CdTe/CdS-罗丹明体系用于铅离子（Ⅱ）的测定

目的建立以CdTe/CdS-RhB荧光共振能量转移体系为荧光探针,设计出一种快速、超灵敏且高选择性的检测Pb（Ⅱ）含量的方法。方法研究CdTe/CdS和罗丹明B（受体）之间FRET的最佳条件利用,Pb2＋能对能量转移体系中CdTe/CdS的荧光峰强产生猝灭作用从而测定Pb2＋的含量。结果在3.85×10-10～9.62×10-9mol.L-1线性关系良好,相关系数r为0.997 5。9组浓度为3.85×10-9mol.L-1的Pb（Ⅱ）对量子点-罗丹明体系荧光猝灭的相对标准偏差（RSD）为0.32%。将该方法用于测定菠菜和柑橘叶中Pb（Ⅱ）的含量分别为69.6和55.2μmol.kg-1,与火焰原子吸收光谱法比较无显著性差异。结论该体系作为荧光探针测定Pb（Ⅱ）简便经济可行,灵敏度高,选择性好。     

盐酸法舒地尔的水溶性CdTe量子点荧光猝灭法测定

以谷胱甘肽(GSH)为稳定剂,在水溶液中制备稳定的CdTe纳米量子点.基于盐酸法舒地尔在pH 7.4的磷酸盐缓冲液中对该量子点的荧光具有较强的猝灭作用,建立了一种简便灵敏的测定盐酸法舒地尔的新方法.考察了缓冲体系、缓冲液pH、反应时间和量子点浓度等对盐酸法舒地尔测定的影响.结果表明,在0.1 mmol/L的磷酸盐缓冲液(pH 7.4)中,CdTe量子点浓度4 μg/ml,反应时间5 min,体系的相对荧光强度与盐酸法舒地尔浓度的线性关系良好,线性范围为0.525～78.7 ug/ml.     

海鞘素简易结构类似物GJ7-1和GJ7-2的抗肿瘤活性及分子靶向研究

目的:研究依照特有DNA靶向性的抗肿瘤海洋天然产物海鞘素(Ecteinascidins,ETs)合成的简易结构类似物GJ7-1和GJ7-2的抗肿瘤活性及分子靶向。方法:MTT法检测0.012 5¹.6μmol/L的GJ7-1、GJ7-2作用72 h对10种体外培养的肿瘤细胞的增殖抑制作用;荧光结合竞争法测定0.011.6μmol/L的GJ7-1、GJ7-2作用72 h对10种体外培养的肿瘤细胞的增殖抑制作用;荧光结合竞争法测定0.01¹00μmol/L的GJ7-1、GJ7-2与DNA的结合情况;流式细胞仪检测0.01、0.1、1μmol/L的GJ7-1、GJ7-2对A549细胞周期(作用24、48 h)的影响;Hochest33342染色和AnnexinⅤ/PI双染检测0.01、0.1、1μmol/L的GJ7-1、GJ7-2对A549细胞凋亡(作用24、48、72 h)的影响。结果:GJ7-1和GJ7-2对10种肿瘤细胞均有增殖抑制作用,但无明显肿瘤类型选择性;浓度增加到100μmol/L时也与DNA无明显结合;仅1μmol/L的GJ7-1、GJ7-2对A549细胞周期有一定的影响,但均不能诱导其明显凋亡。结论:GJ7-1和GJ7-2虽有一定的抗肿瘤活性,但较ETs大幅降低,部分原因是其完全丧失了与DNA的结合活性。     

光谱学手段对芦荟大黄素潜在毒副作用的研究

目的研究芦荟大黄素与DNA的相互作用和结合方式,从光谱学的角度考察芦荟大黄素的潜在基因毒性。方法采用荧光共振散射手段检测1.0 ml的0.756×10-5 mol/L DNA溶液分别加入0.5、1.0和1.5ml的1×10-4mol/L芦荟大黄素溶液,30 min后图谱的变化,并与相同条件下1×10-4 mol/L EB比较。选择480 nm激发波长,扫描纪录495～650 nm波长范围内1.00 ml 1×10-4 mol/L芦荟大黄素溶液加入一定量的DNA溶液后的荧光发射光谱,并通过荧光加强型理论公式计算结合常数。结果芦荟大黄素与DNA在308和537nm处有较强的共振散射峰;芦荟大黄素在545 nm附近有荧光激发峰,其强度随着DNA浓度增大而增强,通过公式计算得出芦荟大黄素与DNA的结合常数为1.43×106 L/mol。结论在该试验条件下,芦荟大黄素与DNA之间存在着嵌插作用,具有潜在基因毒性。     

基于PI3K/Akt/GSK3β信号通路的芥子酸抗Aβ1-42致PC12细胞损伤的机制研究

目的:基于磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(Akt)/糖原合成激酶3β(GSK3β)信号通路探讨芥子酸(SA)抗β淀粉样蛋白1-42(Aβ_1-42)致PC12细胞损伤的作用机制。方法:将大鼠PC12细胞随机分为对照组、Aβ组(Aβ_1-422μmol/L)、Aβ+SA组(Aβ_1-422μmol/L+SA 100μmol/L)、Aβ+SA+LY组[Aβ_1-422μmol/L+SA 100μmol/L+LY294002(PI3K抑制剂)10μmol/L]、Aβ+LY组(Aβ_1-422μmol/L+LY294002 10μmol/L)、LY组(LY294002 10μmol/L)。除对照组、LY组外,其余各组细胞均以Aβ_1-42复制损伤模型。培养24 h后,使用显微镜观察各组细胞的形态,采用MTT法检测各组细胞的存活率;采用Western blotting法检测各组细胞PI3K、p-PI3K、Akt、p-Akt、GSK3β、p-GSK3β蛋白的表达情况...     

阻断Hedgehog信号通路对HepG2.2.15细胞增殖的影响

目的探讨环耙明阻断Hedgehog信号通路对肝癌细胞HepG2.2.15增殖的影响。方法培养肝癌细胞HepG2.2.15,用5μmol/L、15μmol/L、25μmol/L环耙明处理HepG2.2.15细胞24h、48h、72h,采用MTT检测环耙明对细胞活力的影响;EDU法检测细胞DNA合成情况;Real-timePCR法检测细胞Gli1的表达情况。结果用5μmol/L、15μmol/L、25μmol/L浓度的环耙明处理HepG2.2.15细胞24h、48h、72h后,MTT检测结果显示细胞活力降低,较空白组差异明显(P<0.05);EDU法检测结果显示25μmol/L的环耙明作用于细胞不同时间后,HepG2.2.15细胞的DNA合成率下降,与空白组相比差异有统计学意义(P<0.01);Real-time PCR法实验结果显示5μmol/L、15μmol/L、25μmol/L浓度的环耙明处理组与对照组相比,Gli1表达水平明显降低(P<0.05)。结论不同浓度环耙明能抑制HepG2.2.15细胞的增殖,减少HepG2.2.15细胞的DNA合成率;其作用机制可能与HepG2.2.15细胞中Gli1、mRNA的表达水平下降有关。     

白鲜碱对小鼠脾淋巴细胞活力的体外抑制作用及机制研究

目的:研究白鲜碱对小鼠脾淋巴细胞活力的体外抑制作用并探讨其可能的作用机制。方法:分离并培养小鼠原代脾淋巴细胞,以0(空白对照)、50、100、150μmol/L白鲜碱作用细胞24 h后,采用MTT法检测细胞活力,乳酸脱氢酶(LDH)法检测细胞LDH释放率,流式细胞术检测细胞早期凋亡率,Hoechst 33342和碘化丙啶(PI)双染法检测细胞坏死率,Western blot法检测细胞中胱天蛋白酶3(Caspase 3)、剪切体Caspase 3(Cleaved-Caspase 3)蛋白表达水平以及彗星试验法检测细胞DNA损伤(以DNA拖尾区域比例反映)。结果:与空白对照比较,100、150μmol/L白鲜碱可显著抑制细胞活力(P<0.01);150μmol/L白鲜碱可显著增加细胞LDH的释放(P<0.05),释放率达到79.37%;50、100、150μmol/L白鲜碱均可提高细胞的早期凋亡率,但差异均无统计学意义(P>0.05);150μmol/L白鲜碱可显著增加细胞坏死率(P<0.05),坏死率达到78.64%;50、100、150μmol/L白鲜碱均可升高Caspase 3蛋白表达水平,但差异均无统计学意义(P>0.05),然而50、100μmol/L白鲜碱可显著提高Cleaved-Caspase 3蛋白表达水平(P<0.05);白鲜碱可呈剂量依赖性地引起DNA损伤,其中100、150μmol/L白鲜碱可显著增加DNA拖尾区域比例(P<0.01)。结论:白鲜碱可以抑制脾淋巴细胞活力,其作用机制可能与诱导脾淋巴细胞坏死和造成DNA损伤有关。     

苦参碱对乙醛活化的肝星状细胞CFSC-8B增殖和胶原合成的影响及机制研究

目的:研究苦参碱对乙醛活化的大鼠肝星状细胞CFSC-8B增殖和胶原合成的影响,并探讨其可能的作用机制。方法:取体外培养的CFSC-8B细胞,分为空白对照组、模型组、阳性对照组(2.5μmol/L秋水仙碱)和苦参碱低、中、高浓度组(30、60、120μmol/L)。除空白对照组外,其余各组细胞均加入200μmol/L乙醛溶液诱导活化,并同时加入相应药液(空白对照组和模型组加入等体积空白培养液),共同作用24 h后,采用CCK-8法检测各组细胞的存活率。另取细胞分为空白对照组、模型组、阳性对照组(2.5μmol/L秋水仙碱)和苦参碱中、高浓度组(60、120μmol/L),同法活化和加药处理。分别采用酶消化法检测细胞培养液中羟脯氨酸(Hyp)含量;采用酶联免疫吸附法检测细胞培养液中Ⅰ型胶原蛋白(Col-Ⅰ)和Ⅲ型胶原蛋白(Col-Ⅲ)含量;采用实时荧光定量-聚合酶链式反应法检测细胞中α-平滑肌激动蛋白(α-SMA)、转化生长因子β₁(TGF-β₁)、TGF-β₁Ⅰ型受体(TβR-Ⅰ)、TGF-β₁Ⅱ型受...     

芯片显色法检测结核分枝杆菌利福平和异烟肼耐药基因实验条件优化研究

目的优化研究芯片显色法检测结核分枝杆菌利福平和异烟肼耐药基因的最佳条件。方法自制DNA芯片,每条寡核苷酸探针片段长度为19bp,点样浓度在15μmol/L,以痰液为待检标本,分别用不同引物、Mg2 、核苷酸、DNA聚合酶浓度等进行多重聚合酶链反应;分别在不同时间、温度下进行杂交,分别在不同酶作用时间和显色时间进行分析,比较不同条件芯片检测结果。结果引物、Mg2 、核苷酸、DNA聚合酶浓度分别为0.2μmol/L、1mmol/L、0.2mmol/L、5U效果最好,杂交时间和杂交温度分别为0.5h、60℃信号最强,酶作用时间和显色时间均为0.5h时得到最佳结果。结论在最佳条件下,该技术的重复性、特异性、灵敏度与可靠性均较好,适合基层医院推广应用。     

量子点荧光探针用于汉坦病毒重组抗原的检测

目的应用量子点荧光探针对汉坦病毒（Hantavirus,HV）重组抗原进行检测。方法合成水溶性量子点荧光纳米颗粒,并在其表面修饰G蛋白和anti-HV抗体作为量子点荧光探针,对HV重组抗原进行检测并优化检测条件。结果量子点与抗体的最佳偶联条件：pH 6.0、反应时间2 h、抗体浓度为20μg/ml。用本方法检测HV重组抗原的最低检测值为5 ng/ml。结论该探针能有效的识别HV抗原,且操作简便快速,为HV重组抗原的检测和肾出血热综合征的诊断提供了新方法。     

妇炎康胶囊中违法添加诺氟沙星的检测

目的:建立妇炎康胶囊中违法添加诺氟沙星的检测方法。方法:采用高效液相色谱结合二级管阵列紫外光谱检测,色谱柱C18(4.6mm×250mm,5μm);流动相为0.025mol/L甲酸溶液(以三乙胺调节pH至3.0±0.1)-乙腈(90∶10);流速为1mL/min;柱温为35℃;进样体积为10μL;检测波长为278nm。结果:结合高效液相色谱保留时间、紫外光谱等信息分析得出"妇炎康胶囊"中非法添加诺氟沙星。诺氟沙星在1.156～23.12μ/gmL范围内呈良好的线性关系,r=0.9992;检测限为1ng;加样平均回收率为103.51%,RSD为1.15%(n=6),每粒"妇炎康胶囊"中含诺氟沙星量为3.30mg。结论:本方法操作简便、准确、可靠,可用于检测妇炎康胶囊中违法添加诺氟沙星的定性和定量。     

Quantum dots, lighting up the research and development of nanomedicine

Quantum dots (QDs) have proven themselves as powerful inorganic fluorescent probes, especially for long term, multiplexed imaging and detection. The newly developed QDs labeling techniques have facilitated the study of drug delivery on the level of living cells and small animals. Moreover, based on QDs and fluorescence imaging system, multifunctional nanocomplex integrated targeting, imaging and therapeutic functionalities have become effective materials for synchronous cancer diagnosis and treatment. In this review, we will summarize the recent advances of QDs in the research of drug delivery system from the following aspects: surface modification strategies of QDs for drug delivery, QDs as drug nanocarriers, QD-labeled drug nanocarriers, QD-based fluorescence resonance energy transfer (FRET) technique for drug release study as well as the development of multifunctional nanomedicines. Possible perspective in this field will also be discussed. FROM THE CLINICAL EDITOR: This review discusses the role and significance of quantum dots (QDs) from the following aspects: surface modification strategies of QDs for drug delivery, QDs as drug nanocarriers, QD-labeled drug nanocarriers, QD-based fluorescence resonance energy transfer (FRET) technique for drug release study as well as the development of multifunctional nanomedicines.     

An exciplex-based, target-assembled fluorescence system with inherently low background to probe for specific nucleic acid sequences

A novel detection technique, called ExciProbes, has been developed to proof-of-principle level for DNA oligonucleotides. The new approach is based on the use of two short oligonucleotides complementary to a target nucleic acid sequence. Each short-probe oligonucleotide bears the separated parts of a new class of fluorescence detector, an exciplex. These isolated parts of the detector have no inherent signal at the detection wavelength. They are designed to detect biotarget by being assembled by the target itself to give a new molecular entity (the exciplex), with a characteristic fluorescence and very large Stokes shift (typically >150 nm). The technique is not related to fluorescence resonance energy transfer, and can potentially resolve to 1 base pair. ExciProbes can detect single or double mutations in a short sequence of DNA, and can be combined with temperature-filtering to provide allelic discrimination of single nucleotide polymorphism analysis. Compared to other fluorophore systems that have large backgrounds (typically >60%), ExciProbes show backgrounds of <1% under comparable conditions, and can be used with DNA, RNA, or synthetic nucleic acids such as locked nucleic acid.     

Preliminary study of interactivity between mercury and cells labeled with carboxymethyl chitosan coated quantum dots

This paper describes the development of a simplified and rapid method for the aqueous synthesis of quantum dots (QDs) with CdTe cores and gradient CdS external shells (CdTe/CdS QDs) aided by microwave irradiation. In order to improve the biocompatibility of the CdTe/CdS QDs, these QDs were then interacted with carboxymethyl chitosan (CMC) so as they could be used as fluorescent probes in the aqueous phase. As fluorescent probes, these modified QDs were successfully used for imaging live Madin-Darby canine kidney (MDCK) cells. Then mercury was incubated with the micro-system formed by quantum dots labeled MDCK. Fluorescence quenching was occurred in the micro-system after 24 h. The micro-system’s fluorescence quenching caused by mercury(II) was consistent with the fluorescence quenching equation and displayed a good linearity between the quenched fluorescence intensity of mercury(II). The preliminary results indicated that this micro-system can be used for detection of trace amounts of mercury in vivo and interaction process investigation between mercury and cells.     

Ingenious nanoprobes in bioassays

This article has a special focus on the broad range of innovative nanoprobes for signal amplification and new generations of bioassays. Advances in functionalizing gold nanoparticles with oligonucleotides speed up the development of a series of new nucleic acid assays. A biobarcode assay allows signal amplification by utilizing antibody-coated magnetic beads to concentrate the analytes and antibody-coated gold nanoparticle probes to carry a large number of oligonucleotides. Novel signal-amplification technologies, based on either new classes of nanoprobes consisting of releasable fluorophores or with aggregation-induced emission features, can also improve the sensitivity of bioassays. Advances in synthesis and biofunctionalization of quantum dots with unique properties have generated increasingly widespread applications in DNA sorting, multiplexing bioassays and fluorescence resonance energy transfer-based sensing. Ingenious nanoprobes in bioassays can offer PCR-like sensitivity, high selectivity, capacity for massive multiplexing, time efficiency and, most importantly, the ability to be performed at the point- of-care.     

Semiautomated fluorometric analysis of nucleic acids in tissue homogenates

This report describes a technique that was developed to provide an efficient and accurate estimation of RNA:DNA ratios. These ratios have been used as an instantaneous measure of recent growth of individual aquatic organisms where morphometrics are not appropriate (e.g., field-collected species) or insufficiently sensitive (e.g., small life stages or species). In this semiautomated, sensitive method, ethidium bromide fluorescence was used to quantitate total nucleic acids in crude homogenates. Individual concentrations of RNA and DNA were determined by differences in fluorescence before and after elimination of RNA by digestion with RNase. Efficiency of the procedure was enhanced using a computer-driven multiwell plate scanning system (CYTOFLUOR, Millipore Corporation

1 Reference to trade names does not imply endorsement.

) to measure fluorescence at timed intervals and perform data manipulations. Routinely, detection limits of 0.1 μg DNA and 0.4 μg RNA were achieved, allowing the analysis of small, individual organisms. Fluorescence results of split samples were comparable with those obtained using a standard spectro-photometric method to quantitate nucleic acids. Coefficients of variation for replicate samples within an assay (1.6%) and for samples within replicate assays (5.6%) indicated good test reproducibility. Quantitative recoveries of nucleic acid standards spiked into tissue homogenates were generally high, averaging 91.0% for DNA and 119.0% for RNA. Factors affecting the fluorescence of ethidium bromide stained nucleic acids—e.g., nucleic acid source, crude homogenate components, and buffer composition—are discussed relative to assay performance. This method provides a rapid and reliable assessment of individual growth, an important sublethal toxicological end point, that is suitable for both laboratory and field studies. © 1994 by John Wiley & Sons, Inc..     

荧光共振能量转移光谱法测定尿液中美罗培南的含量

目的：建立荧光共振能量转移光谱法测定尿液中美罗培南的含量。方法：采用LS-55型荧光分光光度计,于10 ml比色管中,依次加入美罗培南溶液、荧光素溶液、曙红Y溶液、BR缓冲溶液和十六烷基三甲基溴化铵,于λex=455 nm,λem=547 nm处,测定体系和样品试剂空白的荧光强度F和F0,以ΔF=F-F0作为测定美罗培南的信号响应值,计算其含量。结果：尿液中美罗培南的线性回归方程：ΔF=33.8C＋53.4,r=0.991 7,线性范围为0.5-10μg·ml^-1,检出限为0.13μg·ml^-1,加标回收率为98.9%-103.0%,RSD为0.3%-0.4%。结论：该方法灵敏准确,适用于美罗培南临床药物动力学的研究。     

Determination of ketoprofen based on its quenching effect in the fluorescence of quantum dots

Ketoprofen is a potent nonsteroidal anti-inflammatory drug used for the treatment of inflammatory diseases and musculoskeletal injuries. Taking into account the increasing consumption of this drug, it is important to develop a rapid, easy, and reliable analytical strategy for its quality control. In this work, we present a novel method for ketoprofen determination, based on its quenching effect produced in the fluorescence of CdTe quantum dots modified by mercaptopropionic acid. Under optimized conditions, the method was linear in the range of 7.5–100 μg/mL, with a detection limit of 2.3 μg/mL and relative standard deviations lower than 2%. It was applied to the determination of ketoprofen in pharmaceutical formulations, obtaining results in good agreement with those provided by the manufacturer.