多波长荧光互相关光谱仪(FCCS)研制

项目来源

国家自然科学基金(NSFC)

项目主持人

谭蔚泓

项目受资助机构

湖南大学

立项年度

2013

立项时间

未公开

项目编号

21327009

项目级别

国家级

研究期限

未知 / 未知

受资助金额

350.00万元

学科

化学科学-化学测量学-谱学方法与理论

学科代码

B-B04-B0403

基金类别

专项基金项目-科学仪器基础研究专款

关键词

荧光互相关光谱 ; 多通道 ; 荧光相关光谱 ; 多波长激发 ; 单分子检测 ; FCCS ; FCS ; single-molecule detection ; multi-laser excitation ; multi-channel

参与者

宋又群;庞新宇;李伟;符婷;张晶;周礼义;薛琳;孟红敏

参与机构

长沙理工大学

项目标书摘要:由于其具有检测灵敏度高及分析速度快等优点,荧光相关光谱技术已经成为单分子检测的主要手段之一,是单分子水平上的生物分子研究,揭示了大分子的结构和功能的一个理想的分析工具。为了更好地拓展该高灵敏技术对于活细胞体内分子及分子相互作用的检测和监控,并不受任何分子体积及重量的限制,本项目拟开发:1将单波长荧光自相关光谱仪升级为多波长荧光互相关光谱仪,实现并优化单分子检测技术;2将实现与显微镜的连接以应用于单细胞研究,简化仪器设计,并组装搭建成可携式装置;3优化并应用所改良的多通道荧光互相关光谱仪,构建活细胞内基因表达和信使核糖核酸的分布荧光互相关光谱研究系统;4运用互相关光谱技术监测及追踪蛋白质间的相互作用,建立活细胞内的蛋白质—蛋白质相互作用荧光互相关光谱仪检测平台;5利用荧光互相关光谱技术进行分子扩散速度测量以获取分子量信息,构建系列超灵敏、多元化的活细胞荧光互相关光谱分析系统。

Application Abstract: Due to having a high detection sensitivity and the advantages of rapid analysis,fluorescence correlation spectroscopy technique has become one of the primary means for the detection of single molecules.It is an ideal analysis tool for single molecule level biomolecules research and revealing the structure and function of macromolecular.In order to better develop the highly sensitive technology for the detection and monitoring of molecular and molecular interactions in living cells,and not subject to any molecular size and weight restrictions,the project is intended to develop:1upgrading single wavelength fluorescence autocorrelation spectrometer to multi-wavelength fluorescence cross-correlation spectrometer,realization and optimization of single molecule detection technology with it;2connecting multi-wavelength fluorescence cross-correlation spectrometer with microscope for single-cell research,and simplifying instrument design,assembling and building into a portable device;3optimization and application of the modified multi-channel fluorescence cross-correlation spectrometer;constructing fluorescence cross-correlation spectroscopy study system for gene expression and messenger RNA distribution in living cells researches;4Using the cross-correlation spectroscopy to monitor and track protein interactions,establishing fluorescence cross-correlation spectroscopy detected platform of protein-protein interactions within living cells;5the use of fluorescence cross-correlation spectroscopy for molecular diffusion velocity measurements to obtain molecular weight information,build a series of ultra-sensitive,a wide range of live cell fluorescence cross-correlation spectroscopy system.

项目受资助省

湖南省

项目结题报告(全文)

荧光相关光谱技术因具有检测灵敏度高及分析速度快等优点,已经成为单分子检测的主要手段之一,是在单分子水平上进行生物分子研究、揭示大分子结构和功能的理想分析工具。为了更好地拓展该高灵敏技术对于活细胞体内分子及分子相互作用的检测和监控,并不受任何分子体积及重量的限制,本项目以单波长荧光自相关光谱仪为基础,设计并搭建了高灵敏的多波长荧光互相关光谱仪,并将其与显微镜的连接,实现了单分子检测;简化了仪器设计,并组装搭建成可携式装置FCS,FCCS显微镜。该荧光互相关光谱仪有望建成可应用于一系列超灵敏、多元化的活细胞分析的分析检测平台;利用多波长通道标记不同的生物分子,并研究其互相关信号,达到检测并监控其相互作用的目的。该改进大大地扩大了对生物分子相互作用研究的范围,也扩大了荧光相关光谱仪在生物研究中的应用范围。

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  • 1.DNA-supramolecule conjugates in theranostics

    • 关键词:
    • DNA-supramolecule conjugate; aptamer; supramolecular chemistry; DNAnanotechnology; host-guest interaction;HOST-GUEST INTERACTIONS; IN-VITRO SELECTION; LIVE CELLS; APTAMER;CYCLODEXTRIN; COMPLEXES; BINDING; MOLECULES; CHEMISTRY; RELEASE

    The elegant properties of deoxyribonucleic acid (DNA), such as accurate recognition, programmability and addressability, make it a well-defined and promising material to develop various molecular probes, drug delivery carriers and theranostic systems for cancer diagnosis and therapy. In addition, supramolecular chemistry, also termed "chemistry beyond the molecule", is a promising research field that aims to develop functional chemical systems by bringing discrete molecular components together in a manner that invokes noncovalent intermolecular forces, such as hydrophobic interaction, hydrogen bonding, metal coordination, and shape or size matching. Thus, DNA-supramolecule conjugates (DSCs) combine accurate recognition, programmability and addressability of DNA with the greater toolbox of supramolecular chemistry. This review discusses the applications of DSCs in sensing, protein activity regulation, cell behavior manipulation, and biomedicine.

    ...
  • 2.DNA-supramolecule conjugates in theranostics

    • 关键词:
    • DNA-supramolecule conjugate; aptamer; supramolecular chemistry; DNAnanotechnology; host-guest interaction;HOST-GUEST INTERACTIONS; IN-VITRO SELECTION; LIVE CELLS; APTAMER;CYCLODEXTRIN; COMPLEXES; BINDING; MOLECULES; CHEMISTRY; RELEASE

    The elegant properties of deoxyribonucleic acid (DNA), such as accurate recognition, programmability and addressability, make it a well-defined and promising material to develop various molecular probes, drug delivery carriers and theranostic systems for cancer diagnosis and therapy. In addition, supramolecular chemistry, also termed "chemistry beyond the molecule", is a promising research field that aims to develop functional chemical systems by bringing discrete molecular components together in a manner that invokes noncovalent intermolecular forces, such as hydrophobic interaction, hydrogen bonding, metal coordination, and shape or size matching. Thus, DNA-supramolecule conjugates (DSCs) combine accurate recognition, programmability and addressability of DNA with the greater toolbox of supramolecular chemistry. This review discusses the applications of DSCs in sensing, protein activity regulation, cell behavior manipulation, and biomedicine.

    ...
  • 3.Recent progresses in small-molecule enzymatic fluorescent probes for cancer imaging

    • 关键词:
    • AGGREGATION-INDUCED EMISSION; FIBROBLAST-ACTIVATION PROTEIN;GAMMA-GLUTAMYL-TRANSPEPTIDASE; LIGHT-UP PROBE; BETA-GALACTOSIDASEACTIVITY; METHIONINE SULFOXIDE REDUCTASES; ALKALINE-PHOSPHATASEACTIVITY; CELL-PENETRATING PEPTIDES; INDUCED LIVER-INJURY; TURN-ON PROBE

    Abnormal enzymatic activities are directly related to the development of cancers. Identifying the location and expression levels of these enzymes in live cancer cells have considerable importance in early-stage cancer diagnoses and monitoring the efficacy of therapies. Small-molecule fluorescent probes have become a powerful tool for the detection and imaging of enzymatic activities in biological systems by virtue of their higher sensitivity, nondestructive fast analysis, and real-time detection abilities. Moreover, due to their structural tailorability, numerous small-molecule enzymatic fluorescent probes have been developed to meet various demands involving real-time tracking and visualizing different enzymes in live cancer cells or in vivo. In this review, we provide an overview of recent advances in small-molecule enzymatic fluorescent probes mainly during the past decade, including the design strategies and applications for various enzymes in live cancer cells. We also highlight the challenges and opportunities in this rapidly developing field of small-molecule fluorescent probes for interventional surgical imaging, as well as cancer diagnosis and therapy.

    ...
  • 4.Aptamer-Conjugated Nanomaterials for Specific Cancer Diagnosis and Targeted Therapy

    • 关键词:
    • Aptamer; Nanomaterial; Specific recognition; Cancer diagnosis; Targetedtherapy;UP-CONVERSION LUMINESCENCE; BLOCK-COPOLYMER MICELLES; DRUG-DELIVERY;CELL-SURFACE; DNA APTAMER; PHOTODYNAMIC THERAPY; SIGNAL AMPLIFICATION;PHOTOTHERMAL THERAPY; SENSITIVE DETECTION; SENSING PLATFORM

    Owing to their unique optical, electronic, magnetic, and surface plasmon resonance properties, nanomaterials have attracted significant attention for potential bioanalysis and biomedical applications. Aptamers are single-stranded oligonucleotides, which are generated by a procedure termed as SELEX (Systematic Evolution of Ligands by EXponential Enrichment) and typically demonstrate high affinity and selectivity toward their target molecules. As a result of their unique characteristics, aptamers are promising recognition units that can be conjugated with nanomaterials for cancer cell imaging, diagnosis, and cancer therapy. By integrating the recognition abilities of aptamers with the properties of nanomaterials, aptamer-conjugated nanomaterials can serve as extraordinary tools for bioimaging and cancer therapy. Recently, aptamer-conjugated nanomaterials have attracted significant attention in the field of specific cancer cell targeted therapy owing to their improved efficacy and lower toxicity. In this review, we summarize the progress achieved of aptamer-conjugated nanomaterials as nanocarriers for specific cancer cell diagnosis and targeted therapy. In addition to drug delivery for cancer therapy, the various achievements of the aptamer-conjugated nanomaterials in combination with other emerging technologies to improve the efficiency and selectivity of cancer therapy have also been reviewed.

    ...
  • 5.Aptamer-Conjugated Nanomaterials for Specific Cancer Diagnosis and Targeted Therapy

    • 关键词:
    • Aptamer; Nanomaterial; Specific recognition; Cancer diagnosis; Targetedtherapy;UP-CONVERSION LUMINESCENCE; BLOCK-COPOLYMER MICELLES; DRUG-DELIVERY;CELL-SURFACE; DNA APTAMER; PHOTODYNAMIC THERAPY; SIGNAL AMPLIFICATION;PHOTOTHERMAL THERAPY; SENSITIVE DETECTION; SENSING PLATFORM

    Owing to their unique optical, electronic, magnetic, and surface plasmon resonance properties, nanomaterials have attracted significant attention for potential bioanalysis and biomedical applications. Aptamers are single-stranded oligonucleotides, which are generated by a procedure termed as SELEX (Systematic Evolution of Ligands by EXponential Enrichment) and typically demonstrate high affinity and selectivity toward their target molecules. As a result of their unique characteristics, aptamers are promising recognition units that can be conjugated with nanomaterials for cancer cell imaging, diagnosis, and cancer therapy. By integrating the recognition abilities of aptamers with the properties of nanomaterials, aptamer-conjugated nanomaterials can serve as extraordinary tools for bioimaging and cancer therapy. Recently, aptamer-conjugated nanomaterials have attracted significant attention in the field of specific cancer cell targeted therapy owing to their improved efficacy and lower toxicity. In this review, we summarize the progress achieved of aptamer-conjugated nanomaterials as nanocarriers for specific cancer cell diagnosis and targeted therapy. In addition to drug delivery for cancer therapy, the various achievements of the aptamer-conjugated nanomaterials in combination with other emerging technologies to improve the efficiency and selectivity of cancer therapy have also been reviewed.

    ...
  • 6.Recent progresses in small-molecule enzymatic fluorescent probes for cancer imaging

    • 关键词:
    • AGGREGATION-INDUCED EMISSION; FIBROBLAST-ACTIVATION PROTEIN;GAMMA-GLUTAMYL-TRANSPEPTIDASE; LIGHT-UP PROBE; BETA-GALACTOSIDASEACTIVITY; METHIONINE SULFOXIDE REDUCTASES; ALKALINE-PHOSPHATASEACTIVITY; CELL-PENETRATING PEPTIDES; INDUCED LIVER-INJURY; TURN-ON PROBE

    Abnormal enzymatic activities are directly related to the development of cancers. Identifying the location and expression levels of these enzymes in live cancer cells have considerable importance in early-stage cancer diagnoses and monitoring the efficacy of therapies. Small-molecule fluorescent probes have become a powerful tool for the detection and imaging of enzymatic activities in biological systems by virtue of their higher sensitivity, nondestructive fast analysis, and real-time detection abilities. Moreover, due to their structural tailorability, numerous small-molecule enzymatic fluorescent probes have been developed to meet various demands involving real-time tracking and visualizing different enzymes in live cancer cells or in vivo. In this review, we provide an overview of recent advances in small-molecule enzymatic fluorescent probes mainly during the past decade, including the design strategies and applications for various enzymes in live cancer cells. We also highlight the challenges and opportunities in this rapidly developing field of small-molecule fluorescent probes for interventional surgical imaging, as well as cancer diagnosis and therapy.

    ...
  • 7.Recent advances in DNAzyme-based gene silencing

    • 关键词:
    • DNAzymes; gene-silencing; delivery; stability; cofactors;LOCKED NUCLEIC-ACID; IN-VITRO SELECTION; DRUG-DELIVERY SYSTEMS;MESSENGER-RNA; DNA ENZYMES; METAL-IONS; INTRACELLULAR DELIVERY;AMPLIFIED DETECTION; TAT PEPTIDE; EXPRESSION

    DNAzymes, generated through in vitro selection processes, are single-stranded DNA catalysts that can catalyze a wide variety of reactions, such as RNA or DNA cleavage and ligation or DNA phosphorylation. Based on specific cofactor dependence and potent catalytic ability, DNAzymes have been extensively used to develop highly sensitive and specific sensing platforms for metal ions, small molecules, and biomacromolecules. However, in spite of their multiple strong enzymatic turnover properties, few reports have addressed the potential application of RNA-cleaving DNAzymes as therapeutic gene-silencing agents. The main challenges are being met with low efficiency of cellular uptake, instability and the lack of sufficient cofactors for cellular or in vivo study, which have limited the development of DNAzymes for clinical application. In recent years, substantial progress has been made to enhance the delivery efficiency and stability of DNAzymes by developing variety of methods. Smart metal oxide nanomaterials have also been used to meet the requirement of cofactors in situ. This review focuses on the gene silencing application of DNAzymes as well as their physicochemical properties. Methods of increasing the efficacy of DNAzymes in gene therapy are also discussed: delivery systems to enhance the cellular uptake, modifications to enhance the stability and smart systems to generate sufficient cofactors in situ. Finally, some future trends and perspectives in these research areas are outlined.

    ...
  • 8.Recent advances in DNAzyme-based gene silencing

    • 关键词:
    • DNAzymes; gene-silencing; delivery; stability; cofactors;LOCKED NUCLEIC-ACID; IN-VITRO SELECTION; DRUG-DELIVERY SYSTEMS;MESSENGER-RNA; DNA ENZYMES; METAL-IONS; INTRACELLULAR DELIVERY;AMPLIFIED DETECTION; TAT PEPTIDE; EXPRESSION

    DNAzymes, generated through in vitro selection processes, are single-stranded DNA catalysts that can catalyze a wide variety of reactions, such as RNA or DNA cleavage and ligation or DNA phosphorylation. Based on specific cofactor dependence and potent catalytic ability, DNAzymes have been extensively used to develop highly sensitive and specific sensing platforms for metal ions, small molecules, and biomacromolecules. However, in spite of their multiple strong enzymatic turnover properties, few reports have addressed the potential application of RNA-cleaving DNAzymes as therapeutic gene-silencing agents. The main challenges are being met with low efficiency of cellular uptake, instability and the lack of sufficient cofactors for cellular or in vivo study, which have limited the development of DNAzymes for clinical application. In recent years, substantial progress has been made to enhance the delivery efficiency and stability of DNAzymes by developing variety of methods. Smart metal oxide nanomaterials have also been used to meet the requirement of cofactors in situ. This review focuses on the gene silencing application of DNAzymes as well as their physicochemical properties. Methods of increasing the efficacy of DNAzymes in gene therapy are also discussed: delivery systems to enhance the cellular uptake, modifications to enhance the stability and smart systems to generate sufficient cofactors in situ. Finally, some future trends and perspectives in these research areas are outlined.

    ...
  • 9.Advances in the development of aptamer drug conjugates for targeted drug delivery

    • 关键词:
    • IN-VITRO SELECTION; DNA APTAMER; CANCER-CELLS; MOLECULAR RECOGNITION;SYSTEMATIC EVOLUTION; PHOTOTHERMAL THERAPY; VISUAL DETECTION; GOLDNANORODS; NANOPARTICLES; SELEX

    A key goal of modern medicine is target-specific therapeutic intervention. However, most drugs lack selectivity, resulting in off-target' side effects. To address the requirements of targeted therapy,' aptamers, which are artificial oligonucleotides, have been used as novel targeting ligands to construct aptamer drug conjugates (ApDC) that can specifically bind to a broad spectrum of targets, including diseased cells. Accordingly, the application of aptamers in targeted drug delivery has attracted broad interest due to their impressive selectivity and affinity, low immunogenicity, easy synthesis with high reproducibility, facile modification, and relatively rapid tissue penetration with no toxicity. Functionally, aptamers themselves can be used as macromolecular drugs, and they are also commonly used in biomarker discovery and targeted drug delivery. In this review, we will highlight the most recent advances in the development of aptamers and aptamer conjugates, and discuss their potential in targeted therapy. WIREs Nanomed Nanobiotechnol 2017, 9:e1438. doi: 10.1002/wnan.1438 For further resources related to this article, please visit the .

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  • 10.Molecular engineering of two-photon fluorescent probes for bioimaging applications

    • 关键词:
    • two-photon fluorescence; molecular engineering; small-molecule probes;bioimaging;BOND ENERGY-TRANSFER; TURN-ON PROBE; LIVING CELLS; NITRIC-OXIDE;HYDROGEN-PEROXIDE; TRANSFER CASSETTES; LIVE CELLS; ENDOGENOUSFORMALDEHYDE; RHODAMINE SPIROLACTAM; SUPEROXIDE ANION

    During the past two decades, two-photon microscopy (TPM), which utilizes two near-infrared photons as the excitation source, has emerged as a novel, attractive imaging tool for biological research. Compared with one-photon microscopy, TPMoffers several advantages, such as lowering background fluorescence in living cells and tissues, reducing photodamage to biosamples, and a photobleaching phenomenon, offering better 3D spatial localization, and increasing penetration depth. Small-molecule-based two-photon fluorescent probes have been well developed for the detection and imaging of various analytes in biological systems. In this review, we will give a general introduction of molecular engineering of two-photon fluorescent probes based on different fluorescence response mechanisms for bioimaging applications during the past decade. Inspired by the desired advantages of small-molecule two-photon fluorescent probes in biological imaging applications, we expect that more attention will be devoted to the development of new two-photon fluorophores and applications of TPMin areas of bioanalysis and disease diagnosis.

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