluorescence-based single-molecule localization microscopy(3D TIRF-SLM)imaging technique showed an excellent detection limit of 90 yoctomoles(~54 molecules)and a wide linear dynamic range of 1.14 zmol/L-100 pmol/L for TSH.The detection sensitivity was about 4.4×10^(9)times higher than conventional enzyme-linked immunosorbent assay and could successfully quantify TSH in human serum.The wavelength-dependent 3D TIRF-SLM technique may emerge as a reliable platform for ultrahigh-sensitive nanobiosensors at the single-molecule level and early diagnosis with quantification of disease-related ultra-tracebiomolecules.展开更多
Biosensors featuring single molecule detection present huge opportunities as well as challenges in food safety inspection,disease diagnosis,and environmental monitoring.Single-molecule detection is largely lacking of ...Biosensors featuring single molecule detection present huge opportunities as well as challenges in food safety inspection,disease diagnosis,and environmental monitoring.Single-molecule detection is largely lacking of high enough activity,precision molecule selectivity,and understanding in the exact operating mechanism.Single-atom catalysts(SACs),especially those metals-nitrogen-carbon that mimic the natural metalloenzyme structure,and with well-defined metal atom bond configurations,high level of molecular selectivity,and easy fabrication,endow single molecule detections with practical-use feasibilities.The recent advances in single-atom catalysts also present new pathways in the key mechanism understandings.In this short review,we will first visit the brief history and advantages of SACs that have been explored only recently for molecule-scale biosensors,where they are analogous and also differentiated from those nanozymes and natural metalloenzymes.Their applications in electrochemical,photochemical,and photoelectrochemical sensors are then discussed comprehensively by focusing on the different molecule-scale sensing modes in achieving local coordination-modulated signal amplifications.Finally,we identify new opportunities and challenges faced by these SACs-based single molecule detections in the further development of biosensors.展开更多
基金Supported by Major Projects of Cultivating New Varieties by Trans-genic Technology (2008ZX08012-001)~~
文摘[Objective] The aim was to construct a plasmid reference molecule (PRM) for detection of transgenic soybean MON89788. [Method] the lectin gene sequence,3'-junction and 5'-junction sequence between host plant DNA integrated DNA of MON89788 soybean were amplified independently,and the three fragments were cloned into the cloning vector pMD18-T in order through molecular manipulation method to construct pMD-LM3M5,the applicability of the constructed novel PRM was tested. [Result] Sequencing confirmation result showed that the PRM was 3 700 bp in length,containing 1 029 bp of recombined DNA fragment. The limits of qualitative detection of the PRM were 10 copies. [Conclusion] The PRM constructed in this study was suitable for the identification of MON89788 event.
基金supported by the National Key Research and Development Program of China(No.2021YFA1200104)New Cornerstone Science Foundation,the National Natural Science Foundation of China(Nos.22027807,22034004,and 22078104)+1 种基金the Strategic Priority Research Program of Chinese Academy of Sciences(No.XDB36000000)Tsinghua-Vanke Special Fund for Public Health and Health Discipline Development(No.2022Z82WKJ003).
文摘It is important and challenging to analyze nanocluster structure with atomic precision.Herein,α-hemolysin nanopore was used to identify nanoclusters at the single molecule level by providing two-dimensional(2D)dwell time–current blockage spectra and translocation event frequency which sensitively depended on their structures.Nanoclusters such as Anderson,Keggin,Dawson,and a few lacunary Dawson polyoxometalates with very similar structures,even with only a two-atom difference,could be discriminated.This nanopore device could simultaneously measure multiple nanoclusters in a mixture qualitatively and quantitatively.Furthermore,molecular dynamics(MD)simulations provided microscopic understandings of the nanocluster translocation dynamics and yielded 2D dwell time–current blockage spectra in close agreement with experiments.The nanopore platform provides a novel powerful tool for nanocluster characterization.
文摘DNA sequencers have become increasingly important research and diagnostic tools over the past 20 years.In this study,we developed a single-molecule desktop sequencer,GenoCare 1600(GenoCare),which utilizes amplification-free library preparation and two-color sequencing-by-synthesis chemistry,making it more user-friendly compared with previous single-molecule sequencing platforms for clinical use.Using the GenoCare platform,we sequenced an Escherichia coli standard sample and achieved a consensus accuracy exceeding 99.99%.We also evaluated the sequencing performance of this platform in microbial mixtures and coronavirus disease 2019(COVID-19)samples from throat swabs.Our findings indicate that the GenoCare platform allows for microbial quantitation,sensitive identification of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)virus,and accurate detection of virus mutations,as confirmed by Sanger sequencing,demonstrating its remarkable potential in clinical application.
基金We thank the Army Research Office(56355-CH)National Science Foundation(NSF,No.CBET 0851257)+1 种基金American Chemical Society Petroleum Research Foundation(No.47918-G5)NSF-funded Cornell Center for Materials Research for fi nancial support。
文摘Owing to their structural dispersion,the catalytic properties of nanoparticles are challenging to characterize in ensemble-averaged measurements.The single-molecule approach enables studying the catalysis of nanoparticles at the single-particle level with real-time single-turnover resolution.This article reviews our single-molecule fluorescence studies of single Au-nanoparticle catalysis,focusing on the theoretical formulations for extracting quantitative reaction kinetics from the single-turnover resolution catalysis trajectories.We discuss the single-molecule kinetic formulism of the Langmuir-Hinshelwood mechanism for heterogeneous catalysis,as well as of the two-pathway model for product dissociation reactions.This formulism enables the quantitative evaluation of the heterogeneous reactivity and the differential selectivity of individual nanoparticles that are usually hidden in ensemble measurements.Extension of this formulism to single-molecule catalytic kinetics of oligomeric enzymes is also discussed.
文摘目的：建立乌头霜霉病病原菌快速分子检测方法,为乌头种苗检疫及栽培环境土壤安全性评价提供依据。方法：使用真菌DNA提取试剂盒提取病原菌DNA,采用真菌核糖体内转录间隔区（ITS）通用引物ITS1/ITS4,对病原菌的r DNA-ITS区间序列进行扩增,将聚合酶链式反应（PCR）产物回收纯化并进行序列测定,并将测得的ITS序列同Gene Bank中搜索到的相关ITS序列进行比较,运用DNAMAN比对出特异序列片段,利用特异序列片段通过Primer Premier 5.0设计并筛选特异性引物,建立乌头霜霉病病原菌快速PCR检测方法。结果：从Primer Premier 5.0设计的8对引物中筛选出了灵敏度高、特异性强的引物对L1A/L1B,该引物能够从乌头霜霉病病原菌DNA扩增出670 bp的具有检测价值的明亮条带,利用该引物也能够检测出乌头种苗、成株以及栽培土壤是否存在乌头霜霉病原菌。结论：筛选出的引物对L1A/L1B能够快速、简便、有效地检测出乌头霜霉病病原菌,建立的方法能用于对乌头种苗霜霉病的提前检测或检疫。
基金supported by the National Natural Science Foundation of China(22074005)the Natural Science Foundation of Beijing Municipality(2202038)the Open Research Fund Program of Beijing Key Lab of Plant Resource Research and Development,Beijing Technology and Business University(PRRD-2021-YB6)
文摘Formaldehyde(FA,a typical reactive carbonyl species)is a well-known environmental pollutant and a disease-related biomarker,making its sensitive and selective detection significant.Fluorescent probes have been explored for FA perception in environment,intracellular media and in vivo.In this review,we majorly conclude the recently represented fluorescence FA analysis based on small molecule probes.The general FA sensing mechanisms are first introduced.Regarding the FA detection in various environments,sensing tactics and performances are discussed in order of natural environment,living cells and in vivo.In the end,this review discusses the challenges and future trends of FA detection based on fluorescent probes.
基金supported by National Key Research and Development Program of China(2021YFA0910900)the National Natural Science Foundation of China(32222044,22104147)+5 种基金Shenzhen Municipal Science and Technology Innovation Council(RCYX20210609103823046)Youth Innovation Promotion Association CAS(2021359)Natural Science Foundation of Guangdong(2020A1515111130)Guangdong Provincial Key Laboratory of Synthetic Genomics(2019B030301006)Shenzhen Science and Technology Program(KQTD20180413181837372)Shenzhen Outstanding Talents Training Fund.
文摘Virus is a kind of microorganism and possesses simple structure and contains one nucleic acid,which must be replicated using the host cell system.It causes large-scale infectious diseases and poses serious threats to the health,social well-being,and economic conditions of millions of people worldwide.Therefore,there is an urgent need to develop novel strategies for accurate diagnosis of virus infection to prevent disease transmission.Quantum dots(QDs)are typical fluorescence nanomaterials with high quantum yield,broad absorbance range,narrow and size-dependent emission,and good stability.QDs-based nanotechnology has been found to be effective method with rapid response,easy operation,high sensitivity,and good specificity,and has been widely applied for the detection of different viruses.However,until now,no systematic and critical review has been published on this important research area.Hence,in this review,we aim to provide a comprehensive coverage of various QDs-based virus detection methods.The fundamental investigations have been reviewed,including information related to the synthesis and biofunctionalization of QDs,QDs-based viral nucleic acid detection strategies,and QDs-based immunoassays.The challenges and perspectives regarding the potential application of QDs for virus detection is also discussed.
基金supported by the National Research Foundation of Korea(NRF)grant funded by the Korea government(MSIT)(Nos.2019R1A2C2002556 and 2020R1C1C1009668)supported by Nano-Material Technology Development Program through the NRF funded by the Ministry of Science,ICT and Future Planning(No.2009-0082580).
文摘A wavelength-dependent three-dimensional(3D)superlocalization imaging method on gold nanoislands(GNIs)chip was developed as a supersensitive single-molecule thyroid-stimulating hormone(TSH)nanobiosensor.Scattered and fluorescent signals from gold nanoislands on the substrate and quantum dots(QDs)nanoprobes were simultaneously isolated and acquired within an evanescent field layer generated by total internal reflection(TIR)of incident light using a dual-view device.The 3D TIR fluorescence images of TSH-bound QDs on the GNIs were obtained using z-axis optical sectioning at 10nm intervals before/after immunoreaction to identify the optimal conditions for detection.The localized centroid position of QD nanoprobes and GNI were distinguished at a subdiffraction limit resolution using 3D Gaussian fitting to the point spread function.The QD TSH nanobiosensor using wavelength-dependent 3D TIR fluorescence-based single-molecule localization microscopy(3D TIRF-SLM)imaging technique showed an excellent detection limit of 90 yoctomoles(~54 molecules)and a wide linear dynamic range of 1.14 zmol/L-100 pmol/L for TSH.The detection sensitivity was about 4.4×10^(9)times higher than conventional enzyme-linked immunosorbent assay and could successfully quantify TSH in human serum.The wavelength-dependent 3D TIRF-SLM technique may emerge as a reliable platform for ultrahigh-sensitive nanobiosensors at the single-molecule level and early diagnosis with quantification of disease-related ultra-tracebiomolecules.
基金Prof.Z.Kou acknowledges the financial support of the Fundamental Research Funds for the Central Universities(Grant No.40120631)Natural Science Foundation of Hubei Province(Grant No.2021CFB007)+2 种基金National Natural Science Foundation of China(Grant No.52202291)the support.Prof.J.Wang thanks the support by the Singapore Ministry of Education,and the National Research Foundation(NRF)research conducted at the National University of Singapore(Tier 1,A-8000186-01-00,and CRP NRF-CRP26-2021-0003).
文摘Biosensors featuring single molecule detection present huge opportunities as well as challenges in food safety inspection,disease diagnosis,and environmental monitoring.Single-molecule detection is largely lacking of high enough activity,precision molecule selectivity,and understanding in the exact operating mechanism.Single-atom catalysts(SACs),especially those metals-nitrogen-carbon that mimic the natural metalloenzyme structure,and with well-defined metal atom bond configurations,high level of molecular selectivity,and easy fabrication,endow single molecule detections with practical-use feasibilities.The recent advances in single-atom catalysts also present new pathways in the key mechanism understandings.In this short review,we will first visit the brief history and advantages of SACs that have been explored only recently for molecule-scale biosensors,where they are analogous and also differentiated from those nanozymes and natural metalloenzymes.Their applications in electrochemical,photochemical,and photoelectrochemical sensors are then discussed comprehensively by focusing on the different molecule-scale sensing modes in achieving local coordination-modulated signal amplifications.Finally,we identify new opportunities and challenges faced by these SACs-based single molecule detections in the further development of biosensors.
