量子点荧光技术标记肿瘤细胞中HSP的应用研究

目的探讨量子点荧光技术对人肾癌细胞株(ACHN)中不同HSP进行标记的可行性应用。方法利用量子点的荧光特性,免疫细胞化学方法检测体外培养的ACHN细胞中量子点特异性标记的HSP70、HSP90、HSPgp96的表达情况。结果共聚焦荧光显微镜下可见ACHN细胞中HSP70、HSP90、HSPgp96均有明显表达,呈现均匀分布的橙红色强荧光,且量子点在持续激发30分钟后无荧光淬灭发生。结论量子点荧光标记技术能够对不同HSP进行标记,且与传统的标记方法相比具有显著优点,可作为一种新型的检测技术应用于科研及临床标记检测中。     

基于量子点的荧光共振能量转移探针的应用

量子点因其独特的光学性质,以及可与有机分子所形成的偶联物的特殊性质,在光学生物标记,由其是荧光共振能量转移(Fluorescence resonance energy transfer,FRET)探针的合成与应用等领域具有广泛的应用前景,并因其实时、准确、灵敏的检测优势,在生物及医学领域始终被热切关注。该文以量子点的优势为基础,分别介绍了用于检测核酸、蛋白酶、生物反应及细胞状态的量子点-FRET探针的研究机理研究进展及应用优势。并对量子点-FRET探针的存在问题及研究方向进行了展望,为进一步进行该领域的研究提供理论支撑。     

功能核酸荧光免标记型定量统一化检测技术的研究进展

功能核酸是一类具有特定空间构象、执行特异生物功能的天然或者人工核酸序列,具有易于修饰、价格低廉、稳定性高、特异性强等优势,其搭载免标记荧光传感系统后,功能核酸可起到将多种靶物质统一转为较为稳定的核酸信号,以及通过核酸扩增对检测信号进行扩增等重要作用。而免标记荧光传感技术可以在检测中免去荧光标记时荧光、猝灭基团的筛选和标记过程的繁琐和成本,同时可保证与核酸的特异或非特异性结合后产生荧光信号变化。通过两种技术的优势结合,检测的灵敏度、实时性可进一步提高,逐渐被广泛应用于环境污染物检测、食品风险因子检测、疾病诊断等多个领域。首先明确功能核酸与荧光定量检测技术等相关概念,详细阐述了几种重要的荧光物质的特点以及其与功能核酸的分子识别、作用方式与发光机制,接着紧紧围绕该种传感技术的几个特点,从功能核酸荧光免标记型定量统一化检测技术与其实际应用角度进行分类介绍与评价对比,最后就功能核酸荧光免标记型定量统一化检测技术在多种领域的检测分析中的研究意义以及存在的问题进行讨论,并对未来的发展与应用作出展望。     

用量子点标记活细胞

量子点是一种具有纳米尺寸的半导体晶体。与传统的荧光染料相比,量子点拥有许多独特的光学特性,如宽的吸收谱、窄而对称的发射谱、耐漂白、亮度高和荧光寿命长等。由于其出色的光物理特性和相对较小的尺寸,量子点可作为生物学研究的荧光探针。随着量子点合成与修饰技术的发展,其在生物和医学领域的应用已从探索阶段逐步发展到了应用阶段。将量子点应用于活细胞标记,将为揭示细胞内的复杂生命现象提供全新的视野。该文重点介绍了量子点的荧光特性、用量子点标记活细胞所要克服的障碍及基本的标记策略和方法。     

ZnS量子点的生物合成及对阿萨尔基亚芽胞杆菌的标记

通过微生物合成金属量子点是目前研究的热点。本研究通过白色念珠菌合成ZnS量子点,对合成的量子点用高分辨透射电镜(HR-TEM)和X射线衍射(XRD)进行表征,用生物合成的ZnS量子点标记生防菌阿萨尔基亚芽胞杆菌,为建立生防菌阿萨尔基亚芽胞杆菌的荧光探针标记提供科学依据。实验结果表明,ZnSO4浓度为20 mmol/L时白色念珠细胞内合成ZnS量子点,通过反复冻融破细胞壁提出胞内ZnS量子点,紫外-可见光谱(UV)检测ZnS量子点在348 nm处显示有吸收带,HR-TEM测其粒径约7. 06 nm,荧光分光光谱分析量子点激发波在280 nm和363 nm,XRD显示ZnS量子点特征峰。ZnS量子点中加入1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)偶联剂的芽胞杆菌样本的标记效率明显高于不加偶联剂的样本。白色念珠菌合成的ZnS量子点可以用于微生物标记。     

酪胺信号放大-量子点标记银染增强的基因芯片可视化检测方法的建立

目的:建立一种酪胺信号放大-量子点标记银染增强的基因芯片可视化检测方法,提高基因芯片检测的灵敏度。方法:待测靶基因与固定在玻片上的探针杂交,依次加入链霉亲和素标记的辣根过氧化物酶、生物素标记的酪胺及链霉亲和素标记的量子点,37℃孵育,然后加入银增强试剂显色,最后用可视化生物芯片扫描仪扫描并记录结果;以牛布鲁菌210105株为检测对象,以酪胺信号放大-荧光素Cy3(TSA-Cy3)检测法为对照方法,测定酪胺信号放大-量子点标记银染增强(TSA-QDS)检测法的灵敏度。结果:确定了基因芯片量子点标记银染增强可视化检测方法的检测流程,优化了检测条件,并考察了检测灵敏度。优化的检测条件为:酪胺-生物素稀释比例为1∶4000,链酶亲和素标记的量子点稀释比例为1∶50,37℃孵育时间为25~30 min,银染增强时间为6~7 min。检测牛布鲁菌的灵敏度为103CFU/mL。结论:该方法实现了基因芯片高灵敏度可视化检测,其灵敏度与荧光法相当,并且有可视化的优势。     

量子点对细菌的标记及抗菌作用

目的量子点是近年来发展起来的一种新型的荧光纳米材料,与传统的材料相比具有独特的性质,所以在生物传感器、实时追踪、多色标记及成像等方面有着广泛的应用。本文主要对量子点在细菌标记和抗菌等方面的应用进行了综述。     

固定剂、封片剂、温度及除菌方式对量子点标记细胞的影响

为了为利用量子点标记细胞、组织,进一步研究其功能提供新的方法,本实验观察了3种发射波长的量子点（quautum dost,QDs）对所标记的小鼠腹腔巨噬细胞和正常皮肤的影响。利用发射波长610mm的红色荧光水溶液（量子点610）、发射波长为523mm的绿色荧光水溶液（量子点523）和发射波长576nm的黄色荧光脂溶性溶液（量子点576）的3种量子点（5mg／ml）以及具有吞噬能力的小鼠腹腔巨噬细胞、正常皮肤为载体,观察不同的除菌方式、温度、封片剂及固定剂对量子点标记细胞、组织的影响,为量子点在生物体内的应用及在生物制片过程中对其性能的影响等研究奠定基础。     

功能核酸荧光标记型定量统一化检测技术的研究进展

功能核酸是一类具有特定空间构象、执行特异生物功能的天然或者人工核酸序列,具有易于修饰、价格低廉、稳定性高、特异性强等优势,搭载荧光传感系统,组装成多种荧光生物传感器,被广泛应用于环境污染物检测、食品风险因子检测、疾病诊断等多个领域。首先明确功能核酸与荧光定量检测技术等相关概念,详细阐述了几种重要的荧光物质的特点以及其与功能核酸的分子识别、作用方式与发光机制,再主要从功能核酸荧光标记型定量统一化检测技术与其实际应用角度进行分类介绍与评价对比,最后就功能核酸荧光标记型定量统一化检测技术在多种领域的检测分析中的研究意义以及存在的问题进行讨论,并对未来的发展与应用作出展望。     

量子点在生物学中的研究进展

量子点作为一种新型的荧光标记物近年来已在生物学中获得广泛应用。本文总结了量子点的主要光学特性,其中包括荧光激发和发射光谱特性、量子产额、光漂白特性和荧光寿命等。重点综述了量子点在细胞标记、活体和组织成像、组合标记和光动力学治疗等生物学中的应用及其最新研究进展。同时讨论了量子点在应用中可能存在的细胞毒性等主要问题,最后对量子点在生物学中的应用前景作了展望。     

Enzyme‐triggered fluorescence turn‐off/turn‐on of carbon dots for monitoring β‐glucosidase and its inhibitor in living cells

Energy transfer engineering based on fluorescent probes for directly sensing enzyme activities are in great demand as enzyme‐mediated transformations, which are central to all biological processes. Here, a fluorescence carbon dot (CD)‐based assay exhibiting selective responses to the quantitation of β‐glucosidase and the effect of its inhibitor was developed. The most common substrate, para‐nitrophenyl‐β‐d ‐glucopyranoside (pNPG) was hydrolyzed by β‐glucosidase to release p‐nitrophenol (pNP), which can efficiently quench fluorescence of CDs via an inner filter effect and electron transfer. However, in the presence of inhibitors of β‐glucosidase, the fluorescence intensity gradually recovered as the concentration of inhibitors increased. Therefore, the enzyme‐triggered fluorescence turn‐off/turn‐on of specific CDs successfully achieved sensitive detection of β‐glucosidase and monitored the effect of its inhibitors. This new strategy was applied to detect β‐glucosidase and monitor β‐glucosidase inhibitor in hepatoma cells using cell imaging. All results suggest that the new method is sensitive and promising for use in cancer diagnosis and treatment.     

Effect of Inhibitors of Auxin Transport and of Calmodulin on a Gravisensing-Dependent Current in Maize Roots

Some characteristics of the gravity sensing mechanism in maize root caps were investigated using a bioelectric current as an indicator of gravity sensing. This technique involves the measurement of a change in the current density which arises at the columella region coincidently with the presentation time. Two inhibitors of auxin transport, triiodobenzoic acid and naphthylphthalamic acid, blocked gravitropic curvature but not the change in current density. Two inhibitors of calmodulin activity, compound 48/80 and calmidazolium, blocked both curvature and gravity-induced current. The results suggest that auxin transport is not a component of gravity sensing in the root cap. By contrast, the results suggest that calmodulin plays an intrinsic role in gravity sensing.     

A rapid, sensitive, and selective bioluminescence resonance energy transfer (BRET)-based nucleic acid sensing system

Here we report the design of a bioluminescence resonance energy transfer (BRET)-based sensing system that could detect nucleic acid target in 5 min with high sensitivity and selectivity. The sensing system is based on adjacent binding of oligonucleotide probes labeled with Renilla luciferase (Rluc) and quantum dot (Qd) on the nucleic acid target. Here Rluc, a bioluminescent protein that generates light by a chemical reaction, is employed as an energy donor, and a quantum dot is used as an energy acceptor. Bioluminescence emission of Rluc overlaps with the Qd absorption whereas the emission of Qd is shifted from the emission of Rluc allowing for monitoring of BRET. In the presence of target, the labeled probes bind adjacently in a head-to-head fashion leading to BRET from Rluc to Qd upon addition of a substrate coelenterazine. The sensing system could detect target nucleic acid in buffer as well as in Escherichia coli cellular matrix in 5 min with a detection limit of 0.54 pmol. The ability to detect target nucleic acid rapidly in a cellular matrix with high sensitivity will prove highly beneficial in biomedical and environmental applications.     

Impact of the structure-activity relationship of AHL analogues on quorum sensing in Gram-negative bacteria

With the emergence of microbial resistance pathogens, recent research aims at studying new mechanisms of action of antibiotics. This review discusses the mechanisms and types of quorum sensing (QS) inhibitors in Gram negative bacteria. It illustrates all published data available in literature pertaining to novel compounds that showed activity against different targets in the quorum sensing pathways in Gram negative bacteria. A systemic overview has been conducted by searching PubMed, Medline, and the Cochrane Library and data extraction of all quorum sensing inhibitors with their mechanisms of action have been collected. This review will focus on signaling autoinducer AI-1 in Gram negative bacteria. The biological activity of the antagonists is mainly reported as IC₅₀ (the concentration of an inhibitor where the response is reduced by half).     

分子对接技术在研究群体感应抑制剂中的进展

在大多数致病菌中都存在群体感应系统,而群体感应抑制剂就是以此系统作为靶点,在不影响细菌生长的情况下阻断细菌生物被膜形成或抑制毒力基因表达,不易导致耐药性的产生,是一种理想的抗菌增效剂。分子对接作为虚拟筛选技术之一,其目标具体、效率高、成本低,是药物研发的重要手段。本文重点介绍了分子对接的主要模块及其在研究群体感应抑制剂中的进展。     

Molecular insights into quorum sensing in the human pathogen Pseudomonas aeruginosa from the structure of the virulence regulator LasR bound to its autoinducer

Many Gram-negative bacteria communicate via molecules called autoinducers to coordinate the activities of their populations. Such communication is termed quorum sensing and can regulate pathogenic virulence factor production and antimicrobial resistance. The quorum sensing system of Pseudomonas aeruginosa is currently the most intensively researched, because this bacterium is an opportunistic human pathogen annually responsible for the death of thousands of cystic fibrosis sufferers and many other immunocompromised individuals. Quorum sensing inhibitors can attenuate the pathogenicity of P. aeruginosa. Here we present the crystal structure of the P. aeruginosa LasR ligand-binding domain bound to its autoinducer 3-oxo-C(12)-acylhomoserine lactone. The structure is a symmetrical dimer, with each monomer exhibiting an alpha-beta-alpha fold similar to the TraR and SdiA quorum sensing proteins of Agrobacterium tumefaciens and Escherichia coli. The structure was determined up to 1.8-A resolution and reveals the atomic interactions between LasR and its autoinducer. The monomer structures of LasR, TraR, and SdiA are comparable but display differences in their quaternary organization. Inspection of their binding sites shows some unexpected variations resulting in quite different conformations of their bound autoinducers. We modeled interactions between LasR and various quorum sensing inhibitors, yielding insight into their possible mechanisms of action. The structure also provides a platform for the optimization, or de novo design, of quorum sensing inhibitors.     

具有群体感应抑制活性的海洋细菌的筛选

【目的】从海洋环境中筛选出能有效抑制细菌群体感应的活性菌株,为以致病菌群体感应为靶点的新型疗法提供新的天然产物资源。【方法】以紫色杆菌(Chromobacteriumviolaceum)为报告菌,采用滤纸片法和双层软琼脂法相结合的筛选模型进行群体感应抑制活性菌的筛选。【结果】通过对美国圣璜岛海域海绵中分离出来的272株海洋细菌群体感应抑制活性的筛选,得到了具有抑制紫色杆菌素产生的细菌51株,其中74号菌株抑制效果最好,具有进一步研究的价值。【结论】海洋细菌中有很多具有抑制细菌群体感应效应的菌株,是天然群体感应抑制剂的潜在来源。     

Pyrogallol and its analogs can antagonize bacterial quorum sensing in Vibrio harveyi

Bacteria can coordinate community-wide behaviors through quorum sensing, that is, the secretion and sensing of autoinducer (AI) molecules. Bacterial quorum sensing is implicated in the regulation of pathologically relevant events such as biofilm formation, bacterial virulence, and drug resistance. Inhibitors of bacterial quorum sensing could therefore be useful therapeutics. Herein we report for the first time the discovery of several pyrogallol compounds as single digit micromolar inhibitors of bacterial quorum sensing in Vibrio harveyi.     

Quantum dot-nucleic acid/aptamer bioconjugate-based fluorimetric biosensors

Over the last 10 years, fluorescent semiconductor QD (quantum dot)-biomolecule conjugates have emerged as a powerful new sensing platform showing great potential in a wide range of applications in biosensing, environmental monitoring and disease diagnosis. The present mini-review is a brief account of the recent developments in QD-NA (nucleic acid), particularly NA aptamer, conjugate-based biosensors using the FRET (F?rster resonance energy transfer) readout mechanism. It starts with a brief introduction to the NA aptamer and QD-FRET, followed by example approaches to compact QD-DNA conjugates, target readout strategies and sensing performance, and concludes with challenges and outlook for the QD-NA/aptamer bioconjugate sensors.     

Simulation and investigation of quantum dot effects as internal heat-generator source in breast tumor site

The Pennes bio-heat transfer equation, which introduces the exchange magnitude of heat transfer between tissue and blood, is often used to solve the temperature distribution for thermal imaging and sensing. Near-infrared light has the ability to be used as a non-invasive means of diagnostic imaging within the woman's breast. Due to the diffusive nature of light in different tissue, computational model-based methods are required for functional imaging within the breast. In this article, the time-dependent bio-heat transfer is solved by a numerical method. In our model, the heat generation source (intrinsic and extrinsic) involves laser, metabolism, and quantum dot that the metabolism and heat generated by QDs are considered as intrinsic. We supposed the injected quantum dots would target the tumor site by a passive targeting process and then by interaction of laser radiation and quantum dot, the photoluminescence of quantum dot is converted to heat in the tumor site. The extra generated heat can impact on the extracted heat profile. One of the important applications of this research has led to a sensitivity improvement of the imaging system, which is potentially useful in the diagnosis and detection of breast cancer.