Separation and detection of amino acid metabolites of Escherichia coli in microbial fuel cell with CE

In this work, CE‐LIF was employed to investigate the amino acid metabolites produced by Escherichia coli (E. coli) in microbial fuel cell (MFC). Two peptides, l ‐carnosine and l ‐alanyl‐glycine, together with six amino acids, cystine, alanine, lysine, methionine, tyrosine, arginine were separated and detected in advance by a CE‐LIF system coupled with a homemade spontaneous injection device. The injection device was devised to alleviate the effect of electrical discrimination for analytes during sample injection. All analytes could be completely separated within 8 min with detection limits of 20–300 nmol/L. Then this method was applied to analyze the substrate solution containing amino acid metabolites produced by E. coli. l ‐carnosine, l ‐alanyl‐glycine, and cystine were used as the carbon, nitrogen, and sulfur source for the E. coli culture in the MFC to investigate the amino acid metabolites during metabolism. Two MFCs were used to compare the activity of metabolism of the bacteria. In the sample collected at the running time 200 h of MFC, the amino acid methionine was discovered as the metabolite with the concentrations 23.3 μg/L.     

A Sensitive Nanoporous Gold-Based Electrochemical DNA Biosensor for Escherichia coli Detection

A sensitive electrochemical DNA biosensor based on a mixed monolayer structure self-assembled at nanoporous gold (NPG) electrode surface was prepared for Escherichia coli (E. coli) detection. The NPG was fabricated on gold electrode, onto which thiolated oligonucleotides (SH-DNA) and mercaptohexanol (MCH) were covalently linked forming a mixed self-assembled monolayer (SAM). The hybridization between the SH-DNA/MCH modified biosensor and E. coli DNA was monitored with differential pulse voltammetry measurement using methylene blue (MB) as the hybridization indicator. The biosensor can detect 1 × 10^?12 M DNA target and 50 cfu/μL E. coli without any nucleic acid amplification steps. The detection limit was lowered to 50 cfu/mL after 5.0 h of incubation.     

Fura-2荧光探针研究Ca2+对大肠杆菌细胞的跨膜作用

以Fura-2／AM作为荧光探针,研究了Ca^2 对大肠杆菌HB101细胞的跨膜作用．考察了不同浓度外源钙离子处理不同生长时期细胞的跨膜行为,并采用停流技术测定了荧光动力学．结果表明大肠杆菌在用CaCl2溶液处理后,胞外Ca^2 可大量进入胞内,且进入细胞的钙离子量与胞外钙离子浓度相关;处于对数生长前期的细胞与对数生长后期和稳定期的细胞相比,胞内自身Ca^2 浓度低,但其摄取外源Ca^2 的能力最强,这应该与其生理代谢活性是相关的,而且这一时期是大肠杆菌细胞最易建立人工诱导感受态的时期．该研究对于测定革兰氏阴性菌细胞内Ca^2 浓度及胞外离子的跨膜传导行为,以及探索钙离子诱导的大肠杆菌人工感受态建立的生理机制都具有重要意义．     

Rapid pre-concentration of Escherichia coli in a microfluidic paper-based device using ion concentration polarization

We report a microfluidic paper based analytical device implementing ion concentration polarization (ICP) for rapid pre-concentration of Escherichia coli in water. The fabricated device consists of a paper channel with a Nafion^® membrane and in-built micro wire electrodes to supply electric voltage to induce the ICP effect. E. coli cells were stained with SYTO 9 and fluorescence was used as a sensing method. The device achieved high concentration factor up to 2 × 10⁵ within minutes. The effect of total ion concentration, on ICP and fluorescence intensity was studied. The reported device and method are suitable and effective for detection of E. coli during ballast water quality monitoring, coastal water quality monitoring where high salinity water is present.     

大肠杆菌代谢物组成的核磁共振分析

为了认识重要模式生物大肠杆菌的内源性代谢物组成,本研究综合使用一维和二维高分辨核磁共振波谱技术,系统测定了大肠杆菌的代谢物种类和含量.结果表明:核磁共振方法可检测到分别来自多个代谢途径的氨基酸、有机羧酸、糖类、核苷及其衍生物等40多种高丰度代谢物.葡萄糖是稳定期大肠杆菌中含量最丰富的物质;谷氨酸、甜菜碱、腐胺和核糖-5...     

A New Sensor Method for Studying the Effect of Gentamicin and Cefotaxime Combination Against Escherichia coli

Abstract

A new sensor method based on a multi-channel series piezoelectric quartz crystal (MSPQC) was proposed for studying the effect of gentamicin and cefotaxime combination against Escherichia coli ATCC 25922. The frequency curves under different combination regimens were obtained and compared with each other. When antibiotic had an inhibitory effect to the growth of bacteria, frequency detection time (FDT) would be prolonged. The FDT was used to assess the effect of an antibiotic combination. By using the proposed method, simultaneous administration, different order, and time interval of nonsimultaneous administration were investigated in detail. The MSPQC method can provide the process information in real time. It is simple, rapid, and easy to perform.     

Au纳米标记物增强电化学免疫分析大肠杆菌的研究

通过在Au纳米颗粒表面修饰辣根过氧化酶(HRP)标记的大肠杆菌抗体制备了一种新型的Au纳米标记物, 并将该纳米标记物应用于增强电化学免疫分析大肠杆菌. 经过酶联免疫反应后, Au纳米标记物、免疫磁性颗粒(IMB)和大肠杆菌形成了IMB/抗体-大肠杆菌-Au纳米标记物的三明治式免疫复合物. 以3,3,5,5-四甲基联苯二胺(TMB)溶液作为底物, 采用电化学与流动注射检测(FIA)相结合的技术测定HRP的活性. 检测到的电流大小与免疫复合物上HRP的量成正比, 从而与大肠杆菌的浓度成正比. Au纳米颗粒增加了HRP的负载量, 增强了电化学信号, 大大提高了大肠杆菌的检测灵敏度. 实验结果表明, 大肠杆菌浓度在 1.0×102～5.0×104 cfu•mL－1范围内与电流大小成线性相关, 最低检测限达50 cfu•mL－1, 若对大肠杆菌样品溶液进行预浓缩, 将得到更宽的检测范围和更低的检测限. 本方法总的分析时间比其他方法短, 在1 h内就能完成对大肠杆菌样品的快速检测.     

Metabolic viability of Escherichia coli trapped by dielectrophoresis in microfluidics

The spatial and temporal control of biological species is essential in complex microfluidic biosystems. In addition, if the biological species is a cell, microfluidic handling must ensure that the cell's metabolic viability is maintained. The use of DEP for cell manipulation in microfluidics has many advantages because it is remote and fast, and the voltages required for cell trapping scale well with miniaturization. In this paper, the conditions for bacterial cell (Escherichia coli) trapping using a quadrupole electrode configuration in a PDMS microfluidic channel were developed both for stagnant and for in‐flow fluidic situations. The effect of the electrical conductivity of the fluid, the applied electric field and frequency, and the fluid‐flow velocity were studied. A dynamic exchange between captured and free‐flowing cells during DEP trapping was demonstrated. The metabolic activity of trapped cells was confirmed by using E. coli cells genetically engineered to express green fluorescent protein under the control of an inducible promoter. Noninduced cells trapped by negative DEP and positive DEP were able to express green fluorescent protein minutes after the inducer was inserted in the microchannel system immediately after DEP trapping. Longer times of trapping prior to exposure to the inducer indicated first a degradation of the cell metabolic activity and finally cell death.     

Highly Sensitive in vitro Biosensor for Enterotoxigenic Escherichia coli Detection Based on ssDNA Anchored on PtNPs‐Chitosan Nanocomposite

Detection of Enterotoxigenic Escherichia coli in various biological samples has tremendous importance in human health. In this direction, we have designed a label free electrochemical biosensor for highly selective detection of Escherichia coli through detecting ST gene. The ability of sensor probe to detect ST_G was confirmed using polymerase chain reaction. The biosensor was fabricated based on ST_G specific probes immobilized on platinum nanoparticles chitosan nanocomposite on screen printed carbon electrode, which was characterized by cyclic voltammetry, transmission electron microscopy, and fourier transform infrared spectroscopy. A highly sensitive label free sensing was achieved by analyzing ST_G hybridization using electrochemical impedance spectroscopy (EIS) technique. The EIS analysis showed a significant increase in charge transfer resistance after ST_G interaction with the highly selective ssDNA probe immobilized on the nanocomposite film. The increase in charge transfer resistance was evaluated for varying concentrations of ST_G, which shows a dynamic range between 1.0×10⁻¹² and 1.0×10⁻⁴ with the detection limit of 3.6×10⁻¹⁴ M (RSD<4.5 %). The regeneration of sensor probe was also studied and interference due to non‐target sequences was evaluated to ensure the selectivity of the designed sensor. The practical applicability of sensor probe was also analyzed by detecting the ST_G from the bacteria present in surface water.     

水溶性CdSe量子点的合成及其作为荧光探针对大肠杆菌的快速检测

采用水相法以谷胱甘肽为稳定剂合成高稳定性的CdSe量子点,利用化学偶联剂的作用使得量子点表面基团与菌体之间的成功结合,对偶联的条件进行了优化.基于荧光分析法建立了一种快速简便的大肠杆菌检测定量分析方法.研究结果表明:合成的量子点具有稳定、荧光性能良好等突出优点.通过偶联剂量子点能与大肠杆菌结合,其荧光强度与大肠杆菌浓度...     

亲水作用色谱/质谱联用方法用于大肠杆菌代谢组分析

建立了两性离子亲水作用色谱/质谱联用方法用于大肠杆菌胞内极性代谢物的分离分析。选取52个代表性极性物质对方法进行考察,发现此方法有较好的线性范围,且大部分物质最低检测限均在ng/mL数量级。平行制备6份样品进行分析,结果显示85%以上代谢物峰面积的RSD值小于30%。6个内标物质在低、中、高3个浓度下的日内精密度（RSD）均小于20%,大部分物质的相对回收率都在可接受的范围内（70%~130%）。把此方法用于yfcC基因改造的3株大肠杆菌代谢组分析,发现一些小肽、氨基酸、核苷、有机酸、磷脂等物质在基因改造后发生明显变化。此研究结果表明,建立的两性离子亲水作用色谱/质谱方法检测到的物质化学性质分布广,跨越了极性磷脂到小肽的各个范围,且具有良好的重复性、稳定性和适用性。     

Metabolic profiling of Escherichia coli by ion mobility-mass spectrometry with MALDI ion source

Comprehensive metabolome analysis using mass spectrometry (MS) often results in a complex mass spectrum and difficult data analysis resulting from the signals of numerous small molecules in the metabolome. In addition, MS alone has difficulty measuring isobars and chiral, conformational and structural isomers. When a matrix-assisted laser desorption ionization (MALDI) source is added, the difficulty and complexity are further increased. Signal interference between analyte signals and matrix ion signals produced by MALDI in the low mass region (<1500 Da) cause detection and/or identification of metabolites difficult by MS alone. However, ion mobility spectrometry (IMS) coupled with MS (IM-MS) provides a rapid analytical tool for measuring subtle structural differences in chemicals. IMS separates gas-phase ions based on their size-to-charge ratio. This study, for the first time, reports the application of MALDI to the measurement of small molecules in a biological matrix by ion mobility-time of flight mass spectrometry (IM-TOFMS) and demonstrates the advantage of ion-signal dispersion in the second dimension. Qualitative comparisons between metabolic profiling of the Escherichia coli metabolome by MALDI-TOFMS, MALDI-IM-TOFMS and electrospray ionization (ESI)-IM-TOFMS are reported. Results demonstrate that mobility separation prior to mass analysis increases peak-capacity through added dimensionality in measurement. Mobility separation also allows detection of metabolites in the matrix-ion dominated low-mass range (m/z < 1500 Da) by separating matrix signals from non-matrix signals in mobility space.     

Threonine Facilitates Cd Excretion by Increasing the Abundance of Gut Escherichia coli in Cd-Exposed Mice

Cadmium (Cd) can easily enter the body through the food chain and threaten health since Cd pollution is prevalent in the environment. Gut microbiota is necessary for the reduction of metal ions. To reduce Cd-induced harmful impacts and Cd accumulation in the body, we investigated the effect of amino acids on gut microbiota and Cd excretion in (fecal Cd) Cd-exposed mice. The screening of 20 amino acids showed that threonine (Thr) effectively increased fecal Cd, and reduced Cd-induced intestinal structural damage. The abundance of Escherichia-Shigella genus and KF843036_g significantly increased after the oral administration of Thr. As the type species of the Escherichia-Shigella genus, Escherichia coli exhibited high similarity to KF843036_g species and significantly decreased Cd-induced gut damage. Cd contents in the liver, kidney, and gut of Cd-exposed mice were also significantly (p < 0.05) decreased after E. coli treatment, while the contents in the feces were increased. The results demonstrated the potential roles that gut E. coli might play in Thr-mediated Cd excretion in Cd-exposed mice. The findings may provide important data for better understanding the molecular biological mechanism of Thr in reducing Cd accumulation in the body.     

Inactivation of Escherichia coli on Immobilized CuO/CoFe2O4‐TiO2 Thin‐Film under Simulated Sunlight Irradiation

Composite photocatalysts of CuO/CoFe₂O₄‐TiO₂ were successfully synthesized by a sol‐gel method and fixed on ordinary tiles. The photosterilization of Escherichia coli was examined on CuO/CoFe₂O₄‐TiO₂ thin films under a xenon lamp irradiation. The film was characterized by XRD, and the morphology was observed by SEM. Disinfection data indicated that CuO/CoFe₂O₄‐TiO₂ composite photocatalysts have the much better photocatalytic activity than CuO/CoFe₂O₄ and TiO₂. The optimized composition of the nanocomposites has been found to be m_CuO/CoFe2O4:m_TiO2=3:7, with loadings ranging from 790 to 1400 mg/m². The photocatalytic inactivated rate of E. coli (10⁵ CFU/mL) reached 98.4% under the xenon lamp of 150 W within 30 min.     

Preparation of the Ag2O2-PbO2 Modified Electrode and Its Application towards Escherichia coli Fast Counting in Water

A novel nano crystalline Ag2O2-PbO2 film chemically modified electrode (CME) was prepared and the CME was characterized by X-ray diffractometer (XRD) and atomic focrce microscope (AFM). By chronoamperometry, the nano Ag2O2-PbO2 CME was used as bioelectrochemical sensor to determine the population of Escherichia coli (E.coli) in water. Compared with conventional methods, it is found that the technique we used is fast and convenient in counting E.coil     

A Rapid and Sensitive Aptamer‐Based Electrochemical Biosensor for Direct Detection of Escherichia Coli O111

A sensitive and specific electrochemical biosensor based on target‐induced aptamer displacement was developed for direct detection of Escherichia coli O111. The aptamer for Escherichia coli O111 was immobilized on a gold electrode by hybridization with the capture probe anchored on the electrode surface through Au‐thiol binding. In the presence of Escherichia coli O111, the aptamer was dissociated from the capture probe‐aptamer duplex due to the stronger interaction between the aptamer and the Escherichia coli O111. The consequent single‐strand capture probe could be hybridized with biotinylated detection probe and tagged with streptavidin‐alkaline phosphatase, producing sensitive enzyme‐catalyzed electrochemical response to Escherichia coli O111. The designed biosensor showed weak electrochemical signal to Salmonella typhimurium, Staphylococcus aureus and common non‐pathogenic Escherichia coli, indicating high specificity for Escherichia coli O111. Under the optimal conditions, the proposed strategy could directly detect Escherichia coli O111 with the detection limit of 112 CFU mL^?1 in phosphate buffer saline and 305 CFU mL^?1 in milk within 3.5 h, demonstrated the sensitive and accurate quantification of target pathogenic bacteria. The designed biosensor could become a powerful tool for pathogenic microorganisms screening in clinical diagnostics, food safety, biothreat detection and environmental monitoring.     

Reaction‐Based Ratiometric Chemosensor for Instant Detection of Cyanide in Water with High Selectivity and Sensitivity

A highly selective chemosensor 1 based on an acylhydrazone group as binding site and naphthalene group as the fluorescence signal group were described, which could instantly detect CN^? in water with specific selectivity and high sensitivity. The detection of cyanide was performed via the nucleophilic attack of cyanide anion on the carbonyl group, which could be confirmed by ¹H NMR, ¹³C NMR, ESI‐MS and DFT calculations. The addition of CN^? to sensor 1 induced a remarkable color change from colorless to yellow and generated a blue fluorescence, these sense procedure could not interfered by other coexistent competitive anions (F^?, Cl^?, Br^?, I^?, AcO^?, H₂PO₄^?, HSO₄^?, ClO₄^?, SCN^?, S^2?, NO₃^? and SO₄^2?). The detection limits were 5.0×10^?7 M and 2.0×10^?9 M of CN^? using the visual fluorescent color changes and fluorescence spectra changes respectively, which is far lower than the WHO guideline of 1.9×10^?6 M . Test strips based on sensor 1 were fabricated, which could act as a convenient and efficient CN^? test kit to detect CN^? in pure water for “in‐the‐field” measurement.     

嗜盐古菌染色体DNA片段在大肠杆菌中的启动子功能

以启动子探针质粒pKK232-8为载体, 用微量量热法研究了源自盐生盐杆菌R1染色体的RM13 DNA片段在大肠杆菌HB101中的真细菌启动子功能, 该启动子RM13 DNA片段的大小为1000bp(碱基对), 它能启动探针质粒pKK232-8上的氯霉素乙酰水解酶基因(即:氯霉素抗性基因Cmr), 氯霉素抗性水平可达到150 mg•L－1, 抗性水平较高, 启动活性较大.研究结果表明, 在盐生盐杆菌染色体上可能存在具有双功能或多功能的启动子DNA片段, 这对系统发育、微生物遗传学和生物热化学等均有重要的意义.     

铂纳米颗粒修饰电极对大肠杆菌的电化学快速检测

本文采用了电化学沉积法制备了铂纳米颗粒化学修饰电极（PtNP/GCE）,并将它应用于大肠杆菌的检测。原理是基于检测大肠杆菌溶液中酶与底物的反应产物,对氨基酚,实现了对大肠杆菌的快速检测。采用了铂纳米颗粒修饰电极,并对检测系统进行优化,提高大肠杆菌的检测灵敏度。大肠杆菌浓度在50—1.0×10⁵cfu/ml与响应电流成良好的线性关系,最低检测限为20 cfu/ml,检测时间在4个小时以内。与传统方法相比,该电化学方法能很好地满足食品安全、环境监控和临床医学等领域中快速检测的要求。