SPR生物传感器快速检测大肠杆菌O157:H7的研究

建立了一种基于表面等离子体共振(surface plasmon resonance,SPR)原理的生物传感器方法,实现了快速检测大肠杆菌O157:H7.研究选用BIACORE 3000系统及葡聚糖修饰的CM5芯片,先用EDC/NHS将芯片活化,然后抗体直接通过酰胺键固定在金表面,再用乙醇胺封闭,这样处理之后的芯片就可用于检测大肠杆菌O157:H7.利用NaOH溶液对芯片再生,实现对多个不同浓度样品检测,采用时间对响应单位(RU)记录数据.该法检测大肠杆菌O157:H7的检测限为3×105 CFU·mL-1,RU变化值和大肠杆菌O157:H7的浓度在一定范围内相关性良好,相关系数达到0.99.检测时间短,一个样品仅需5～7 min,再生效果好,芯片可重复使用50次以上.可快速、在线、稳定地检测大肠杆菌O157:H7,有望成为一种在线检测食品致病性微生物的有力手段.     

Inactivation of Escherichia coli by ultrasonic irradiation

Ultrasonic inactivation of Escherichia coli XL1-Blue has been investigated by high-intensity ultrasonic waves from horn type sonicator (27.5 kHz) utilizing the "squeeze-film effect". The amplitude of the vibration face contacting the sample solution was used as an indication of the ultrasonic power intensity. The inactivation of the E. coli cells by ultrasonic irradiation shows pseudo first-order behavior. The inactivation rate constant gradually increased with increasing amplitude of the vibration face and showed rapid increase above 3 microm (p-p). In contrast, the H2O2 formation was not observed below 3 microm (p-p), indicating that the ultrasonic shock wave might be more important than indirect effect of OH radicals formed by ultrasonic cavitation in this system. The optimal thickness of the squeeze film was determined as 2 mm for the E. coli inactivation. More than 99% of E. coli cells was inactivated within 180-s sonication at the amplitude of 3 microm (p-p) and 2 mm of the thickness of the squeeze film.     

基于硒化锌薄膜法的食源性致病菌原位红外光谱检测

食源性致病菌是引发和威胁公众健康的主要因素之一。由于食源性致病菌种类繁多,常规检测方法复杂耗时要求高,因此迫切需要一种更加快速精确的致病菌检测技术。在传统红外光谱检测致病菌的流程中,如经典的溴化钾压片法,除了压片本身的操作之外通常还需对样品进行冷冻干燥（约需2 d）等耗时前处理过程,因而不利于高通量快速检测。本研究利用硒化锌薄膜法,在硒化锌窗片上直接滴加菌液、低温（48 ℃）烘干后进行原位检测,无需漫长的冻干处理,整个检测过程在50 min之内。同时,检测所需样品量少（10 μL）无需研磨等物理破坏性的制样过程,避免了常规溴化钾压片法中研磨颗粒粗细、制片厚薄误差及易碎片、吸潮等的不利影响。四种常见食源性致病菌（大肠杆菌DH5α;沙门氏菌CMCC 50041;霍乱弧菌SH04;金黄色葡萄球菌SH10）的硒化锌薄膜法与溴化钾压片法红外谱图对比分析表明：在相同的峰值检测阈值下（透过率大于0.05%）,本研究所采用的方法获得的二阶导数图谱在900～1 500 cm-1范围内可被识别的特征峰个数比溴化钾压片法明显增多（硒化锌薄膜法共计81个,溴化钾压片法共计58个）,特征峰在多个位置强度显著增加(1 119,1 085和915 cm-1等),且可将溴化钾压片法中较宽的单峰或不明显的双峰显示为较明显的双峰（大肠杆菌DH5α：1 441,1 391和1 219 cm-1等; 沙门氏菌CMCC 50041：1 490,1 219和1 025 cm-1;霍乱弧菌SH04：1 441和1 219 cm-1;金黄色葡萄球菌SH10：1 491,1 397和1 219 cm-1）,说明硒化锌薄膜法可以提高图谱分辨率及信噪比。基于硒化锌薄膜法的原位红外光谱法对常见食源性致病菌整体快速高通量检测将具有巨大的应用前景。     

Frequency-dependent Escherichia coli chemotaxis behavior

We study Escherichia coli chemotaxis behavior in environments with spatially and temporally varying attractant sources by developing a unique microfluidic system. Our measurements reveal a frequency-dependent chemotaxis behavior. At low frequency, the E.?coli population oscillates in synchrony with the attractant. In contrast, in fast-changing environments, the population response becomes smaller and out of phase with the attractant waveform. These observations are inconsistent with the well-known Keller-Segel chemotaxis equation. A new continuum model is proposed to describe the population level behavior of E.?coli chemotaxis based on the underlying pathway dynamics. With the inclusion of a finite adaptation time and an attractant consumption rate, our model successfully explains the microfluidic experiments at different stimulus frequencies.     

Temperature-dependent magnetization in (Mn, N)-codoped ZnO-based diluted magnetic semiconductors

The influences of Mn doping on the structural quality of the Zn_xMn_1−xO:N alloy films have been investigated by XRD. Chemical compositions of the samples (Zn and Mn content) and their valence states were determined by X-ray photoelectron spectrometry (XPS). Hall effect measurements versus temperature for Zn_xMn_1−xO:N samples have been designed and studied in detail. The ferromagnetic transitions happened at different T_C should explain that the magnetic transition in field-cooled magnetization of Zn_1−xMn_xO:N films at low temperature is caused by the strong p-d exchange interactions besides magnetic transition at 46 K resulting from Mn oxide, and that the room temperature ferromagnetic signatures are attributed to the uncompensated spins at the surface of anti-ferromagnetic nano-crystal of Mn-related Zn(Mn)O.     

Low intensity infrared laser effects on Escherichia coli cultures and plasmid DNA

Biostimulative effect of low intensity laser in tissues has been described on a photobiological basis and clinical protocols are recommended for treatment of various diseases. The aim of this work was to evaluate effects of laser exposure on the survival of Escherichia coli cultures and plasmid topological forms. Escherichia coli cultures and plasmids were exposed to infrared laser to study bacterial survival and electrophoretic profile, respectively. Data indicate low intensity infrared laser: (i) had no effect on E. coli wild type, endonuclease IV, exonuclease III, formamidopyrimidine DNA glycosylase/MutM protein and endonuclease III deficient cultures, but decreased the survival of E. coli UvrA protein deficient cultures; (ii) there was no alteration in the electrophoretic profile of plasmids. Exposure to low intensity infrared laser decreases survival of Escherichia coli cultures deficient in nucleotide excision repair of DNA and this effect could depend on fluences, wavelength and tissues conditions.     

免疫磁分离技术在E.coli O157:H7检测中的应用

免疫磁分离(immunomagnetic separation)技术因其具有选择性好、特异性强、能起到浓缩的作用,已与其他快速检测方法如电化学、光学等方法连用,应用于食品、环境卫生检测等领域.免疫磁分离技术是目前最有推广价值的技术之一.文章通过比较不同的E.coli O157:H7-免疫磁珠(immunomagnetic beads,IMB)浓度配比、E.coli O157:H7与Bacillus subtilis不同的体积比下的免疫磁分离的捕获率,得出当E coli O157:H7-IMB浓度配比为1:30时,捕获率近100%,即所有的目标菌均可被捕获到;在总体积不变、IMB的加入量一定的情况下,随着Bacillus subtilis比例的增加,捕获率出现先下降后上升的情况.同时,将IMS与ATP生物发光法结合起来,对不同浓度的E coli O157:H7进行了检测,得出该方法与传统的平板培养法具有很好的线性相关性,则R2=0.988 2,检测限可低达102 CFU·mL-1.     

A radial mode ultrasonic horn for the inactivation of Escherichia coli K12

Tuned cylindrical radial mode ultrasonic horns offer advantages over ultrasonic probes in the design of flow-through devices for bacterial inactivation. This study presents a comparison of the effectiveness of a radial horn and probe in the inactivation of Escherichia coli K12. The radial horn is designed using finite element analysis and the predicted modal parameters are validated using experimental modal analysis. A validated finite element model of the probe is also presented. Visual studies of the cavitation fields produced by the radial horn and probe are carried out using luminol and also backlighting to demonstrate the advantages of radial horns in producing a more focused cavitation field with widely dispersed streamers. Microbiological studies show that, for the same power density, better inactivation of E. coli K12 is achieved using the radial horn and, also, the radial horn offers greater achievable power density resulting in further improvements in bacterial inactivation. The radial horn is shown to be more effective than the probe device and offers opportunities to design in-line flow-through devices for processing applications.     

Molecular regulatory mechanisms of Escherichia coli O157:H7 in response to ultrasonic stress revealed by proteomic analysis

The antimicrobial effects of ultrasonic filed have been studied for years at the phenotypic level, but there is little research to reveal the molecular regulatory mechanisms underlying the phenotypes. In this study, isobaric tag for relative and absolute quantification (iTRAQ) proteome was applied to analyze the regulatory networks of Escherichia coli O157:H7 in response to ultrasonic stress in whole-genome scale. A total of 1856 differentially expressed proteins were identified, of which 1141 were significant up-regulated and 715 down-regulated compared with live control cells. The comprehensive proteome coverage analysis showed that ultrasonic filed influenced various metabolic pathways in Escherichia coli O157:H7 cells. The ultrasound-induced up-regulation of global stress response regulator RpoS, bacterial mechanosensitive channels and SOS response protein RecA were described from the molecular level for the first time. In addition, we proposed a possible action mechanism that the free radicals produced by acoustic cavitation might enter into cells via the activated mechanosensitive channels, leading to the elevated intracellular ROS level and subsequent cell death. Last but not the least, we illustrated the all-or-nothing phenomenon of power ultrasound might due to the destruction of crucial cell defensive systems, including heat shock proteins and oxidative response regulators. These new findings can let us understand the ultrasonic effects more deeply and will contribute to this area.     

以银纳米棒/氯化银为SERS基底用维多利亚蓝B分子探针检测大肠杆菌

加入增敏剂AgNO₃和NaCl,在银纳米棒(AgNRs)表面吸附了较牢固的AgCl并形成高SERS活性的AgNR/AgCl溶胶基底,维多利亚蓝B(VBB)分子探针在1 611 cm-1处有一较强的SERS峰。用VBB做大肠杆菌(EC)的染色剂,使染色的大肠杆菌具备VBB分子探针的SERS特性,即VBB染色大肠杆菌也在1 611 cm-1处有一较强的SERS峰。在最优条件下,该SERS峰强与大肠杆菌浓度在5×106～3×109 cfu·mL-1 范围内成正比,检出限为2×106 cfu·mL-1,用于水样和饮料中大肠杆菌的分析,具有简便、快速、灵敏等优点。     

Ultrasound pasteurization: the effects of temperature, soluble solids, organic acids and pH on the inactivation of Escherichia coli ATCC 25922

The objectives of this research were to study the effects of temperature, sugar concentration (8, 12, and 16 g/100ml), organic acids (citric and malic acids) and pH (2.5 and 4.0) on ultrasound pasteurization. The model organism used for the research was Escherichia coli ATCC 25922, and ultrasound treatment times were conducted to achieve a 5 log (base 10) reduction. Ultrasound generates heat, therefore the study involved removing the heat using a jacketed beaker with refrigerant (-30 degrees C) to maintain processing temperature at or below 30 degrees C and eliminate the thermal inactivation effects. Overall, ultrasound increased the sensitivity of E. coli to thermal inactivation. The presence of soluble solids had a protective effect where the sonication time requirement increased. Similar to heat sensitivity, the lower pH environment resulted in E. coli having less resistance to sonication. The type of organic acid had the least significant effect on ultrasound inactivation of E. coli.     

Lasing from Escherichia coli bacteria genetically programmed to express green fluorescent protein

We report on lasing action from colonies of Escherichia coli bacteria that are genetically programmed to synthesize the green fluorescent protein (GFP). When embedded in a Fabry-Perot type cavity and excited by ns-pulses of blue light (465 nm), the bacteria generate green laser emission (～520 nm). Broad illumination of pump light yields simultaneous lasing over a large area in bacterial colonies.     

A population-level model from the microscopic dynamics in Escherichia coli chemotaxis via Langevin approximation

Recent extensive studies of Escherichia coli (E. coli) chemotaxis have achieved a deep understanding of its mi- croscopic control dynamics. As a result, various quantitatively predictive models have been developed to describe the chemotactic behavior of E. coli motion. However, a population-level partial differential equation (PDE) that rationally incorporates such microscopic dynamics is still insufficient. Apart from the traditional Keller-Segel (K-S) equation, many existing population-level models developed from the microscopic dynamics are integro-PDEs. The difficulty comes mainly from cell tumbles which yield a velocity jumping process. Here, we propose a Langevin approximation method that avoids such a difficulty without appreciable loss of precision. The resulting model not only quantitatively repro- duces the results of pathway-based single-cell simulators, but also provides new inside information on the mechanism of E. coli chemotaxis. Our study demonstrates a possible alternative in establishing a simple population-level model that allows for the complex microscopic mechanisms in bacterial chemotaxis.     

A Novel RBF Neural Network Training Methodology to Predict Toxicity to Vibrio Fischeri

Summary This work introduces a neural network methodology for developing QSTR predictors of toxicity to Vibrio fischeri. The method adopts the Radial Basis Function (RBF) architecture and the fuzzy means training strategy, which is fast and repetitive, in contrast to most traditional training techniques. The data set that was utilized consisted of 39 organic compounds and their corresponding toxicity values to Vibrio fischeri, while lipophilicity, equalized electronegativity and one topological index were used to provide input information to the models. The performance and predictive ability of the RBF model were illustrated through external validation and various statistical tests. The proposed methodology can be used to successfully model toxicity to Vibrio fischerifor a heterogeneous set of compounds.     

Microbial inactivation in cloudy apple juice by multi-frequency Dynashock power ultrasound

The study determined the efficacy of Dynashock wave power ultrasound as an alternative processing technique for apple juice against a number of pathogenic and spoilage microorganisms. The effects of several implicit, intrinsic and extrinsic properties on the Dynashock wave inactivation of the microorganisms were also investigated. Results showed that acid adaptation increased the resistance of Escherichia coli O157:H7 and Salmonella spp. but decreased that of Listeria monocytogenes. Spoilage yeast mixed inoculum composed of Debaryomyces hansenii, Torulaspora delbrueckii, Clavispora lusitaniae, Pichia fermentans and Saccharomyces cerevisiae was found to be more resistant than any of the adapted or non-adapted pathogens. Among the individual, acid-adapted E. coli O157:H7, the MN-28 isolate was found most resistant; while three other individual isolates had greater resistance than the composited E. coli inoculum. Increased in pulp content decreased the efficacy of Dynashock waves, but co-treatment with ultraviolet-C rays significantly enhanced inactivation in the cloudy apple juice. The results demonstrated the potential of Dynashock wave technology, together with other antimicrobial hurdles as alternative juice processing technique/s.     

Oxygen flux influence on the morphological, structural and optical properties of Zn1−xMgxO thin films grown by plasma-assisted molecular beam epitaxy

The Zn_1−xMg_xO thin films were grown on Al₂O₃ substrate with various O₂ flow rates by plasma-assisted molecular beam epitaxy (P-MBE). The growth conditions were optimized by the characterizations of morphology, structural and optical properties. The Mg content of the Zn_1−xMg_xO thin film increases monotonously with decreasing the oxygen flux. X-ray diffractometer (XRD) measurements show that all the thin films are preferred (0 0 2) orientated. By transmittance and absorption measurements, it was found that the band gap of the film decreases gradually with increasing oxygen flow rate. The surface morphology dependent on the oxygen flow rate was also studied by field emission scanning electron microscopy (FE-SEM). The surface roughness became significant with increasing oxygen flow rate, and the nanostructures were formed at the larger flow rate. The relationship between the morphology and the oxygen flow rate of Zn_1−xMg_xO films was discussed.     

Persistence of pierisin-1 activities in the adult cabbage white butterfly,Pieris rapae,during storage after killing

Crude extracts from larvae, pupae and adults of cabbage white butterflies, Pieris rapae and Pieris brassicae, and green-veined butterfly, Pieris napi, have an ability to induce apoptosis in the human cancer cell lines. As apoptosis inducing protein, pierisin-1 and -2 have been isolated from pupae of P. rapae and P. brassicae, respectively, and shown to exhibit DNA ADP-ribosylating activity. Although the highest activity was detected in the late phase of larvae and early phase of pupae, certain activity was found in adult butterflies. In order to investigate distribution of substances having pierisin-like activities in butterflies, many species need to be analyzed. However, fresh samples of larvae and pupae are hard to obtain, especially if samples are of scarce species or from overseas. The usage of adult butterflies is practical to examine the distribution of pierisin-like activity in many species. In this study, we examined the cytotoxicity of crude extracts from adults of P. rapae against HeLa cells and DNA ADP-ribosylation ability during storage for 1, 2 and 8 weeks at room temperature after killing adult butterflies after eclosion. Body weights decreased to 18% for 8 weeks through dehydration. Cytotoxicity of samples from butterfly kept for 1, 2 and 8 weeks decreased to 47, 39 and 22%, respectively, of the control value. DNA ADP-ribosylating activity of the samples also decreased to 30, 27 and 23%. Similar reduction was observed on western blot analysis with anti-pierisin-1 antibody. Fortunately, these results suggest that cytotoxic and DNA ADP-ribosylating activity persists to some extent in the body after killing, at least for 8 weeks. Thus, butterfly adult samples kept for two months at room temperature can still be useful for examination of the presence of substance having pierisin-like activity.