荧光原位杂交技术在活性污泥菌群识别中的研究进展

荧光原位杂交技术(FISH)广泛地应用于分析环境微生物的群落组成,能够同时对微生物进行定性、定量分析,并确定微生物群落空间分布情况.本文阐述了FISH法应用于活性污泥处理系统的重要作用,综述了FISH法在活性污泥及生物膜微生物菌群的组成、结构与功能的研究和应用,同时讨论了其在应用中存在的不足,并在此基础上总结了一系列可...     

Universal liposomes: preparation and usage for the detection of mRNA

Dye-encapsulating liposomes can serve as signaling reagents in biosensors and biochemical assays in place of enzymes or fluorophores. Detailed here is the use and preparation of streptavidin-coupled liposomes which offer a universal approach to biotinylated target detection. The universal approach provides two advantages, i.e. only one type of liposome is necessary despite varying target and probe sequences and the hybridization event can take place in the absence of potential steric hindrance occurring from liposomes directly conjugated to probes. One objective of this work was to optimize the one-step conjugation of SRB-encapsulating liposomes to streptavidin using EDC. Liposome, EDC, streptavidin concentrations, and reaction times were varied. The optimal coupling conditions were found to be an EDC:carboxylated lipid:streptavidin molar ratio of 600:120:1 and a reaction time of 15 min. The second goal was to utilize these liposomes in sandwich hybridization microtiter plate-based assays using biotinylated reported probes as biorecognition elements. The assay was optimized in terms of probe spacer length, probe concentration, liposome concentration, and streptavidin coverage. Subsequently, the optimized protocol was applied to the detection of DNA and RNA sequences. A detection limit of 1.7 pmol L⁻¹ and an assay range spanning four orders of magnitude (5 pmol L⁻¹−50 nmol L⁻¹) with a coefficient of variation ≤5.8% was found for synthetic DNA. For synthetic RNA the LOQ was half that of synthetic DNA. A comparison was made to alkaline phosphatase-conjugated streptavidin for detection which yielded a limit of quantitation approximately 80 times higher than that for liposomes in the same system. Thus, liposomes and the optimized sandwich hybridization method are well suited for detecting single-stranded nucleic acid sequences and compares favorably to other sandwich hybridization schemes recently described in the literature. The assay was then used successfully for the clear detection of mRNA amplified by nucleic acid sequence-based amplification (NASBA) isolated from as little as one Cryptosporidium parvum oocyst. The detection of mRNA from oocysts isolated from various water sample types using immunomagnetic separation was also assessed. Finally, to prove the wider applicability and sensitivity of this universal method, RNA amplified from the atxA gene of Bacillus anthracis was detected when the input to the preceding NASBA reaction was as low as 1.2 pg. This highly sensitive liposome-based microtiter plate assay is therefore a platform technology allowing for high throughput and wide availability for routine clinical and environmental laboratory applications.     

借助聚丙烯酸制备铜粒子修饰电极并用于双氧水的电催化性能研究

利用羧基对金属离子的络合作用在电极表面原位制备金属粒子用于电催化过氧化氢(H2O2). 首先在电极表面电沉积聚丙烯酸, 然后化学吸附铜离子, 再用水合肼将其还原成零价铜. 铜粒子簇均匀分散在聚丙烯酸网络中, 其尺寸可通过吸附铜离子的数量和吸附-还原次数来控制. 由于铜微粒的电催化作用, 该修饰电极对H2O2表现出了良好的电流响应. 运用该方法可以在电极表面制备多种金属微粒, 并且聚丙烯酸的自由羧基可以与氨基反应, 从而可共价固定带氨基的酶和抗体, 有望构建多种新型的化学和生物传感器.