荧光能量转移体系在生化分析中的应用-吖啶橙(AO)-中性红(NR)荧光探针测定DNA研究

本文简述了以荧光能量转移体系测定ＤＮＡ方法的发展过程,对近几年这一领域的新成果作了介绍;同时对吖啶橙（ＡＯ）－中性红ＮＲ为荧光探针测定ＤＮＡ的条件进行分析,证明了该法具有灵敏度高、线性范围宽、检出限低、选择性好、抗干扰能力强等,该法为微量ＤＮＡ定量分析提供了一个新的途径。     

铁(Ⅲ)-依诺沙星-SDS三元体系的荧光特性及应用

喹诺酮类药物是一类化学合成抗菌素，在临床上有重要用途。由于其分子结构的特殊性，它们可与许多金属离子发生络合反应，根据其分子吸收或荧光光谱的变化可对药物或金属含量进行测定。依诺沙星（Erloxacin）是临床上广泛应用的第三代喹诺酮类药物，其分子本身具有内源性荧光。本文在实验中发现，在表面活性剂十二烷基硫酸钠（SDS）的存在下，微量铁（Ⅲ）的加入可使依诺沙星的内源性荧光显著猝灭，据此建立了铁（Ⅲ）-依诺沙星-SDS三元体系测定微量铁（Ⅲ）的荧光分析方法。该法灵敏、准确，直接用于水样和食品中微量铁的测定，结果满意。     

水中痕量钼(Ⅵ)的4-(2-吡啶偶氮)-间苯二酚螯合活性炭吸附富集分光光度法测定

提出了一种简单快速预富集水中痕量钼（Ⅵ）的新方法，方法的机理在于4－（2－吡啶偶氮）－间苯二酚（PAR）与钼（Ⅵ）螯合后被活性炭中附，然后用NaOH解吸下来，再用水杨基荧光酮与钼（Ⅵ）显色，分光光度法测定钼（Ⅵ）的含量；详细研究了影响Mo-PAR螯合物定量吸附于活性炭上的各种参数，找出了最佳吸附－解吸条件，包括吸附酸度、PAR用量、吸附时间、解吸酸度等；该法的主要优点是操作简单、快速，干扰小，避免了常规方法中用浓硝酸消化活性炭费时和污染大的解吸方法，该法已用于测定水中痕量钼（Ⅵ），加标回收率达到94％－97％。     

4-酰基-双(1,3-二苯基-5-吡唑啉酮)铽(Ⅲ)配合物的和成及荧光性质

合成了3种4－酰基－双（1，3－二苯基－5－吡唑啉酮），1，5－双（1′，3′－二苯基－5′－吡唑啉酮－4′－基）－1，5－戊二酮；1，6－双（1′，3′－二苯基－5′－吡唑啉酮－4′－基）－1，6－己二酮和1，10－双（1′，3′－二苯基－5′－吡唑啉酮－4′－基）－1，10－癸二酮，通过元素分析，红外光谱和核磁共振氢谱对产物组成进行了表征，合成了它们的Tb(Ⅲ）二元和三元[1,10-二氮杂菲（Phen)或2，2′－联吡啶（Dipy)]配合物，测定了配合物的荧光光谱，对其荧光性质进行了研究，结果表明，配合物发射Tb(Ⅲ）的特征荧光，4－酰基0双（1，3－二苯基－5－吡唑啉酮）配体的三重态能级与Tb(Ⅲ）的最低激发态（5D4）能级匹配较好，配合物荧光强度随4－酰基－双（1，3－二苯基－5－吡唑啉酮）配体2个吡唑环间碳链的增长而减弱，第2配体Phen和Dipy具有荧光增强作用，且前者优于后者。′     

在线还原富集-导数火焰原子吸收光谱法测定环境水样中铬(Ⅲ)和铬(Ⅵ)的形态

采用单阀双阳离子交换树脂微柱并联，设计了双路采样逆向洗脱在线分离富集系统，该系统与原子吸收导数测量技术相结合，实现了在线分离富集．导数火焰原子吸收光谱法同时测定水中Cr（Ⅲ）和Cr(Ⅵ），导数仪用2mV／min档位，富集lmin时，分析速度为60样/h，测定Cr（Ⅲ）和Cr(Ⅵ）的特征浓度分别为0．448μg/L和0．793μg/L(相当于1％导数吸收度)，线性范围分别为0-90和0-180μg/L；对浓度分别为10、20μg/LCr（Ⅲ）和Cr(Ⅵ）测定的相对标准偏差分别为2．85％和2．85％；检出限分别为0．855和1．7lμg/L．该法对实际水样加标回收率在94．7％．104％之间。     

修饰聚合物液-固亲和萃取体系连续分离铜(Ⅱ)、锌(Ⅱ)、钴(Ⅱ)、镍(Ⅱ)

用亚胺基二乙酸（IDA）修饰了聚乙二醇PEG8000，并构建了修饰聚合物-盐-水液-固亲和萃取体系，用于高选择富集与分离金属离子。控制最佳分离条件，成相聚合物吐温80浓度；5%～10%；PEG-（IDA）2含量0.1%；（NH4）2SO4浓度；1.14～1.67mol/L；溶液酸度pH3.20～7.00。连续萃取分离了Cu(Ⅱ）、Zn(Ⅱ）、Co（Ⅱ）及Cu（Ⅱ）、Zn（Ⅱ）、Ni（Ⅱ），并探讨了该萃取体系的萃取机理。从水样、发样等多种样品中，成功地富集并测定了痕量Cu(Ⅱ)和Zn（Ⅱ），结果满意。     

多联吡啶螯合物反相高效液相色谱法分离测定铜(Ⅱ)、钴(Ⅱ)、汞(Ⅱ)

以三联吡啶衍生物６，６”－二甲基－４＇－苯基－２，２＇：６＇，２”－三联吡啶（ＴＰＹ）作柱前显色剂，于ＡｃｃＱ－Ｔａｇ柱上，用内含２．０×１０～（－６）ｍｏｌ／Ｌ ＴＰＹ和０．６ ｍｏｌ／Ｌ　ＮａＡｃ－ＨＡｃ缓冲溶液（ｐＨ＝３．５）的甲醇－水溶液（５５：４５，Ｖ／Ｖ）作流动相，流速为１．０ ｍＬ／ｍｉｎ，并以紫外－可见检测器于３１０ｎｍ处进行检测，开发了一种 ＲＰ－ＨＰＬＣ法同时分离测定铜（Ⅱ）、钴（Ⅱ）、汞（Ⅱ）的方法。该方法简便快速，灵敏度高，对于铜、钴、汞的检测限分别是０．００２０、０．００５５和０．００４０ｍｇ／Ｌ。用于实际样品测定，结果满意。     

一种测定痕量铜(Ⅱ)的荧光新体系

茚三酮和过氧化氢反应能产生微弱的荧光,微量铜（Ⅱ）的存在能大大增敏其荧光强度。基于此现象,建立了测定痕量铜（Ⅱ）的催化动力学新方法。该方法具有极高的灵敏度,检出限达7．4×10－12g／mL,线性范围为10－11~10-6g／mL,且选择性好,已成功地应用于人发和中药中痕量铜（Ⅱ）的测定。     

4-(S)-[2-(N-甲基)吗啉基 ]-5-(R )-(l-薄荷烷氧基 )-丁内酯结构分析

N－甲基吗啉对5－（l－薄荷烷氧基）－2（5H）－呋喃酮的光催化不对称共轭加生成了4－（S）－[2－（N－甲基）吗啉基]－5－（R）－(l－薄荷烷氧基）－丁内酯,在四氢呋喃（THF）对比实验、参比物^13CNMR对照及不同溶剂^13CNMR测定的基础上,该新化合物的结构用高分辨率的子核磁共振谱、碳核磁共振谱、质谱、红外光谱及元素分析、旋光度等数据进行了确证。     

卡尔曼滤波在极谱分析中的应用——Sn(Ⅳ)、Pb(Ⅱ)、In(Ⅲ)、Cd(Ⅱ)体系的同时测定

利用卡尔曼滤波（ＫＦ）技术，对互相干扰的Ｓｎ（Ⅳ）、Ｐｂ（Ⅱ）、Ｉｎ（Ⅲ）、Ｃｄ（Ⅱ）体系进行了定量测试研究。在５．００×１０５～３．００×１０４ｍｏｌ·Ｌ１的浓度范围内，各组分测试的相对误差为－４．０％～３．８５％。对大理石抛光粉管理样中的Ｓｎ（Ⅳ）、Ｐｂ（Ⅱ）进行了测定，结果与ＩＣＰＡＥＳ结果相符。回收率在９５．０％～１０４％之间。为保证ＫＦ的精度，使用了两个监控出口。数据说明效果良好     

基于寡核苷酸链的汞离子荧光生物传感器

基于G-四链体结构和卟啉类化合物N-甲基卟啉二丙酸IX(NMM)结合产生强烈的荧光,利用T-Hg(Ⅱ)-T错配对汞离子(Hg2+)的特异性识别,建立了一种简单、灵敏、高效的Hg2+检测新方法.在富含鸟嘌呤(G)寡核苷酸链中,引入了大量胸腺嘧啶(T).在没有Hg2+存在时,可以自发形成G-四链体结构,与NMM结合产生强烈的荧光;在Hg2+存在时,可与另一条富含T序列的互补链通过T-Hg(Ⅱ)-T特异性结合,形成双链DNA分子,从而导致G-四链体结构不能产生.优化后最佳实验条件为:缓冲溶液的pH=6.7,20 mmol/LKCl,2.5 μmol/L NMM,反应时间为2h.在优化条件下,体系的荧光强度变化值与Hg2+浓度呈现良好的线性关系,线性范围为50～ 1000 nmol/L,检出限为22.8 nmol/L(30).此生物荧光传感器对Hg2+具有良好的选择性.实际水样中Hg2+的加标回收率为106.1％ ～ 107.8％,可以满足实际水样品中Hg2+的检测要求.     

Label-free aptamer biosensor for selective detection of thrombin

We fabricated a novel fluorescence biosensor for the selective detection of thrombin by using bovine serum albumin-capped CdS quantum dots (BSA-CdS QDs). Two kinds of designed DNA (DNA1 and DNA2) could bind to CdS QDs through the electrostatic interaction between DNA and Cd²⁺ on the surface of CdS QDs. The obtained DNA/BSA-CdS QDs kept stable in the solution with the fluorescence intensity obviously enhanced. Hairpin structure of DNA1contained two domains, one is the aptamer sequence of thrombin and the other is the complementary sequence of DNA2. When thrombin was added, it would bind to DNA1 and induce the hairpin structure of DNA1 changed into G-quadplex structure. Meanwhile, DNA2 would transfer from the surface of CdS QDs to DNA1 via hybridization, which resulted in the removal of DNA1 and DNA2 from the surface of CdS QDs, and led to the fluorescence intensity of CdS QDs reduced. Thus, the determination of thrombin could be achieved by monitoring the change of the fluorescence intensity of CdS QDs. The present method is simple and fast, and exhibits good selectivity for thrombin over other proteins. We have successfully detected thrombin in human serum samples with satisfactory results.     

Graphene Oxide-Based Homogeneous Fluorescence Sensor for Multiplex Determination of Various Targets by a Multifunctional Aptamer

A sensitive and selective fluorescent aptasensor for the determination of metal ions, small molecules, sequence-specific DNA, and proteins was fabricated. The analytical approach was based on target-induced conformational changes of the probe and the self-assembled probe-graphene oxide architecture. The probe employed fluorescence “on/off” switching in a single step in solution. This approach was simple to prepare and had little background with good sensitivity and rapid response. In the absence of targets, the probe adsorbed on the surface of graphene oxide through π–π stacking and quenched fluorescence of the probe. Upon the addition of analyte, the random coil of the probe bonded to the targets, reducing the interaction between the probe and graphene oxide, which disrupted the energy transfer from the probe to graphene oxide and therefore increased the fluorescence. This approach was employed for the determination of Ag⁺, Hg²⁺, cysteine, sequence-specific DNA, and thrombin. The results demonstrated that the probe-graphene oxide architecture was an excellent and versatile platform for the determination of multiple analytes.     

三聚氰胺对DNA潜在损伤作用的研究

在生理酸度条件下(pH 7.4),采用溴化乙锭(EB)为荧光探针的荧光光谱法、I-离子荧光猝灭效应、DNA熔点和粘度效应等手段,研究了三聚氰胺与DNA的相互作用。随着DNA的加入,三聚氰胺的荧光强度明显减小而且三聚氰胺能够猝灭DNA-EB复合物的荧光,说明三聚氰胺能够竞争置换EB而与DNA作用;三聚氰胺的加入使得DNA的粘度增大,DNA-EB的熔点降低;DNA的加入减小了I-对三聚氰胺荧光的猝灭程度。三聚氰胺以嵌插方式作用于DNA的亲核位点,意味着三聚氰胺进入生物体后有可能通过形成DNA加合物的形式造成DNA损伤,从而最终导致基因突变。     

Surface modified microarray chip and laser induced fluorescence microscopy to detect DNA cleavage

This work describes a quantitative method to detect DNA damage in the presence of Pb and Cd ions using a surface modified microarray chip and a laser induced fluorescence microscopy (LIFM). The detection was carried out by the immobilization of a single-stranded DNA oligomer, tagged with a Cy5 fluorophore on a polydimethylsiloxane (PDMS) microarray chip followed by LIFM. Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC) was attached as a cross-linker via the formation of covalent amide bonds. Then, the single-stranded DNA oligomer containing Cy5 as a fluorophore and thiol functional groups at both terminals, was bonded to the linker by reaction with sulfhydryl group. As the DNA oligomers were reacted with metal ions of Pb and Cd, the un-cleaved DNA oligomers were quantitatively identified by monitoring Cy5 fluorescence. Cadmium showed a quenching constant of 0.84 in the Stern–Volmer plot, whereas lead gave 0.22, indicating that cadmium ions suppress fluorescence more than lead ions. When optimized, fluorescence reductions of 23% (± 2.1) for Pb and 25% (± 1.4) for Cd were observed in air and decreased to almost < 5.0% in a radical scavenger of 5 mM. The cleaved DNA was also confirmed by MALDI-TOF-MS. In result, this experimental method using a microarray chip with surface modification provided quantitative determination of DNA oligomer damage with reproducible results, significantly reduced sample volumes and analysis times.     

Enhancement in fluorescence response by a quencher for amiloride upon binding to thymine opposite an abasic site in a DNA duplex

By using iodide (I⁻) as a quencher, we successfully improve the fluorescence response of amiloride when binding to thymine opposite an AP site in a 21-meric DNA duplex. From fluorescence measurements, as compared to the NaCl solutions, the addition of NaI as a quencher as well as salt to adjust the ionic strength effectively suppresses the background fluorescence from unbound amiloride in a solution. The Stern-Volmer analysis shows that the bound amiloride to the nucleobase at the AP site is unexposed to NaI quencher. Therefore the high signal-to-background fluorescence response of amiloride is obtained. Such enhancement in fluorescence response of amiloride by using the quencher can provide the significant improvement of the detection limit for DNA duplexes carrying T target base. The method presented in this study is simple and effective. The present method could be applicable to other detection system where microenvironment of fluorophores changes at a recognition event.     

A fluorescent molecular switch for room temperature operation based on oligonucleotide hybridization without labeling of probes or targets

A molecular switch was prepared by self-assembly. Neutravidin served as a template that allowed for a biotinylated probe oligonucleotide to be placed adjacent to a biotinylated long-chain linker that was terminated with thiazole orange (TO). Hybridization of probe oligonucleotide with target to form double-stranded DNA resulted in intercalation of the adjacent TO probe. This was a reversible process that could be tracked by fluorescence intensity changes. Formamide was used as a denaturant for double-stranded DNA, and could be used to depress thermal denaturation temperatures. In this work formamide had a dual function, providing for control of hybridization selectivity at room temperature, while concurrently ameliorating non-specific adsorption to improve signal-to-noise when using thiazole orange as a fluorescence signalling agent to determine oligonucleotide hybridization. Room temperature single nucleotide polymorphism (SNP) discrimination for oligonucleotide targets was achieved both in solution and for molecular switches that were immobilized onto optical fibers. In solution, a concentration of 18.5% formamide provided greater than 40-fold signal difference between single-stranded DNA and double-stranded DNA, in contrast to only a 2-fold difference in the absence of formamide. Selectivity for SNP determination in solution was demonstrated using targets of varying lengths including a 141-base PCR amplicon. The improved signal-to-noise achieved by use of formamide is likely due to preferential displacement of dye molecules that are otherwise electrostatically bound to the polyanionic nucleic acid backbone.     

Fluorimetric determination of nucleic acid using the enhancement of terbium-gadolinium-nucleic acid-cetylpyridine bromide system

It is found that the fluorescence intensity of Tb-cetylpyridine bromide (CPB)-nucleic acid system can be enhanced by La³⁺, Gd³⁺, Lu³⁺, Sc³⁺ and Y³⁺, of which Gd³⁺ has the greatest enhancement. The experiments indicate that under the optimum condition, the fluorescence intensity of the system is in proportion to the concentration of nucleic acids in the range from 9×10⁻⁸ to 1×10⁻⁵ g ml⁻¹ for yeast RNA, from 1×10⁻⁷ to 1×10⁻⁵ g ml⁻¹ for fish sperm DNA. The detection limits are 3.2 and 4.1 ng ml⁻¹, respectively. This method has been used satisfactorily for the determination of both synthetic and actual samples. In comparison with most fluorescence method for the determination of nucleic acids, this method has higher sensitivity and stability.     

稳态荧光猝灭法确定胶束聚集数的研究

选用芘作为荧光探针, 二苯酮作为猝灭剂, 以稳态荧光猝灭法测定了十二烷基硫酸钠、十二烷基磺酸钠和十六烷基三甲基演化铵的胶束聚集致, 并对其测定胶束聚集数方法的有效范围进行了讨论. 实验结果表明, 选择的探针-猝灭剂体系适用于稳态荧光猝灭法确定阴离子和阳离子表面活性剂的胶束浓度和聚集数.     

基于CdSe-CdTe量子点能量转移荧光猝灭法测定前列腺抗原

研究了CdSe-CdTe量子点间发生的荧光共振能量转移,并用于荧光猝灭法测定超痕量前列腺抗原(PSA)。在pH 8.0的Tris-HCl缓冲溶液中,CdSe-CdTe间发生有效能量转移,使CdTe荧光大大增强。PSA抗原与CdTe标记的PSA抗体发生特异性反应,使能量转移体系的CdTe上的荧光强度降低,即发生猝灭。建立了CdSe-CdTe能量转移荧光猝灭法测定PSA抗原的方法。在优化的实验条件下,PSA抗原的线性范围为0.28～10μg/L,相关系数r=0.9992,检出限达1.5×10-2μg/L(n=11)。